Fig. 3: LNPs efficiently delivered small circRNA to lymph nodes and APCs to elicit T cell responses.
From: Small circular RNAs as vaccines for cancer immunotherapy
a, Design of nanocarrier screening for small circRNA vaccines, using circRNA-SIINFEKL as a model and 5moU-mRNA-OVA as a control. RNA: 5 μg, subcutaneous (s.c.) injection at the tail base of C57BL/6 mice (n = 5) on days 0 and 14. b, Tetramer staining on day 21 showed that SM-102 LNPs of circRNA-SIINFEKL elicited the highest frequency of PBMC SIINFEKL+CD8+ T cells among all these RNA nanoformulations. c, Luminex results of normalized serum cytokine and chemokine levels 12 h after booster immunization (day 14). SM-102 LNP-circRNA-SIINFEKL induced relatively low reactogenicity-associated chemokines. Each cytokine or chemokine level (X) was respectively normalized as follows: \({X}_{\mathrm{normalized}}=\frac{X-{X}_{\min }}{{X}_{\max }-{X}_{\min }}\). d–f, Upon s.c. injection at the foot pad of BALB/c mice (n = 5), SM-102 LNPs efficiently delivered IR800-circRNA-SIINFEKL (0.5 nmol) to draining popliteal lymph nodes (dLNs) (circled in d), as shown by whole-body IVIS imaging (d), quantified IR800 fluorescence intensities of popliteal dLNs (e) and tissue fluorescence intensities quantified from ex vivo IVIS imaging (f). g, Flow cytometry results showing the Cy5-circRNA+ APC subsets among total CD45+ cells in draining lymph nodes 24 h after s.c. injection of free Cy5-circRNA or Cy5-circRNA LNPs, respectively. h, Confocal microscopy images showing efficient LNP delivery of Cy5-circRNA to DC2.4 cells and rapid endosome escape of Cy5-circRNA, the latter of which was indicated by the cytosolic Cy5-circRNA outside endolysosomes (circRNA, 100 nM; treatment, 0.5 h). Inset: one cell. i, As quantified from the above confocal microscopy images, the Cy5-circRNA fluorescence signal intensity ratios of outside/inside (O/I) endolysosome suggest the rapid endosome escape of LNP-circRNA in DC2.4 cells. Liposomal circRNA (lipo-circRNA) served as a control.
