Microencapsulation

Microencapsulation of Single Cells for Delivery Applications and Microenvironmental Control

Microencapsulation describes a technique of surrounding cells and tissue with a synthetic, possibly porous, matrix. The matrix is used to isolate the cells from the surrounding tissue while still allowing chemical interaction. One common use application is the microencapsulation of pancreatic islets. The function of these clusters is to secrete insulin in response to changes in glucose levels. Because of the short supply of donors, people are examining the potential of pig pancreatic islets for xenotransplantation in humans suffering from type II diabetes. Microencapsulation is essential to prevention of the severe immune response. The encapsulated islets usually range in size from 300-4500 microns. Common materials used to encapsulate the cells are agarose, alginate, poly-L-lysine, chitosan, glycosaminoglycans, polyacrylates, carboxymethylcellulose.

A novel material based on the polymerization of a photomonomer, such as acrylic acid, will be designed to coat the surface of the cells (Figure 1). A photomonomer will be covalently bonded to a short segment of poly-L-Lactide (PLA) which, in turn, is attached to the amino acid sequence arginine-glycine-glutamate (RGD). This sequence is specific to cell adhesion proteins (integrins) located on the cell surface. This compound will be allowed to attach to the cell surface before photopolymerization is initiated. Segments of PM-PLA-PM and PM alone will be included as part of the polymerization. The ratio of these components will determine the physical characteristics of the matrix such as thickness, mechanical strength, and diffusivity. The length of the PLA segments will determine the degradation rate. Since only one linkage needs to be broken in order to degrade the matrix, the degradation rate increase as the segment increase in length.

Figure 1: Encapsulation of individual cells by photopolymerization.The encapsulation matrix is formed by the polymerization of a photomonomer (PM). PM is attached to poly-L-lactide (PLA) and the tripeptide RGD. The RGD motif will attach to the integrins found on the cell surface. The matrix will degrade as the PLA is hydrolyzed.

Encapsulation is usually accomplished by either emulsion formation of a two-phase system or by dropwise extrusion through a needle syringe. Emulsions can be formed with agarose within paraffin oil. Shaking is done to obtain the desired droplet size of the agarose. With dropwise extrusion, the islets are transferred from one solution into a different one where some form of polymerization occurs.

Both of these methods will be evaluated for their capacity to separate single cells and encapsulate them. In order to mimic dropwise extrusion, cells will be passed through a device akin to a flow cytometer (Figure 2). With this device, the cells will move through a nozzle where a sheath fluid is introduced to reduce the shear stresses on the cell. A piezoelectric transducer will be used to vibrate the stream and form the individual drops. An appropriate choice for the photomonomer can be made to ensure the polymerization reaction will occur during the time the cell falls to the collection vessel. The small droplet size will assist in transferring heat that is evolved during the exothermic reaction. Alternatively, cells can be separated within a liquid-liquid phase emulsion. The choice of emulsifier and agitation will be chosen for their capacity to separate the cells without placing mechanical and chemical stresses on them. Once separated, the light source will initiated] the photopolymerization reaction. In either case, the system will need to be optimized with respect to photopolymer (PM), PM-PLA-PM, and PM-PLA-RGD concentrations. These concentrations can be adjusted to affect the thickness and mechanical strength of the encapsulation as well as the transport characteristics of molecules that pass through the membrane.

Figure 2: Dropwise separation of single cells. Cells flowing through the nozzle will be isolated in individual drops by vibration s from the piezoelectric transducer. Sheath fluid will help to reduce shear stresses as the cells exit the nozzle.

Encapsulating single cells will provide a method for delivering the cells to specific targets as well as controlling a local microenvironment. For example, the encapsulation will protect type II alveolar cells during aerosolization for delivery into the lungs. The cells will be protected from the shear stresses that occur during aerosol formation. In addition, chemical factors such as vascular endothelial growth factor can be incorporated into the biodegradable matrix to stimulate blood vessel formation. This therapy can be used to increase the surface area in the lungs available for oxygen transfer for patients suffering from alveolar degenerative diseases such as emphysema.

Another application is the control of the microenvironment of nerve tissue regenerating within a three dimensional matrix. The surface of encapsulated Schwann cells can be modified to promote binding to the three-dimensional matrix. Growth factors secreted by the Schwann cells will encourage axon extension and control the direction by chemotaxis. Surface modification can be used to attach ligands that are specific to certain tissue within the body. For example, cardiac muscle cells can be encapsulated and target to the heart to replace damaged tissue.

Specific Projects:

• Synthesis organic molecule (RGD-PLA-PM) as well as (PM-PLA-PM) linker molecules

• Encapsulate cells using the emulsion method

• Test cells for survival rate

• Test polymer for degradation rate