Biologic Activity of Nerve Growth Factor Slowly Released from Microspheres

ABSTRACT

Efficient and sustained delivery of neurotrophic factors to the regenerating nerve in biologically active form presents a challenge. Protein delivery in biodegradable microsphere vehicles has been difficult, based on destabilization and breakdown during both the loading and release phases. This study examines the extravasation and stability of Nerve Growth Factor (NGF) in polylactic-co-glycolic acid (PLGA) microspheres, via both ELISA and PC-12 bioassays.

PLGA microspheres co-loaded with bovine serum albumin (BSA) and NGF were prepared by a water-in-oil-in-water (W/O/W) technique, using chloroform for the organic phase and 1 percent polyvinyl alcohol (PVA) for the emulsion step. Aliquots of lyophilized microspheres were incubated in double distilled water (dd H₂O) at 37° C, and the supernatants assayed over time for NGF activity. ELISA was utilized for quantitative determination of NGF concentration, and a PC-12 cell neurite outgrowth assay assessed biologic activity. Both ELISA and PC-12 assays demonstrated the extravasation of NGF from microspheres. Over time, the predicted concentration of NGF via the two assays differed, suggesting possible preservation of recognizable epitopes for ELISA, but loss of biologic activity. NGF can be stored and released from microspheres. Extravasation studies should include biologically relevant assays for activity.

KEYWORDS

NGF - microspheres - slow release

REFERENCES

• 1 Saltzman W M, Olbricht W L. Building drug delivery into tissue engineering.  Nat Rev Drug Discov . 2002;  1 177-186
• 2 Haller M F, Saltzman W M. Nerve growth factor delivery systems.  J Control Release . 1998;  53 1-6
• 3 Kanje M, Lundborg G, Edstrom A. A new method for studies of the effects of locally applied drugs on peripheral nerve regeneration in vivo.  Brain Res . 1988;  439 116-121
• 4 Van der Zee E C, Brakkee J H, Gispen W H. Alpha-MSH and Org.2766 in peripheral nerve regeneration: different routes of delivery.  Eur J Pharmacol . 1988;  147 351-357
• 5 Laird J M, Mason G S, Thomas K A. Acidic fibroblast growth factor stimulates motor and sensory axon regeneration after sciatic nerve crush in the rat.  Neuroscience . 1995;  65 209-216
• 6 Sterne G D, Brown R A, Green C J, Terenghi G. Neurotrophin-3 delivered locally via fibronectin mats enhances peripheral nerve regeneration.  Eur J Neurosci . 1997;  9 1388-1396
• 7 Worseg A, Zeng L, Leichtfried G. A combination of fibrin sealant and nerve growth factor accelerates the regeneration of leading sensory fibers in experimental nerve repair. In: Schlag G, Holle J, eds. Plastic Surgery Nerve Repair Burns Berlin: Springer Verlag 1995: 73-78
• 8 Saltzman W M, Mak M W, Mahoney M J. Intracranial delivery of recombinant nerve growth factor: release kinetics and protein distribution for three delivery systems.  Pharm Res . 1999;  16 232-240
• 9 Truong-Le V L, August J T, Leong K W. Controlled gene delivery by DNA-gelatin nanospheres.  Hum Gene Ther . 1998;  9 1709-1717
• 10 Hanes J, Sills A, Zhao Z. Controlled local delivery of interleukin-2 by biodegradable polymers protects animals from experimental brain tumors and liver tumors.  Pharm Res . 2001;  18 899-906
• 11 Lewis D. Controlled release of bioactive agents from lactide/ glycolide polymers. In: Chasin M, Langer R, eds. Biodegradable Polymers as Drug Delivery Systems New York: Marcel Dekker, Inc. 1990: 1-41
• 12 Sofroniew M V, Howe C L, Mobley W C. Nerve growth factor signaling, neuroprotection, and neural repair.  Annu Rev Neurosci . 2001;  24 1217-1281
• 13 Camarata P J, Suryanarayanan R, Turner D A. Sustained release of nerve growth factor from biodegradable polymer microspheres.  Neurosurgery . 1992;  30 313-319
• 14 Tabata Y, Gutta S, Langer R. Controlled delivery systems for proteins using polyanhydride microspheres.  Pharm Res . 1993;  10 487-496
• 15 Levi-Montalcini R, Meyer H, Hamburger V. In vitro experiments on the effects of mouse sarcomas 130 and 37 on the spinal and sympathetic ganglia of the chick embryo.  Cancer Res . 1954;  14 49-57
• 16 Krewson C E, Dause R, Mak M, Saltzman W M. Stabilization of nerve growth factor in controlled release polymers and in tissue.  J Biomater Sci Polym Ed . 1996;  8 103-17
• 17 Pean J M, Venier-Julienne M C, Boury F. NGF release from poly(D,L-lactide-co-glycolide) microspheres. Effect of some formulation parameters on encapsulated NGF stability.  J Control Release . 1998;  56 175-187
• 18 Pean J M, Boury F, Venier-Julienne M C. Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres?.  Pharm Res . 1999;  16 1294-1299
• 19 Castellanos I J, Carrasquillo K G, Lopez J D. Encapsulation of bovine serum albumin in poly(lactide-co-glycolide) microspheres by the solid-in-oil-in-water technique.  J Pharm Pharmacol . 2001;  53 167-178
• 20 Cao X, Shoichet M S. Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders.  Biomaterials . 1999;  20 329-339
• 21 Castellanos I J, Cuadrado W O, Griebenow K. Prevention of structural perturbations and aggregation upon encapsulation of bovine serum albumin into poly(lactide-co-glycolide) microspheres using the solid-in-oil-in water technique.  J Pharm Pharmacol . 2001;  53 1099-1107
• 22 Morita T, Sakamura Y, Horikiri Y. Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethylene glycol) as a protein micronization adjuvant.  J Control Release . 2000;  69 435-444
• 23 Carrasquillo K G, Stanley A M, Aponte-Carro J C. Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein.  J Control Release . 2001;  76 199-208
• 24 Carrasquillo K G, Carro J C, Alejandro A. Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation.  J Pharm Pharmacol . 2001;  53 115-120