Targeting Delivery Of Simvastatin Using Anti-ICAM-1 Conjugated Nanostructured Lipid Carriers For Acute Lung Iniury Therapy

Acute lung injury (ALI) is a critical illness without effective therapeutic modalities currently. Recent studies indicated that statins showed potential endothelium-protective and anti-inflammatory properties, which contributed to the improvement of ALI. High-dose statins was suggested to be significant for attenuating inflammation in vivo, while excessive statins relative to human dosing regimens may increase the risk of dose-dependent side effects. In this research, simvastatin (SV) was used as the model drug. The intercellular adhesion molecule 1(ICAM-1)-mediated lung-targeted anti-ICAM-1 modified nanostructured lipid carriers (NLCs) loaded with simvastatin was developed, attempting to improve ALI with a decreased dose and minimize the potential adverse effects.Monostearin was used as solid lipid, medium-chain triglycerides as liquid lipid, polyethylene glycol monostearate as amphiphilic lipid. Using solvent diffusion method, blank nanostructured lipid carriers NLCs-1 (1 mg/mL), NLCs-2 (3 mg/mL) and NLCs-3 (9 mg/mL) of different diameters were prepared through modulating total lipid materials concentration. The diameters of prepared blank NLCs were about 43.3 nm,85.3 nm and 198.3 nm respectively, which increased with the rise of lipid materials concentration. The Balb/c mice were used as model animals to investigate the in vivo distribution of blank NLCs. The NLCs with large diameter showed obvious advantage of pulmonary distribution.With the same formulation of blank NLCs, solvent diffusion method was carried out to prepare SV loaded nanostructured lipid carriers SV/NLCs-1 (1 mg/mL), SV/NLCs-2 (3 mg/mL) and SV/NLCs-3 (9 mg/mL) with different diameters through the addition of 5% SV and modulating total lipid materials and drug concentration. The diameters of SV loaded NLCs were about 143.7 nm,259.6 nm and 337.8 nm respectively, which were larger than the homologous blank NLCs. Meanwhile, the diameters of SV loaded NLCs also increased with the rise of total lipid materials and drug concentration. The drug encapsulated efficiency increased from 66.70% to 91.04% with the rise of total lipid materials and drug concentration. In vitro release assay indicated that the NLCs showed sustained release of SV, which released from NLCs could be prolonged to 36 h, and the release rate decreased with the rise of diameter.The human vascular endothelial cell line EAhy926 (EAs) was used as the model cell to investigate the cytotoxicity of blank NLCs and SV loaded NLCs. The 50% cellular growth inhibitions (IC50) of blank NLCs in EAs were determined above 400 ?g/mL, while the IC50 of SV loaded NLCs in EAs were determined above 220 ?g/mL. The IC50 increased with the rise of the diameter. In vitro cellular uptake indicated the SV loaded NLCs showed excellent time-dependent cellular uptake ability, the internalization rate decreased along with the diameter increasing.Amino-terminated polyethylene glycol monostearate (NH2-PEG2000-SA) was synthesized as the amphiphilic lipid material to partially replace polyethylene glycol monostearate in the original prescription of NLCs. NH2-PEG2000-SA modified drug loaded NLCs (9 mg/mL) were prepared using solvent diffusion method. The anti-ICAM-1 modified SV loaded NLCs (anti-ICAM/SV/NLCs) were prepared, mediated by N,N'-Disuccinimidyl carbonate. The diameter, zeta potential and drug encapsulation efficiency of anti-ICAM/SV/NLCs were about 354.7 nm,-32.1 mV and 96.78% respectively. In vitro drug release showed a prolonged release of SV during 36 h. Anti-ICAM/SV/NLCs exhibited similar physicochemical characteristics with SV/NLCs-3. The LPS-stimulated EAs was used to investigate the cellular uptake of nano-particles quantitatively. Anti-ICAM/SV/NLCs showed higher uptake than control SV/NLCs-3 in the activated EAs. The cellular uptake of anti-ICAM/SV/NLCs was decreased after ICAM-1 blockage. The Balb/c mice were used as model animals, which were challenged with the intra-tracheal instillation of LPS to establish the mice model of ALI, followed by investigating the in vivo distribution of nano-particles via intravenous injection. In the ALI model mice, anti-ICAM/SV/NLCs exhibited obviously higher pulmonary distribution than SV/NLCs-3 without anti-ICAM-1 modified. And the pulmonary distribution of anti-ICAM/SV/NLCs was also higher in the ALI model mice than that in the normal mice.The Balb/c mice were used as model animals, which were challenged with the intra-tracheal instillation of LPS to establish the mice model of ALI. The in vivo pharmacodynamics of free simvastatin, SV/NLCs-3 and anti-ICAM/SV/NLCs were investigated subsequently after i.v. administration. Anti-ICAM/SV/NLCs attenuated pulmonary TNF-a and IL-6 more effectively than free simvastatin and SV/NLCs-3 at 48 h after administration. As for the inhibition of pulmonary inflammatory cells infiltration, anti-ICAM/SV/NLCs showed higher efficacy than other treatments at both 24 h and 48 h after administration. Besides, H&E stain showed that anti-ICAM/SV/NLCs exhibited significant amelioration of inflammatory cells infiltration, congestion and alveolar wall thickening compared with other treatments at 24 h after administration.The study suggests that, anti-ICAM/SV/NLCs exhibited improved pulmonary distribution in the ALI model mice via the considerable binding on the pulmonary vascular endothelium, thereby improving the long-term efficacy of drug. Hopefully the safe and effective improvement of ALI could be realized via anti-ICAM/SV/NLCs through the potential of targeted pulmonary accumulation of drugs and the decreased dosing frequency.