Protein encapsulation methods

Counterion extraction Due to the relative slowness of back extraction based on the methods above, the back-extraction of proteins encapsulated in AOT reverse micelles was evaluated by adding a counterionic surfactant, either TOMAC or DTAB, to the reverse micelles [33]. This novel backward transfer method gave higher backward extraction yields compared to the conventional method. The back-extraction process with TOMAC was found to be 100 times faster than back-extraction with the conventional method, and as much as three times faster than forward extraction. The 1 1 complexes of AOT and TOMAC in the solvent phase could be efficiently removed using adsorption onto montmorillonite so that the organic solvent could be reused. 

There are a wide variety of encapsulating agents available on the market. Modified food starches, maltodextrins, gums, proteins, corn syrups and sugars are popular choices (4-7). The selection of an encapsulating agent depends upon the chemical composition of the flavor, the encapsulation method, the desired properties of the final microcapsule and its end uses. Other considerations include cost and availability. 

In addition, tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communicating with the external medium. The inventors claimed that these results constitute a step ahead in the improvement of an existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple solvent evaporation method [215]. 

In terms of oral delivery, under normal conditions, only negligible amounts of proteins are absorbed intact through the gastrointestinal (GI) tract [70,71]. Ingested proteins are naturally fragmented into amino acids and short peptides by various enzymes present in the gut [72]. These proteolytic enzymes are found both in the lumen and in the mucosal epithelium therefore, although proteins can be protected from the luminal enzymes by common encapsulated methods, they are still susceptible to degradation at the absorption site by various mucosal enzymes located in the intestinal walls. 

Physical immobilization methods do not involve covalent bond formation with the enzyme, so that the native composition of the enzyme remains unaltered. Physical immobilization methods are subclassified as adsorption, entrapment, and encapsulation methods. Adsorption of proteins to the surface of a carrier is, in principle, reversible, but careful selection of the carrier material and the immobilization conditions can render desorption negligible. Entrapment of enzymes in a cross-linked polymer is accomplished by carrying out the polymerization reaction in the presence of enzyme the enzyme becomes trapped in interstitial spaces in the polymer matrix. Encapsulation of enzymes results in regions of high enzyme concentration being separated from the bulk solvent system by a semipermeable membrane, through which substrate, but not enzyme, may diffuse. Physical immobilization methods are represented in Figure 4.1 (c-e). 

PLGA is the most widely used polymer for encapsulating proteins for pulmonary delivery. The most commonly used method for preparing protein-encapsulated PLGA microspheres is the solvent evaporation technique based on the formation of a double emulsion (w/o/w). Incorporation of protein into the microspheres could be done by two methods [15, 16]. 

Protein preparation method (e.g., immobilization, lyophilization, deriva-tization, and encapsulation)... 

So far, little work has been done to encapsulate therapeutic proteins. Hence, the encapsulation of recombinant insulin was a challenging test of therapeutic protein encapsulation in PEG-based polymersomes [241], Encapsulation of insulin in neutral and biologically stable PEG-PBD polymersomes provides a promising method to increase therapeutic efficiency by maintaining protein structure. 

A different approach of protein encapsulation is reported by Morel, Gasco, and Cavalli [38], These authors describe a method of applying a warm multiple microemulsion in which the peptide is dissolved in an aqueous solution and added to a mixture of melted stearic acid, egg lecithin, and butyric acid at 70°C. This primary microemulsion is then added at 70°C to an aqueous solution of egg lecithin, butyric acid, and taurodeoxycholate sodium salt. Addition of warm multiple microemulsions to water at 2°C leads to precipitation of the lipid phase, forming solid lipospheres. 

In summary, the tested encapsulation method is applicable for other components and different concentrations (aromas, antioxidants, or other oil substances). Other possible supports may be proposed to replace maltodextrin and acacia gum as modified starch (Buffo et al., 2002), proteins, gums, or protective agents. 

Kato M, Kumiko SK, Matsumoto N, Toyo oka T (2002) A protein-encapsulation technique by the sol-gel method for the preparation of monolithic columns for capillary electrochromatography. Anal Chem 74 1915-1921... 

Li YK, Chou MJ, Wu TY, Jinn TR, Chen-Yang YW (2008) A novel method for preparing a protein-encapsulated bioaerogel using a red fluorescent protein as model. Acta Biomater 4 725-732 Gao S, Wang Y, Wang T, Luo G and Dai Y (2009) ImmobUization of hpase on methyl-modified silica aerogels by physical adsorption. Bioresour Technol 100 996-999... 

Takahiro, M., Yumi, S., Yuji, H., Takehiko, S., and Yoshino, H. 2000. Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethylene glycol) as a protein micronization adjuvant. / Control Release, 69,435 4. 

Usually, the encapsulation methods for proteins in PLA are modifications of two basic techniques solvent extraction/solvent evaporation and spray drying [41,42],... 

---