Characterizing PEG Chains Tethered onto Micelles and Liposomes

Chapter 5

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Characterizing PEG Chains Tethered onto Micelles and Liposomes Applied as Drug Delivery Vehicles Using Scattering Techniques Shota Fujii,1,† Mina Sakuragi,2,† and Kazuo Sakurai*,1 1Department

of Nanoscience, Faculty of Engineering, Sojo University, 4-22-1 Ikeda, Nishi, Kumamoto 860-0082, Japan 2Department of Chemistry and Biochemistry, University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan *E-mail: [email protected]. †These authors equally contributed to this manuscript.

A polyethylene glycol (PEG)-coated surface provides biocompatibility to nanoparticles including polymeric micelles and liposomes for injectable drugs and other biomaterials. Although this technology seems well established and the term PEGylation is now commonly used, how to analyze it quantitatively, namely, determining how densely PEG chains are coated on the surface and its relationship to biocompatibility, is still not fully understood. This chapter reviews our recent studies of quantitative characterization of PEG chains tethered onto the nanoparticle surface by using light scattering and small-angle X-ray scattering.

Introduction In recent decades, many efforts have been made to develop drug-carrying nanoparticles, including polymeric micelles and liposomes. Among them, improving pharmacokinetics after intravenous administration is a key issue. Unfavorable interactions and binding with plasma proteins are the primary mechanisms for the reticuloendothelial system to recognize the circulating nanoparticles. This eventually causes major loss of the injected dose due to clearance. Therefore, controlling or prolonging the period during which nanoparticles circulate in the blood has been one of the major research targets © 2017 American Chemical Society Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


in the development of efficient drug delivery systems (DDSs) (1–3). To confer such biocompatibility on DDS nanoparticles, controlling the properties of the nanoparticle surface is essential. Such biocompatibility is sometimes called the “stealth effect,” meaning that the particles become undetectable to the reticuloendothelial system. Polyethylene glycol (PEG) is one of the biocompatible polymers approved by the US food and drug administration (FDA) as a material that can be injected into the human body (4, 5). DDS nanoparticles including micelles bearing PEG corona phase in the shell exhibit the ability to eliminate other macromolecules and particles sterically. This is due to the high flexibility of PEG and the large exclusion volume in water, and is a property useful for suppressing protein adsorption and cell adhesion (5). PEG coating prevents the particles from aggregating and stabilizes them even in complex biological media by its steric and hydration repulsions (6–8). Suppression of non-specific adsorption of plasma proteins and cells (called the antifouling effect) is most important for determining the fate of particles injected into human bodies (9). Such antifouling properties promote the reduction of cellular uptake by the mononuclear phagocyte system (MPS), which leads to a longer half-life in the blood by lowering the degradation and elimination rates. This inhibition of protein binding depends on the coverage density of PEG chains on the particle surfaces, which is usually achieved by a regime with a high coverage of polymeric brushes (3, 10). The optimum coverage rate has been under discussion, but the antifouling properties have been observed even at low coverage in other studies (11). Furthermore, tethering the PEG layer onto the particle surface also increases the drug encapsulation rate due to the physicochemical barriers for drug release (12). Many studies have attempted to characterize PEG tethered onto nanoparticles quantitatively, including the coverage density and conformations, which are assumed to be related to the stealth effect. However, it is difficult to obtain structural information by using direct observation tools such as transmission electron microscopy and atomic force microscopy. These techniques do not give statistical and quantitative information about PEG chains. Although 1H NMR (13, 14) and X-ray photoelectron spectroscopy (XPS) (15, 16), are used to directly assess the quantity of PEG on nanoparticle surfaces, the qualification is always relative not absolute. In this chapter, we present our recent work (17–20) on the quantitative characterization of PEG chains on nanoparticles by using light scattering (LS) and synchrotron small-angle X-ray scattering (SAXS) and then discuss the relationship between the PEG chains on nanoparticles and their biocompatibility.

Scattering Techniques Basic Equation of Static Scattering Small-angle scattering is a term used to describe the techniques of small-angle neutron (SANS), X-ray (SAXS), and light (SLS) scattering. Among these techniques, the physics is almost identical, namely, radiation is elastically scattered by a sample and the resulting scattering pattern is analyzed to provide 116 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


information about the size, shape, and molecular weight of the sample. The excess scattering intensity [I(q)] from the solutions that contain uniformly dispersed scattering objects (in our case, specifically DDS nanoparticles) can be written as follows:

Here, KC is the optical constant and Mw is the weight-averaged molecular weight. The magnitude of the scattering vector q is related to the scattering angle θ through with the wavelength of the radiated light (λ). P(q) and S(q) are the form and structural factors, which relate to inter- and intra-practice diffractions, respectively. When the concentrations (C) of the sample are low enough, we can regard S(q) = 1. When q is small enough, P(q) ~ exp[−(qRg)2/3] = 1 − (qRg)2/3 + ..., where Rg is the radius of gyration of the scattering object. Therefore, by extrapolating C → 0 and q → 0 (i.e., θ → 0), Mw can be obtained and the angular dependence of I(q gives Rg. Dynamic light scattering seems to be more commonly used to determine the particle size in the DDS community. This gives the hydrodynamic radius (Rh); however, there are several assumptions needed to obtain Rh and it should be noted that the average value of Rh and its distribution sometimes lead to incorrect results and these may not be absolute values, depending on the instrument. On the other hand, Mw and Rg are absolute values. Carrying out reliable measurements to obtain accurate values of Mw and Rg requires the use of dust-free solutions and removing secondary aggregation from the sample. In particular, these procedures are essential in SLS measurements. However, they are troublesome to perform for aqueous solutions.

Static Light Scattering Combined with Fractionation Devices When SLS is combined with a particle fractionation system such as size exclusion chromatography (SEC) or asymmetric flow field flow fractionation (aFFF) (21, 22), SLS becomes a powerful analytical tool for aqueous solutions. This is because a major advantage of static light scattering combined with a fractionation system is that the fractionation device also removes out or segregates dust and secondary aggregation. SEC/SLS can also be combined with a viscometer to give the intrinsic viscosity [η]. From the plots [η] vs. Mw and Rg vs. Mw, which are called “conformational plots,” we can obtain the conformational information of the solutes (23). aFFF is another fractionation method (Figure 1). Here, the injected samples flow into a thin channel chamber with parabolic velocity and another flow is applied perpendicular to the sample flow (24). Smaller particles can diffuse faster than larger ones and thus they can reach a more central portion of the sample flow. The first central flow can elute out the smaller particles earlier than the larger ones. This system can basically be used for any sample, while SEC cannot be applied to micelles due to adsorption or interaction with the SEC column. 117 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 1. (A) An aFFF channel and (B) a schematic illustration of the fractional system inside the channel. The outflow is led to a differential refractive index (RI) detector, a multi-angle light scattering detector, a viscometer, and a UV spectrometer, yielding the solute concentration, compositions, Mw, [η], and Rg. SAXS and Contrast Variation Technique SAXS is a powerful method of studying the inner structure of DDS particles in solutions and basically can be applied to any type of particles even to aggregates. In particular, the synchrotron X-ray source gives a much stronger X-ray beam than conventional ones and thus we can reduce the sample concentration or exposure time. The bright beam makes it possible to carry out dynamic and micro-beam measurements (25, 26). The obtained I(q) vs. q profiles are fitted by a theoretical curve calculated from an appropriate model and the iteration of fitting can provide information on the internal structure, including the sizes and the electron densities (27). However, it sometimes becomes difficult to determine the structural parameters because the models normally contain multiple fitting parameters. For example, core-shell models that consist of two concentric spheres with different electron densities are normally used to analyze spherical micelles or polymeric micelles (see Figure 2a). The core-shell models contain five parameters: the core radius (RC), the core plus shell radius (Rg), and three electron densities: core, shell, and solvent (ρs, ρc, and ρsol). Here, ρsol can be changed and its absolute value can be calculated. As far as changing the solvent conditions does not alter the solute structures, a series of I(q) measurements obtained by systematically changing ρsol make it possible to determine the structural parameters. Figure 2(b) illustrates how the scattering profile is changed upon changing only ρsol . This method is called the contrast variation technique and is very useful for determining the structures of scattering objects in neutron scattering (28, 29). In neutron scattering, the scattering contrast is due to the mass of the atomic nucleus. Therefore, mixing H2O with D2O can change ρsol and normally no 118 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


structural changes are induced by mixing D2O. For SAXS, changing ρsol can be performed by the addition of sucrose or PEG to the solvent and we always have to be careful about the fact that these additives may change the structure of the scattering objects. Fortunately, such undesirable perturbation in the PEG-coated particles may be avoided by the addition of low-molecular-weight PEG to the solvent because of their chemical identity.

Figure 2. A conceptual drawing of the contrast variation technique in the case of the core-shell sphere model. (A) A typical spherical micelle and a core-shell model to represent the micelle, and (B) changes of the scattering profiles upon changing the value of (ρs − ρsol) when the core and micelle volumes (i.e., where X = C or S are fixed at 1 and 5 nm3, respectively. Tethered PEG Chains on Polymeric Micelles In aqueous solutions, amphiphilic block copolymers self-assemble into spherical micelles called polymeric micelles containing a core-shell structure in which the insoluble hydrophobic block forms a core and the hydrophilic block becomes a shell. The surface activity and micellization behavior of these block polymers are particularly useful in the biotechnology field for a colloidal dispersion, surface modification, and drug carrier (30–32). As we mentioned in introduction, the PEG layer plays various important roles in determining the particle behavior in bio-media, which especially depends on the coverage density of PEG in the shell. For this reason, block copolymers containing PEG are of considerable importance in the development of biomaterials and can be expected to play an important role in cell and organ engineering, bio-sensing, and DDS. Quantitative analysis of the PEG chain crowding on PEG-tethered particles is very important, but it has been extremely challenging because of experimental difficulties in obtaining accurate data on Mw. SEC or aFFF makes it possible to determine Mw of polymeric micelles accurately. Once Mw is obtained and we know 119 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the molecular weight of the constituent block copolymer, the aggregation number of polymeric micelles (Nagg) can be determined. Nagg is directly related to how many PEG chains are tethered on the micellar core. When the SAXS profiles of the polymeric micelles can be treated by a core-shell or core-corona model, RC and RS can be determined. By combining these parameters, we can derive the following two parameters to describe the PEG chain crowding (17, 18).

Here, Rg.PEG is a radius of gyration of the free (i.e., unperturbed state) PEG single chain with the same molecular weight as the tethered one. σ represents how the tethered chains are crowding on the core/shell interface surface (33). DOS is PEG density at the outermost shell surface. When this index is less than 4, the tethered chains are not interacting with each other (called the mushroom region). As shown in Figure 3, in the case of σ < 1, the tethered chains are not interacting with each other (called the mushroom region). When σ = 1, a tethered chain occupies the same value on the surface as when the chain is present in the unperturbed state. For σ = 4–10, the chains are crossed over each other and the conformation of each chain deviates from the sphere (called the brush region). In the range of σ > 10, the chains are highly stretched normal to the surface. Therefore, σ represents how the tethered PEG can cover the hydrophobic core surface to prevent water molecules from contacting the core.

Figure 3. Schematic illustration of the relationship between σ and the tethered PEG crowding on the nanoparticle/water interface. We measured a series of micelles made from poly(ethylene glycol)block-poly(partially benzylesterified aspartic acid), denoted PEG-P[Asp(Bzl)], containing PEG and P[Asp(Bzl)] with different chain lengths and different benzylation rates (17, 18). This system is the most examined block copolymer for DDS. We carried out aFFF/MALS and synchrotron SAXS to determine σ and DOS. As shown in Figure 4, we found that σ is closely related to the micellar stability, which is evaluated by determining what fraction of the injected micelles are not trapped by a hydrophobic column. The value of σ is over 1, indicating that 120 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the adjacent chains of PEG starts to contact and confer a much more covered the hydrophobic core surface. Yokoyama et al. reported that this stability is related to the initial burst of the PEG-P[Asp(Bzl)] micelles when they are injected into mouse blood. The immediate and substantial micellar burst just after injection may cause an abrupt release of drug and thus may push up the drug concentration to a toxic level. Therefore, it is better to reduce the initial burst. The initial burst may be caused by a jump in osmotic pressure and unfavorable interaction with serum proteins. From the strong relationship between σ and stability, we can conclude that greater PEG coverage of the hydrophobic core leads to a lower probability of interactions between serum proteins and the core, which might cause a reduction of the initial burst.

Figure 4. Panel A illustrates the structural meaning of DOS and σ. Relationships between the two PEG crowding parameters (DOS and σ) of the PEG-P[Asp(Bzl)] polymeric micelles and the AUC (micellar concentration in blood area under the curve in vivo) is described in panel B. The micellar stability parameter determined by recovery ratio of samples after passing a gel permeation chromatography (GPC) column is shown in panel C. Another important finding is that the blood circulation time is correlated to DOS, meaning the PEG density on the surface of the micelles. The greater the density of PEG chains on the surface, the more the AUC (micellar concentration in blood area under the curve in vivo) increases. This tendency can be interpreted by the micelle surface being the first portion to contact serum proteins and greater PEG crowding preventing the proteins from invading into the micelles and binding between them. DOS and σ are positively correlated to each other, although 121 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


we found that AUC is more strongly correlated to DOS than σ. Therefore, the invaded proteins are also prevented from undergoing unfavorable interactions with the hydrophobic core. The PEG nanoparticles are considered to accumulate in solid tumors by the enhanced permeability and retention (EPR) effect (34). This is the major mechanism by which anti-cancer drugs are selectively delivered to tumors. The longer blood circulation accelerates the accumulation of the drug to cancer cells. This means that increasing DOS is important, and DOS can be increased by increasing the PEG chain length as well as the benzylation ratio of the P[Asp(Bzl)] chain because Nagg exponentially increases with an increase in this ratio. However, a large benzylation ratio results in an increase in the micellar size as well as large Nagg. The EPR effect is quite sensitive to the particle size. According to Kataoka et al., the particle size has to be less than 50 nm in radius for efficient accumulation in tumors (35). Therefore, DOS or the benzylation ratio would have an optimal condition to compensate between the size accumulation effect and the blood circulation.

Competition of the Tethered PEG Chains and the Targeting Ligand Molecule Fukuda et al. presented a more quantitative assessment of the ligand recognition ability as a function of σ (19). The ion complex of p-DNA with a two-component micelle containing an α-mannose-modified aromatic lipid and dioleoyl trimethylammonium propane (DOTAP) showed high transfection efficiency to RAW264 cells through the mannose lectins of these cells (36). To evaluate how PEG density on DDS particle surfaces influences the α-mannose recognition of the lectin, they prepared PEG-tethered liposomal nanoparticles (PLNs) by adding a PEG-bearing lipid, 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (denoted DSPE-PEG2000) to the micelles. The structures of the PLNs composed of various ratios of DSPE-PEG2000 were characterized using SAXS and LS techniques. The obtained σ seems to have a linear relationship with the composition ratio of DSPE-PEG2000 (X). By using a quartz-crystal microbalance (QCM), the interactions between α-mannose on the PLNs and its lectin, concanavalin A (ConA), immobilized on a gold substrate in a QCM sensor cell were examined. Figure 5 shows that the mannose recognition was dramatically suppressed at σ = 1.0 where PEG chains start to overlap with each other (see Figure 3). As mentioned above, the tethered PEG chains are isolated at σ < 1.0; thus, the mannose moiety on the PLNs is exposed to the particle surface/water interface without being shielded by PEG, which promotes the interaction between the PLNs and ConA. This clearly demonstrated the importance of controlling the PEG density for the biocompatibility of DDS particles.

122 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 5. Relationship between σ and the α-mannose ligand recognition ability determined with QCM.

Characterization of PEGylated Vesicles Structural Transition Induced by Addition of a Cationic Lipid Cationic surfactants interact with DNA through the combination of electrostatic interactions and hydrophobic interactions, leading to the formation of a polyion complex (lipoplex) used as a carrier for gene delivery (37). Benzamidine derivatives (denoted by TRX shown in Figure 6) exhibit better efficiency and less toxicity than commercial products as transfection agents (38, 39). However, there is a major problem that cationic gene carriers exhibit a short blood circulation time due to the non-specific electrostatic binding with proteins. Adding PEGylated lipids to cationic liposomes improves the half-life in blood circulation (40). This is the most commonly used method for formulating liposomal drugs with a prolonged circulation time. It is usually believed that the addition of PEG-liposome just adds the tethered PEG chain on the surface. However, this also has a high possibility of changing the entire structure of the liposome itself. Here, we present an example of the characterization of PEG-bearing liposomes composed of TRX using SAXS with contrast variation techniques (20). HSPC liposome was prepared, and then filtrated with an extrusion process (Extruder T-10; Lipex Biomembranes) using a 0.2 m membrane filter. Subsequently, DSPE-PEG5000 was then added at 1.5 mol% into the liposome solution, and then extra DSPE-PEG5000 was removed by gel filtration. In this study, phosphate buffered saline (pH = 7.4) was used as a solvent. The PEGylated liposomes composed of HSPC and DSPE-PEG5000 (Figure 6a) were mixed with TRX solution. Figure 6b shows the SAXS profiles for the three samples with different TRX compositions. The profiles of 12- and 19-TRX show typical scattering from unilamellar vesicle (ULV); namely, the scatterings from the outermost sphere and bilayer membrane are reflected around q < 0.3 nm−1 and 123 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


0.3 < q < 2 nm−1, respectively. The sharp minimum at q > 2 nm−1 indicates the narrow size distribution. The SAXS profile of 0-TRX has almost the same feature as the others, except for a shoulder peak around q = 1.0 nm−1 (shown with an arrow). This type of shoulder was caused by the diffraction between liposomes due to infinite concentrations. However, as shown in Figure 6c, the shoulder did not disappear at a low concentration (0.02 wt%), which should be sufficiently low to eliminate the inter-particle diffraction. This leads to the conclusion that the shoulder can be ascribed to the multilamellar vesicle (MLV) structure. This observation is consistent with the findings of electron microscopy (Figure 6d). We can conclude that the HSPC/DSPE-PEG5000 liposome forms MLV and the addition of TRX changes its structure from MLV to ULV, as depicted in Figure 6e. This change may be induced by the addition of cationically charged TRX potentially creating an electrostatic repulsion between adjacent lamellae and increasing the free energy.

Figure 6. (a) Chemical structures of TRX, HSPC, and DSPE-PEG5000. (b) SAXS profiles of TRX/HSPC solutions at several compositions. (c) Comparison of the concentration dependence of the SAXS profiles of 0-TRX. (d) TEM images after staining with 1 wt% ammonium molybdate for 12-TRX and 0-TRX; scale bar: 20 nm. (e) Schematic drawing of structural transition from ULV to MLV. Contrast Variation Analysis of SAXS from 12-TRX The form factor S(q) in Eq. 1 of ULV can be expressed by the following equation (41):

where j1(x) is the first-order spherical Bessel function, while ρi, Vi , and Ri are the electron density, the volume, and the radius of the i-th sphere, respectively. In this equation, ρk+1 is the electron density of solvent and the width of the i-th layer 124 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


(ti) is given by ti = Ri+1 − Ri. For ULV, the layers of the outer head of the lipid, alkyl chain, inner head of the lipid, and water drop are considered. Therefore, the total number of layers, k, is 4 (42, 43). Additionally, since we suppose that the PEG block of DSPE-PEG5000 forms a layer at the outermost surface due to the high hydrophilicity, these two parameters, the PEG layer width (t5) and its electron density (ρ5), can be introduced as presented in Figure 7a. However, there were too many adjustable parameters, which sometimes mislead us to inaccurate structure. To solve this problem, we applied the contrast variation technique. PEG400 was added to adjust the electron density of the solvent and the values of ρsol can be calculated from the composition. If the addition of PEG did not alter the liposome structures, we would be able to fit all of the data with the same parameters, except for ρsol (44). Figure 7b presents how the scattering profile of 12-TRX was changed by the addition of PEG400. The best fitting curves are shown as black lines in Figure 7b. The small undulation at the high-q region reflects a more detailed atomic structure and cannot be represented by the simple multilayer model.

Figure 7. (a) A schematic of the multi-spherical model (k = 5 in Eq. 4) used to fit 12-TRX. (b) Changes of the scattering profiles of 12-TRX when PEG400 was added to solution at a fixed lipid concentration (gray lines) and their best fit curves (black lines) calculated from Eq. 4. When the concentration of PEG400 in the solvent was 0.96 M, we found that ρsol became almost identical to ρ5. Since all of the data could be fitted with the same parameters, we concluded that the addition of PEG400 did not alter the original structures of the liposomes. When we fitted the data at ρ2 = ρ4, the resultant fitting curve was not good enough compared to that at ρ2 ≠ ρ4. This may be explained by the outer head group incorporating the phosphoester moiety of DSPE-PEG5000 or the inner head group incorporating the hydrophobic tails. Although we did not find any evidence of DSPE-PEG5000 anchors on the inner layer, we found that PEG400 can be located inside ULV (ρ1 = ρsol). The width of the head group and its electron density, and those of the alkyl domain take reasonable values in terms of their 125 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

molecular structures (45). On the basis of the above discussion, a five-layer model is adequate to describe the present system. Many studies have reported that PEG chain crowding on the liposomal surface is closely related to the stealth effect of PEGylated liposomes. We calculated the parameter (σ) of tethered PEG chains crowding on the flat surface by using Eq. (4). We estimated that σ values of 12-TRX and 0-TRX were 1.53 and 1.51, respectively, which indicates that the shell region of the liposomes is completely covered with PEG chains but not crowded enough to cause significant stretching, indicating that the present systems have a sufficient stealth effect.


Concluding Remarks In this chapter, we discussed the relationship between PEG chain behavior on the surface of DDS nanoparticles and the biocompatibility in vivo, showing that scattering techniques including SLS and SAXS are useful to obtain quantitative data. By using the contrast variation technique in SAXS, we can determine the accurate fitting parameters of SAXS analysis. For application of the nanoparticles to DDS, rigorous characterization using scattering techniques is needed to evaluate the properties of nanoparticles and improve their design.

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