The Effect of Millisecond Pulsed Electric Fields (msPEF) on Intracellular Drug Transport with Negatively Charged Large Nanocarriers Made of Solid Lipid Nanoparticles (SLN): In Vitro Study

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 (i.e., hydrodynamic GM-SLN-glycerol monostearate, GM, as the lipid and ATO5-SLNs-glyceryl palmitostearate, ATO5, as the lipid) with diameters DH of 379.4 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 CHO-K1 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 alpha-actin and beta-tubulin. 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 lines.

Keywords: Coumarin-6; Drug delivery; Electroporation; Millisecond pulsed electric field; Solid lipid nanocarriers.

Fig. 1

Molecular structure of coumarin 6 (C6)

Fig. 2

SEM images of a GM-SLN, b ATO5-SLN

Fig. 3

AFM images of a GM-SLN, b ATO5-SLN

Fig. 4

DSC profiles of the melting process of C6, lipids, and C6- loaded SLNs

Fig. 5

Top row the effect of EP parameters on cell viability (MTT test): a CHO-K1 cells; b LoVo cells. Middle row the viability of cells after treatment with free C6, empty, and C6-loaded nanoparticles (GM-SLN and ATO5-SLN); SLNs and free C6 added before EP: c CHO-K1 cells, d LoVo cells. Bottom row FACS analysis, uptake of PI and free C6, added before EP, and SLNs added post-EP: e CHO-K1 and LoVo cells—dependency of PI uptake (percent of cells) on EP parameters; f CHO-K1 cells—uptake of free and encapsulated C6 (fluorescence intensity); g LoVo cells—uptake of free and encapsulated C6 (fluorescence intensity)

Fig. 6

The fluorescence microscopy analysis of C6 and PI uptake together with DAPI staining indicating cells nuclei. Top row free C6 uptake (without EP and EP at 500 V/cm, C6 added before EP): a CHO-K1 cells; b LoVo cells; Middle row C6-loaded into GM-SLNs, nanoparticles added immediately post-EP (100, 500 and 1000 V/cm): c CHO-K1 cells; d LoVo cells; Bottom row C6-loaded into ATO5-SLNs, nanoparticles added immediately post-EP (100, 500 and 1000 V/cm): e CHO-K1 cells; f LoVo cells

Fig. 7

The evaluation C6 distribution in the cells using CLSM analysis. C6-loaded in nanoparticles (GM-SLN and ATO5-SLN) added before EP at 500 V/cm (5 pulses, 1.5 ms each) or no electroporation applied. Images obtained 10 min after the procedure: a CHO-K1 cells and b LoVo cells; and images obtained 24 h after the procedure: c CHO-K1 cells and d LoVo cells

Fig. 7

The evaluation C6 distribution in the cells using CLSM analysis. C6-loaded in nanoparticles (GM-SLN and ATO5-SLN) added before EP at 500 V/cm (5 pulses, 1.5 ms each) or no electroporation applied. Images obtained 10 min after the procedure: a CHO-K1 cells and b LoVo cells; and images obtained 24 h after the procedure: c CHO-K1 cells and d LoVo cells

Fig. 8

The analysis of fluorescence intensity from CLSM analysis. C6-loaded nanoparticles (GM-SLN and ATO5-SLN) added before EP at 500 V/cm (5 pulses, 1.5 ms each) or no electroporation applied. Images obtained 10 min after the procedure: a CHO-K1 cells and b LoVo cells; and images obtained 24 h after the procedure: c CHO-K1 cells and in d LoVo cells