Bioprocessing Methods to Prepare Biobased Surfactants for Pharmaceutical Products

• University of Tennessee

Summary

Surfactants have numerous uses in pharmaceuticals, for solubilization of hydrophobic drugs in aqueous media, as components of emulsion or surfactant self-assembly vehicles for oral and transdermal drug delivery, as plasticizers in semisolid delivery systems, and agents to improve drug absorption and penetration. Many pharmaceutical-grade surfactants consist of saccharide- or polyol-fatty acid esters or fatty alcohol ethers. There is increased interest and opportunity to produce the surfactants from renewable resources rather than employing petrochemical feedstocks, to improve sustainability. With the cost of petroleum anticipated to increase, biobased surfactants will become cost-competitive in the near term. Bioprocessing-based manufacture of saccharide- and polyol-based surfactants using enzymes to direct manufacture will become increasingly attractive compared to chemical-based preparation due to the reduction of by-products and the lowering of energy and downstream purification costs, which will offset the increased input costs for the biocatalysts.

Surfactants: Important Components in Pharmaceutical Products

Surfactants, chemical species which lower the surface energy at interfaces between liquid, solid, and/or gas phases, play numerous roles in pharmaceutical products [1, 2]. Surfactants are amphiphilic; moreover, their chemical structure contains both hydrophilic and lipophilic domains. Their major function in pharmaceutical processing is to improve the solubility of drugs, particularly those which are poorly soluble in water, which includes an increasing number of new and developing bioactive agents (e.g., small molecular therapeuticals, peptides, proteins, vitamins, vaccines, and oligonucleotides), to enable their in vivo delivery. They also improve the stability of encapsulated drugs, and possibly the thermodynamic activity, rate of diffusion. They are particularly important to enable the penetration of drugs across cell walls and membranes, skin, and other biological interfaces. Surfactants are also important plasticizers, needed to improve the fluidity and in vivo dissolution of semisolid delivery vehicles and viscous excipients such as those employed for suppositories. For example, sucrose-fatty acid esters are important lubricants for tabletting [3]. They can also serve as wetting agents to enable drug incorporation into delivery vehicles and dispersants for powders, granules, and nanoparticles.

The most common use of surfactants is for self-assembly systems as drug delivery vehicles. Common surfactant monolayer-based, selfassembly structures are emulsions, dispersions of oil-in-water (O/W-) or vice versa (W/O-). Emulsions are not thermodynamically stable, often requiring agitation for their long-term stability, and are relatively large in size, typically on the order of microns to millimeters. Emulsions are also employed in the preparation of aerosols and microencapsulation media. High-pressure homogenation can be employed to prepare nanoemulsions (also referred to as “miniemulsions”), of average size 0.05-1.0 μm, which due to their smaller size can be sterilized by microfiltration, are more likely to avoid physiological clearance, and penetrate interfaces in vivo. Nanoemulsions are commonly used in parenteral delivery.

Figure 1 - Surfactant self-assembly structures formed in a. watersurfactant and b. water-surfactant-oil systems as a function of the surfactant’s hydrophilic-lipophilic balance. Reprinted from [6] with permission from Elsevier.

Figure 1a depicts thermodynamically stable surfactant self-assembly structures for surfactant-water systems (reviewed in [4-6]). The figure traces the evolution of different nanostructures that form as the surfactant’s structure changes from polar to nonpolar. Two different parameters are employed to designate the relative degree of hydrophilicity of surfactants. The HLB, or hydrophilic-lipophilic balance refers to the weight percent of the surfactant’s hydrophilic group for nonionic surfactants. The second parameter is the surfactant packing parameter v a_o^-1 l_c^-1, where v, ao, and lc refer to the volume of the surfactant tail region, the interfacial surface area per surfactant polar, or “head”, group, and the surfactant chain length, respectively. This parameter models the curvature of the surfactant monolayer, where an increase of the numerator v reflects an increase of the surfactant tail region, leading to the encapsulation of water in oil, and an increase of a_o reflects an increased spacing of surfactant head groups, leading to the encapsulation of oil in water. Surfactant possessing balanced hydrophilicity and lipophilicity yield lamellae, anisotropic phases containing layers of water separated by bilayers of surfactant. Micelles and inverse micelles, isotropic solutions of spherical nanoparticles, form when surfactants become highly polar or apolar, respectively. The self-assembly systems of Figure 1a contain surfactant monomer; the fraction of surfactants that form self-assembly structures increases as the surfactant concentration surpasses a “critical” concentration. Intermediate between micellar and lamellar phases are anisotropic rod-like hexagonal and cubic phases, the latter having structure and properties intermediate of lamellae and rod-like structures. Hexagonal, cubic, and lamellar phases are examples of “lyotropic liquid crystals,” and are commonly formed by monoacylglycerols (MAG) and phospholipids.

Water-oil-surfactant mixtures often form thermodynamically-stable microemulsions, characterized by nanometer-sized architectures (reviewed in [4, 5]). Hydrophilic and lipophilic surfactant systems form O-W- and W/O-microemulsions, respectively, typically consisting of spherical nanodroplets (Figure 1b). Surfactant systems possessing balanced hydrophilicity and lipophilicity form either swollen lamellae or bicontinuous microemulsions, dynamic intertwined networks of oil and water separated by surfactant monolayers (Figure 1b). Microemulsions (and emulsions) are often formed in vivo by delivering a water-free mixture of the components that self-microemulsify (emulsify) upon contact with water.

Surfactants are also employed as therapeutical agents. For instance, saccharide-fatty acid esters, amino acid-based surfactants, and glycolipid biosurfactants possess antimicrobial activity [3,7,8]. Glycolipid biosurfactants and polyunsaturated fatty acid MAGs possess anticancer activity [8, 9]. Sophorolipid biosurfactants are effective modulators of immune response [8]. Surfactants are prominent components of several dermatological, cosmetics, and personal care products.

Figure 2 - Commonly employed surfactants in pharmaceutical products.

The chemical structures of common surfactants employed in pharmaceutical preparations are given in Figure 2 (reviewed in [2, 10, 11]). The surfactants shown are highly biocompatible and nonionic. (Cationic surfactants are needed for the delivery of oligonucleotides. Biobased cationic derivatives of arginine have recently been shown to be potentially effective as biocompatible, delivery agents [12]). As shown, the surfactants generally contain fatty acyl or alcohol lipophiles and polyhydric alcohols, or polyols, as hydrophiles: either glycerol or saccharide derivatives, with the lipophile and hydrophile conjugated via ester or ether bonds. (An exception is α-tocopheryl PEG succinate, TPGS, which is a derivative of vitamin E.) Fatty acyl groups generally consist of linear alkyl chains of 8-16 carbons, with oleic (18:1- 9^cis) or ricinoleic (R-18:1-9^cis -12-OH) acyl groups also employed. Often, chains of poly(ethylene glycol), or PEG, also referred to as “ethoxylate” groups, are conjugated to hydroxyls of polyhydric alcohol to increase hydrophilicity, and to reduce their adsorption onto surfaces or to proteins or other biological molecules, such as polysorbates. In addition to those depicted in Figure 2, other pharmaceutical surfactants include sodium dodecyl sulfate, fatty acid soaps, fatty acid and alcohol ethoxylates, amino acid surfactants [12], block copolymers [e.g., PEG-poly(propylene glycol)], and glycolipid biosurfactants (e.g., sophorolipids, rhamnolipids, and mannosoylerythritol lipids [8]), and bile salts. Polar cosolvents such as ethanol, glycerin, propylene glycol (PG), and (PEG) are commonly employed with surfactants, while common biocompatible oils employed include mixture of long- and medium-chain triaclyglycerols, TAG, often accompanied by smaller amounts of diacylglycerol, DAG, and fatty acid esters such as isopropyl myristate, the latter more prominent in dermatological products.

Another common surfactant category is phospholipids. They are the major components of synthetic lung surfactant, used in the treatment of acute and neonatal distress syndrome [13], and of spherical vesicles known as liposomes, which can consist of 1 or more concentric phospholipid bilayers (uni- or multi-lamellar, respectively), which are common delivery vehicles [14, 15].

Surfactants Employed in Pharmaceuticals are Primarily Biobased

The development of “biobased” surfactants is on the rise due mainly to the increased feedstock cost for petroleum compared to oleochemical starting materials (due to increased global demand and decreased production and availability), and the enhancement of sustainability for utilizing renewable feedstocks [16]. Moreover, dependence upon dwindling production of petroleum (exacerbated by increased global demand) has been linked to environmental damage: the leakage of the “Deepwater Horizon” off-shore oil well in the Gulf of Mexico in 2010 (the largest environmental disaster in US history) and the generation of CO₂ and other greenhouse gases and their impact upon climate change. These factors have increased consumer demand for more sustainable products. In general, the processing cost for preparing biobased surfactants is not significantly different from the production cost of petroleum-derived surfactants. Therefore, the market share of biobased surfactants has increased in recent years, with this trend anticipated to continue.

The majority of the surfactants described in the previous section are at least partially derived from renewable resources [16, 17]. The fatty acyl components of saccharide esters, polysorbates, MAG, and fatty acid ethoxylates are derived from oleochemicals, with fatty acid methyl or ethyl esters, the major component of biodiesel, serving as the principal starting material. Therefore, the production of biobased surfactants integrates well with the development and growth of oleochemical biorefineries to produce fuels, chemical intermediates, and biobased products from oilseed crops [16, 17]. Feedstocks enriched in C10-C16 saturated fatty acyl groups include palm, palm kernel (particularly palm stearine, a palmitic acyl-rich byproduct from the fractionation of palm kernel oil), coconut, and cuphea oils. Inexpensive sources of 16:0, 18:0, 18:1 and 18:2 fatty acyl groups include tallow, used cooking oils, algal oils, jatropha oil, soapnut oil, and soapstock. Ricinoleic acid is derived from castor oil, grown in India, Brazil, and several other countries worldwide. Medium-chain fatty alcohols, the lipophilic group of APGs, can be derived either from petroleum or from fatty acid methyl ester via heterogeneous catalytic reactions [18]. Phospholipids are directly obtained from soapstock, gums, and other oleochemical processing co-products.

Saccharide polar or “head” groups are also derived from inexpensive renewable feedstocks: cellulose, hemicellulose, starch, and other natural polysaccharides. The sorbitan moiety of sorbitan esters and polysorbates (5-member ring that contain an O atom, Figure 2) is derived from the dehydration of sorbitol, a sugar alcohol in turn derived from glucose via hydrogenation, electrochemical reduction, or microbial transformation utilizing oxidoreductases [17] . Glycerol, the head group of MAG, is an abundant and inexpensive co-product produced via transesterification of TAG to prepare biodiesel. However, MAG is often obtained directly from TAG via glycerolysis [17]. Many nonionic surfactants employed in pharmaceuticals contain poly(ethylene glycol), or equivalently, poly(ethylene oxide), as a component of their hydrophile (e.g., polysorbate, Figure 2), through conjugation with fatty acyl or -OH groups. This hydrophile is mostly formed from petroleum-derived ethylene with ethylene oxide serving as a chemical intermediate, the latter being carcinogenic, mutagenic, highly flammable, volatile, and reactive. However, recently biobased ethylene has been derived from bioethanol derived from sugar cane [19].

Advantages of Bioprocessing to Prepare Surfactants for Pharmaceuticals

The surfactants depicted in Figure 2 are typically prepared chemically under harsh conditions (e.g., temperatures between 100 and 250oC), and often are produced at low yield with a broad product distribution and the presence of byproducts, the latter requiring additional purification steps, such as energy-intensive molecular distillation [20, 21]. Either heterogeneous catalysts, acids, or bases are employed, with environmentally unfriendly polar solvents such as dimethylformamide or dimethylsulfoxide often used [20, 21]. An exception is the manufacture of APGs, which are readily produced under solvent-free conditions and relatively mild reaction conditions; however, purification via high-temperature molecular distillation is required to remove excess fatty alcohol reactant. Therefore, chemical processing of surfactants for pharmaceuticals possesses many disadvantages when examined from a sustainability, or life cycle assessment (LCA) -based, perspective. LCA refers to a quantitative analysis of the costs involved with a product “from cradle to grave,” which includes cost factors for their environmental “footprints:” greenhouse gases and toxicants which persist in ecosystems [22]. First, the harsh conditions can lead to product degradation and byproduct formation. Second, the high temperatures lead to excessive energy usage, hence to increased production of CO₂ and other greenhouse gases. Third, heterogeneous catalysts, acid/bases, and/or toxic solvents yield waste products that can harm the environment and induce safety hazards for workers. Fourth, often these reactions produce broad product distributions of desired and undesired products, which can impair product performance, biocompatibility and biodegradability.

Enzymes can potentially play an important role in the manufacture of many biobased surfactants [17]. Bioprocessing provides many advantages compared to chemical processing, particularly for improving sustainability: lower energy use (due to lower temperatures), lower amounts of waste and byproducts, the absence of toxic metal catalysts or acids/bases, and safer operating conditions. The major disadvantages are the prohibitive costs for enzymes compared to chemical catalysts (although this concern is reduced when enzymes are immobilized to enable reuse) and the lower reaction rates that accompany many enzymatic reactions. In addition, due to the need to reduce any inhibitory agents, the starting materials must be pre-purified; for instance, fatty acyl-containing material must not contain phospholipids, aldehydes/ketones, peroxides, and other contaminants. But, as energy costs increase (as anticipated), the importance of sustainability increases (due to government regulation and/or consumer demand), and the capabilities of enzymes and their production systems increase (due to improved biotechnologies), enzymatic bioprocessing is anticipated to become more cost-competitive and attractive.

Enzymatic Preparation of Monoacylglycerols (MAG)

A recent review provides several examples where enzymes have, or potentially can, catalyze formation of biobased surfactants [17]. Herein, a few examples of enzymatic synthesis of biobased surfactants used in pharmaceuticals will be given. Of note, glycolipid biosurfactants are also produced via bioprocessing, specifically, through fermentation.

Preparation of MAG using lipases is well known and can occur via several different routes [23]. Key aspects for bioprocessing are the need to enable miscibility between acyl donor and glycerol substrates and retain low water concentrations for reactions involving ester bond formation. One approach is esterification between glycerol and fatty acid using a molar excess of glycerol. The reaction is typically operated using a thermophilic lipase at 40-75oC, enabling the free evaporation of the co-product water, hence increasing conversion by reducing the extent of the reverse reaction, hydrolysis. Solvents possessing slight polarity that cannot serve as acyl acceptor (e.g., tert-butanol or –pentanol) are often employed to enhance miscibility. The lipase type typically employed possesses either 1,3-positional selectivity (catalyzing ester bonds only at glycerol’s 1- and 3- positions) or is selective to forming partial glycerides (e.g., Penicillum cambertii lipase), to increase selectivity toward MAG and less toward DAG and TAG. (Several free and immobilized lipases from thermophilic organisms are commercially available.) Yet, this reaction typically yields a mixture of MAG and DAG as product, and contains ~10-15% unreacted FFA and perhaps a small amount of TAG. The latter occurs as a result of “acyl migration,” reversible isomerization of 1-(3-)MAG into 2-MAG and 1,2-(2,3-)DAG into 1,3-DAG, with 1-(3-) MAG and 1,3-DAG typically predominating. Glycerolysis using 1,3-selective lipases is conducted under similar conditions, except that TAG rather than FFA serves as acyl donor. MAG is formed both via transesterification and from the original TAG after release of the fatty acyl groups from the 1- and 3- positions. A third approach is 1,3-selective lipolysis of TAG using oil-in-water emulsions at 50oC and in the presence of calcium at pH 10, the latter to irreversibly producing soaps from the released acyl groups, which after saponification cannot participate further in the reaction [24].

Enzymatic Preparation of Saccharide-Fatty Acid Esters

Lipase-catalyzed conjugation of fatty acyl groups and saccharides has been recently reviewed [25]. The reactions utilize mono- or di-saccharides (e.g., fructose, glucose, maltose, ribose, sucrose, and xylose) or sugar alcohols (e.g., mannitol and sorbitol) as acyl acceptor substrate and yield primarily mono- or di-esters due to the regioselectivity of lipases. Moreover, ester bonds are formed almost exclusively from primary –OH groups, with regioselectivity toward a particular –OH group when multiple primary hydroxyls exist. For instance, for the lipase-catalyzed esterification of sucrose, the 6-OH group of the latter’s pyranose ring is selectively acylated over the 1’ and 6’ primary hydroxyls of its furanose ring (Figure 2). As described above for MAG manufacture, the key goals for the successful biotransformation are to enhance miscibility between the acyl donor and acceptor, usually achieved via polar co-solvent, and to maintain low water concentration in the reaction system.

Recently, solvent-free media has successfully been employed: metastable 10-100 μm-sized suspensions of saccharide crystals dispersed in mixtures of acyl donor and the main reaction product, monoester, with the latter being present initially at 5-25 wt% to increase the loading of suspended saccharide crystals in the media [26]. For instance, for the reaction between fructose and oleic acid, the maximum fructose concentration achieved increased from 0.7 wt% up to 2.5 wt % as the ester concentration increased from 5 wt % (initially) to 93% ester during the time course of reaction. Using this approach, the final product consisted of 85-90% ester, of which 85-90% is monoester and the remainder diester. The final product may serve as a technical-grade product not requiring further purification.

Enzyme-catalyzed Synthesis of Alkylpolyglycosides (APG)

Chemical synthesis of alkyl polyglycosides, or APGs, yields a mixture of α- and β-pyranoside isomers. Anomerically pure glycosides may lead to more consistent and reliable performance of APGs from batch to batch; their chemical synthesis is complex, requiring several protection and deprotection steps [27]. Enzymatic synthesis of APG possessing high anomeric purity can be achieved by combining two steps: the synthesis of alkyl (mono)glycoside, or AG, via glycosidases [27] and the elongation of the AG’s oligosaccharide group catalyzed by cyclodextrin glycosyl transferase, CGT [28]. For the first step the medium-chain fatty alcohol serves both as substrate and co-solvent, with mono- or di-saccharide serving as second substrate (e.g., glucose or lactose). Use of the latter disaccharide yielded only AGs due to the concurrent cleavage of glycosidic bonds between the monosaccharide units. A key parameter for operating the glucosidase-catalyzed reaction is the water activity of the reaction medium: a sufficiently high water concentration is needed to retain enzyme activity; however, high water concentration will promote hydrolysis. Typical yields are ≤50%. For the second step, an alkyl glycoside, α-cyclodextrin (a source of additional saccharide units, present at 800% stoichmetric excess), and CGT were dissolved in pH 5.2 buffer at 60oC. This reaction yielded mainly the alkyl β-D-polyglycoside with an overall yield of 50%, with the degree of glycosylation increasing by 6 units, corresponding to the 6 units present in a α-cylcodextrin ring.

Conclusions

Although the technological development is in its infancy, significant potential value for bioprocessing approaches to prepare biobased surfactants for use in pharmaceuticals, as well as foods and commercial products, has been demonstrated. Biocatalytic manufacturing is particularly attractive due to its enhancement of sustainability, its higher selectivity toward desired products, and its lower production of byproducts. However, most of the published work has occurred on the laboratory-to-preparative (0.1-1 kg) scale. Therefore, further work is required for process scale-up and bioreactor design. Biotechnology will need to produce more robust enzymes at lower cost to enable this approach. Scientists and engineers will continue to improve enzymatic bioprocess design and perhaps develop new biobased surfactants for pharmaceutical application as the interest and availability of renewable feedstocks increases.

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Author Biography

Douglas G. Hayes received his B.Sc. and Ph.D. in chemical engineering from Iowa State Univ. (1986) and Univ. Michigan (1991), respectively. He currently serves as a Professor of Biosystems Engineering at Univ. Tennessee, and as Associate Editor for J. Surfactants Deterg., J. Am. Oil Chem. Soc. and Biol. Eng. Trans. He has coauthored over 60 research articles, book chapters, and books. His research areas consist of complex fluids, biocatalysis, lipid chemistry, and biofuels and biobased products. To contact the author please email him directly at: [email protected]