An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

Abstract

Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future.

Keywords: long circulation; lysosome escape; nanocarrier; pH-sensitive liposomes; pharmacokinetics.

Figure 1

(A) The synthetic route of PHS. (B) HPLC spectrum of PHS. (C) ¹H NMR spectrum of PHS, D₂O was used as a solvent. (D) The chemical structure of mPEG-Hz-PE. Abbreviations: PHS, mPEG2000-hydrazone-stearate (mPEG2000-Hz-STR); HPLC, high-performance liquid chromatography; PEG, polyethylene glycol; min, minutes.

Figure 2

Scheme of various liposomes preparations including CL, CCL, PL, PSL, CPL, and CPPL. Abbreviations: CL, conventional liposomes; CCL, CPP-modified conventional liposomes; PL, PEGylated liposomes; PSL, pH-sensitive PEGylated liposomes; CPL, CPP-modified PEGylated liposomes; CPPL, CPP-modified pH-sensitive PEGylated liposomes; CPP, cell-penetrating peptide; PEG, polyethylene glycol; SPC, soybean phosphatidylcholine; STR, stearic acid; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; mPEG, methoxy(polyethyleneglycol); DSPE, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine.

Figure 3

(A) HPLC/ELSD analysis of PEG cleavage from PSL at different pH. (I) pH 7.0, (II) pH 6.5, (III) pH 6.0, (IV) pH 4.5. (B) pH sensitivity of PSL measured with flow cytometer. CL and PL served as control groups. Coumarin-6 was selected as a model drug to indicate the cellular uptake of each liposomal carrier at different pH on MCF-7 cells due to its fluorescence emission (λ_ex=466 nm, λ_em=504 nm [n=3]). **P<0.05. Abbreviations: HPLC, high-performance liquid chromatography; CL, conventional liposomes; PL, PEGylated liposomes; PSL, pH-sensitive PEGylated liposomes; PEG, polyethylene glycol; min, minutes.

Figure 4

Effect of CPP density on cell uptake of coumarin-6-CPPL at pH 6.0 and 7.0 (n=3). Notes: (A) Effect of CPP density on cell uptake of coumarin-6-CPPL at pH 6.0 and 7.0. (B,C) The cells distribution with fluorescence intensity of CPPL detected by FCM at pH 7.0 and pH 6.0, respectively. Abbreviations: CPP, cell-penetrating peptide; CPPL, CPP-modified pH-sensitive PEGylated liposomes; PEG, polyethylene glycol.

Figure 5

(A) CLSM of DOX loaded with 4% CPP-modified CPPL at different pH. CPL (4% CPP modified, acid-insensitive) and PSL (non-CPP-modified and acid-sensitive) are negative control groups and CCL is served as positive control. (C) Effects of incubation time on cell uptake of DOX loaded by 4% CPPL at pH 6.0 and 7.0. The Hoechst33258 was excited by a 345-nm laser. Those in blue represent the nuclei stained with Hoechst33258, and those in red represent DOX fluorescence (λ_ex=480 nm, λ_em=540 nm). Scale bar is 50 μm. Quantitative analysis via ImageJ is shown in (B) and (D), *P<0.05, **P<0.01 (n=15). Abbreviations: CLSM, confocal laser-scanning microscope; PSL, pH-sensitive PEGylated liposomes; CPL, CPP-modified PEGylated liposomes; CPPL, CPP-modified pH-sensitive PEGylated liposomes; CCL, CPP-modified conventional liposomes; CPP, cell-penetrating peptide; PEG, polyethylene glycol; DOX, doxorubicin; h, hours.

Figure 6

Colocalization of coumarin-6 and lysosomes observed with CLSM after cell internalization with CCPL and endosome escaping for 2 hours. PL is a control group. Co-location description was indicated by the white arrow. Abbreviations: CLSM, confocal laser-scanning microscope; PL, PEGylated liposomes; CPPL, CPP-modified pH-sensitive PEGylated liposomes; CPP, cell-penetrating peptide; PEG, polyethylene glycol.

Figure 7

Ex vivo images of interested organs after 2 hours, 4 hours, and 8 hours intravenous administrated FITC-CPPL and FITC-CPL in tumor-bearing nude mice. Notes: (A) Heart, (B) liver, (C) spleen, (D) lung, (E) kidney, (F) brain, and (G) tumor (from left to right). Abbreviations: CPL, CPP-modified PEGylated liposomes; CPPL, CPP-modified pH-sensitive PEGylated liposomes; CPP, cell-penetrating peptide; PEG, polyethylene glycol; h, hours.

Figure 8

Scheme of CPPL in vivo tumor targeting process. Notes: (A) PEG was kept on the surface of CPPL in the neutral environment of blood. (B) CPPL accumulated at the tumor site via EPR effect, and then CPP was exposed on the surface of liposomes as a result of the missing of PEG caused by the degradation of hydrazone bond at low pH tumor area. (C) Improved cellular uptake due to the exposed CPP. (D) Internalization of CCL into tumor cells. (E) CPP adsorbed more H⁺ in endosome. (E) CPP adsorbed more H⁺ in endosome and Electrostatic attraction between the cationic CPP-H+ and the anionic components in the endosomal membrane happened. (F) Fusion pore formed and the liposomal cargo was released into the cytosol. (G) The released cargoes has more opportunity entering into nuclei. Abbreviations: PEG, polyethylene glycol; CPP, cell-penetrating peptide; CPPL, CPP-modified pH-sensitive PEGylated liposomes; EPR, enhanced permeability and retention; CCL, CPP-modified conventional liposomes; CPP, cell-penetrating peptide; SPC, soybean phosphatidylcholine; STR, stearic acid; DOX, doxorubicin.