In vivo gene silencing with novel siRNA loaded polypeptoide nanoparticles for anti-stromal therapy in hepatocellular carcinoma

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Background and Aims Cancer associated fibroblasts (CAF) support tumor growth and metastasis in the tumor microenvironment (TME) and are therefore promising target cells for anti-stromal therapy in solid tumors [Kaps, Schuppan; Cells 2020]. We have designed a novel polypeptoide nanoparticle (NP) with improved endosomal escape for small interfering RNA (siRNA) delivery into stroma cells of hepatocellular carcinoma (HCC) [Birke et al., Prog. Polym. Sci. 2018]. NPs loaded with CAF targeting siRNA were tested in a murine model of primary liver cancer. Method and Results In vitro screening for CAF relevant target genes revealed that the CAF derived microfibrillar-associated protein 5 (MFAP-5) was highly upregulated in fibroblasts (3T3 fibroblasts and MHSC-SV40 hepatic stellate cells) when co-cultured with HCC cells (Dt81Hepa1-6). NPs have been designed utilizing the triblock copolymers polysarcosine-b-poly(-benzyl glutamic acid)-b-polylysine, which enable co-loading of siRNA and desloratidin, an antihistamine that triggers endosomal release of the siRNA after cell uptake. Anti-MFAP-5 siRNA loaded NPs induced a robust knockdown (<50%) at low siRNA concentrations (≤5 nM) in fibroblasts as assessed on RNA level (qPCR). For the HCC model, B6 mice were intrasplenically injected with syngeneic 500.000 HCC cells (Dt81Hepa1-6) to develop macroscopic tumor lesions exclusively in their livers after 28 days. After intravenous injection, fluorescence labeled Cy5.5 siRNA loaded NPs distributed preferentially to the liver (>80%), while biodistribution did not differ between healthy and tumor mice. Ex vivo FACS analysis of digested livers confirmed a cellular uptake of NPs in CAF (FAP+)>macrophages (CD45+, F4/80+, CD11b+)>dendritic cells (CD45+, F4/80+, CD11c+). For in vivo anti-stromal therapy, tumor mice (n=5) received three intravenous injections of NPs loaded with anti-MFAP-5 siRNA (corresponding to 0.5 or 1 mg/kg siRNA) in week four, while controls received equal concentrations of scramble siRNA (scsiRNA) loaded NPs. Histological analysis and liver weight of mice treated with anti-MFAP-5 siRNA revealed significantly (*p<0.05) less hepatic tumor burden compared to mice treated with encapsulated scsiRNA. In vivo knockdown of MFAP-5 (>50%) was confirmed both on RNA (qPCR)- and protein-level (FACS), while controls had similar MFAP-5 levels like healthy mice. The treatment was well tolerated by the mice and safety serum parameters were in the normal range. Histological analysis of liver sections revealed that markers of tumor vascularization (e.g. CD34, CD105) were downregulated by the siRNA treatment in the TME, suggesting that the knockdown of MFAP-5 may inhibit angiogenesis. Conclusion Liver targeting NPs loaded with anti-MFAP-5 siRNA induced a gene specific knockdown of CAF derived MFAP-5 and demonstrated a convincing antitumor effect by interference with angiogenesis in the TME of HCC. Publication History Article published online: 28 August 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

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