Dual Functionalized Liposomes for Selective Delivery of Poorly Soluble Drugs to Inflamed Brain Regions

Abstract

Dual functionalized liposomes were developed to cross the blood–brain barrier (BBB) and to release their cargo in a pathological matrix metalloproteinase (MMP)-rich microenvironment. Liposomes were surface-functionalized with a modified peptide deriving from the receptor-binding domain of apolipoprotein E (mApoE), known to promote cargo delivery to the brain across the BBB in vitro and in vivo; and with an MMP-sensitive moiety for an MMP-triggered drug release. Different MMP-sensitive peptides were functionalized at both ends with hydrophobic stearate tails to yield MMP-sensitive lipopeptides (MSLPs), which were assembled into mApoE liposomes. The resulting bi-functional liposomes (i) displayed a < 180 nm diameter with a negative ζ-potential; (ii) were able to cross an in vitro BBB model with an endothelial permeability of 3 ± 1 × 10−5 cm/min; (iii) when exposed to functional MMP2 or 9, efficiently released an encapsulated fluorescein dye; (iv) showed high biocompatibility when tested in neuronal cultures; and (v) when loaded with glibenclamide, a drug candidate with poor aqueous solubility, reduced the release of proinflammatory cytokines from activated microglial cells.

SPRi analysis of molecular interactions of mApoE-functionalized liposomes as drug delivery systems for brain diseases

“…Signals obtained on APOER2, LDL-R, LRP1 and VLDL-R have comparable intensities, suggesting that, being all expressed at brain level, they collaborate to the BBB crossing of LPs, as already verified in vitro . 25 Moreover, at these tested concentrations, the signal intensities on TLR2, VLA4 and TLR4 are lower than what we obtained on APOER2, LDL-R, VLDL-R, LRP1 and VCAM-1, with some aspecific signals on TLR4. Indeed, when we injected only PBS without liposomes, we collected a signal although there is no specific binding on that receptor in those conditions.…”

“…The dual functionalization is necessary for BBB crossing and for MMP sensitivity that guarantee a localized release of the encapsulated drug in diseased areas, as recently published. 25…”

“…LPs were prepared as described previously. 25 Briefly, the 17-mer lipopeptide SKK(stearate)SGPLGIAGQSK(stearate)KS (SG-17) was prepared in good yields and high purity (MS-LC) by microwave (MW)-assisted Fmoc solid-phase peptide synthesis (Fmoc-SPPS) on Wang resin. LPs composed of cholesterol, sphingomyelin, and 1,2-distearoyl- sn-glycero -3-phosphoethanolamine- N -[maleimide(polyethylene glycol)-2000] in a 48.75 : 48.75 : 2.5 molar ratio were prepared by probe sonication technique.…”

Raman Spectroscopy Characterization of Multi-Functionalized Liposomes as Drug-Delivery Systems for Neurological Disorders

“…We synthetically targeted SG-17 ( Figure S1 ), centered on an MMP2-responsive sequence (GPLGIAGQ)20 flanked on both sides by Ser and Lys as polar and protonated residues to increase its hydrophilicity, and connected with two hydrophobic tails; this hybrid peptide–lipopeptide sequence, once embedded into a hybrid liposome, provides sensitivity to MMP2 and selective cargo release in pathogenic microenvironments [ 25 ]. The seventeen-aminoacid-membered SG-17 lipopeptide was successfully prepared in good yields and purity (>90%) by microwave-assisted solid-phase peptide synthesis (MW-SPPS), using standard Fmoc-based automated protocols, as described in the Materials and Methods; N-Fmoc Ser, Lys, Ile, Ala, Gly, Pro and Leu were used as building blocks together with Nα-Fmoc-(Nε-stearoyl)-Lys, prepared according to [ 30 ].…”

“…The ability of mApoE to increase the BBB penetration of LPs has already been demonstrated in in vitro and in vivo models [ 21 , 22 , 23 ], while surface modification with SG-17 has been shown to determine liposomal destabilization and cargo release [ 24 ]. Moreover, the ability to cross the BBB of LPs functionalized with both mApoE and SG-17 has already been demonstrated in vitro [ 25 ]. As the chemical characterization of LPs is a crucial step for their development and validation in a clinical setting, we tested RS as a sensitive, fast and cheap method to evaluate and optimize the efficacy of LP synthesis, by collecting the specific Raman fingerprint of each formulation.…”