Dynamic and structural aspects of PEGylated liposomes monitored by NMR

Journal of Colloid and Interface Science 325 (2008) 485–493

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Dynamic and structural aspects of PEGylated liposomes monitored by NMR Cecília Leal a,∗ , Sibylla Rögnvaldsson b , Sigrid Fossheim b , Esben A. Nilssen b , Daniel Topgaard c a b c

Radiation Biology, Norwegian Radium Hospital, Montebello N-0310 Oslo, Norway Epitarget AS, PO Box 7159 Majorstuen, N-0307 Oslo, Norway Physical Chemistry 1, Lund University, POB 124, SE-221 00 Lund, Sweden

a r t i c l e

i n f o

a b s t r a c t Proton-detected NMR diffusion and 31 P NMR chemical shifts/bandwidths measurements were used to investigate a series of liposomal formulations where size and PEGylation extent need to be controlled for ultrasound mediated drug release. The width of the 31 P line is sensitive to aggregate size and shape and self-diffusion 1 H NMR conveys information about diffusional motion, size, and PEGylation extent. Measurements were performed on the formulations at their original pH, osmolality, and lipid concentration. These contained variable amounts of PEGylated phospholipid (herein referred to as PEGlipid) and cholesterol. At high levels of PEG-lipid (11.5 and 15 mol%) the self-diffusion 1 H NMR revealed the coexistence of two entities with distinct diffusion coefficients: micelles (1.3 to 3 × 10−11 m2 /s) and liposomes (≈5 × 10−12 m2 /s). The 31 P spectra showed a broad liposome signal and two distinct narrow lines that were unaffected by temperature. The narrow lines arise from mixed micelles comprising both PEG-lipids and phospholipids. The echo decay in the diffusion experiments could be described as a sum of exponentials revealing that the exchange of PEG-lipid between liposomes and micellar aggregates is slower than the experimental observation time. For low amounts of PEG-lipid (1 and 4.5 mol%) the 31 P spectra consisted of a broad signal typically obtained for liposomes and the diffusion data were best described by a single exponential decay attributed solely to liposomes. For intermediate amounts of PEGlipid (8 mol%), micellization started to occur and the diffusion data could no longer be fitted to a single or bi-exponential decay. Instead, the data were best described by a log-normal distribution of diffusion coefficients. The most efficient PEG-lipid incorporation in liposomes (about 8 mol%) was achieved for lower molecular weight PEG (2000 Da vs 5000 Da) and when the PEG-lipid acyl chain length matched the acyl chain length of the liposomal core phospholipid. Simultaneously to the PEGylation extent, selfdiffusion 1 H NMR provides information about the size of micelles and liposomes. The size of the micellar aggregates decreased as the PEG-lipid content was increased while the liposome size remained invariant. © 2008 Elsevier Inc. All rights reserved.

Article history: Received 7 April 2008 Accepted 24 May 2008 Available online 3 June 2008 Keywords: PEGylated liposomes PEG-lipid micelles Self-diffusion 1 H NMR 31 P NMR Drug delivery Ultrasound

1. Introduction The hydrophobic/hydrophilic versatility and biocompatibility of lipid molecules that readily form closed lipid bilayers with an aqueous interior (liposome) has made these materials one of the most studied systems for drug delivery. Liposomes form via noncovalent self-assembly rendering them relatively easy to tailor in order to meet certain physico-chemical properties like charge density, membrane “fluidity,” size, and others. However, it also means that the liposome, namely the lipid bilayer, is very sensitive to external conditions. One important example is their inconveniently short lifetime in the blood stream. There are ways to remedy some of these problems and all liposomal formulations intended for in vivo drug delivery need to comprise a number of stabilizers. Two

*

Corresponding author. Currently at Materials Department, University of California Santa Barbara, CA 93106, USA. E-mail address: [email protected] (C. Leal). 0021-9797/$ – see front matter doi:10.1016/j.jcis.2008.05.051

©

2008 Elsevier Inc. All rights reserved.

classical examples are the inclusion of cholesterol to reduce passive drug leakage [1] and polymeric lipids to increase the blood circulation time [2]. Even if the mechanisms involved in extended circulation times are somewhat unclear [3] most formulations nowadays include polymeric phospholipids, the most well known being phosphoethanolamines grafted with polyethylene glycol (PE-PEG). The amounts of PEG grafted phospholipids and the molecular weight of the PEG unit can be modulated to optimize the circulation time and as little as 2 mol% of PE-PEG (2000 Da) is enough to prolong the circulation time of liposomes [4]. Triggered drug delivery where the drug payload is only released at the required site has great advantages as the integrity of healthy tissue is maintained during treatment. In order to achieve this, drugs need to be incorporated in a carrier that accumulates at the diseased site and finally responds to external stimuli that trigger the release. The development of liposomal formulations exploits the responsive lipid phase behavior and liposomes sensitive to pH, temperature, and other physiological parameters [5] have

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been previously described. Ultrasound (US) has been suggested as an alternative method to trigger drug release [6]. The concept relies on liposomes being disrupted by US after accumulation at the target site, as well as acoustically induced increase in cell membrane permeability. Hence, the effect is twofold: improved drug release and intracellular uptake [7,8]. Nearly all studies including US sensitive carriers involve gas-containing particles which are too big to efficiently accumulate at the target site, but recently there has been an effort to tailor classical drug delivery particles to be US sensitive [9,10]. Interestingly, it has been demonstrated that the inclusion of PEG-lipid increases liposome sonosensitivity [11] and the authors have recently found out a synergistic interplay between PEG-lipid content and size [12]. Therefore, to maximize the sonosensitivity of the liposomal formulation the liposome size has to be controlled and the amount of PEG-lipid should be as high as possible. Moreover, the PEG-lipid should remain within the liposome membrane for a reasonable amount of time. However, PEGlipid is a micellar forming entity and only limited amounts can be incorporated within the liposome membrane [13]. Polymer chain interactions induce lateral pressure in the liposome bilayer and at some point the PEG-lipid departs from the liposome because it is energetically more favorable to form lipid/polymer-lipid mixed micelles. The threshold for the onset of micellization depends on the PEG-lipid acyl chain length [14] and on the PEG molecular weight [15,16]. The micellization is initiated by the formation of large discoidal micelles that become smaller as the PEG-lipid content is increased [17–19]. The fact that the PEG-lipid (similarly to common detergents) is able to partially solubilize the liposome, urges to control the level of PEG-lipid included in the formulation. The micellization process of conventional surfactants is extremely sensitive to minor changes in the external environment such as the presence of salts, buffers, co-surfactants, etc. [20]. One can anticipate that the micellization involving PEG-lipids is equally responsive to the same factors. Hence, it is important to evaluate the extent of PEG-lipid micellization in the formulations at their final state of concentration, pH and osmolality. A number of different methods have been employed to study PEG-lipid incorporation in liposomes including differential scanning calorimetry combined with dynamic light scattering [16,21], X-ray [22], electron spin resonance [14,15], amongst others. These methods require high concentration of the samples or the inclusion of probes in the system. Cryo-transmission electron microscopy is a method that has shown to be very useful for assessing the structural information of PEGylated liposomes, micelles and intermediate structures [17–19]. In this case, the observation of many micrographs is required to compensate for the poor sampling. In most cases a combination of techniques is required to asses both the size of the liposome and the efficiency of PEG-lipid incorporation. In addition, while structural studies in these systems are abundant, the dynamic aspects are scarce. NMR is a non-invasive method widely used within colloidal science conveying both structural and dynamic information at the molecular level [23]. Self-diffusion 1 H NMR makes use of the 1 H chemical shift resolution to assess the self-diffusion coefficients (and conversely the sizes) of different species in a mixture. Simultaneously, the fraction of components exhibiting a certain diffusion coefficient can be estimated. The method does not require any sample manipulation and the experimental time is on the minute scale. Within the field of drug delivery in particular, exploiting the fact that molecules incorporated in liposomes diffuse much slower than in solution, self-diffusion NMR has been used to evaluate the efficiency of drug incorporation [24] and vesicle membrane-water partition coefficients of anesthetics [25]. Liposomal formulations are usually composed of long chain saturated phospholipids with a high phase transition temperature (Tm); this imposes limitations on the resolution of the 1 H NMR spectra, however PEG-lipids yield intense resonances in the proton dimension making the diffusion

Fig. 1. Illustration of a liposome and a micellar aggregate with the self-diffusion coefficients differing in an order of magnitude: micelle (fast), liposome (slower) obtained in a 1 H pulse gradient spin echo (PGSE) NMR experiment. To the right is an illustration of 31 P NMR lineshapes typically obtained for micelles (narrow) and liposomes (broader).

experiment easy to perform [26]. Odeh and co-workers have been measuring the size of phospholipid liposomes by light scattering and diffusion NMR. The latter measurements were facilitated by the incorporation of species that “fluidize” the liposome membrane such as hydrotropes and/or PEG-lipids [27]. In this work we investigated a selection of liposome formulations at their original pH, osmolality and lipid concentration. The formulations are intended to be used in ultrasound mediated drug release for which liposome size and PEG-lipid partition between micelles and liposomes need to be controlled. Self-diffusion 1 H NMR simultaneously monitors size and PEG-lipid incorporation while 31 P NMR linewidths and lineshapes provide additional structural information. Fig. 1 illustrates the main features making the two NMR methods suitable for investigating the dynamic and structural features of PEGylated liposomes and/or micellar aggregates. The diffusion coefficient of micelles and liposomes typically differs by an order of magnitude [28], as for the 31 P NMR spectra, micellar aggregates give rise to substantial narrower lines when compared to those originating from liposomes. 2. Materials and methods 2.1. Materials Poly(ethylene glycol)-grafted phospholipids: 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- X ], where X is the molecular weight (M w ) of the polyethylene glycol moiety, were purchased from Genzyme Pharmaceuticals (Liestal, Switzerland). Polyethelyne glycols with M w of 5000 and 2000 Da are conjugated with lipid diacyl chain lengths of 16 (DPPE) and 18 (DSPE) carbons. Specifically: DSPE-PEG2000, DPPE-PEG2000, and DPPE-PEG5000. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was supplied by Genzyme Pharmaceuticals (Liestal, Switzerland). Cholesterol, HEPES, sodium azide, and sucrose were obtained from Sigma–Aldrich. The liposomes were passively loaded with a drug model substance calcein. Calcein was purchased by Sigma–Aldrich and used to prepare a 50 mM stock solution in HEPES (10 mM), sodium azide (0.02 wt% vol/vol) and sucrose resulting in a solution of pH 7.4 and osmolality 313 mosmol/kg. For reference material a PEGylated liposomal commercial product, Caelyx (ScheringPlough, US), was supplied from the pharmacy at the Norwegian Radium Hospital and used as received. 2.2. Liposome preparation and characterization Liposomes with a nominal lipid concentration of 16 mg/mL were prepared using the thin film hydration method [29]. In short,

C. Leal et al. / Journal of Colloid and Interface Science 325 (2008) 485–493

all lipid components were dissolved in chloroform/methanol (9:1 v/v ratio) and the solvents were slowly removed under reduced pressure in a rotary evaporator until a thin lipid film was deposited on the wall of a round-bottomed flask. The liposomes were passively loaded with calcein by hydrating the lipid film with the aqueous solution containing calcein at 65 ◦ C and allowing the swelling of the resulting liposome dispersion for at least 1 h at the above temperature. The liposome dispersion was then freezethawed three times. The liposomes were sized down to a target size of 85 ± 5 nm by sequential extrusion (Lipex, Biomembrane Inc., Canada) at 65 ◦ C through polycarbonate (Nucleopore) filters of consecutive smaller size. Extraliposomal calcein was removed by exhaustive dialysis against isosmotic 10 mM HEPES buffered sucrose solution (pH 7.4) containing 0.02% (w/v) sodium azide. The dialyzed liposomes were stored in the fridge protected from light. The concentration of the different components in the formulation as given in the text regard the nominal contents added during sample preparation. Liposomes were characterized with respect to particle size, pH, and osmolality. All the formulations were also tested with respect to US responsitivity and shelf-life stability (results not shown here). The average particle size (intensity weighted) and size distribution were determined by photon correlation spectroscopy (PCS) at a scattering angle of 173◦ and 25 ◦ C (Nanosizer, Malvern Instruments, Malvern, UK). Prior to sample measurements, the instrument was tested by running a latex standard (60 nm). For the PCS measurements, 10 μL of liposome dispersion was diluted with 2 mL 0.2 μfiltered isosmotic 10 mM HEPES buffered sucrose solution (pH 7.4) containing 0.02% (w/v) sodium azide. Osmolality was determined on undiluted liposomes by freezing point depression analysis (Fiske 210 Osmometer, Advanced Instruments, MA, US). Prior to sample measurements, a reference sample with an osmolality of 290 mosmol/kg was measured; if not within specifications, a three step calibration was performed. 2.3. Self-diffusion 1 H NMR Pulsed gradient spin echo (PGSE) experiments (Fig. 2) were performed on a Bruker DMX-200 spectrometer with a Bruker DIFF-25 gradient probe. For studies of liposomes and micelle diffusion, preliminary experiments were performed where the strengths G of δ = 4.1 ms gradient pulses were linearly incremented in 32 steps from 0% to the maximal value of 9.6 T/m. After this preliminary experiment, the gradient strength was set to evolve in 64 steps from 6 to 75% of the maximal value of 9.6 T/m. This was done in order to concentrate most data points in the region of diffusion coefficients that turned out to be of most importance. An effective diffusion time Δ − δ/3 of 23.0 ms results from τ1 = 5.7 and τ2 = 13.2 ms. Typical acquisition parameters were as follows: a 90◦ pulse length of 12 μs, a recycle delay of 1 s, and a spectral width of 3 kHz. 2.4.

31

P NMR 31

Static P NMR spectra with 40.7 kHz spectral width were acquired at 202.4 MHz 31 P resonance frequency (11.7 T) on a Bruker AVII-500 spectrometer using a Bruker broadband observe (BBO) probe. 8 k scans were accumulated using 9 μs 90◦ pulses, 4.5 μs receiver dead time and 4 s recycle delay. The temperature was controlled to 65 and 25.0 ◦ C with a precision of ±0.1. 30 Hz line broadening was applied prior to Fourier transformation. No 1 H decoupling was applied during 31 P acquisition. This results in an enhanced broadening of the liposome 31 P line which becomes useful when comparing it with the narrow signal arising from micellar aggregates.

487

Fig. 2. Pulse sequence for the PGSE experiment: A stimulated spin-echo is produced at 2τ1 + τ2 after three 90◦ pulses. A pair of gradient pulses with amplitude G and duration δ encodes the signal for molecular displacements. The effective diffusion time is Δ − δ/3, where Δ is the time between the onset of the gradient pulses.

2.5. Differential scanning calorimetry (DSC) DSC thermograms were acquired with a high-sensitivity differential scanning calorimeter VP-DSC (Microcal Inc., Northampton, MA), equipped with reference and sample cells of 0.5 mL. The solutions were degassed prior to use using a Nueva II stirrer (Thermolyne) and inserted in the cells with a Hamilton syringe. The scan rate used was of 60 ◦ C/h. For baseline subtraction a reference sample without liposomes was measured before the experiments. 3. Results and discussion In this section the results obtained for a series of liposomal formulations differing in nominal PEG-lipid content/type and also in cholesterol content are presented and discussed. For reference purposes a commercial product Caelyx and its “home made” analogue loaded with calcein were also tested. The section is outlined as follows: the self-diffusion 1 H NMR results are presented, followed by 31 P NMR lineshapes of some selected formulations at different temperatures. To facilitate the discussion of the NMR results, a DSC thermogram for a selected formulation is also shown. 3.1. Self-diffusion 1 H NMR Fig. 2 shows a schematic illustration of the pulsed gradient spin echo (PGSE) NMR pulse sequence used for measuring diffusion coefficients. During the duration of the first gradient pulse (δ) the nuclear spins are encoded with a phase shift. The magnetization will be equally rephased after the second gradient pulse if no diffusion occurs during the diffusion time (Δ). In case of diffusion events, the magnetization is incompletely rephased resulting in proportional echo attenuation. In a PGSE experiment, the decay of the echo intensity I relates to the isotropic diffusion coefficient D of the spin system, the longitudinal relaxation time T 1 , and transverse relaxation time T 2 as [30] I=

I0 2



exp −

2τ1 T2

−

τ2 T1

 − bD ,

(1)

where b = (γ G δ)2 (Δ − δ/3) and I 0 is the signal intensity after the first 90◦ pulse. By measuring I as a function of G (at constant values of τ1 , τ2 , δ , and Δ), the diffusion coefficient D is obtained by a non-linear fit to I as a function of b. Plotting log(I ) vs b results in a straight line of slope D, this is known as the Stejskal–Tanner plot. The liposome formulations are stored in the fridge prior to the diffusion experiments conducted at 25 ◦ C. Temperature fluctuations during the first gradient steps can cause variations in the shape of the 1 H lines. To avoid this problem, and given that the

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Table 1 Micelle and liposome diffusion coefficients ( D LIP , D MIC ) and diameters (dLIP , dMIC ). Liposome diameter obtained by PCS (dPCS ), PEG-lipid content (C PEG ) in the liposomes and fraction of micelles (χMIC ). For distributions of diffusion coefficients, D av is displayed between brackets   and the standard deviation in parenthesis ( ) (Eq. (4)). A: DPPE-PEG5000 B: DPPE-PEG2000 C: DSPE-PEG2000

(a)

(b) Fig. 3. (a) 1 H NMR spectra (25 ◦ C) of a liposomal formulation containing DSPC:DSPEPEG2000:cholesterol (7C-60:15:25 mol%) as a function of gradient strength (G ) from 0% to the maximal value (9.6 T/m) increased in 32 steps. Resolved resonances: water (4.7 ppm), sucrose (3.7 ppm), and the overlapping DSPC/DSPE-PEG peaks (3.5 ppm). After the first gradient pulses, all resonances decay quickly except for the DSPC/DSPE-PEG peak and a residual peak for water entrapped inside the liposomes. (b) 1 H NMR spectra (25 ◦ C) of a liposomal formulation containing DSPC:DSPE-PEG2000:cholesterol (7C-60:15:25 mol%) as a function of gradient strength (G ) from 6 to 75% of the maximal value (9.6 T/m) increased in 64 steps. A few initial 1 H spectra were removed due to uncertainties in the lineshapes during temperature equilibration. Only the water peak and the DSPC/DSPE-PEG resonance are present.

experiment was performed with a total of 64 steps, a few initial points were skipped for the fitting of the data. This procedure is equivalent to a few minutes of temperature equilibration prior to the actual experiment. Fig. 3a shows a series 1 H NMR spectra of a liposome composed of DSPC:DSPE-PEG2000:cholesterol at the nominal mole percentages of 60:15:25 (sample 7C in Table 1) as a function of gradient strength (G ) in the PGSE experiment. A few resonances can be resolved, three of higher intensity corresponding to water (4.7 ppm), sucrose (3.7 ppm) and a third at 3.5 ppm that corresponds to the ethylene oxides of the PEG groups in DSPE-PEG overlapping with the choline methyls of DSPC [31]. Here are shown spectra for a sample containing 15 mol% of DSPE-PEG; for samples containing lower amounts the integrated intensity of the resonance at 3.5 ppm is decreasing accordingly. Other resonances corresponding for instance to the methylenes groups of the phospholipid hydrocarbons chains are absent due to their short T 2 relative to the value of the echo delay used in the experiment (2 × τ1 = 11.4 ms). One can observe that the water resonance decays rapidly for the first set of G, this corresponds to the rapid water diffusion outside the liposomes. Interestingly, as G is increased further more, it is evident that the water resonance does not decay completely persisting almost until the end of the diffusion experiment. This corresponds to the diffusion of water entrapped inside the liposomes under a regime of water exchange that is longer than the diffusion time (23 ms). Slow water exchange across the liposome membrane is experienced when the latter is in the gel-state (rigid)

DSPC:PEG:Chol (mol%)

C PEG (mol%)

D LIP /1012 (m2 /s)

dLIP (nm)

dPCS (nm)

1A-78:4.5:17.5 2A-71:11.5:17.5 3A-63:4.5:32.5 4A-56:11.5:32.5 5A-67:8:25 6A-67:8:25 6B-67:8:25 7A-60:15:25 7B-60:15:25 7C-60:15:25 8A-52.8:40 8B-52:8:40 9A-52:8:10 9B-52:8:10 10A-74:1:25 10B-74:1:25 Caelyx-57:5:38 CC3-57:5:38

4.5 5.8 4.5 5. 8 <8 <8 <8 4.5 6.3 8.3 <8 <8 <8 <8 1 1 5 5

4.7 5 4.3 4.4 5.8 6.1 7.0 4.4 4.6 5.2 5.6 5.9 7.0 8.0 4.7 5.3 5.0 4.7

93 87 102 99 75 72 60 99 96 84 77 74 62 55 93 82 87 93

97 87 98 89 86 96 83 87 85 84 88 86 85 83 89 81 85 92

(0.4) (0.3) (0.5)

(0.3) (0.4) (0.6) (0.5)

D MIC /1011 (m2 /s)

dMIC (nm)

χMIC

1.3

34

0.5

1.3

34

0.5

1.9 3.0 1.9

23 15 23

0.69 0.58 0.45

at the experimental temperature. The resonance attributed to sucrose decays within the first values of G, as opposed to the peak assigned to DSPE-PEG/DSPC that decays slowly. In order to focus on the diffusion associated with the liposomes and not on other free components in solution the number of steps in the diffusion experiments was increased and the G was concentrated between 6–75% of the maximal value. As shown in Fig. 3b, the only remaining resonances for this diffusion experiment are the water resonance (4.7 ppm) and the DSPE-PEG/DSPC (3.5 ppm) reflecting the species with relatively slow diffusion incorporated within liposomes and/or micellar aggregates as will be presented below. To obtain the diffusion coefficients of each component with resolved resonances in Fig. 3b one can produce a Stejskal–Tanner plot as described above. The water resonance gives rise to a diffusion coefficient of 4 × 10−12 m2 /s corresponding to the diffusion of water essentially within the liposome interior. The focus of this work is however on the diffusion of the PEG-lipid component. While studying the US release of a number of liposomal formulations, we observed that the ones containing high amounts of PEG-lipid were very sonosensitive but displayed a dramatic decrease in US responsitivity during storage (results not shown). We attributed this to a loss of PEG-lipid from the liposome membrane over storage time. In order to investigate the extent of PEG-lipid incorporation in the liposomal formulations used for US release we performed diffusion experiments as described above on a collection of formulations where the contents of PEG-lipid and cholesterol were varied at 5 levels. For some selected formulations, other types of PEG-lipid (PEG M w and PE chain length) were also tested. The results are summarized in Table 1. Representative results at high, low, and intermediate contents of PEG-lipid are shown in the following sections. 3.1.1. High content of PEG-lipid (11.5 and 15 mol%) Fig. 4a shows the Stejskal–Tanner plot obtained for a sample containing 15 mol% of DSPE-PEG 2000 (sample 7C in Table 1). The individual data points are the diffusion data and the lines represent different fits to the experimental data: single exponential (dot line) and bi-exponential (full lines). It is clear that the data is better described by a bi-exponential fit, indicating that DSPE-PEG is associated with species of different diffusion coefficients. The fitting gives a fast diffusion coefficient of 1.8 × 10−11 m2 /s and a slower one of 5.2 × 10−12 m2 /s. The hydrodynamic radius ( R ) of a

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489

particle with a diffusion coefficient D in a free volume depends on the Boltzmann constant (k), the temperature ( T ) and the viscosity (η) according to the Stokes–Einstein equation as D=

(a)

(b)

(c)

Fig. 4. (a) Echo intensity vs b in the PGSE diffusion experiment for a liposomal formulation containing DSPC:DSPE-PEG2000:cholesterol (7C-60:15:25 mol%). The experimental data (individual data points) are plotted with a single-exponential fit (dot line) and a bi-exponential fit (full line). The echo decay is better described with the bi-exponential fit yielding a diffusion coefficient for micelles (fast, 1.9 × 10−11 m2 /s) and another one for liposomes (slow, 5.2 × 10−12 m2 /s). (b) Echo intensity vs b in the PGSE diffusion experiment for a liposomal formulation containing DSPC:DPPE-PEG5000:cholesterol (3A-63:4.5:32.5 mol%). The data points are described by a single-exponential fit (full line), for liposomes with diffusion coefficient of 4.3 × 10−12 m2 /s. (c) Echo intensity vs b in the PGSE diffusion experiment for a formulation containing DSPC:DPPE-PEG2000:cholesterol (8B-52:8:40 mol%). The single-exponential fit (full line) is not appropriate. A bi-exponential fit (dot line) with a small fraction (0.1) of fast decaying components (2.6 × 10−11 m2 /s) gives a reasonable fit but the best alternative is a log-normal distribution of the diffusion coefficients.

kT 6πη R

.

(2)

The two diffusion coefficients give rise to particles with average diameters of 23 nm (fast diffusion) and 84 nm (slow diffusion). We attribute the slow diffusion component to DSPE-PEG incorporated in the liposomes and the fast diffusion component to DSPE-PEG in the form of micelles. The average size of the liposome used in the experiment was measured by PCS to be 84 nm and the size of PEG-lipid micelles in aqueous solution can vary from 15 to 30 nm depending on the polymer size and content [32,33]. The fraction of liposomes is obtained from the fitting procedure and used to calculate the actual PEG-lipid content (CPEG in Table 1). The fraction of the fast diffusion component (micelles) was 0.45. This is assuming that T 1 and T 2 for the PEGylated part of the lipids are similar when present in micelles and liposomes. This is a reasonable assumption since the PEGylated part of the lipids senses the same relaxation environment whether is protruding out from a micelle or a liposome. In addition, T 1 and T 2 relaxation times for PEGylated aggregates were measured in a previous work to be longer than the delays τ1 and τ2 in the diffusion experiment [34]. If there is an associated error due to significant dissimilarities between the relaxation times it is towards an overestimation of the fraction of liposomes. It is noteworthy to mention that the bi-exponential decay reveals not only that micelles and liposomes are present in the sample but also that the exchange of PEG-lipid molecules between the two is slower than the diffusion time (23 ms). PEG-lipid is a micellar forming amphiphile that incorporates in the liposome at limited amounts. Normally the PEG-lipid is mixed with the other phospholipids during the lipid film preparation, however, even if to a lower extent PEG-lipid molecules can be incorporated after liposome preparation [17]. One could therefore expect that PEG-lipid molecules exchange rapidly in and out of micelles and liposomes. In samples containing species with different diffusion coefficients (e.g., micelles and liposomes), in the event of fast exchange between the two entities only a single-exponential echo decay can be detected in a diffusion NMR experiment. In the case of slow exchange within the observation time, the echo decay on the other hand is given by a sum of exponentials each describing the diffusion coefficient of a given entity. It is well reported that at a nominal concentration of DSPEPEG2000 of 15 mol%, the PEG-lipid component is partitioned between liposomes and micellar aggregates [2,3,13] and to this particular formulation we can estimate that the maximum incorporation efficiency of DSPE-PEG2000 in DSPC based liposome is 8 mol%. For all formulations containing nominal 15 or 11.5 mol% of PEGlipid, the Stejskal–Tanner plot revealed a bi-exponential decay indicating the presence of micelles and liposomes. However, the fraction and size of the micellar aggregates was different depending on PEG-lipid type (results displayed in Table 1). The diffusion coefficient of the micellar aggregates increases with PEG-lipid content (see sample 2A vs 7A in Table 1) and thus the micelles become smaller as the PEG-lipid content is increased. The diameter calculated by the diffusion coefficient drops 10 nm when the PEG-lipid content is increased from 11.5 to 15 mol%. For the samples 7A, 7B, and 7C we can observe that the micelles formed with DPPE-PEG5000 are larger than the ones formed with DPPE-PEG2000, which is of course due to the smaller polymer size in the latter case. Micelles of DSPE-PEG2000 are larger than the ones of DPPE-PEG2000 due to the longer chain length of DSPE. Most of the formulations were prepared using DPPE-PEG (2000 and 5000) mixed with DSPC and cholesterol, this choice of PEGlipid relates with other studies performed on this particular for-

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Fig. 5. DSC thermogram obtained for a formulation containing DSPC:DPPE-PEG5000: cholesterol (1A-78:4.5:17.5 mol%). The integration of the peak yields a T m of 51.4 ◦ C and H m = 7 kcal/mol. The peak is divided showing that the phospholipids in the PEG-lipid rich domains undergo the main transition at slightly lower temperatures than the domains poor in PEG-lipid.

mulation design. However, we wanted to evaluate the effect of matching the PEG-lipid chain length with the core PC of the liposome. Samples 7A, 7B, and 7C have the same mole composition (DSPC 60%, cholesterol 25%, and PEG-lipid 15%) but different PEG-lipids were used: A-DPPE-PEG5000, B-DPPE-PEG2000, and C-DSPE-PEG2000. We observe that the best efficiency of incorporation was obtained when the chain length of the PEG-lipid matched the one of the PC in the liposome. In this case, the incorporation was estimated to be 8.3 mol%. As described above, at these nominal PEG-lipid contents the DSPC liposomes containing DPPE-PEG (2000 and 5000) already showed signs of the first micellar aggregates. It is well know that the mixing of two phospholipid species is better achieved if the chain lengths match. The mixing of a 18 carbon chain length with a 16 carbon chain length is not totally efficient, in fact, there is a rather large area of phase separation in the temperature vs composition phase diagram [35]. The mixing is even less efficient at room temperature; therefore, it is natural that the DPPE-PEG molecules are more readily departing DSPC liposomes than DSPE-PEG. The incorporation of the DPPE-PEG5000 is also less efficient than DPPEPEG2000 [15]. The PEG chains experience van der Waals attraction forces inducing considerable lateral pressure in the liposome membrane. The longer the polymer chain, the stronger the lateral pressure and this may result in membrane phase separation into domains which are PEG-lipid rich and PEG-lipid poor [16,36]. Naturally, domain formation facilitates the departure of DPPE-PEG5000 from the liposome when compared to DPPE-PEG2000. To evaluate the situation of phase separation in the liposome membrane we conducted DSC on a DSPC:DPPE-PEG5000:cholesterol at the molar percentages of 78:4.5:17.5 (sample 1A in Table 1) which did not contain any micellar aggregates. The thermogram is shown in Fig. 5. The integration of the peak gives a transition temperature of 51.4 ◦ C and a melting enthalpy H m = 7 kcal/mol which is consistent for a DSPC membrane with some “impurities” (e.g., PEG-lipids) [35]. We also observe that the melting peak is split. This has been observed for other liposome formulations containing PEG-lipids with PEG of 5000 M w [36] and indicates that there is PEG-lipid segregations within the liposome membrane. The phase separation can occur in the bilayer with domains rich in PEG-lipid undergoing the phase transition at a slightly lower temperature than the domains poor in PEG-lipid. Alternatively, the segregation can occur with an uneven distribution of PEG-lipid molecules in the outer and inner monolayers constituting the liposome membrane, this would also result in a split DSC thermogram [37].

3.1.2. Low content of PEG-lipid (1 and 4.5 mol%) Fig. 4b shows the Stejskal–Tanner plot for a formulation containing DSPC:DPPE-PEG5000:cholesterol at the nominal mole percentages of 63:4.5:32.5 (sample 3A in Table 1). The individual data points are plotted with the best fit (full line). In this case, where the content of PEG-lipid is 4.5 mol% the individual data points can be very well fitted to a single exponential decay, giving a diffusion coefficient of 4.2 × 10−12 m2 /s corresponding to a liposome of 104 nm (the PCS data gives an average size of 98 nm). In this case, it is clear that all the nominal content of PEG-lipid could be incorporated in the liposome. This result was obtained for all formulations in the regime of low PEG-lipid content. A single-exponential decay was also obtained for Caelyx and its calcein-analogue-CC3. The efficiency of PEG-lipid incorporation seems not to be affected by the amount of cholesterol [17]. The CC3 liposome was also prepared without calcein to evaluate the possible interference of the dye in the diffusion experiments and exactly the same results were obtained for the liposome with and without calcein. 3.1.3. Intermediate content of PEG-lipid (8 mol%) Fig. 4c shows the Stejskal–Tanner plot for a formulation containing DSPC:DPPE-PEG2000:Cholesterol at the nominal molar percentages of 52:8:40 (sample 8B in Table 1). The data points are plotted together with a number of different fits: single exponential (full line), bi-exponential (dot line), and a log-normal distribution fit [38,39] (dash line). The single exponential fit adjusts poorly to the data. The best fit is either the bi-exponential alternative yielding a small fraction (0.1) of the fast decaying component (micellar aggregates) or the log-normal distribution fit. The latter case applies for polydisperse samples comprising particles with diffusion coefficients which are not dissimilar enough to be separated. The fitting procedure yields an average diffusion coefficient that is defined as



D av = D 0 exp

σln2 D 2

 ,

(3)

where D 0 and σln D are the mass weighted median diffusion coefficient and the standard deviation of the logarithm of the distribution coefficient, respectively. The standard deviation σD in diffusion coefficients is defined as the width of the distribution and is given by







1/2

σD = D av exp σln2 D − 1

.

(4)

The fitting procedure for this particular sample gives a D av = 5.8 × 10−12 m2 /s (equivalent to an average size of 75 nm), and a σln D = 0.4. Edwards and co-workers [17,18] report that when PEGlipid is included in liposomal formulations at increasing amounts, the onset of PEG-lipid micelle formation is characterized by the formation of PEG-lipid/PC discoidal micelles, and this can initiate already at 5–8 mol% depending on the type of PEG-lipid. The structural evolution of the aggregates formed with increasing PEG-lipid content can be summarized as: liposomes → discoidal micelles → spherical micelles and there is a region where liposomes and disks coexist. The average size of discoidal micelles can be half the size of the liposomes, and as the PEG-lipid content is further increased the disks become smaller. Ultimately at around 60 mol% of PEGlipid, one obtains particles of around 10 nm, which are attributed to be pure PEG-lipid micelles. The fact that the data can be fitted to a distribution of diffusion coefficients and/or to a bi-exponential fit with a small fraction of micelles is consistent with a sample where liposomes coexist with a small fraction of large (discoidal) micelles having a diffusion coefficient comparable to that of a liposome. For all samples at intermediate PEG-lipid content similar results were obtained. For all the samples containing A: DPPE-PEG5000 or B: DPPEPEG2000 (6A vs 6B, 8A vs 8B, and 9A vs 9B) the average diffu-

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491

(a)

(b)

(c)

(d)

Fig. 6. (a) 31 P NMR spectrum (30 Hz line broadening) obtained at 65 ◦ C (and 25 ◦ C in the inset) for a liposomal formulation containing DSPC:DPPE-PEG2000:cholesterol (7B-60:15:25 mol%). Narrow peaks at both temperatures indicate the presence of micelles. Two peaks, one for DPPE-PEG 2000 and another one for DSPC can be detected revealing that the micellar aggregates comprise both phospholipids. (b) 31 P NMR spectrum obtained at 65 ◦ C for a liposomal formulation containing DSPC:DPPE-PEG5000: cholesterol (1A-78:4.5:17.5 mol%). Only a broad peak for liposomes is present. (c) 31 P NMR spectrum obtained at 65 ◦ C for a liposomal formulation containing DSPC:DPPEPEG2000:cholesterol (6B-67:8:25 mol%). Only a broad peak for liposomes is present. (d) 31 P NMR spectrum obtained at 65 ◦ C for a liposomal formulation containing DSPC: DPPE-PEG2000:cholesterol at 15 mol% PEG-lipid (gray line) and 11.5 mol% of PEG-lipid (black line). The 31 P line for 11.5 mol% is wider than that for 15 mol% of PEG-lipid, this is consistent with smaller micelles in the latter case.

sion coefficient is higher in the case of samples containing DPPEPEG2000 or, conversely, the average size is smaller than in the case of DPPE-PEG5000 containing samples. This further confirms that at 8 mol% of PEG-lipid the samples may contain a small fraction of micelles and the average size is smaller for the case of samples with DPPE-PEG2000 because the micelles are smaller. The size of the liposomes estimated by diffusion NMR and by PCS are generally in good agreement, the discrepancy in a few cases was around 10 nm. Small differences in the average sizes calculated with scattering methods and NMR have been observed for other liposomal systems and is related to how the average sizes are estimated [40]. 3.2.

31

P NMR

Phospholipids arranged in a bilayer structure exhibit characteristic 31 P chemical shift anisotropy (CSA) powder patterns. The lineshape of 31 P NMR spectra of phospholipid bilayers is determined by the principal elements of the CSA tensor, and by the orientation of phospholipid molecules relative to the applied magnetic field. The tensor is affected by molecular and intramolecular motional averaging. For 31 P spectra of phospholipids one normally refers to the CSA as

σ = σ − σ⊥ ,

(5)

where σ and σ⊥ are the values of the 31 P shielding of the bilayers oriented parallel or perpendicular relative to the magnetic field. This results in a powder 31 P NMR spectrum exhibiting a characteristic lineshape with a high-field peak and low-field shoulder. In liquid ordered phospholipid bilayers, where reorientation around the molecular axis is the only mechanism of averaging, σ is approximately 100 ppm [41]. For the same phospholipid in the liquid disordered phase, where the molecular axis is rapidly moving with respect to the bilayer normal, σ is reduced about 50%. Diffusion along a curved bilayer leads to further averaging of the 31 P CSA tensor and for phospholipid vesicles in the liquid disordered state bigger than 200 nm one typically obtains σ ∼ 40 ppm [42,43]. The 31 P NMR signal of phospholipids depends on the size of the molecule and the molecular order in the particle in which the molecule is embedded. The CSA of phospholipids in small bilayer liposomes is further averaged by tumbling of the entire aggregate to about 10 ppm. If no decoupling is applied during 31 P acquisition the liposome line can be wider than this due to 1 H dipolar couplings. For phospholipids in micellar aggregates fast isotropic averaging yields narrow symmetric spectra [44]. 31 P NMR can be used to distinguish micellar and liposomal aggregates and relies on the fact that a bigger entity (e.g., a liposome) produces a wider 31 P NMR signal than a smaller micellar particle [45].

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Selected formulations at low, high, and medium content of PEG-lipid were assessed by 31 P NMR above the transition temperature (65 ◦ C) and also at 25 ◦ C. Fig. 6a shows the 31 P NMR spectrum obtained for a sample with high PEG-lipid content containing DSPC:DPPE-PEG2000:cholesterol at the nominal mole percentages of 60:15:25 (sample 7B in Table 1) measured at 65 ◦ C. Two narrow peaks and a lower field bump can be detected. The narrow peaks originate from phospholipids embedded in small aggregates and the broader bump arises from phospholipids within larger aggregates. The 31 P CSA for phospholipids at temperatures below the phase transition temperature ( T m ) is broader compared to above T m . Micellar phospholipids are however unaffected by this, as in micelles the acyl chains are disordered at temperatures even below room temperature [16,46] and there is fast diffusion along the aggregate surface. The inset in Fig. 6a shows the effect of lowering the temperature on the same sample. Clearly, the sharp peaks are unaffected by lowering the temperature from 65 to 25◦ C, as opposed to the lower-field bump that gets broader. As presented above in the diffusion data, for high PEG-lipid contents, the sample comprises liposomes and micelles so the sharp peaks arise from phospholipids within the micelles and the broader signal is attributed to liposomes. Free PEG-lipid monomers in solution could also give rise to sharp lines but in these samples the PEG-lipid concentration is 1000× higher than the PEG-lipid critical micellar concentration (CMC ≈ 1 μM) [47] so it is not likely that there is a large amount of free monomers in solution. The fact that there are two narrow signals further indicates that there are two phospholipid species in the micellar aggregate. In this system we have two phospholipid entities: DSPC and DPPE-PEG2000. The assignment of the peaks is however uncertain because the spectra of the phospholipids are a result of motional averaged CSA rather than isotropic chemical shifts. In addition, the CSA tensors of the phosphate groups of phosphatidylethanolamine and phosphatidylcholine are similar [48]. However, liquid crystalline bilayers of pure phosphatidylethanolamine display a smaller CSA than phosphatidylcholine [49] and, as a result, a mixture of these two phospholipids can be resolved by a few ppm difference. Here the higher field peak is attributed to DSPC and the peak a few ppm lower to DPPE-PEG2000 [26]. Clearly the micellar aggregates comprise both DSPC and DPPE-PEG2000 [2,17]. Fig. 6b shows the 31 P NMR obtained at 65 ◦ C for a low PEG-lipid content sample containing DSPC:DPPE-PEG5000:cholesterol at the nominal mole percentages of 78:4.5:17.5 (sample 1A in Table 1). In this case, no sharp peaks due to micelles could be detected and only the motional averaged CSA for liposomes smaller than 200 nm [44] is observed. The spectrum obtained for a sample having intermediate amounts of PEG-lipid, specifically DSPC:DPPE-PEG2000:cholesterol at the nominal mole percentages of 67:8:25 (sample 6B in Table 1) is shown in Fig. 6c. Also in this case, no micellar peaks were observed. This corroborates the diffusion data suggesting that at the intermediate levels of PEG-lipid the fraction of micellar aggregates is low and the size is not small enough to give rise to a narrow isotropic signal. Large discoidal micelle tumbling is similar to that of a liposome resulting in similar lineshapes for both aggregates. As suggested by Edwards and co-workers [17,18], the first micelles appearing are big discoidal PC/PEG-lipid micelles and their size is progressively reduced as the PEG-lipid content is increased. Fig. 6d shows the spectra obtained for two formulations comprising both micellar and liposomal aggregates: one containing 11.5 mol% of DPPE-PEG2000 and another one containing 15 mol%. Unfortunately, the spectra obtained lack the desired signal-to-noise ratio. Nevertheless, one can observe that at 11.5 mol% DPPE-PEG2000 the micellar peaks are wider than at 15 mol%, suggesting that the higher the PEG-lipid content, the smaller the micelles are. This was also observed by self-diffusion NMR.

4. Concluding remarks Proton-detected NMR diffusion and 31 P NMR chemical shifts/ bandwidths measurements were successfully employed to study the structure and dynamic properties of a collection of liposomal formulations containing variable amounts of DSPC, PEG-lipid, and cholesterol. The size and PEGylation of the liposomes need to be controlled for successful use in ultrasound drug delivery. DiffusionNMR was demonstrated to be a quick, non-invasive method to simultaneously determine the extent of PEG-lipid incorporation and liposome size. Advantageously, the measurements could be done at the formulation’s original conditions of composition and concentration. Additional structural information was obtained by 31 P NMR. Liposomal formulations are normally manufactured with long chain saturated phospholipid rendering the liposomal membrane rather rigid at room temperature. This can limit the resolution of the 1 H NMR spectrum and diffusion experiments become difficult to execute. This is not the case if the liposome contains PEG-lipids. For all the formulations tested here the PEG-lipid moiety gives rise to sharp resonances on the 1 H NMR spectrum and the self-diffusion 1 H NMR experiment could be therefore easily conducted. For formulations containing high nominal concentrations of PEG-lipid (11.5 and 15 mol%) diffusion coefficients (and fractions) of micelles and liposomes could be measured. This entails important structural and dynamic aspects. Specifically: (i) we observed that the fraction of PEG-lipid residing in the liposome is roughly half of the nominal content and (ii) the fact that two diffusion coefficients could be resolved indicates that during the observation time (Δ = 23 ms), there is slow exchange of PEG-lipid molecules between micelles and liposomes. The diffusion coefficients allow for the calculation of liposome and micelle size. The liposome size is in good agreement with the one obtained by scattering methods. The micelles are larger in size the lower the amounts for PEG-lipid while the liposome size remains invariant. For intermediate nominal amounts of PEG-lipid (8 mol%), the diffusion data were better described by a log-normal distribution of diffusion coefficients and at this regime it is assumed that the first micelles start to appear in coexistence with the liposomes. For low PEGlipid contents (1 and 4.5 mol%) a single-exponential echo decay was obtained yielding diffusion coefficients characteristic of exclusively liposomes. The presence of micelles for high contents of PEG-lipid could also be detected by the presence of narrow 31 P NMR lines. Two sharp peaks (one for DSPC and one for PEG-lipid) could be detected, indicating that the micellar aggregates were composed of DSPC and PEG-lipid. For intermediate and low contents of PEGlipid no micellar peaks could be detected. The PEG-lipid incorporation is most efficient (maximum of ∼8 mol%) when the acyl chain length of the PEG-lipid matches the one of the core liposomal phospholipid. This is due to the fact that, especially at room temperature, the mixing of two phospholipids with a slight difference in chain length is suboptimal. Thus, if the acyl chain length of the PEG-lipid is shorter than the core phospholipid in the liposome, the incorporation is less efficient. PEG-lipids with larger PEG M w are also incorporated in the liposome but at lower extent. For high PEG M w (e.g., 5000 Da) the attractive van der Waals interactions between polymer chains induce segregation in PEG-rich and PEG-poor domains facilitating PEG-lipid depletion from the liposome membrane. Acknowledgments This work was funded by the Norwegian Research CouncilNANOMAT programme (C.L., S.R., S.F., and E.N.) and the Swedish research council (D.T.).

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