New classes of products are being tested for use in humans and animals, all sharing genes as common targets. Products based on antisense technology directed toward neutralizing messenger RNA are probably being pursued most vigorously; gene therapy through permanent alteration of chromosomes might hold the greatest potential for treatment of diseases like cancer and for correction of genetic disease. The products depend either on classes of compounds that are related to nucleic acids (oligonucleotides and oligonucleotide analogues), on cells that have been genetically altered, of on viruses that bear appropriate nucleic acids.
For the large-scale production of nucleotides and nucleotide analogues, new molecular techniques must be developed. There are now no procedures for making substantial quantities of these types of materials in high purity and with appropriate chirality. Basic chemical and biochemical techniques must be developed for their preparation; new techniques (probably based on high-pressure chromatography) will be required for large-scale purification, and biological methods might be required for preparation of precursors and perhaps for formation of bonds.
For genetically modified cells and viruses, the usual techniques for mammalian-cell culture and molecular biology will be required, as will additional measures for safety and for economical, patient-specific production.
BIOTECHNOLOGY
Microencapsulation for Cell Delivery
Microencapsulation is currently the most widely used form of cell delivery with preparation methods including:
1. Gelation and polyelectrolyte complexation,
2. Interfacial polymerization/phase inversion and
3. Conformal coating.
Microencapsulation involves surrounding a collection of cells with a thin generally micrometer sized, semipermeable membrane. Its primary purpose is to protect the encapsulated cells from the host’s immune system, while allowing the exchange of small molecules and thereby ensuring cell survival and function. There are several requirements for polymer capsules or hydrogels used as components of microcapsules:
# Noncytotoxicity to the encapsulated cells
# Biocompatibility with the surrounding environment where capsules are to be implanted (e.g., minimal fibrotic response)
# Adequate permeability for diffusion of essential nutrients (e.g., oxygen and glucose for islets of Langerhans) and cell secretory products (such as insulin, metabolic waste)
# Impermeability to secreted antibodies of the host’s immune system (e.g., immunoglobulins and glycoproteins after complement activation
# Chemical and mechanical stability
From the technological point of view, the requirements for microencapsulation include:
# Small capsule diameters to ensure sufficient diffusion and internal organ transplantability (depending on application, <>
# Minimum shrinking/swelling due to changes in osmotic conditions upon transplantation
# Uniform wall thickness for optimum transport of molecules across the membrane and effective immunoprotection.
In addition, the technology used for encapsulation must be nontraumatic to the encapsulated cells. This includes minimizing the mechanical stress during encapsulation and solvent toxicity (if any), as well as optimizing temperature, viscosity, pH and ionic strength. This, in turn, limits the concentration and molecular mass which can be employed. In addition, the ionic content of the polymer backbone (density distribution of charges in the polymer chain), the chemistry and location of functional group attachment, the chain rigidity, aromaticity, conformation and extent of branching were identified as important variables in the type of complex produced. The presence of secondary hydrogen bonding interactions was also found to be significant.
Several problems may prevent wide scale application of microcapsules in the clinic. The capsules can clump together, in which case the cells towards the center may suffer severely from limited diffusion of oxygen and nutrients. A substantial fraction of the capsules may also adhere to tissue. If the capsules degrade, the liberated islet cells, even if nonviable, would greatly increase the antigenic burden on the patient. Semipermeable polymeric membranes have been developed with the aim of permitting the transplantation of xenogenic cells thus removing the need for immunosuppression therapy. However, early clinical implementations is not likely to involve xenografts or genetically modified cells but rather auto- and allografts supplemented by immunosuppression when necessary.
1. Gelation and polyelectrolyte complexation,
2. Interfacial polymerization/phase inversion and
3. Conformal coating.
Microencapsulation involves surrounding a collection of cells with a thin generally micrometer sized, semipermeable membrane. Its primary purpose is to protect the encapsulated cells from the host’s immune system, while allowing the exchange of small molecules and thereby ensuring cell survival and function. There are several requirements for polymer capsules or hydrogels used as components of microcapsules:
# Noncytotoxicity to the encapsulated cells
# Biocompatibility with the surrounding environment where capsules are to be implanted (e.g., minimal fibrotic response)
# Adequate permeability for diffusion of essential nutrients (e.g., oxygen and glucose for islets of Langerhans) and cell secretory products (such as insulin, metabolic waste)
# Impermeability to secreted antibodies of the host’s immune system (e.g., immunoglobulins and glycoproteins after complement activation
# Chemical and mechanical stability
From the technological point of view, the requirements for microencapsulation include:
# Small capsule diameters to ensure sufficient diffusion and internal organ transplantability (depending on application, <>
# Minimum shrinking/swelling due to changes in osmotic conditions upon transplantation
# Uniform wall thickness for optimum transport of molecules across the membrane and effective immunoprotection.
In addition, the technology used for encapsulation must be nontraumatic to the encapsulated cells. This includes minimizing the mechanical stress during encapsulation and solvent toxicity (if any), as well as optimizing temperature, viscosity, pH and ionic strength. This, in turn, limits the concentration and molecular mass which can be employed. In addition, the ionic content of the polymer backbone (density distribution of charges in the polymer chain), the chemistry and location of functional group attachment, the chain rigidity, aromaticity, conformation and extent of branching were identified as important variables in the type of complex produced. The presence of secondary hydrogen bonding interactions was also found to be significant.
Several problems may prevent wide scale application of microcapsules in the clinic. The capsules can clump together, in which case the cells towards the center may suffer severely from limited diffusion of oxygen and nutrients. A substantial fraction of the capsules may also adhere to tissue. If the capsules degrade, the liberated islet cells, even if nonviable, would greatly increase the antigenic burden on the patient. Semipermeable polymeric membranes have been developed with the aim of permitting the transplantation of xenogenic cells thus removing the need for immunosuppression therapy. However, early clinical implementations is not likely to involve xenografts or genetically modified cells but rather auto- and allografts supplemented by immunosuppression when necessary.
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