The Effect of Millisecond Pulsed Electric Fields

Authors: Julita Kulbacka Agata Pucek Kazimiera Anna Wilk Magda DubińskaMagiera Joanna Rossowska Marek Kulbacki Małgorzata Kotulska

Publish Date: 2016/05/12

Volume: 249, Issue: 5, Pages: 645-661

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Abstract

Drug delivery technology is still a dynamically developing field of medicine The main direction in nanotechnology research nanocarriers nanovehicles etc is efficient drug delivery to target cells with simultaneous drug reduction concentration However nanotechnology trends in reducing the carrier sizes to several nanometers limit the volume of the loaded substance and may pose a danger of uncontrolled access into the cells On the other hand nanoparticles larger than 200 nm in diameter have difficulties to undergo rapid diffusional transport through cell membranes The main advantage of large nanoparticles is higher drug encapsulation efficiency and the ability to deliver a wider array of drugs Our present study contributes a new approach with large Tween 80 solid lipid nanoparticles SLN ie hydrodynamic GMSLN—glycerol monostearate GM as the lipid and ATO5SLNs—glyceryl palmitostearate ATO5 as the lipid with diameters DH of 3794 nm and 547 nm respectively They are used as drug carriers alone and in combination with electroporation EP induced by millisecond pulsed electric fields We evaluate if EP can support the transport of large nanocarriers into cells The study was performed with two cell lines human colon adenocarcinoma LoVo and hamster ovarian fibroblastoid CHOK1 with coumarin 6 C6 as a fluorescent marker for encapsulation The biological safety of the potential treatment procedure was evaluated with cell viability after their exposure to nanoparticles and EP The EP efficacy was evaluated by FACS method The impact on intracellular structure organization of cytoskeleton was visualized by CLSM method with alphaactin and betatubulin The obtained results indicate low cytotoxicity of both carrier types free and loaded with C6 The evaluation of cytoskeleton proteins indicated no intracellular structure damage The intracellular uptake and accumulation show that SLNs do not support transport of C6 coumarin Only application of electroporation improved the transport of encapsulated and free C6 into both treated cell linesOne of the main objectives of nanomedicine technology is the delivery of poorly soluble drugs and/or supporting their bioavailability improvement of drug targeting to the target cells with simultaneously reduced toxicity of normal cells and additional control of the location and the rate of drug release Currently nanosized carriers up to 200 nm in diameter are selected for pharmaceutical and medical applications Small sizes of nanoparticles increase the probability of their efficient transport into the cells However intracellular delivery system may benefit from various types of carriers ranging in diameter from 30 nm to several micrometers The main advantage of all nanocarriers is their ability to provide separate chemical environments which can protect the loaded cargo from a potential damage Lamch et al 2014 Szczepanowicz et al 2014 Lima et al 2013 Puglia et al 2014 Wissing et al 2004 Bazylińska et al 2012 2014b Nanosized carriers are currently regarded as one of the most promising directions in pharmaceutical research They allow encapsulation of a complex cargo and its selective delivery to cells enabled with a variety of carrier surface modifications Lamch et al 2014 Bazylińska et al 2012 2014a b Paliwal et al 2014 Tran et al 2014 de Morais et al 2014 Saadeh et al 2014 Large nanoparticles have many advantages but there is still a great challenge concerning their effective delivery into targeted cells New approaches are being developed but new research in this field is still required Lai et al 2007 suggested additional coating of large nanoparticles to enable their rapid penetration into physiological human mucus So far there is no method suitable for effective delivery of large carriers or vesicles thus enhancement of their intracellular transport is needed SLNs are considered the next generation of delivery system after liposomes Positively charged nanoparticles are advantageous in targeted drug delivery with regard to negatively charged cell surface However the research of liposomes showed that both positive and negative charges could enhance the delivery of liposomes to cells through adsorptive endocytosis and the extension of the halflife clearance of liposomes from the blood The halflife clearance can take from a minute to hours and the distribution to organs can be controlled in part by changing the physical properties of nanoparticles such as size charge and fluidity However some authors indicate that positively charged lipids are not approved by FDA for clinical use Makholf et al 2011 Zeta potential of liposomes or nanoparticles is an important factor for their cytotoxicity and in some cases cationic charge is demanded to increase the cytotoxicity Liu et al2012 Müller et al 2001In this study solid lipid nanoparticles SLNs were applied SLNs are nanocarriers composed of a lipid which is solid both at body and room temperatures stabilized by a suitable surfactant Aqueous SLN dispersions are alternative colloidal systems to polymeric nanoparticles liposomes and emulsions exhibiting many advantages such as good stability high drug payload possibility of controlled drug release and drug targeting low production costs Mehnertet al 2001 SLNs can be an alternative system to emulsions liposomes microparticles and their polymeric derivatives Lipid nanoparticles with a solid matrix have been proposed for encapsulation of many substances such as chemotherapeutics Wissing et al 2004 fluorescent dyes CalderónColón et al 2015 photosensitizers Lima et al 2013 and chemical UVfilters Puglia et al 2014 SLNs are considered to be the next generation delivery system after liposomes Their most advantageous features include the solid state of the particle matrix the ability to protect chemically unstable cargo against chemical decomposition and the ability to modulate drug release Műller et al 2000The work reported here extends our recent studies on delivery of large nanocarriers with fluorescent cargo their fabrication and imaging drug encapsulation release profiles and biological impact Szczepanowicz et al 2014 Bazylińska et al 2012 Bazylińska et al 2014a b Lamch et al 2014 This research is focused mainly on the effects of combination of largetype SLNs with electroporation EP on molecular transport of a loaded substance into normal and cancer cells EP is induced with a pulsed millisecond electric field applied either before or after the addition of the nanocarriers into the solution with cells in which the first mode enables an additional effect of electrophoresis Such nanocarriers combined with EP may lead to more effective transport of multidrug component and its enlarged dose to the target cells and simultaneous cargo protectionSLN dispersion was obtained with the ultrasound technique coupled with previous hot homogenization Bazylińska et al 2014a b The total amount of lipid phase GM or ATO5 was kept constant in all lipid nanoparticle suspensions 4  w/w The lipid nanoparticles in suspension were stabilized using 05  w/w concentration of surfactant T80 Coumarin 6 was used in a concentration of 1  w/w with regard to the solid lipid matrix

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