Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4- d]pyrimidines for Targeting Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds.

Figure 1

Molecular structures of compounds SI113, S29, and SI163, encapsulated into liposomes and TRP (plasmin-binding tripeptide-linker), used to decorate liposomal surface.

Scheme 1. Preparation of Tripeptide Portion d-Ala-Phe-Lys

Reagents and conditions: (a) SOCl₂, MeOH, r.t., 16 h; (b) Boc-Phe-OH, DMAP, EDC·HCl, CH₂Cl₂ dry, r.t., 16 h; (c) 10% TFA, CH₂Cl₂ dry, r.t., 1 h; (d) Boc-d-Ala-OH, DMAP, EDC·HCl, CH₂Cl₂ dry, r.t., 16 h; (e) H₂, Pd/C 10%, MeOH, HCl conc., r.t., 12 h; (f) Boc₂O, THF, 5% NaOH, r.t., 16 h.

Scheme 2. Preparation of TRP

Reagents and conditions: (g) 3,4-dihydro-2H-pyran, p-TsOH cat, CH₂Cl₂ dry r.t., 5 h; (h) d-Ala-Phe-Lys (7), DMAP, EDC·HCl, CH₂Cl₂ dry, r.t., 16 h; (i) 10% TFA, CH₂Cl₂ dry, r.t., 16 h.

Figure 2

Schematic representation of TRP (R₁ = CH₃; R₂ = CH₃–Bn; R₃ = CH₃–(CH₂)₄–NH₂) and the sites that undergo MS/MS fragmentation, their m/z (black), and the corresponding collision energy in volts (red).

Figure 3

Left: Fluorescence microscopy images of HepG2 cells following LPs and TLPs administration: cells after 5 min of incubation with LPs (A) and TLPs (B) and cells after 1 h of incubation with LPs (C) and TLPs (D). Green, liposomes; red, cell marker; blue, nuclei. Right: Relative fluorescence intensity per cell area histograms for each formulation.