Hyaluronidase is an effective adjuvant in the permeabilization of muscle fibers.?

OLAIZ N; SIGNORI E; GUERRA L; SUAREZ, ; VILLAVERDE M. S.; COLOMBO L; MICHISNKI S; MARSHALL G.

Conferencia; 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies; 2015

INTRODUCTIONSince 1950, hyaluronidase has been known as a ?spreading factor? favoring better diffusion of traditional vaccines in the site of inoculation. Nowadays, hyaluronidase is used in the clinical practice as adjuvant therapy to increase the absorption and dispersion of injected drugs. In our laboratory we are evaluating the optimal conditions for effective gene electrotransfer (GET) protocols for the delivery of plasmid vectors. We have theoretically predicted by in silico modeling that optimal efficiency in GET protocols could be achieved when the applied electric field is near 158 V/cm, using hyaluronidase as an adjuvant (Olaiz N et al, Bioelectrochemistry, 100: 105-11, 2014). Here, we evaluated in vivo our previous theoretical predictions.MATERIALS AND METHODSSix to eight-weeks-old Balb-C mice (weight: 21.75 + 1.53 g) were injected in both of their tibialis anterior muscles (TAM) with 25 ug bgalactosidase vector followed by different GET protocols (10 pulses of 20 ms and 200, 175, 150, 50 or 0 V/cm, at 1Hz). In the left TAM of these animals, a pretreatment with 25 ug bovine hyaluronidase was performed. The animals were euthanized 8 days after the treatment, TAM was excised and their fibers evaluated by X-gal stained dark blue. All staining was performed on 160 m thick cross-sections cut (cryostat-microtome) through a plane perpendicular to the surface of the electrodes at the center of the treated region. Hematoxylin and eosin (H&E) staining was performed to study the morphology of the affected cells and to delineate the margin between normal and damaged cells. The H&E-stained section was first cut from the tissue block, and another parallel slice was separately taken for the X-gal staining. RESULTSIn previous similar studies with GET protocols, we observed pH fronts in the animal skin with values reaching pH 2 at the anode and pH 10 at the cathode. With these extreme pH values, we could expect tissue damage in muscle fibers affecting vector expression. Here, we found that vector expression was negative with GET protocols of 150, 50 and 0 V/cm, with no observed tissue damage concluding that pretreatment with hyaluronidase had no effect in those cases. GET protocols with electric fields higher than 150 V/cm without hyaluronidase pretreatment allowed less than 4% of positive cells for vector expression in each muscle section. With these protocols, only 10% of all sections from the whole mouse TAM evaluated were positives. Nevertheless, with hyaluronidase pretreatment , electric fields higher than 150 V/cm induced near 8% of positive cells for vector expression in each muscle section, with more than 30% of positive sections from the whole mouse TAM. These GET protocols with hyaluronidase pretreatment induced a specific positive-cell pattern extending from the edges to the center of the muscle. GET protocols at 200 V/cm and hyaluronidase pretreatment also produced high damage in each evaluated section. CONCLUSIONSOur results show that: a) the presence of pH fronts in mice skins when electric fields are applied is experimentally corroborated; b) enhanced level of the expression vector in tissues pretreated with hyaluronidase and electric fields 150 V/cm is experimentally corroborated; c) evidence that electric fields between 150 and 200 V/cm would be optimal for GET protocols based on pretreatment with hyaluronidase is experimentally corroborated; d) the presence of tissue damage when the electric field applied is near 200V/cm is experimentally corroborated; e) theoretical predictions from (1) are experimentally corroborated here.