BTK 4102Advanced Enzyme Technology

Assoc. Prof. Dr. Lai Oi Ming

Topic 8:

Microencapsulation

Learning Outcomes

At the end of the topic, you should know :1. The advantages and disadvantages of

microencapsulation2. Different techniques used to perform

microencapsulation

Introduction• Enzyme in solution is entrapped

in a membrane system such that it creates an intracellular environment for the enzyme.

• Usually, the membrane is semi-permeable, does not allow the enzyme out but allows low molecular weight substrates to diffuse inside to interact with the enzyme.

E

EE

E

E

Semi-permeable membrane

Other molecules that can stabiliseenzyme

Other molecules impermeable to membrane

S

P

“Artificial cells”

• Besides enzymes, cells, vaccine Ag, antiserum, cofactor, hormone can also be immobilised using this technique.

• This method provides a means of utilising an enzyme in its native statecontinuously over a long period of time and no bonds are formed.

• This technique is simple, cheap, used mostly in medical applications and to be effective, the biocatalyst must be stable in solution form.

• In particular, this technique has been used in vivo where the capsules protects the enzyme from the host’s immune system.

• Materials that have been microencapsulated are from hemoglobin to entire cell contents of erythrocytes. However, practical applications of these systems have not been utilised.

Figure 1. Optical photomicrograph of Hb-loaded alginate microspheres (magnification 200 X).

• Microencapsuation is nevertheless used for the slow release of oils, perfumes, dyes and hormones.

• By comparisons, the conditions required for the encapsulation of nonbiologicalmaterials, those for microencapsulation of enzymes etc. must be carefully controlled.

Types of membrane materials

• Synthetic membranes• cellulose nitrate

membrane• nylon membrane• dialysis membrane• epoxy resins• butyl rubber

• Biological membranes (biodegradable)

• ultra thin spherical lipid membrane (single lipid layer membrane) - like RBC

• liposome-phospholipidbilayer membranes

• Reverse micelles

Basic Procedures for Microencapsulation

• There are three basic procedures:

a) coacervation (or phase separation)

b) interfacial polymerisation

c) liquid drying

• a) Coacervation or Phase Separation Process

• An aqueous solution of enzyme is dispersed in a water-immiscible organic solvent containing the polymer in question. The types of polymer that can be used include cellulose nitrate, cellulose acetate or butyl rubber.

• To this, a second water immiscible organic solvent is added with stirring.

• Phase separation of the colloidal particles of the polymer which then takes place causes them to associate around the small aqueous droplets which contain the enzymes.

Aqueous solution of enzyme

Organic solvent

Organic solvent containngpolymer

• A continuous membrane is formed by coalescence of the droplets.

coalescence

of droplets

Enzyme solution

Enzyme solution

polymer

b) interfacial polymerisation• A common example of this technique is

the formation of nylon.• An aqueous solution of enzyme is

emulsified with a hydrophilic monomer in a water-immiscible organic solvent.

• A hydrophobic monomer solution dissolved in the same water immiscible organic solvent is then added and the mixture is stirred.

Hydrophilic monomer

Hydrophobic monomer

Aqueous solution of enzyme

• The polymerisation that occurs at the interface between the aqueous and organic phases results in the enzymes being encapsulated by a membrane of polymer.

• In this procedure, the size of the capsules can be varied by adjusting the stirringrates.

c) Liquid drying

• In this procedure, a suitable polymer (eg polystyrene) is dissolved in a water immiscible solvent (eg benzene, chloroform) with a boiling point lower than water.

• An aqueous solution of the enzyme to be encapsulated is then dispersed in the organic phase to form an emulsion.

• This emulsion is then dispersed in an aqueous phase containing protective colloidal substances such as gelatin, to form a second emulsion.

• While the emulsion is being stirred, the organic solvent is being removed by warming in vacuo.

• During this process, the enzymes are encapsulated by a membrane of the polymer.

Enzyme solution

polymer

Enzyme solution

Microencapsulation Using Reverse Micelles

• Reversed micelles provide a mechanism of suspending a low volume of aqueous phase in a large volume of organic phase.

Detergent monolayer

Organic phase

Aqueous phase (enzyme solution)

• A correctly proportioned mixture of buffered enzyme solution, immiscible organic solvent and detergent (egAOT= aerosol diisoocytl sulphosuccinate) will spontaneously transform into a microheterogenoussystem in which a uniformly sized "droplets" of aqueous phase surrounded by a monolayer of detergent are dispersed in the bulk organic phase.

• Reversed micelles are easy to generate, reproducibly stable and uniform and the enzyme is protected from the organic phase.

• As for the substrates, a finite concentration of substrate must be able to dissolve in the detergent monolayer in an amount accessible to the solvated enzyme.

Microencapsulation with Liposomes

• Liposomes are formed when a water insoluble polar lipid such as phosphatidylcholine meets with water and upon sonification (agitation), these structures rearrange to form a closed-sealing solvent filled vesicles that are bounded by only a single bilayer of lipid molecules.

• During the period of arrangement of the lipid molecules, protein or other materials in the aqueous solution are trapped within the liposomes.

• Liposome entrapped proteins have enjoyed considerable success in therapeutic use where the entrapped proteins are released into the circulator system.

• The general process for preparation of protein containing liposomes consists of dissolving the lipid in organicsolvent with subsequent elimination of the solvent under reduced pressure.

• This leaves a thin lipid layer on the walls of the container. This is dispersed with the protein solution. Liposomes containing the protein forms spontaneously

• Microencapsulation of enzymes in liposomes a) Negatively charged liposomes (PO3-)

b)Positively charged liposomes (NH3+)• Similar to (a) except• Stearylamine, a +ve charged• Lipid is used.

+ +

++++++

• Microencapsulation of Asparaginase Enzyme in Red Blood Cells (RBC)

Disadvantages of Injecting Free Enzyme Solutions Into Human Body

a) May cause immunological reactions - rejection followed by the rapid removal or inactivation and destruction of the free enzymes by proteolytic enzymes.

b) Free enzymes cannot be kept at target sites for long and will be carried away by blood stream/body fluids to other parts of the body

• Enzymes in free solution is not stable and activity will rapidly decrease after injection.

• Thus, microencapsulating enzyme in RBCis one way to bring the enzyme to target sites, cause no immunological response while maintaining the activity of the enzymes, without being destroyed by proteolytic enzymes.

Advantages of Microencapsulationa) The enzymes/cells are protected from the

changes in temperature, pH or ionic strength of the bulk phase.

b) The microcapsule can also protect the enzymes/cells from the body’s immune system.

c) If biological capsules such as RBC are used with a patient, possible rejection and removal from the body is prevented.