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. 2016 Jun 29:11:3035-48.
doi: 10.2147/IJN.S107292. eCollection 2016.

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

Mireia Campos-Martorell  1 , Mary Cano-Sarabia  2 , Alba Simats  1 , Mar Hernández-Guillamon  1 , Anna Rosell  1 , Daniel Maspoch  3 , Joan Montaner  4
Affiliations

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

Mireia Campos-Martorell et al. Int J Nanomedicine. .

Abstract

Background and aims: Although the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain.

Materials and methods: In order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS(®) Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography.

Results: Whereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood-brain barrier, when administered either as a free drug or encapsulated into liposomes.

Conclusion: This study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects.

Keywords: brain; delivery; liposomes; rat; simvastatin; stroke.

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Figures

Figure 1
Figure 1
Diagram summarizing the number of animals included (Incl) and excluded (Excl) per substudy. Abbreviations: MCAO, middle cerebral arterial occlusion; h, hours.
Figure 2
Figure 2
CryoTEM images of liposomes formed by the lipid mixtures. Notes: (A) DLPC/CHOL/CHOL–PEG/CHOL (+), (B) DLPC/CHOL/CHOL–PEG (=), and (C) DLPC/CHOL/CHOL–PEG/DOPA (−). Scale bar: 200 nm. Abbreviations: CryoTEM, cryogenic transmission electron microscopy; DLPC, 1,2-didodecanoyl-sn-glycero-3-phosphocholine; CHOL, cholesterol; CHOL–PEG, cholesteryl–polyethylene glycol 600 sebacate; DOPA, 1,2-dioleoyl-sn-glycero-3-phosphoric acid monosodium salt.
Figure 3
Figure 3
Representative ex vivo rat brain images and fluorescent signal quantifications captured by an imaging system (Xenogen IVIS® Spectrum). Notes: (A) Whole brain of representative sham and infarcted ischemia rats receiving different net surface charged liposomes. (B) Representative sliced rat brain after being submitted to an MCAO and receiving neutral liposomes. Fluorescent signal colocalized with infarcted area. (C) Quantifications of liposome-emitted signal in sham and MCAO rat brain expressed as radiant efficiency. Images are adjusted to the scale positioned beside. Data present four independent experiments in which three to four animals per group were included. Median values (interquartile range) are represented and significant differences are indicated as *P<0.05. Abbreviations: LIP1+, positive liposomes; LIP2=, neutral liposomes; LIP3−, negative liposomes. MCAO, middle cerebral arterial occlusion; p, photons; sec, seconds; sr, steradian.
Figure 4
Figure 4
Fluorescent signal quantifications of images captured by Imaging system (Xenogen IVIS® Spectrum) considering distribution of differentially charged liposomes within the body in sham and MCAO rats. Notes: Liposome accumulation in (A) liver, (B) spleen, (C) kidneys, and (D) lungs. In each group, three to four animals were included. Mean ± SD or median values (interquartile range) are represented depending on normality data distribution and significant differences are indicated as *P<0.05, **P<0.01. Abbreviations: +, positive; =, neutral; −, negative; MCAO, middle cerebral arterial occlusion; SD, standard deviation.
Figure 5
Figure 5
Presence of liposomes in plasma. Notes: (A) Plasma collected from sham or ischemic rats 90 minutes after receiving liposomes. Median values (interquartile range) are represented and significant differences are indicated as *P<0.05. (B) Representative plasma image of one experiment. (C) Liposome kinetics in plasma. Blood was collected from naïve rats (with no surgical procedure) after 1.5 hours, 4 hours, and 24 hours of receiving neutral or negatively charged liposomes. Symbols indicated mean ± SD. (D) Image representing data on graph (C). Images are adjusted to the scale positioned beside. In all experiments, three to four animals per group were considered. Significant differences are represented as *P<0.05, and ***P<0.001. Abbreviations: +, positive; =, neutral; −, negative; MCAO, middle cerebral arterial occlusion; h, hours; p, photons; sec, seconds; sr, steradian; SD, standard deviation.
Figure 6
Figure 6
Simvastatin-loaded liposome characterization. Notes: (A) Structure and composition of neutral liposome compounds used in detection experiments. (B) CryoTEM image of liposomes with simvastatin encapsulated formed by the lipid mixture DLPC/CHOL/CHOL–PEG (scale bar: 200 nm). (C–E) UHPLC chromatographic profile of peaks corresponding to (C) active simvastatin (also named acid simvastatin), (D) IS (simvastatin hydroxy acid ammonium salt), and (E) inactive simvastatin (also named lactone). For each compound analyzed, retention time (expressed in minutes) and transition are presented. Abbreviations: CryoTEM, cryogenic transmission electron microscopy; DLPC, 1,2-didodecanoyl-sn-glycero-3-phosphocholine; CHOL, cholesterol; CHOL–PEG, cholesteryl–polyethylene glycol 600 sebacate; UHPLC, ultra-high-protein liquid chromatography; IS, internal standard; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; simv, simvastatin; min, minute; PEG, polyethylene glycol.
Figure 7
Figure 7
Simvastatin detection in brain tissue through UHPLC technique after intravenous administration of 1 mL of free simvastatin or simvastatin encapsulated into neutral liposomes. Notes: All rats were treated with the same simvastatin dose (20 mg/kg). Rats were submitted to sham or MCAO surgery, treated 90 minutes later, and killed 2 hours or 4 hours after treatment administration. (A) Detection of simvastatin in its active form when rats were treated with free simvastatin and euthanized 2 hours or 4 hours later. (B) Detection of simvastatin in its lactone form (inactive) when rats were treated with free simvastatin and killed 2 hours or 4 hours later. (C) Detection of simvastatin in its active form in IP hemispheres when rats were treated with free or encapsulated simvastatin and killed 2 hours or 4 hours later. (D) Detection of simvastatin in its lactone form (inactive) in IP hemispheres when rats were treated with free or encapsulated simvastatin and killed 2 hours or 4 hours later. In all experiments, three to five animals per group were included. Box plots indicated median (interquartile range). After applying Mann–Whitney test, significant differences are represented as *P<0.05 for comparisons between sham and ischemic animals, #P<0.05 for comparisons between IP and CL hemispheres (A) and Free and Lipos treatments (C, D) and †P<0.05 for comparisons between 2 hours and 4 hours. Broken lines represent background signal detected through UHPLC technique when brains of naïve animals without receiving treatment were analyzed. Abbreviations: UHPLC, ultra-high-protein liquid chromatography; MCAO, middle cerebral arterial occlusion; IP, ipsilateral; CL, contralateral; IS, internal standard; simv, simvastatin; h, hours; Isch, ischemic; Lipos, liposomes.
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