Coacervation

For preparative purposes batch fractionation is often employed. Although fractional crystallization may be included in a list of batch fractionation methods, we shall consider only those methods based on the phase separation of polymer solutions fractional precipitation and coacervate extraction. The general principles for these methods were presented in the last section. In this section we shall develop these ideas more fully with the objective of obtaining a more narrow distribution of molecular weights from a polydisperse system. Note that the final product of fractionation still contains a distribution of chain lengths however, the ratio M /M is smaller than for the unfractionated sample. 

Figure 8.5 illustrates the sort of separation this approach predicts. Curve A in Fig. 8.5 shows the weight fraction of various n-mers plotted as a function of n. Comparison with Fig. 6.7 shows that the distribution is typical of those obtained in random polymerization. Curve B shows the distribution of molecular weights in the more dilute phase-the coacervate extract-calculated for the volumes of the two phases in the proportion 100 1. The distribution in the concentrated phase is shown as curve C it is given by the difference between curves A and B. 

Coa.cerva.tlon, A phenomenon associated with coUoids wherein dispersed particles separate from solution to form a second Hquid phase is termed coacervation. Gelatin solutions form coacervates with the addition of salt such as sodium sulfate [7757-82-6] especially at pH below the isoionic point. In addition, gelatin solutions coacervate with solutions of oppositely charged polymers or macromolecules such as acacia. This property is useful for microencapsulation and photographic apphcations (56—61). 

Commercial lecithin is insoluble but infinitely dispersible in water. Treatment with water dissolves small amounts of its decomposition products and adsorbed or coacervated substances, eg, carbohydrates and salts, especially in the presence of ethanol. However, a small percentage of water dissolves or disperses in melted lecithin to form an imbibition. Lecithin forms imbibitions or absorbates with other solvents, eg, alcohols, glycols, esters, ketones, ethers, solutions of almost any organic and inorganic substance, and acetone. It is remarkable that the classic precipitant for phosphoHpids, eg, acetone, dissolves in melted lecithin readily to form a thin, uniform imbibition. Imbibition often is used to bring a reactant in intimate contact with lecithin in the preparation of lecithin derivatives. 

Table 1 Hsts representative examples of capsule shell materials used to produce commercial microcapsules along with preferred appHcations. The gelatin—gum arabic complex coacervate treated with glutaraldehyde is specified as nonedible for the intended appHcation, ie, carbonless copy paper, but it has been approved for limited consumption as a shell material for the encapsulation of selected food flavors. Shell material costs vary greatly. The cheapest acceptable shell materials capable of providing desired performance are favored, however, defining the optimal shell material for a given appHcation is not an easy task.

Complex Coacervation. This process occurs ia aqueous media and is used primarily to encapsulate water-iminiscible Hquids or water-iasoluble soHds (7). In the complex coacervation of gelatin with gum arabic (Eig. 2), a water-iasoluble core material is dispersed to a desired drop size ia a warm gelatin solution. After gum arabic and water are added to this emulsion, pH of the aqueous phase is typically adjusted to pH 4.0—4.5. This causes a Hquid complex coacervate of gelatin, gum arabic, and water to form. When the coacervate adsorbs on the surface of the core material, a Hquid complex coacervate film surrounds the dispersed core material thereby forming embryo microcapsules. The system is cooled, often below 10°C, ia order to gel the Hquid coacervate sheU. Glutaraldehyde is added and allowed to chemically cross-link the capsule sheU. After treatment with glutaraldehyde, the capsules are either coated onto a substrate or dried to a free-flow powder. 

Eig. 2. Elow diagram of a typical encapsulation process based on the complex coacervation of gelatin with gum arabic. 

Any pair of oppositely charged polyelectrolytes capable of forming a Hquid complex coacervate can be used to form microcapsules by complex... 

A wide variety of capsules loaded with water-immiscible or water-iasoluble materials have been prepared by complex coacervation. Capsule size typically ranges from 20—1000 p.m, but capsules outside this range can be prepared. Core contents usually are 80—95 wt %. Complex coacervation processes are adversely affected by active agents that have finite water solubiUty, are surface-active, or are unstable at pH values of 4.0—5.0. The shell of dry complex coacervate capsules is sensitive to variations ia atmospheric moisture content and becomes plasticized at elevated humidities. 

The presence of heat-sensitising or delayed-action coacervants. 

Xylans from beech wood, corncobs, and the alkaline steeping liquor of the viscose process have been shown to be applicable as pharmaceutical auxiliaries [3]. Micro- and nanoparticles were prepared by a coacervation method from xylan isolated from corncobs [150]. The process is based on neutralization of an alkaline solution in the presence of surfactant, which was shown to influence both the particle size and morphology. They are aimed at applications in drug delivery systems. 

Xylan-based micro- and nanoparticles have been produced by simple coacervation (Garcia et al., 2001). In the study, sodium hydroxide and chloride acid or acetic acid were used as solvent and non-solvent, respectively. Also, xylan and surfactant concentrations and the molar ratio between sodium hydroxide and chloride acid were observed as parameters for the formation of micro- and nanoparticles by the simple coacervation technique (Garcia et al., 2001). Different xylan concentrations allowed the formation of micro- and nanoparticles. More precisely, microparticles were found for higher concentrations of xylan while nanopartides were produced for lower concentrations of the polymer solution. When the molar ratio between sodium hydroxide and chloride acid was greater than 1 1, the partides settled more rapidly at pH=7.0. Regarding the surfactant variations, an optimal concentration was found however, at higher ones a supernatant layer was observed after 30 days (Garda et al., 2001). 

Qv, X. Y., Zeng, Z. P. Jiang, J. G. (2011). Preparation of lutein microencapsulation by complex coacervation method and its physicochemical properties and stability. Food Hydrocolloids, Vol. 25, 6, (August 2011), pp. (1596-1603), ISSN 0268-005X... 

In spite of the overwhelming evidence suggesting that recombinant resilin is amorphous, there are some results that suggest that a level of defined stmcmre cannot be completely ruled out. In particular, the fact that the protein solution coacervates when cooled (Figure 9.7) suggests that there is a degree of self-association between protein molecules. 

Coacervation occurs in tropoelastin solutions and is a precursor event in the assembly of elastin nanofibrils [42]. This phenomenon is thought to be mainly due to the interaction between hydro-phobic domains of tropoelastin. In scanning electron microscopy (SEM) picmres, nanofibril stmc-tures are visible in coacervate solutions of elastin-based peptides [37,43]. Indeed, Wright et al. [44] describe the self-association characteristics of multidomain proteins containing near-identical peptide repeat motifs. They suggest that this form of self-assembly occurs via specific intermolecular association, based on the repetition of identical or near-identical amino acid sequences. This specificity is consistent with the principle that ordered molecular assembhes are usually more stable than disordered ones, and with the idea that native-like interactions may be generally more favorable than nonnative ones in protein aggregates. 

FIGURE 9.7 Coacervated purified recombinant resilin. The lower phase of the aqueous solution contains about 300 mg/mL of purified recombinant resilin protein. The upper aqueous phase contains about 20 mg/mL protein. The protein solution separates at 4°C into a protein-rich lower phase. 

Vrhovski, B., Jensen, S., and Weiss, A.S., Coacervation characteristics of recombinant human tropoelas-tin, Europ. J. Biochem., 250, 92-98 1997. 

Phase separation microencapsulation procedures are suitable for entrapping water-soluble agents in lactide/glycolide excipients. Generally, the phase separation process involves coacervation of the polymer from an organic solvent by addition of a nonsolvent such as silicone oil. This process has proven useful for microencapsulation of water-soluble peptides and macromolecules (48). 

The coacervation of tropoelastin plays a crucial role in the assembly into elastic fibers. This coacervation is based on the LCST behavior of tropoelastin, which causes tropoelastins structure to become ordered upon raising the temperature. The loss of entropy of the biopolymer is compensated by the release of water from its chain [2, 18, 19]. This release of water results in dehydration of the hydrophobic side chains, and this is the onset of the self-assembly leading to the alignment of tropoelastin molecules. 

This coacervation process forms the basis for the self-assembly, which takes place prior to the crosslinking. The assembly of tropoelastin is based on an ordering process, in which the polypeptides are converted from a state with little order to a more structured conformation [8]. The insoluble elastic fiber is formed via the enzymatic crosslinking of tropoelastin (described in Sect. 2.1). Various models have been proposed to explain the mechanism of elasticity of the elastin fibers. 

Fig. 5 Coacervation curve of 40 mg/mL tropoelastin in 10 mM sodium phosphate pH 7.4, containing 150 mM NaCl. Maximal coacervation was obtained at 37 °C. Reproduced from [20] with permission from John Wiley and Sons, copyright 1997...

LLP-assisted local delivery of dmgs is realized via several strategies. In the first strategy, soluble LLPs are injected and their coacervation is triggered by the body temperature. Here, the dmg can be covalently attached to the ELP or it can just be mixed with the ELP. In another approach, crosslinked ELP depots containing a dmg can be produced and then implanted to generate a stable release of the dmg. 

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