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. 2021 Sep 1;1(1):35-49.
doi: 10.1002/EXP.20210008. eCollection 2021 Aug.

Membrane-destabilizing ionizable lipid empowered imaging-guided siRNA delivery and cancer treatment

Shuai Guo  1 , Kun Li  1 , Bo Hu  1 , Chunhui Li  1 , Mengjie Zhang  1 , Abid Hussain  1 , Xiaoxia Wang  2 , Qiang Cheng  3 , Feng Yang  4 , Kun Ge  5 , Jinchao Zhang  5 , Jin Chang  6 , Xing-Jie Liang  7 , Yuhua Weng  1 , Yuanyu Huang  1
Affiliations

Membrane-destabilizing ionizable lipid empowered imaging-guided siRNA delivery and cancer treatment

Shuai Guo et al. Exploration (Beijing). .

Abstract

One of the imperative medical requirements for cancer treatment is how to establish an imaging-guided nanocarrier that combines therapeutic and imaging agents into one system. siRNA therapeutics have shown promising prospects in controlling life-threatening diseases. However, it is still challenging to develop siRNA formulations with excellent cellular entry capability, efficient endosomal escape, and simultaneous visualization. Herein, we fabricated multifunctional ionizable lipid nanoparticles (iLNPs) for targeted delivery of siRNA and MRI contrast agent. The iLNPs comprises DSPC, cholesterol, PEGylated lipid, contrast agent DTPA-BSA (Gd), and ionizable lipid termed iBL0104. siRNA-loaded iLNPs (iLNPs/siRNA) could be decorated with a tumor targeting cyclic peptide (c(GRGDSPKC)) (termed GARP), or without targeting modification (termed GAP). Data revealed that GARP/siRNA iLNPs exhibited significantly higher cellular entry efficiency than GAP/siRNA iLNPs. GARP/siRNA iLNPs rapidly and effectively escaped from endosome and lysosome after internalization. Compared with GAP/siPLK1, GARP/siPLK1 exhibited better tumor inhibition efficacy in both cell-line derived xenograft and liver cancer patient derived xenograft murine models. In addition, GARP formulation displayed ideal MRI effect in tumor-bearing mice, and was well tolerated by testing animals. Therefore, this study provides an excellent example for achieving imaging-guided and tumor-targeted siRNA delivery and cancer treatment, highlighting its promising potential for translational medicine application.

Keywords: MRI; cancer treatment; hepatocellular carcinoma; lipid nanoparticle; lipid‐like material; siRNA.

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Conflict of interest statement

S.G. and Y.H. declare that a patent relative the study has been filed. The remaining authors declare no competing interests.

Figures

SCHEME 1
SCHEME 1
Schematic illustration of proposed ionizable lipid formulations, which enable MRI‐guided and tumor‐targeted siRNA delivery, as well as effective cancer treatment
FIGURE 1
FIGURE 1
Formulation screening by evaluating the MRI effect and gene silencing activity in vitro. (A) GARP iLNP formulations prepared with various molar ratios of iBL0104, D‐B(Gd), DSPC, cholesterol, and DSPE‐PEG‐cRGD. D‐B(Gd) is short for DTPA‐BSA (Gd). (B) The imaging effects of GAP iLNPs with different amounts of Gd agent. (C) Particle sizes of various GAP/siRNA and GARP/siRNA complexes as detected by DLS. (D and E) Relative PLK1 mRNA expression in HepG2‐luc cells after treated with GAP iLNPs (D) and GARP iLNPs (E), respectively. **P < 0.01; ***P < 0.001, ****P < 0.0001
FIGURE 2
FIGURE 2
In vitro siRNA transfection mediated by GAP35 and GARP35 iLNPs on HepG2‐luc cells. (A) Viability of HepG2‐luc cells received various iLNPs treatment. (B) Relative PLK1 mRNA expression as detected by qRT‐PCR. siRNA was transfected 50 and 150 nM, respectively, for both GAP iLNPs and GARP iLNPs. (C) PLK1 protein level as analyzed by Western blotting. (D) Quantitative analysis of (C) with Image J software. (E) Confocal imaging of HepG2‐luc cells transfected with GAP35/Cy5‐siRNA and GARP35/Cy5‐siRNA for 4 h at the final siRNA concentration of 50 and 150 nM, respectively. (F) Cellular uptake of GAP35/Cy5‐siRNA and GARP35/Cy5‐siRNA formulations as recorded by fluorescence‐activated cell sorting (FACS). (G) Quantitative analysis of (F). (H) Internalization mechanism exploration of GAP35/Cy5‐siRNA and GARP35/Cy5‐siRNA complexes by using three inhibitors involved in three different pathways. The siRNA transfection concentration was 50 nM. (I) Quantitative analysis of (H). Each bar represents the mean ± SEM of three replicates. **P < 0.01; ***P < 0.001; ****P < 0.0001
FIGURE 3
FIGURE 3
Intracellular trafficking of GARP35 iLNPs and pKa‐driven endosomal escape process in HepG2‐luc cells. (A–F) Confocal laser scanning microscopy (CLSM) imaging and quantitative analysis of HepG2‐luc cells transfected with GARP35/Cy5‐siRNA (A–C) and Lipo/Cy5‐siRNA (D–F) at indicated time points after transfection. (A and D) Confocal images of cells received the treatments of GARP35/Cy5‐siRNA (A) and Lipo/Cy5‐siRNA (D), respectively. (B and E) MFIs of GARP35/Cy5‐siRNA iLNPs (B) and Lipo/Cy5‐siRNA (E), respectively. (C and F) Pearson correlation analysis of (A) and (D), respectively. (G and H) pKa values calculated from the TNS fluorescence titration curves of iLNP W/O Gd NPs (G) and GARP NPs (H). (I) Proposed membrane destabilization mechanism of GARP iLNPs. (J and K) Results of hemolytic assay in vitro
FIGURE 4
FIGURE 4
In vivo evaluation of tumor targeting behavior of GARP/siRNA iLNPs. (A) Whole body imaging at given time points after intravenous injection. (B) Grouping information. (C) Fluorescence detection of isolated organs and tumor tissues. (D) Quantitative analysis of the tumors isolated at 6, 10, and 24 h after injection. (E) Confocal observation of cryosections of tumors collected at 10 h after injection. The nucleus was stained with DAPI (blue), and cytoskeleton was stained with FITC‐labeled phalloidin (green). Scale bar, 20 µm
FIGURE 5
FIGURE 5
Tumor growth inhibition on HepG2‐luc cell derived xenograft (CDX) model. (A) Schematic illustration of the treatment course and animal grouping. (B) Tumor growth curves recorded in various groups. (C) Tumor weight recorded at day 18 when the animals were sacrificed. (D) PLK1 mRNA expression in tumors. (E) Body weight monitored during the treatment course. (F and G) Organ coefficients of the liver (F) and the spleen (G) at the end of the experiment (day 18). (H) Serum biochemistry parameters measured at the end of the experiment. (I) H&E staining of the heart, liver, spleen, lung, kidney and tumor in GARP/siPLK1 group. Data are shown as mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ****P < 0.0001
FIGURE 6
FIGURE 6
MRI and anticancer effects of iLNP formulations in PDX model. (A) Schematic illustration of the treatment protocol and grouping information. (B) MRI images acquired before administration and at 1, 5, 10, and 24 h after intravenous or intratumoral administration of lipid/siRNA formulations. The tumors were marked with white ellipse, and intratumorally treated tumors were indicated with red arrows. (C) Quantitative analysis of the tumor tissues shown in (B). (D) Tumor growth inhibition after treated with various formulations in PDX model. (E) Optical images of the tumors isolated on day 5. (F) Average tumor weights recorded on day 5. (G) Expression of PLK1 mRNA in tumor tissues. (H) Survival curves of the tumor‐bearing mice. (I) Body weights of the mice during the treatment course. (J and K) Organ coefficients of the liver (J) and the spleen (K), which were calculated by dividing the weight of the liver to the weight of the brain, and the weight of the spleen to the weight of the brain, respectively. (L) Serum biochemistry parameters examined at the end of experiment. (M) H&E staining of the main organs and the tumor tissue in the G4 group. Data were shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs PBS group
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