Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.
OBJECTIVE
Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein.


METHODS
A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay.


RESULTS
In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth through RhoA inhibition. Characterization of TAT-C3 encapsulation in various blends of capped/uncapped PLGA polymer revealed the 30:70 formulation to be optimal in attaining a mild initial burst release of 25%, followed by a subsequent average daily release of 2.3% of encapsulated protein over one month, matching the change in RhoA level in severed brain and spinal cord. Importantly, TAT-C3 released from the microspheres remained active up to the first three weeks of incubation.


CONCLUSIONS
Enhanced cell entry of TAT-C3 circumvents the need to administer high dose of the protein to site of injury. The encapsulation of TAT-C3 in different blends of capped/uncapped PLGA microspheres allows adjustment of protein release profile to suit the pattern of RhoA expression in injured CNS.
Tan
Elaine Y M
EY
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD9, 2 Medical Drive, Singapore 117597, Singapore.
Law
Janice W S
JW
Wang
Chi-Hwa
CH
Lee
Alan Y W
AY
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001672:Biocompatible Materials; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D002626:Chemistry, Pharmaceutical; D003692:Delayed-Action Preparations; D004337:Drug Carriers; D004339:Drug Compounding; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005240:Feasibility Studies; D007700:Kinetics; D019344:Lactic Acid / Q000737:chemistry*; D051379:Mice; D008863:Microspheres; D041681:NIH 3T3 Cells; D009416:Nerve Regeneration / Q000187:drug effects*; D016501:Neurites / Q000187:drug effects / Q000201:enzymology; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D018696:Neuroprotective Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010316:Particle Size; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers / Q000737:chemistry*; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012995:Solubility; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D001672:Biocompatible Materials; D003692:Delayed-Action Preparations; D004337:Drug Carriers; D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D010446:Peptide Fragments; D011108:Polymers; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D000246:Adenosine Diphosphate Ribose; D011100:Polyglycolic Acid; D019344:Lactic Acid; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein