(W1030-02-10) Delivery of Carbonic Anhydrase 9 siRNA by Lipid Nanoparticles

Purpose: Tumor cells express special proteins to help them maintain normal survival or increase their adaptability to adversity. The abnormal expression of these proteins poses a great challenge to cancer treatment but also makes these proteins potential therapeutic targets. Carbonic anhydrase 9 (CA9) is a transmembrane zinc metalloenzyme, which catalyzes the reversible hydration of carbon dioxide to bicarbonate ions and protons. It has been shown that overexpression of CA9 in cancer cells promotes the acidification of tumor microenvironment, cancer metastasis, and often leads to increasing resistance to chemotherapy and radiotherapy. In this study, we encapsulated small interfering RNA (siRNA) against CA9 into lipid nanoparticles (LNPs) and investigated the biological consequences of cancer cells with CA9-knockdown.

Methods: The lipid components of LNP are DLin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 which were dissolved in ethanol. This lipid mixture was mixed with CA9 siRNA using NanoAssemblr^® Benchtop. The prepared LNPs were dialyzed against PBS using Spectra/Por^® Float-A-Lyzer^® dialysis tubes. The size, polydispersity index and ζ-potential of LNPs were measured by dynamic light scattering using SZ-100 nanoparticle analyzer. The siRNA encapsulation efficiency (EE%) was determined with Quant-iT RiboGreen RNA Assay Kit. The structure of LNP-siRNA was examined using FEI Tecnai G2 F20 TWIN TEM. Cells were cultured in DMEM containing 10% FBS at 37°C in a CO₂ incubator. For hypoxic condition, cells were incubated in a humidified atmosphere of 1% O₂, 5% CO₂ and 94% N₂. To evaluate CA9 mRNA expression, total cellular RNA was extracted using GENEzol^TM TriRNA Pure Kit. RT-qPCR was performed using RevertAid RT Reverse Transcription Kit and 2× SYBR Green qPCR Mix. To evaluate CA9 protein expression, the cells were lysed with RIPA buffer. Protein concentration was measured by BCA Protein Assay Kit. Cell extracts were subjected to SDS-PAGE and transferred to a PVDF membrane. Membrane was blocked with 5% skim milk and incubated with primary antibody against CA9 and β-actin at 4°C overnight, then incubated with HRP-conjugated secondary antibody at room temperature. Chemiluminescent substrate was added to the membrane. The signal was detected with a UVP Biospectrum Imaging System. The survival of cancer cells treated with LNP-siRNA was evaluated by colony formation assay. Cells were cultured on 35-mm dish. LNP-siRNA were added to the cells for two weeks. Cells were fixed with glacial acetic acid and methanol, and fixed with 0.5% crystal violet. The cytotoxicity of cells treated with LNP-siRNA and doxorubicin was determined by MTT assay.

Results: The LNP particle size was 63.3 ± 1.9 nm with the PDI of 0.049 ± 0.028, and ζ-potential of -5.8 ± 8.2 mV. The EE% of LNP-siRNA was 93.7 ± 5.2%. Morphology of LNP-siRNA was observed. Cryo-EM observation confirmed that LNPs encapsulating CA9 siRNA were monodisperse nanoparticles and had a solid core structure (Fig. 1). RT-qPCR was performed to assess the mRNA expression level of CA9 in HuH7, Hela, CL1-5 and MDA-MB-231 cells under hypoxic and normoxic conditions. The mRNA level of CA9 was significantly higher in HuH7, Hela, CL1-5 and MDA-MB-231 cells under hypoxia, showed that hypoxia promote CA9 expression in different cancer cell lines (Fig. 2A). HuH7 cells were transfected with LNPs encapsulating CA9 siRNA. Then, CA9 mRNA and protein expression was evaluated by RT-qPCR and western blotting. Both CA9 mRNA and protein expression in HuH7 cells decreased in a time-dependent manner, and ~90% knockdown of CA9 mRNA was achieved 72 h after transfection. These results clearly indicated that siRNA delivered by LNP could efficiently knockdown the increased expression of CA9 under hypoxic conditions (Fig. 2C, D). MTT assay was performed to examine the cytotoxicity of CA9-knockdown cells treated with doxorubicin. As shown in Figure 3A, HuH7 cells became resistance to doxorubicin under hypoxia condition. Furthermore, there was no significant difference in doxorubicin-induced cellular cytotoxicity in HuH7 cells with or without CA9-knockdown under normoxic conditions. This result was expected because CA9 was not upregulated in cells under normoxia. However, under hypoxia, the viability of doxorubicin treated cells with CA9-knockdown was significantly lower compared to HuH7 cells treated with 0.25 μM doxorubicin alone. These results suggested that the increased CA9 under hypoxia might induce the resistance of HuH7 cells to doxorubicin and CA9 knockdown could reduce the drug resistance (Fig. 3A). Figure 2B showed the CA9-knockdown efficiency in HepG2/C3A cells treated with LNP-siRNA. Colony formation assay was further performed to evaluate the effect of CA9-knockdown on cellular survival. The number of colonies formed by HepG2/C3A after treated with LNP-siRNA was significantly lower, demonstrated that CA9-knockdown had negative impact on the ability of HepG2/C3A cells forming colonies (Fig. 3B). Presently, the possibility of CA9-knockdown in suppressing cellular growth and migration are under investigation.

Conclusion: The level of CA9 mRNA and protein was significantly upregulated in cancer cells under hypoxia condition, and the reduced expression of CA9 inhibits the cellular growth and resistance to doxorubicin, suggesting that CA9 might be a prospective therapeutic target for cancer treatment.

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Figure 1. Cryo-EM images of CA9 siRNA-encapsulated LNPs.

Figure 2. CA9 mRNA and protein expression in cancer cells. (A) Relative CA9 mRNA expression level in different cancer cell lines under normoxia and hypoxia. (B) Relative CA9 mRNA expression level in HepG2/C3A cells treated with different dose of LNP-siRNA. Knockdown of (C) CA9 mRNA and (D) protein in HuH7 cells under hypoxia after the transfection with LNP-siRNA. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3. The effect of CA9-knockdown on the growth of cancer cells. (A) The viability of HuH7 cells after treatment of LNP-siRNA and doxorubicin. (B) The number of HepG2/C3A cell colonies formed under hypoxia after treatment of LNP-siRNA. (*P < 0.05).