Surfactant

Chapter 17

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Biophysical Methods for the Studies of Protein-Lipid/Surfactant Interactions Shuo Sun,1,3 Caleb I. Neufeld,2 Ramil F. Latypov,3 Bernardo Perez-Ramirez,2,3 and Qiaobing Xu*,1,2 1Department

of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States 2Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States 3BioFormulations Department, Global BioTherapeutics, Sanofi, 1 The Mountain Road, Framingham, Massachusetts 01701 United States *E-mail: [email protected].

Comprehensive characterization of protein-lipid/surfactant interaction is crucial for both biopharmaceutical formulation development and drug delivery research. Revealing the mechanism of this interaction will facilitate novel ideas for drug delivery system design, improve protein stability, and optimize protein formulation. Protein/lipid interaction must be analyzed by many different techniques, using various perspectives, in order to approach the full view of conformational, stoichiometric and calorimetric changes accompanying different interaction stages. Among these methods of analysis, biophysical technology is well-suited to monitor the atomic and molecular changes resulting from two-component interactions. The purpose of this chapter is to present some of the most popular biophysical methods employed for such investigations. The basic principles, advantages and limitations of each technique, their roles, applications and limitations, and a few case studies of each are discussed.

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Introduction Surfactant molecules are organic compounds that are amphiphilic: they contain hydrophobic groups, usually an alkyl tail, and hydrophilic head groups, typically a polar moiety. At a sufficiently high aqueous concentrations (the critical micelle concentration or CMC), surfactant molecules associate via their hydrophobic chains to form micelles, composed of a hydrophobic inner phase and a hydrophilic water-exposed exterior (1). Surfactants have a wide range of applications from agriculture and the food industry, to cosmetics and the pharmaceutical industry (2, 3). In pharmaceutics, surfactants can reduce surface tension and thus a formulation’s free energy, reducing protein-protein and protein surface interaction (4, 5). They are commonly used in protein formulation to prevent physical instability during purification, filtration, transportation, freeze-drying, spray drying, storage, and delivery (6–12). Interaction of surfactants with biomolecules depends on the charge on surfactant head groups, hydrophobic tail lengths, and the nature of the biomolecule interacted with (13). Additionally, proteins interact differently with the monomeric and the micellar forms of surfactants (1). In order to evaluate their properties, roles and mechanisms in protein formulation, protein–surfactant interactions must be studied by different biophysical techniques, providing full view of the structural, stoichiometric and calorimetric changes accompanying different interaction stages. Nonionic surfactants bind weakly to proteins. They are widely used as stabilizers in protein formulations to prevent protein aggregation due to transportation or sorption. Polysorbate 20 (PS20) and Polysorbate 80 (PS80) are the two most frequently used non-ionic surfactants in protein formulations (7). PS20 and PS80 are used to stabilize monoclonal antibodies including Rituxan®, ReoPro®, and Humira® (14). However, due to the potential formation of peroxides in PS20 and PS80, they can oxidize proteins they are formulated with, negatively impacting their stability (15, 16). Moreover, recent studies showed that polysorbate raw material can give rise to free fatty acid (FFA) particles which are granular in shape and several microns in size (17, 18). The degree of particle formation tested under recommended storage conditions was found to be dependent on polysorbate type and concentration. Because therapeutic protein formulations must exhibit sufficient stability over a long period of time to be commercially appealing, polysorbate’s propensity to form particles is an undesirable formulation characteristic. New, FFA-free surfactants are intensely needed for use in place of polysorbates. However, only a few surfactants are currently FDA-approved as excipients for inclusion in parenteral medications (19). One example is Poloxamer 188, (known as Pluronic F68) which is found in commercial formulations, many of which are delivered intravenously or subcutaneously, at concentrations up to 0.6% w/v. Another promising surfactant is Triton X-100, which is used mainly in topical formulations such as gels and ointments. A notable exception is Fluarix® from GSK, an FDA-approved injectable product which contains Triton X-100 at 0.0085% w/v (19). Ionic surfactants are usually not used to stabilize proteins because they can bind to both polar and nonpolar groups in proteins, causing denaturation (20, 21). 356 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


For example, sodium dodecyl sulfate (SDS) has been extensively used to denature proteins for subsequent electrophoresis (from whence the term SDS-PAGE). However, these surfactants have been reported to posess a protein-stabilizing effect as well. SDS has been found to play two opposite roles in the folding and stability of proteins: At low surfactant/protein molar ratios, it acts as a structure-stabilizing additive, but at increased amounts, it behaves as a destabilizer (20, 22, 23). The intravenous protein drug Proleukin™/Aldesleukin (Novartis) is an example of this interaction. At 0.18 mg per 1.2 mL (between 95-250 µg SDS per mg of interleukin-2), SDS acts to mitigate ionic and hydrophobic interactions, which would otherwise lead to the formation of non-covalent microaggregates (24). Cationic surfactants, such as cetyltrimethylammonium bromide (CTAB), bezalkonlum chloride (BZK) and cetrimide, have been widely applied as penetration enhancers in transdermal drug delivery. Despite their potential to damage human skin, they cause a larger level of transdermal flux than anionic surfactants (25–28). Cationic surfactants act on the keratin fibrils of cornified cells, resulting in a disrupted cell/ lipid matrix. Additionally, they interact with skin proteins via polar interactions and hydrophobic binding. These interactions result in pendant ionic head groups and subsequently swelling of the stratum corneum (26). Lipids play a key role in biological, pharmaceutical and medical research (29, 30). Drugs and biologically active molecules present in the extracellular medium must either cross the membrane to act inside the cell, or bind to membrane receptors or the lipid matrix of the membrane, (31). Liposomes were first described in 1965 (32). They are lipid bilayer nanoparticles or colloidal carriers, usually 50-500nm in diameter, which form spontaneously when certain lipids are hydrated in aqueous media (32, 33). Due to the “designable” nature of lipids, liposomes as drug delivery systems have undergone significant improvement. Liposomes are one of the few systems that have successfully translated to the clinic and the market (34). For example, DaunoXome® (daunorubicin citrate liposome injection), launched in 1996 in the US, is a prescription drug indicated as a first line cytotoxic therapy for advanced HIV-associated Kaposi’s sarcoma, and is delivered in a liposomal formulation (35). Protein delivery is also carried out in liposomal formulations: For example, PEGylated liposomal delivery of streptokinase dramatically improved its pharamacokinetics in a rat model (36). The activity of many biomolecules and drugs depend upon binding to biological membranes or translocation to the inner lipid leaflet (31). Furthermore, surfactants used as a protein excipient must remain stable over a substantial period of time to effectively be marketed. Understanding of the mechanisms involved in protein and lipid/surfactant interaction provides the basis for rational strategies to optimize these results. Thus, in the following section, we will discuss the principles of some key biophysical characterization methods essential to mechanistic characterization of protein/lipid interactions, presenting a few interesting examples of each technique, as well as their limitations. This review will serve the protein-focused investigator in choosing appropriate analytical techniques for each interaction he wishes to characterize. 357 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Methods


Circular Dichroism Spectroscopy (CD) Plane-polarized light can be viewed as vectors made up of 2 circularlypolarized components of equal magnitude, one rotating counter-clockwise (left handed, L) and the other clockwise (right handed, R). When a light source passes through a sample, due to the differences in absorbance of left and right circularly-polarized light by the chromophores, elliptical polarization can be determined. The numerical relationship between the difference in absorbance of the L and R circularly-polarized components (ΔA = AL − AR) and ellipticity (θ, in degrees), is θ= 32.98 ΔA. In proteins, chromophores of interest include the peptide bond, aromatic amino acids, and disulfide bonds. Detailed information on their absorption range is described in Table 1.

Table 1. Summary of Absorbent Information of CD Spectroscopy Absorbent

Wavelength measured

Structure of protein

Comments

Peptide bond

Below 240 nm

Secondary structure

α-helix, β-sheet and turns can be measured in the far UV region

Aromatic amino acid

Between 260-320 nm

Tertiary structure

Absorbent aromatic amino acids: Phenylalanine, tyrosine, tryptophan

Disulfide bonds

Around 260 nm

Tertiary structure

Weak broad absorption bands around 260 nm

Peptide bonds are measured mainly by far-UV CD, with a wavelength between 240-180 nm. Different secondary structures (α-helix, β-sheet, random coil etc.) of proteins have their own particular CD absorbance signals due to their unique structural properties. Figure 1 shows the CD fingerprints of different secondary protein structure components. α-Helical structure displays dual minima at 222 nm and 208 nm, while β-sheet topology possesses a minimum around 216 nm, providing a secondary probe for protein conformations. By applying an empirical database-based algorithm to deconvolute protein spectra, estimation of the identified secondary structure compositions can be calculated. SELCON, VARSLC, CDSSRR and CONTIN have been widely used in various applications (37–40). 358 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 1. Far UV CD spectra associated with various types of secondary structures. Solid line, α-helix; long dashed line, anti-parallel β-sheet; dotted line, type I β-turn; cross dashed line, extended 31-helix or poly (Pro) II helix; short dashed line, irregular structure. Reproduced with permission from reference (41). Copyright 2005 Elsevier.

Recombinant human growth hormone (rhGH) contains only α-helix and random coil structures. Thus, the 222-nm signal in the CD spectra is a good indication of changes in α-helix content. Thus, CD may be used to asses the stability of the protein upon interaction with surfactants. When rhGH was spray dried without any excipients, the α-helix content was decreased in comparison to that of bulk rhGH. By formulating rhGH with PS20(0.05%, w/w) for spray drying process, however, α-helix content remained close to that of the control. Thus, the interaction of PS20 can effectively protect the protein against aggregation and 359 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


maintain the conformational stability of rhGH by excluding protein molecules from exposure to the air–liquid interface at the surface of droplets (42). The interaction between human serum albumin (HSA)/bovine serum albumin (BSA) and various lipids has been evaluated by CD spectrum to examine the effects of lipid complexation on protein conformational stability (40, 43). It has been reported that there were no major changes in α-helix content for cholesterol (Chol) and dioleoylphosphatidylethanolamine (DOPE)–protein complexes; however, a major secondary structural transition from α-helix to β-sheet was noted for 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP) and (dioctadecyldimethyl)ammonium bromide (DDAB)-protein complexes. These observations indicate that the interaction between DDAB/DOTAP and protein resulted in partial protein unfolding, but cholesterol and DOPE stabilized protein conformation, confirming the complex nature of protein/lipid interaction. CD spectroscopy has been widely used to analyze structural changes in biomolecules, in qualitative and even semi-quantitative fashions. Applications of CD are not limited to conformational assessments of protein, thermodynamics and kinetics of folding-unfolding of macromolecules; protein-lipid/surfactant interaction studies also benefit from CD. However, due to the low sensitivity to structural changes, relatively higher sample concentrations are required. In some cases, this may lead to precipitation of complexes, resulting in a paradoxical reduction of signal intensity. Nevertheless, circular dichroism remains an essential method of biophysical protein analysis. Ultraviolet–Visible Absorption Spectroscopy (UV-vis) In spite of the vigorous development of more sophisticated techniques, UVVis spectroscopy remains an indispensable tool for obtaining an initial insight into the interaction between protein and surfactants or lipids. UV-Vis is based on the absorption of energy by the studied molecule upon interaction with specific light sources (e.g. xenon flashing lights and deuterium lamps). The energy of the light promotes electrons from the ground state to an excited state, leading to a decrease in transmitted light. Figure 2 shows the various kinds of electronic excitation that may occur in organic molecules. Of the six transitions outlined, only the two lowest energy ones (n→π* and π→π*) are achieved by the energies available in the UV-Vis spectrum, and are thus the transitions observed in protein absorption spectra. (44). Spectra are obtained by measuring the absorption of light as a function of its wavelength. Molecules with electrons in delocalized aromatic systems often absorb light in the near-UV (150–400 nm) or the visible (400–800 nm) region. One example of UV-vis spectroscopy in protein structural characterization can be observed in the interaction between SDS and acid-denatured cytochrome c (45, 46). At low SDS concentration (1.0 would imply enthalpic governed binding, and slope

Surfactant

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