Characterization of a polymer-stabilized liposome system

Reactive & Functional Polymers 48 (2001) 181–191 www.elsevier.com / locate / react

Characterization of a polymer-stabilized liposome system N. Heldt a , M. Gauger a , J. Zhao a , G. Slack b , John Pietryka b , Y. Li a , * a

Department of Chemistry and Center for Advanced Materials Processing, Clarkson University, Potsdam, New York, NY 13699 -5810, USA b DuPont Pharmaceuticals Company, Medical Imaging, 331 Treble Road, N. Billerica, MA 01862, USA Received 1 October 2000; received in revised form 19 February 2001; accepted 10 March 2001

Abstract Polymer-stabilized liposome systems and non-polymer-stabilized (conventional) liposome systems were investigated for particle size, lipid association, micropolarity, and migration of a fluorescent probe between lipid bilayers. The size difference between the two types of liposome systems prepared with identical procedure was determined using light scattering method. NMR spectroscopic analysis using deuterium-labeled lipids showed that under the set conditions all lipids in both polymer-stabilized and conventional liposome systems become associated. Environmental micropolarity assessment of pyrene showed that the probe localizes within the lipid bilayers of both polymer-stabilized and conventional liposome systems. Further investigation revealed that the bilayers of both systems were mobile enough to permit migration of the probe into neighboring unlabeled liposome bilayers.  2001 Published by Elsevier Science B.V. Keywords: Micropolarity; Excimer; Pyrene; Particle size; NMR

1. Introduction The pharmaceutical industry utilizes liposomes for a variety of applications, such as drug delivery [1–3]. In this capacity, liposomes can serve as carriers for compounds to enter the body and also as protective shells around these compounds to prevent their degradation upon entry into the body’s environment [4]. The chemical properties that lend liposomes to use in drug delivery include osmotic activity, membrane permeability, solubility, hydrodynamics of membranes, and the ability to form various self assembled aggregates in solution [3]. In *Corresponding author. Fax: 11-315-268-6610. E-mail address: [email protected] (Y. Li).

order to have effective pharmaceutical applications, liposome systems must be relatively stable [5] and the characteristics of the systems (i.e. size, shape, polydispersity, and dynamic properties) must be known [6]. Liposomes are generally formed with amphiphilic lipids, often phospholipids [7]. These lipids have two main sections: a nonpolar tail, often a long hydrocarbon chain, and a polar head group [3]. In aqueous media, these lipids arrange themselves according to hydrophobic / hydrophilic interactions to form bilayered vesicles [3,7,8] (Fig. 1). These vesicles are lined on both the outer and inner surface with lipid polar head groups, making the liposome ideal for carrying water-soluble substances in their inner aqueous compartments, and retaining lipid-solu-

1381-5148 / 01 / $ – see front matter  2001 Published by Elsevier Science B.V. PII: S1381-5148( 01 )00055-4

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Fig. 1. Conventional bilayer liposome (drawing not to scale).

ble or amphiphilic compounds in their bilayer walls [9,10]. Fig. 1 shows a conventional liposome. As these liposomes are cell-like in nature, but not biosynthetic, the body’s immune system regards these vesicles as foreign bodies. Degradation of the vesicle can also occur upon mere movement through the harsh conditions of the body’s inner environment [9]. Liposomes previously used in drug delivery studies had the disadvantage of lasting only a few minutes in the body before the blood and immune systems destroy them [3,9,10]. In order to combat this potential problem and enhance the lifetime of a liposome in the body, liposomes can be sterically stabilized through the addition of a polymer, such as polyethylene glycol (PEG) [11–13]. One way to accomplish this is to add lipids with polymer attached to the polar head groups [3] (Fig. 2). These sterically-stabilized liposomes seem to tolerate the harsh conditions of the body much better than conventional liposomes [14]. A sterically-stabilized liposome displays the characteristics of a conventional liposome. However, the addition of polyethylene glycol chains attached to the polar heads of the lipids creates a ‘brush border’ around the liposome

Fig. 2. Sterically-stabilized liposome (drawing not to scale, the lipid bilayer is enlarged to show detail).

that acts as a barrier to protect the liposome from immunal attack [3,9,15]. The main objective of this research is to characterize a polymer-stabilized system for its similarities and differences comparing to a nonpolymer-stabilized (conventional) liposome system, in terms of relative size, lipid association, and dynamic properties. More specifically, two model systems were investigated: one system composed of lipids with polyethylene glycol chains attached to the polar head groups, and the other system composed of lipids without polyethylene glycol chains. This paper will describe how polymer-stabilized systems differ in particle formation, but correspond in complete lipid association and lipid mobility to conventional liposome systems prepared in the same manner.

2. Experimental section

2.1. Liposome preparation The lipids used in this experiment were: 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), purchased from Lipoid GmbH (Ludwigs-

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hafen, Germany); 1,2-dipalmitoyl-sn-glycero-3phosphate monosodium (DPPA), purchased from Avanti Polar Lipids, Inc. (Alabaster, AL); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine monosodium salt (DPPE), purchased from Sygena, Inc. (Cambridge, MA); and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamineN-[methoxypoly(ethyleneglycol)5000] monosodium salt (DPPE-MPEG), purchased from Shearwater Polymers, Inc. (Huntsville, AL). All lipids were stored at 2 208C. Polymer-stabilized liposome systems (PSLS) composed of phospholipids with polyethylene glycol chains attached to the polar head groups were prepared by dissolving a blend of DPPC, DPPA, and DPPE-MPEG (9:1:1 molar ratio) in propylene glycol, sonicating at 698C, and then mixing with a solution of glycerin in 0.9% aqueous NaCl. Liposome systems without polyethylene glycol chains (NPSLS) were prepared in the same manner as the PSLS, except DPPE was used in place of DPPE-MPEG.

2.2. Particle size analysis The PSLS and NPSLS were prepared and then extruded with Avanti  mini extruder by passing the systems through a 0.1 mm Whatman Nucleopore  polycarbonate membrane 10 times each at 65628C. All samples were analyzed for average particle size using Brookhaven Instrument Systems Quasielastic Light Scattering (QELS). This system consists of a water-cooled argon ion laser light source (Model 85, Lexel Laser) that operated at a wavelength of 514.5 nm. The sampling set-up consists of a BI-DS photomultiplier tube, a stepper-motor-controlled goniometer (BI-2000SM) and a BI-2030AT digital correlator. The QELS was performed at a 908 angle while the sample was maintained at a constant temperature of 23.060.58C. The cumulants method of the experimental autocorrelation functions was used to determine the average particle diameter: ln [g1 (t )] 5 2 K1t K2 (t / 2!), where t equals the mean decay constant, and the cumulants K1 and K2 represent

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the average diameter of the particle and the relative width of the particle distribution, respectively.

2.3. D2 O NMR experiments A control sample was prepared by adding 3 ml of chloroform to 1.2 mg of deuterated DPPC (L-a phosphatidylcholine DI(palmitoyl — d31)), purchased from Sigma (St. Louis, MO) and placed in a second 10-mm NMR tube. A small capillary tube (1.5–1.8390 mm) was filled with 0.9 ml of D 2 O and enough HPLCgrade water to fill the capillary tube to the same level as that of the liquid in the 10-mm NMR tube. The capillary tube was then placed inside the NMR tube with the deuterated DPPC in chloroform. A 1-D spectrum was run on a Bruker AC250 NMR to produce the two peaks, one from the D 2 O and the other from the deuterated DPPC in chloroform. A deuterated PSLS was prepared by adding 1.2 mg of deuterated DPPC to a blend of DPPEMPEG and DPPA in propylene glycol. The lipid mixture was sonicated at 698C until clear, added to a solution of glycerin in 0.9% aqueous NaCl, and then placed in the third 10-mm NMR tube. A second capillary tube containing 0.9 ml D 2 O and enough HPLC-grade water to fill to the same level as that of the liquid in the NMR tube was placed inside the NMR tube with the deuterated PSLS. A 1-D spectrum was run and the peaks generated were compared to the control. A deuterated NPSLS was prepared in the same manner as the deuterated PSLS described above, except DPPE was used instead of DPPEMPEG in the lipid blend. A 1-D spectrum was run and the peaks generated were compared to the peaks produced from the deuterated PSLS and the control.

2.4. Use of a fluorescence probe Samples were prepared by injecting 25 ml of 10 23 M of pyrene in methanol into control vials

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containing 1.5 ml of the saline–glycol mixture, PSLS vials containing 1.5 ml of the prepared PSLS, and NPSLS vials containing 1.5 ml of the prepared NPSLS and allowing samples to sit for at least 10 min. For each sample set, dilutions of 23 (v / v) and 43 (v / v) were prepared using 0.9% aqueous NaCl. Mixed systems were prepared by combining equal volumes of pyrene-labeled and unlabeled PSLS, as well as equal volumes of pyrenelabeled NPSLS mixed with unlabeled NPSLS. All samples were analyzed for fluorescence at room temperature (228C) over a 48-h period using a Perkin Elmer  Luminescence Spectrometer LS50B. Emission spectra for intensity peaks I, III, and V, at wavelengths of ca. 372, ca. 385, and ca. 395 nm, respectively, and the excimer peak at wavelength ca. 470 nm were recorded. Peak ratios III / I and excimer / monomer (Ex / M) were then calculated from spectral data. The settings used on the fluorescence machine were as follows: range: 350–600 nm, emission: 600 v, excitation: 340 nm, excitation slit: 4.0 nm, emission slit: 2.5 nm.

3. Results and discussion

3.1. Particle size analysis Size and size distribution of liposomes are essential parameters for evaluating the integrity and usefulness of a liposome system. It has been shown size influences blood clearance and the elimination process of liposomes [3]. Larger multilamellar conventional liposomes are removed from the blood in two phases due to their heterogeneity [3]. The larger multilamellar liposomes are removed at a faster rate by macrophages, while the smaller liposomes are removed not only by macrophages, but also at a slower rate by extravasation into surrounding tissue. For PEGylated liposome systems, the rate of removal is not affected by the presence of macrophages due to the protective PEG border [4]. Therefore, it is important to develop

a system that has a known particle size and narrow size distribution. For a heterogeneous liposome system, accurate determination of size and size distribution of all components can be difficult. One of the most acceptable techniques for particle size and size distribution determination is quasi-elastic light scattering (QELS) [5,16]. The QELS used in this study is capable of analyzing the fluctuations arising from the Brownian motion of submicron particles using photon correlation spectroscopy to give mean diameter and size distribution [17]. Two liposome systems were prepared in the same manner as described in the Experimental section and denoted as conventional non-polymer-stabilized liposome system (NPSL) and polymer-stabilized system (PSLS). Figs. 3 and 4 show the particle size distribution for each system. The average particle sizes from multiple runs were used to generate the size distribution for each type of liposome system. The PSLS prepared from a blend of DPPC, DPPE-MPEG, and DPPA in an aqueous mixture of saline, propylene glycol, and glycerin ranged in size from ca. 80 to 240 nm, with the largest percent of the samples having a size distribution between 121 and 140 nm. The experimental results on PSLS particle sizes is consistent with the structure illustrated in Fig. 2. It is estimated that a fully stretched PEG chain is 80 nm in length according to a simple molecular mechanic calculation (Spartan 5.0, Wavefunctions, CA). The bilayer is typically sized at 4–5 nm. Assuming a random coiling would reduce the length of the PEG chain to 1 / 4 of its fully stretched distance (i.e. 20 nm), a typical PSLS would have a minimum diameter of 88 nm depending upon the construction of the inner core, the amount of PEG chain and the extent of the PEG chain folding. Comparing this size distribution with that of the NPSLS (Fig. 4), the size distribution for the NPSL is much wider and unevenly distributed. The NPSLS prepared for this experiment had a greater variable size distribution, with larger particles dominating the sample size. Measurements made using QELS have the disadvantage

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Fig. 3. Particle size distribution for polymer-stabilized liposome vesicles prepared from a lipid blend of DPPC, DPPE-MPEG, and DPPA in an aqueous mixture of 0.9% NaCl, propylene glycol, and glycerin at 23.060.58C.

Fig. 4. Particle size distribution for conventional (non-polymer-stabilized) liposome systems made from a lipid blend of DPPC, DPPE, and DPPE in an aqueous mixture of 0.9% NaCl, propylene glycol, and glycerin at 23.060.58C.

of favoring the larger particles in a mixed system [17]. Therefore, the size distribution may be distorted, with smaller particles being discriminated against. The large particles depicted in Fig. 4 (from 360 nm and up) may not be as abundant as they appear in respect to the

smaller particles. However, it is evident that larger particles were more likely to form from the NPSLS than the PSLS prepared for this experiment. This may originate from the fact that conventional liposomes are formed from multilamellar vesicles or sheets of liquid crys-

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tals. The experimental condition and procedure may play a significant role in the formation kinetics and the size of the liposome. For PSLS, however, it appears that the presence of the PEG chains in the PSLS determined the relative size of the PEGylated liposomes. The experimental procedure seems to have much less impact on the final product formation. One possible explanation is that the PEG chains

of the lipid molecules pre-associate to form a polymer aggregate, which serves as the core for the liposome. The additional non-PEGylated lipid molecules then adsorb onto the droplet. A complementary lipid layer then forms outside of this droplet as shown in Fig. 5. Therefore, the size of the liposome is mainly determined by the length of the PEG chains and the relative molar ratio of PEGylated lipid to non-

Fig. 5. Formation of polymer-stabilized liposomes. (a) Formation of polymer aggregate from PEGylated lipids followed by (b) the incorporation of non-PEGylated lipids. (c) Formation of the lipid bilayer from free lipids.

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PEGylated lipid molecules. In other words, the size difference between PSLS and NPSLS originates from the formation mechanism.

3.2. Determination of free lipids using D2 O NMR The key objective of this experiment is to determine the proportion of free lipids and associated lipids in a liposome system. This is significant, because the amount of free lipids yields information on the equilibrium between associated lipid and free lipids and the amount of PEGylated lipid needed for the formation of a polymer-stabilized liposome to keep the ‘stealth’ property. In addition, the amount of dissolved PEGylated lipid may be influenced by the presence of less polar solvent, such as glycols and glycerol. Firstly, deuterated lipids are placed in a solvent that prevents the lipids from associating (aggregating into liposomes). NMR is used to produce a 1-D (standard) spectrum of the free lipids. The spectrum produced by the free deuterated lipids in chloroform (control) can be compared against the spectrum produced by the same quantity of deuterated lipids in a liposome medium. The difference in the two spectra will represent the difference in the amount of lipids that is tied up in the liposome. Deuterated lipids in the liposomes will not give a signal because the liposome structure is too rigid; the relaxation times for the deuterated lipids in the liposomes would be too brief to produce a significant peak [18,19]. The signal produced by an internal standard of deuterium oxide (D 2 O) is used to integrate the signal produced by deuterated

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lipids in solution. For each individual sample, the integration of the signal produced by the internal D 2 O standard is compared to that produced by the free deuterated lipids in solution to produce a ratio. The ratios in each of the lipid samples can be compared against that of the control sample to determine the percentage of free lipids in solution in each of the different types of samples. Table 1 lists the signal peak values for deuterium oxide (D 2 O) and deuterated DPPC in the control sample, the PSLS, and the NPSLS. In all NMR spectra, D 2 O provided a large signal peak at 5.0 ppm, while the deuterated lipids produced a smaller signal at 1.2 ppm. The sample containing free deuterated DPPC in chloroform (the control) was used to set the integral of D 2 O at 1.00. In reference to the amount of deuterium in the insert, only 7% of the total amount of D 2 O is in the deuterated lipids (Table 1). Any change in the integral of the deuterated lipid signal corresponded to a change in the amount of lipids that are free (unassociated). When the value of the integral of the deuterium in the NPSLS was set at 1.00, it was found that the amount of deuterium in the deuterated lipids was nearly zero. Considering the fact that the minimum micelle concentration of these lipids are very low, it is not surprising that nearly all deuterium-labeled lipids are in associated structures. Taking into account the detection limit of the NMR technique, the amount of the free lipids must be well below micromolar concentration. The presence of long PEG chains and the high concentration of glycerol and glycol in the matrix may lead to a

Table 1 Percent of free deuterated DPPC in non-polymer-stabilized systems (NPSLS), polymer-stabilized liposome systems (PSLS) with polyethylene glycol chains, and a control solution of unassociated deuterated lipids Sample

D 2 O integrated signal value

Deuterated DPPC integrated signal values (60.01)

% Free lipid (61.0)

Control Deuterated NPSLS Deuterated PSLS

1.00 1.00 1.00

0.07 0.00 0.00

7.0 0 0

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significantly higher level of lipid dissolution into the matrix. When the value of the integral of deuterium was set at 1.00 for the deuterated PSLS, the integral of the deuterium in the free lipids was also close to zero. This clearly shows that even for the PSLS, the dissolution of the lipids is limited.

3.3. Characterization of hydrophobic domains using a fluorescent probe Fluorescence results from photon excitation of a fluorophore (usually polyaromatic hydrocarbon, such as pyrene) to its singlet state S 1 . This singlet state lasts for a brief period of time (10 26 –10 29 s [20]) before the energy is emitted and the fluorophore is returned to its ground state S 0 . The energy emitted is longer in wavelength and lower in energy than the excited state. This energy difference is called a Stokes Shift [21]. Some fluorophores, such as pyrene, can form dimers (excimers) in the excited state, which produce red shifted emission peaks (ca. 470 nm) [20,22]. The purpose of this experiment is to use a fluorescent probe, pyrene, to characterize the hydrophobic domains in a regular and polymerstabilized vesicle. The use of such a probe can serve as a model for hydrophobic drug delivery. In addition, the probe also helps to assess the mobility of a guest molecule in liposome systems with and without polyethylene glycol chains. The mobility of the guest molecule within a liposome corresponds to the mobility of the lipids within the liposome. Pyrene was used to label both liposome systems. The micropolarity and the local concentration were determined from the peak III / I and Ex / M ratios, respectively. It has been shown that variations in the polarity of solvents relates to changes in the vibronic band spectra (intensity variations of symmetry-forbidden bands) of pyrene, also known as the HAM effect [23–26]. These spectral changes are due to a reduction in symmetry when pyrene is perturbed by surrounding solvent molecules to form ground state

complexes with polar solvents [22,27–29]. Low peak III / I ratios indicate polar micro-environments whereas high peak III / I ratios indicate non-polar microenvironments. When a pyrene molecule is excited, it will fluoresce and emit two types of signals. One set of peaks originated from excited monomers. The broad peak that forms in the red zone is the result of pyrene– pyrene* complexes (pyrene dimers) that formed. If the excimer peaks are compared with the monomer peaks, the relative local concentration of pyrene can be determined [22,30–32]. If the excimer / monomer (Ex / M) ratio is relatively high, this indicates that there are more pyrene molecules in close proximity to one another. Therefore, lower Ex / M ratios would indicate that pyrene molecules are more dispersed in a system. From the micropolarity and the local concentration, one can determine the localization of pyrene in the liposomes. Table 2 shows peak III / I ratios over a 48-h period for the bulk solution, PSLS, and NPSLS that were injected with pyrene and diluted to various factors. Peak III / I ratios indicate environmental micropolarity of pyrene [29]. The peak III / I ratios for the PSLS and the NPSLS range from 0.77 to 0.83. This range indicates that pyrene is in a relatively nonpolar environTable 2 Micropolarity upon dilution: fluorescent emission spectral ratios III / I for pyrene-labeled non-polymer-stabilized (NPSLS) and polymer-stabilized (PSLS) liposome system with polyethylene glycol chains at various dilution factors. Dilutions were prepared using 0.9% aqueous NaCl and run at 228C. (All data reflect the results from more than five measurements, the experimental error is estimated at 60.04) Sample conditions Bulk solution

NPSLS

PSLS

Time

Peak III / I

(h)

No dilution

23 dilution

43 dilution

0 24 48 0 24 48 0 24 48

0.66 0.62 0.65 0.83 0.77 0.76 0.79 0.79 0.80

0.62 0.62 0.44 0.81 0.85 0.89 0.83 0.84 0.84

0.54 0.63 0.70 0.76 0.81 0.77 0.80 0.81 0.81

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ment, which may suggest that pyrene is localized within the hydrophobic bilayer. In the PSLS, it is possible that pyrene could localize in the polyethylene glycol (PEG) chains, as this region of hydrocarbon chains is fairly non-polar. However, the micropolarity of the pyrene molecules in the PSLS is similar to that of the NPSLS. This points to both systems having pyrene in the lipid bilayer. If pyrene were in the PEG chains, the micropolarity should be lower in the NPSLS. Since the data show no significant variation in the micropolarity between the two lipid systems, it can be concluded that pyrene is in the same environment in the NPSLS as in the PSLS. There seems to be no significant difference in micropolarity of any of the samples (bulk solution, PSLS, and NPSLS) upon dilution or over a 24-h period. This trend indicates that pyrene remains in the same environment. However, this stability in micropolarity over time does not rule out the possibility that pyrene could migrate from liposome to liposome. The peak ratios for Ex / M indicate the local concentration of pyrene. From experimental data, the Ex / M ratios for the NPSLS were lower than the Ex / M ratios for the PSLS (Table 3). This difference may be due to the higher packing order of the lipid bilayer region in the NPSLS. The packing order of the lipid bilayer system in the PSLS is lower due to the presence

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of PEG chains and by the mechanism for which these liposomes were formed (Fig. 5). In a similar microenvironment, a more fluid matrix such as PSLS allows for higher frequency of collisions between ground state and excited state pyrene molecules, thus leading to a higher probability for excimer formation. Therefore, this will yield a higher Ex / M ratio for a PSLS sample. In a mixed system of pyrene-labeled vesicles and unlabeled vesicles, an exchange of pyrene between the two types of vesicles will result in a drop in the local concentration of pyrene (Ex / M ratio), as compared to the local concentration of the labeled vesicles alone [22]. By labeling the liposomes with pyrene and diluting this system with an equal amount of unlabeled liposome system, the mobility of pyrene in the original system was assessed. The drop in the Ex / M ratios in the mixed PSLS and NPSLS systems as compared to the systems that contained only pyrene-labeled PSLS or pyrenelabeled NPSLS signifies that pyrene leaves the original labeled liposomes to incorporate into the lipid bilayer of the unlabeled liposomes (Table 3). Since the micropolarity of the pyrene molecules in both the labeled and 50:50 mixture of labeled and unlabeled systems remains unchanged (see Table 4), this suggests that pyrene is still localized in a lipid bilayer. The ability of pyrene to leave a lipid bilayer is a condition of

Table 3 Pyrene migration: fluorescent spectral ratios Ex / M for pyrene-labeled non-polymer-stabilized liposome systems (NPSLS) and polymerstabilized liposome systems (PSLS) with polyethylene glycol chains attached to the polar head group of the phospholipids, and for pyrene-labeled systems diluted with an equal amount of unlabeled systems, over a period of 48 h at 228C (all data reflect the results from more than five measurements, the experimental error is estimated at 60.04) Sample condition

Time (h)

Pyrene-labeled system

Pyrene-labeled system diluted equally with unlabeled system

Bulk solution

0 24 48 0 24 48 0 24 48

0.27 0.21 0.20 0.41 0.34 0.32 0.62 0.45 0.51

0.14 0.02 0.02 0.20 0.17 0.15 0.32 0.36 0.23

NPSLS

PSLS

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Table 4 Micropolarity of mixed systems: fluorescent spectral ratios III / I for pyrene-labeled non-polymer-stabilized liposome systems (NPSLS) and polymer-stabilized liposome systems (PSLS) with polyethylene glycol chains attached to the polar head group of the phospholipids, and for pyrene-labeled systems diluted with an equal amount of unlabeled systems, over a period of 48 h at 228C (all data reflect the results from more than five measurements, the experimental error is estimated at 60.04) Sample condition

Time (h)

Pyrene-labeled system

Pyrene-labeled system diluted equally with unlabeled system

Bulk solution

0 24 48 0 24 48 0 24 48

0.66 0.62 0.65 0.86 0.83 0.82 0.78 0.78 0.80

0.58 0.59 0.59 0.78 0.77 0.77 0.88 0.88 0.79

NPSLS

PSLS

the mobility of the lipids in the bilayer of the liposomes, the concentration of pyrene, and the concentration of liposomes in solution. Transfer of a probe suggests transfer of phospholipids either as monomers through an aqueous solution or as the result of collisions between vesicles [33,34]. It can be concluded that the presence of the PEG border does not affect the exchange of encapsulated molecules between liposomes.

polymer-stabilized systems. When pyrenelabeled liposome systems were mixed with unlabeled systems, pyrene was able to move from labeled vesicles to neighboring unlabeled vesicles, either through monomer diffusion or collisional contact. The experimental results are consistent with the hypothesis that the PSLS are formed via a mechanism that is different from conventional liposomes.

4. Conclusion

References

The purpose of this investigation was to characterize a polymer-stabilized liposome system for relative particle size, lipid association, and membrane polarity / mobility and compare to a non-polymer-stabilized system. It was found that PEGylated liposome systems produced vesicles with narrower size distribution than conventional liposome systems (without PEG) prepared in the same manner. The presence of PEGylated lipids in a liposome system helps to regulate the size of the particles that will form. Based on the NMR results from this experiment, practically all the lipids are associated into liposomes, and the proportion of free lipids that remain unassociated is below the detection limit. A fluorescent probing study indicated that pyrene localizes in the lipid bilayer of both polymer-stabilized and non-

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