Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon

Abstract

For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

Keywords: Accelerated blood clearance (ABC) phenomenon.; Cationic liposomes; Poly(carboxybetaine); Polyethylene glycol; siRNA therapeutics.

Scheme 1

Schematic illustration of the ABC process of the PEGylated and PCBylated lipoplexes.

Figure 1

Characterization of cationic liposomes and lipoplexes. (A) The chemical structure of DSPE-PCB₂₀. (B) Mean particle diameter and zeta potential of cationic liposomes in DMEM. (C) Cryo-TEM images of cationic liposomes and lipoplexes at N/P ratio of 5/1. (D) The siRNA encapsulation efficiency of lipoplexes evaluated by Quant-iT^TM RiboGreen^® RNA Reagent. (E) The mean diameter of lipoplexes at various N/P ratios in DMEM. (F) The zeta potential of lipoplexes at various N/P ratios in DMEM. Data are shown as the mean ± S.D. of three independent experiments.

Figure 2

The changes of lipoplexes sizes in the presence of 10% FBS with the extension of time. Data are shown as the mean ± S.D. of three independent experiments.

Figure 3

Cytotoxicity of DSPE-PEG and DSPE-PCB₂₀ cationic liposomes with siNonsense to Hela cells.

Figure 4

Pharmacokinetic and biodistribution study of DSPE-PEG and DSPE-PCB₂₀ lipoplexes in rats after two dose of injection with time interval of 5 days. (A) Blood clearance profile of DSPE-PEG lipoplexes. (B) Blood clearance profile of DSPE-PCB₂₀ lipoplexes. (C) Distribution of DSPE-PEG lipoplexes in liver and spleen. (D) Distribution of DSPE-PCB₂₀ lipoplexes in liver and spleen. Lipoplexes at N/P ratio of 5/1 were administered to SD rats with Cy5-labeled siRNA concentration of 1 mg/kg via the tail vein. Data are shown as the mean ± S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.005 (n=3).

Figure 5

(A) IgM level in plasma of SD rats following administrated with DSPE-PEG and DSPE-PCB₂₀ lipoplexes 5 days later. (B) IgG level in plasma of SD rats following administrated with two injections of DSPE-PEG and DSPE-PCB₂₀ lipoplexes. The time interval between the two injections was 7 days and the plasma was obtained two weeks after the second injection. Lipoplexes at N/P ratio of 5/1 were administered to SD rats with Cy5-labeled siRNA concentration of 1 mg/kg via the tail vein. Data are shown as the mean ± S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.005 (n=5).

Figure 6

IgM level in plasma after incubation with DSPE-PEG and DSPE-PCB₂₀ lipoplexes at 37 °C for 15 min. The plasma was obtained from SD rats administrated with DSPE-PEG lipoplexes with Cy5-labeled siRNA concentration of 1 mg/kg. Data are shown as the mean ± S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.005 (n=3).

Figure 7

In vivo distribution of lipoplexes in Hela tumor-bearing nude mice after 24 h of two injections. (A) The fluorescence images of major organs and tumor after 24 h of the first injection. (B) The fluorescence images of major organs and tumor after 24 h of the second injection. (C) The mean fluorescence intensity of Cy5-labeled siRNA of major organs and tumor of Figure A. (D) The mean fluorescence intensity of Cy5-labeled siRNA of major organs and tumor of Figure B. (E) Quantitative results of Cy5-labeled siRNA in liver and tumor of the first injection. (F) Quantitative results of Cy5-labeled siRNA in liver and tumor of the second injection. Lipoplexes at N/P ratio of 5/1 were administered to mice at a dose of 0.5 mg Cy5-labeled siRNA/kg via the tail vein. Data are shown as the mean ± S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.005 (n=3).

Figure 8

Flow cytometric analyses of cellular internalization of cationic liposome/FAM-labeled siRNA lipoplexes in Hela cells after incubation for different times. (A) Comparation of cellular internalization between DSPE-PEG and DSPE-PCB₂₀ lipoplexes with the extension of time. (B) Mechanistic probes of the intracellular kinetics of the DSPE-PEG and DSPE-PCB₂₀ lipoplexes by monitoring the cellular uptake level at 4 ºC or in the presence of various endocytic inhibitors. Data are shown as the mean ± S.D. of three independent experiments.

Figure 9

Assessment of cellular internalization and endosomal/lysosomal escape of lipoplexes in Hela cells after incubation for different time points. (A) Images detected by confocal laser scanning microscopy (CLSM). Cell nuclei and endosomes/lysosomes were stained with DAPI (blue) and LysoTracker Red (red). Scale bar is 10 μm. (B) Co-localization ratio of FAM-labeled siRNA and LysoTracker Red after 2 h and 8 h incubation. (C) The percentage of siRNA released from lipoplexes after incubation with the extension of time. (D) The changes of zeta potential of lipoplexes in different pH values PBs. Data are shown as the mean ± S.D. of three independent experiments.

Figure 10

In vivo anti-tumor study of lipoplexes in Hela tumor-bearing nude mice after intravenous injection with PBS, siPlk1, DSPE-PEG and DSPE-PCB₂₀ lipoplexes with siPlk1 at N/P ratio of 5/1 at siPlk1 dose of 2.5 mg/kg (n=5). (A) Relative tumor volume-time curve. (B) Body weight-time curve. (C) The weights of the removed tumors. (D) The image of the solid tumors removed from different treatment groups. *P<0.05, **P<0.01, ***P<0.005.

Figure 11

In vivo gene silencing efficiency of DSPE-PEG and DSPE-PCB₂₀ lipoplexes. (A) Expression of Plk1 mRNA. (B, C) Expression of Plk1 protein. (D) TUNEL analyses of tumor tissue after treatments. The tumor tissues were collected 24 h after the last injection. *P<0.05, **P<0.01, ***P<0.005.