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. 2023 Mar 30:18:1615-1630.
doi: 10.2147/IJN.S402418. eCollection 2023.

Effects of PEG-Linker Chain Length of Folate-Linked Liposomal Formulations on Targeting Ability and Antitumor Activity of Encapsulated Drug

Chaemin Lim #  1 , Yuseon Shin #  2 , Kioh Kang  2 , Patihul Husni  2 , Dayoon Lee  2 , Sehwa Lee  2 , Han-Gon Choi  3 , Eun Seong Lee  4 , Yu Seok Youn  5 , Kyung Taek Oh  1   2
Affiliations

Effects of PEG-Linker Chain Length of Folate-Linked Liposomal Formulations on Targeting Ability and Antitumor Activity of Encapsulated Drug

Chaemin Lim et al. Int J Nanomedicine. .

Abstract

Introduction: Ligand-conjugated liposomes are promising for the treatment of specific receptor-overexpressing cancers. However, previous studies have shown inconsistent results because of the varying properties of the ligand, presence of a polyethylene glycol (PEG) coating on the liposome, length of the linker, and density of the ligand.

Methods: Here, we prepared PEGylated liposomes using PEG-linkers of various lengths conjugated with folate and evaluated the effect of the PEG-linker length on the nanoparticle distribution and pharmacological efficacy of the encapsulated drug both in vitro and in vivo.

Results: When folate was conjugated to the liposome surface, the cellular uptake efficiency in folate receptor overexpressed KB cells dramatically increased compared to that of the normal liposome. However, when comparing the effect of the PEG-linker length in vitro, no significant difference between the formulations was observed. In contrast, the level of tumor accumulation of particles in vivo significantly increased when the length of the PEG-linker was increased. The tumor size was reduced by >40% in the Dox/FL-10K-treated group compared to that in the Dox/FL-2K- or 5K-treated groups.

Discussion: Our study suggests that as the length of PEG-linker increases, the tumor-targeting ability can be enhanced under in vivo conditions, which can lead to an increase in the antitumor activity of the encapsulated drug.

Keywords: PEG-linker length; PEGylated liposome; folate receptor; ligand-conjugated liposome.

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Conflict of interest statement

There are no conflicts of interest to declare.

Figures

None
Graphical abstract
Scheme 1
Scheme 1
Structure of folate-conjugated liposomes (FLs) with different PEG-linker chain lengths and schematic of the behavior of FLs in vivo.
Figure 1
Figure 1
Preparation and characterization of FITC-labeled folate-conjugated liposomes (FITC-FLs). (a) Composition of liposomes (molar ratio %), particle size, polydispersity index (PDI), and zeta potential of liposomes. (b) Flow cytometric analysis of cellular uptake 5 h after sample treatment.
Figure 2
Figure 2
Physicochemical characterization of folate-conjugated liposomes (FL). (a) Morphology and size distribution of Dox-loaded folate-conjugated liposomes (Dox/FL) at various PEG-linker lengths. (b) Loading content, loading efficiency, particle size, polydispersity index (PDI), and zeta potential of prepared liposomes. (c) Stability of FLs or Dox/FLs at various PEG-linker lengths.
Figure 3
Figure 3
In vitro evaluation of FLs. (a) Confocal assay of cellular uptake of Dox/FLs and (b) flow cytometric analysis of cellular uptake of FITC-FLs 5 h after sample treatment. (c) Cytotoxicity of Dox in KB cell line. (d) Tryptophan fluorescence quenching assay by adding either Free Dox, Dox/FL-2K, Dox/FL-5K, or Dox/FL-10K at a given concentration of Dox.
Figure 4
Figure 4
Distribution of Ce6-loaded PEG-Lipo formulations (Ce6/NTL or Ce6/FLs) in KB tumor-bearing mice. (a) Non-invasive whole-body imaging at given time points. (b) Fluorescence image of harvested organs (tumor, liver, heart, lung, kidney, and spleen) 48 h after sample injection. (c) Quantitative fluorescence intensities of tumors and main organs.
Figure 5
Figure 5
In vivo characterization. (a) In vivo antitumor activity, and (b) body weight changes in KB tumor-bearing nude mice after intravenous administration of saline, Free Dox, and Dox-loaded liposomes. (c) Optical images of tumor-bearing mice at days 1 and 24.
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