Using ultrasound activating contrast agents to induce sonoporation is a potential strategy for effective lesion-targeted gene delivery. Previous reports have proven that submicron nanodroplets have a better advantage than microbubbles in that they can pass through tumor vasculature endothelial gaps by passive targeting; however, they cannot achieve an adequate dose in tumors to facilitate ultrasound-enhanced gene delivery. Additionally, a few studies focused on delivering macromolecular genetic materials (i.e. overexpression plasmid and CRISPR plasmid) have presented more unique advantages than small-molecular genetic materials (i.e. miRNA mimics, siRNA and shRNA etc.), such as enhancing the expression of target genes with long-term effectiveness. Thereby, we constructed novel plasmid-loadable magnetic/ultrasound-responsive nanodroplets, where superparamagnetic iron oxide nanoparticle dispersed perfluoropentane was encapsulated with lipids to which plasmids could be adhered, and branched polyethylenimine was used to protect the plasmids from enzymolysis. Furthermore, in vitro and in vivo studies were performed to verify the magnetic tumor-targeting ability of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets and focused ultrasound enhanced intracellular plasmid delivery. The plasmid-loadable magnetic/ultrasound-responsive nanodroplets, carrying 16–19 plasmids per droplet, had desirable diameters less than 300 nm, and integrated the merits of excellent magnetic targeting capabilities and phase transition sensitivity to focused ultrasound. Under programmable focused ultrasound exposure, the plasmid-loadable magnetic/ultrasound-responsive nanodroplets underwent a phase-transition into echogenic microbubbles and the subsequent inertial cavitation of the microbubbles achieved an ∼40% in vitro plasmid delivery efficiency. Following intravenous administration, T2-weighted magnet resonance imaging, scanning electron microscopy and inductively coupled plasma optical emission spectrometry of the tumors showed significantly enhanced intratumoral accumulation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets under an external magnetic field. And a GFP ELISA assay and immunofluorescence staining indicated that focused ultrasound-induced inertial cavitation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets significantly enhanced the intracellular delivery of plasmids within the tumor after magnet-assisted accumulation, while only lower GFP levels were observed in the tumors on applying focused ultrasound or an external magnet alone. Taken together, utilizing the excellent plasmid-loadable magnetic/ultrasound-responsive nanodroplets combined with magnetism and ultrasound could efficiently deliver plasmids to cancer cells, which could be a potential strategy for macromolecular genetic material delivery in the clinic to treat cancer.

Inefficient and low-precision delivery of exogenous nucleic acids (ENA) severely limits gene therapy on ischemic stroke (IS). Two problems need to be urgently addressed to improve the efficacy of gene therapy; first, the blood brain barrier (BBB) should be open to promote the accumulation of ENA or genetic material carriers in the ischemic brain parenchyma, and second, the efficient delivery of ENA into the ischemic cells. Previous studies applied ultrasonic cavitation either for opening BBB or for inducing sonoporation to deliver genetic materials into cells. However, the effectiveness of the two-step ultrasonic cavitation to deliver ENA in the brain remains unclear, let alone the genetic materials to be controllably delivered into the ischemic brain parenchyma of the IS. This study systematically explored the BBB opening and ENA delivery by the two-step ultrasonic cavitation using artificial acoustic-cationic-polymeric-nanodroplets (ACPNs). The results demonstrated that the first focused ultrasound (FUS), set at parameters of 3.3 MPa, 20 Hz, 200 cycles and 5 s, stimulating intravascular ACPNs cavitation effectively opened BBB to allow nonactivated ACPN extravasation and accumulation into the ischemic brain parenchyma. Then, the extravascular ACPNs enhanced the second ultrasonic cavitation that noninvasively and efficiently controlled ENA delivery to the ischemic cells through sonoporation, particularly applying 3.3 MPa, 60 Hz, 200 cycles and 9 s to control FAM-eNA delivery, and 3.6 MPa, 20 Hz, 200 cycles and 7 s for pEGFP-C1 controlled delivery. Overall, the two-step ultrasonic cavitation represented a potential strategy for IS-targeted ENA controlled delivery. Graphical abstract

The spleen, as the largest immune organ, plays a pivotal role in modulating immune responses, particularly in the context of carcinogenesis and tumor progression. Non-pharmacological manipulation, particularly splenic ultrasound stimulation (SUS), has demonstrated significant immunomodulatory efficacy in alleviating chronic inflammatory diseases, suggesting its potential to revitalize splenic immunocompetence suppressing tumor proliferation, yet remains underexplored. This study applied low-frequency pulsed focused ultrasound (FUS) noninvasively stimulating the spleen (FUS sti. spleen) to investigate the efficacy in enhancing antitumor immunity and suppressing hepatocellular carcinoma (HCC). The results showed that FUS sti. spleen significantly suppressed tumor proliferation, achieving a suppression rate of >70% for H22-HCC and >83% for Hepa1-6-HCC, along with significantly prolonged survival. Comprehensive flow cytometry, single-cell RNA sequencing (scRNA-seq) and cytokine analyses demonstrated that SUS profoundly reshaped the splenic and intratumoral immune landscape, specifically activating cytotoxic CD8 ⁺ T cells and NK cells while suppressing immunosuppressive cell populations. Mechanistically, FUS facilitated calcium influx in splenic immunocytes, activating multiple signaling pathways, such as TNF, NFκB, MAPK, HIF-1, and ErbB, thereby counteracting tumor-driven immunosuppressive polarization while potentiating robust immune activation that impedes malignant progression and neoplastic proliferation. Leveraging above insights, we developed spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STNDs@Ca²⁺), which, upon FUS stimulation, undergo cavitation-mediated controlled release of Ca²⁺, further amplifying immunocyte activation and tumor suppression, achieving a remarkable H22-HCC suppression rate of over 90%. This study highlights the therapeutic potential of ultrasound-mediated splenic immunomodulation, both as a standalone intervention and in synergy with STNDs@Ca²⁺, as a novel and noninvasive strategy for cancer immunotherapy.

The spleen, as the largest immune organ, plays a pivotal role in modulating immune responses, particularly in the context of carcinogenesis and tumor progression. Non-pharmacological manipulation, particularly splenic ultrasound stimulation (SUS), has demonstrated significant immunomodulatory efficacy in alleviating chronic inflammatory diseases, suggesting its potential to revitalize splenic immunocompetence suppressing tumor proliferation, yet remains underexplored. This study applied low-frequency pulsed focused ultrasound (FUS) noninvasively stimulating the spleen (FUS sti. spleen) to investigate the efficacy in enhancing antitumor immunity and suppressing hepatocellular carcinoma (HCC). The results showed that FUS sti. spleen significantly suppressed tumor proliferation, achieving a suppression rate of >70% for H22-HCC and >83% for Hepa1-6-HCC, along with significantly prolonged survival. Comprehensive flow cytometry, single-cell RNA sequencing (scRNA-seq) and cytokine analyses demonstrated that SUS profoundly reshaped the splenic and intratumoral immune landscape, specifically activating cytotoxic CD8+ T cells and NK cells while suppressing immunosuppressive cell populations. Mechanistically, FUS facilitated calcium influx in splenic immunocytes, activating multiple signaling pathways, such as TNF, NFκB, MAPK, HIF-1, and ErbB, thereby counteracting tumor-driven immunosuppressive polarization while potentiating robust immune activation that impedes malignant progression and neoplastic proliferation. Leveraging above insights, we developed spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STNDs@Ca²⁺), which, upon FUS stimulation, undergo cavitation-mediated controlled release of Ca²⁺, further amplifying immunocyte activation and tumor suppression, achieving a remarkable H22-HCC suppression rate of over 90%. This study highlights the therapeutic potential of ultrasound-mediated splenic immunomodulation, both as a standalone intervention and in synergy with STNDs@Ca²⁺, as a novel and noninvasive strategy for cancer immunotherapy.