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. 2021 Jan 12;13(1):91.
doi: 10.3390/pharmaceutics13010091.

New Nanoparticle Formulation for Cyclosporin A: In Vitro Assessment

Amandine Gendron  1 , Natalie Lan Linh Tran  1   2 , Julie Laloy  2 , Romain Brusini  1 , Aurélie Rachet  1   3 , Frédéric Gobeaux  4 , Valérie Nicolas  5 , Pierre Chaminade  6 , Sonia Abreu  6 , Didier Desmaële  1 , Mariana Varna  1
Affiliations

New Nanoparticle Formulation for Cyclosporin A: In Vitro Assessment

Amandine Gendron et al. Pharmaceutics. .

Abstract

Cyclosporin A (CsA) is a molecule with well-known immunosuppressive properties. As it also acts on the opening of mitochondrial permeability transition pore (mPTP), CsA has been evaluated for ischemic heart diseases (IHD). However, its distribution throughout the body and its physicochemical characteristics strongly limit the use of CsA for intravenous administration. In this context, nanoparticles (NPs) have emerged as an opportunity to circumvent the above-mentioned limitations. We have developed in our laboratory an innovative nanoformulation based on the covalent bond between squalene (Sq) and cyclosporin A to avoid burst release phenomena and increase drug loading. After a thorough characterization of the bioconjugate, we proceeded with a nanoprecipitation in aqueous medium in order to obtain SqCsA NPs of well-defined size. The SqCsA NPs were further characterized using dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryoTEM), and high-performance liquid chromatography (HPLC), and their cytotoxicity was evaluated. As the goal is to employ them for IHD, we evaluated the cardioprotective capacity on two cardiac cell lines. A strong cardioprotective effect was observed on cardiomyoblasts subjected to experimental hypoxia/reoxygenation. Further research is needed in order to understand the mechanisms of action of SqCsA NPs in cells. This new formulation of CsA could pave the way for possible medical application.

Keywords: bioconjugate; cardiac cell line; cellular uptake; cyclosporin A; cytotoxicity; squalene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Synthetic scheme of the squalene cyclosporin A (SqCsA) conjugate. The squalene chain was introduced on the side chain of the MeBmt residue through a glycolate linker by chloroacetylation of the CsA followed by SN2 displacement of the chloride using squalenic acid cesium salt.
Figure 2
Figure 2
1H NMR of SqCsA (400 MHz, C6D6). The AB system of the methylene system; the glycolate linker is enlarged.
Figure 3
Figure 3
SqCsA nanoparticles (NPs) characterization by using cryogenic transmission electron microscopy (cryoTEM) (A,B) and dynamic light scattering (DLS) (C,D) of NPs suspensions. Stability assessment was performed for 28 days (D).
Figure 4
Figure 4
Remaining SqCsA bioconjugate in fetal bovine serum for 48 h. We see no degradation of the bioconjugate. Data represent mean ± SD of three replicates.
Figure 5
Figure 5
Cell viability assessment of Mouse Cardiac Endothelial Cells (MCEC) (A,C) and H9c2 (B,D) cell lines treated with SqCsA NPs. Cell viability is expressed as a percentage relative to the viability of untreated cells. Use of an equivalent (eq) concentration in free CsA (C,D). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Figure 6
Figure 6
SqCsA NPs uptake assessment. The H9c2 cell line was incubated with two concentrations of nanoparticles 12 or 60 µg/mL loaded with Cholesteryl 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-Indacene-3-undecanoate (CholEsteryl BODIPY™, red). After different times, cells were rinsed, fixed, and incubated with phalloidin (green). The cells were mounted with medium containing 4’,6-diamidino-2-phénylindole (DAPI, blue), analyzed under confocal microscope, and imaged. Cells incubated with only CholEsteryl BODIPY™, at an equivalent concentration to 60 µg/mL of NPs, were used as control and imaged at 24 h. Insert 90× magnification. Scale bar = 50 µm.
Figure 7
Figure 7
SqCsA NPs uptake assessment. The MCEC cell line was incubated with two concentrations of nanoparticles 12 or 60 µg/mL loaded with CholEsteryl BODIPY™ (red). After different times, cells were rinsed, fixed, and incubated with phalloidin (green). The cells were mounted with medium containing DAPI (blue), analyzed under confocal microscope, and imaged. Cells incubated with only CholEsteryl BODIPY™, at an equivalent concentration to 60 µg/mL of NPs, were used as control and imaged at 24 h. Insert 90× magnification. Scale bar = 50 µm.
Figure 8
Figure 8
Cardioprotective effect assessment on H9c2 cell line. Cells were incubated with SqCsA NPs for 24 h. Controls (untreated cells, free CsA, or Sq NPs) were done under the same conditions. After incubation time, cells were rinsed with Phosphate-Buffered Saline (PBS) and incubated in hypoxic condition (1% O2 and restraint medium without fetal bovine serum (FBS) and glucose). Cardioprotection was assessed by using thiazolyl blue tetrazolium bromide (MTT) test (upper panel) and lactate dehydrogenase (LDH) test (lower panel). For the MTT tests, the higher the percentage, the more viable the cells. For the LDH tests, the higher the percentage of LDH-released protein, the more damaged the cells. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Figure 9
Figure 9
Cardioprotective effect assessment on MCEC cell line. Cells were incubated with SqCsA NPs for 24 h. Controls (untreated cells, free CsA, or Sq NPs) were done under the same conditions. After incubation time, cells were rinsed with PBS and incubated in hypoxic condition (1% O2 and restraint medium without FBS and glucose). Cardioprotection was assessed by using MTT test (upper panel) and LDH test (lower panel). For the MTT tests, the higher the percentage, the more viable the cells. For the LDH tests, the higher the percentage of LDH-released protein, the more damaged the cells. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
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