Vascular reactions to in vivo electroporation : characterization and consequences for drug and gene delivery Julie Gehl a ; *, Torben Skovsgaard a , Lluis M. Mir b; 1 a Department of Oncology 54B1, University of Copenhagen in Herlev Hospital, 75 Herlev Ringvej, 2730 Herlev, Denmark b Laboratory of Physicochemistry and Pharmacology of Biological Macromolecules, UMR 8532 CNRS, Institut Gustave-Roussy, 94805 Villejuif, France Received 11 July 2001; received in revised form 12 October 2001; accepted 19 October 2001 Abstract In vivo electroporation (EP) is gaining momentum for drug and gene delivery. In particular, DNA transfer by EP to muscle tissue can lead to highly efficient long-term gene expression. We characterized a vascular effect of in vivo EP and its consequences for drug and gene delivery. Pulses of 10^20 000 Ws and 0.1^1.6 kV/cm were applied over hind- and forelimb of mice and perfusion was examined by dye injection. The role of a sympathetically mediated vasoconstrictory reflex was investigated by pretreatment with reserpine. Expression of a transferred gene (luciferase), permeabilization (determined using 51 Cr-EDTA), membrane resealing and effects on perfusion were compared to assess the significance of the vascular effects. Above the permeabilization threshold, a sympathetically mediated Raynaud-like phenomenon with perfusion delays of 1^2 min was observed. Resolution of this phase followed kinetics of membrane resealing. Above a second threshold, irreversible permeabilization led to long perfusion delays. These vascular reactions (1) affect kinetics of drug delivery, (2) predict efficient DNA transfer, which is optimal during short perfusion delays, and (3) might explain electrocardiographic ST segment depressions after defibrillation as being caused by vascular effects of EP of cardiac muscle. ß 2002 Elsevier Science B.V. All rights reserved. Keywords : Electroporation ; Blood £ow ; Non-viral gene delivery ; Drug delivery ; Muscle ; Cardiac ischemia 1. Introduction After the ¢rst description of in vitro gene transfer to living cells by electroporation (EP) [1] this technique has been widely used. Innovation and sophistication have tak- en EP into a second era, where pulses well controlled with regard to both amplitude and duration can be applied, also in the in vivo setting [2]. Clinical trials with EP for delivery of chemotherapy have shown that EP of tissues in humans is feasible, e¤- cient and tolerable [3^7]. The perspective of this is that many di¡erent compounds, such as drugs, genes or oligo- nucleotides, could be delivered to tissues in patients. Gene delivery by EP has proven e¤cient in various tis- sues including tumor [8], liver [9], skin [10] and muscle [11,12]. In particular for muscle, high level and long-term gene expression has been observed after gene delivery us- ing EP [12], indicating new perspectives for DNA vaccines and gene therapy. We have previously investigated EP of muscle tissue, optimizing conditions for gene transfer, determining thresholds for EP and describing a quantitative method using 51 Cr-EDTA to evaluate permeabilization as a func- tion of uptake [12^15]. We decided to determine time to membrane resealing in muscle tissue in vivo, and it was observed that uptake of a tracer was a¡ected by a vascular e¡ect of EP. We proceeded to characterize this vascular e¡ect in detail, and examine the mechanisms be- hind it. Generally, drug delivery is performed using short high amplitude pulses (e.g. 100 Ws pulses of 1.3 kV/cm [4]) and gene delivery is done using long low amplitude pulses (e.g. 20 ms pulses of 0.2 kV/cm [12]). Therefore, a wide range of conditions was tested. By combining data on drug uptake, transgene expression, permeabilization, pore closure and perfusion, the signi¢cance of the vascular e¡ects could be assessed. 0304-4165 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII:S0304-4165(01)00233-1 * Corresponding author. Fax: +45-4453-3077. E-mail addresses : juge@herlevhosp.kbhamt.dk (J. Gehl), luismir@igr.fr (L.M. Mir). 1 Also corresponding author. Biochimica et Biophysica Acta 1569 (2002) 51^58 www.bba-direct.com