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Single-cell analysis reveals effective siRNA delivery in brain tumors with microbubble-enhanced ultrasound and cationic nanoparticles

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Science Advances  30 Apr 2021:
Vol. 7, no. 18, eabf7390
DOI: 10.1126/sciadv.abf7390
  • Fig. 1 In vitro and in vivo characterization of transcellular and transvascular (BBB) penetration of cationic lipid-polymer hybrid nanoparticles loaded with siRNA.

    (A) Size distribution and morphology of the cationic LPH and LPH:Cy5-siRNA nanoparticles. (B) Surface charge of the cationic (Cat) LPH and LPH:Cy5-siRNA. P values were determined by unpaired t test. (C) Quantification of the LPH and Cy5-siRNA uptake by GL261 glioma cells using fluorescence microscopy. P values were determined by one-way analysis of variance (ANOVA). (D) In vitro cellular uptake kinetics of the cationic LPH:Cy5-siRNA into GL261 glioma cells. (E) Live and dead cell staining at 2 and 8 hours after LPH:Cy5-siRNA exposure. (F) In vivo experimental protocol for the delivery of cationic LPH in the brain of healthy, immunocompetent mice. Inset: Representative contrast-enhanced T1-weighted MR image after MB-FUS. i.v., intravenous. (G) Representative fluorescent microscopy data of LPH extravasation in the brain of healthy mouse at 10 min after LPH administration for non-FUS region (top) and FUS-treated region (bottom). (H) Quantification of the LPH extravasation in non–FUS-treated region and FUS-treated region 10 min after treatment (12-fold; P < 0.0001, unpaired t test). (I) Representative H&E staining images at 10 min after LPH administration. Plots show means ± SEM (N = 3). ****P ≤ 0.0001.

  • Fig. 2 Improved cationic LPH extravasation in the GL261 glioma mouse tumors using MB-FUS.

    (A) In vivo experimental protocol for the delivery of cationic LPH in GL261 glioma mouse tumor model. LPH distribution is analyzed at 10 min after LPH administration. (B) Representative contrast-enhanced T1-weighted MR images before and after MB-FUS treatment. (C) Quantification of the LPH extravasation in non–FUS-treated and FUS-treated tumor at 10 min after treatment (5.4-fold, P = 0.032). (D) Representative fluorescent microscopy data of LPH extravasation in tumor at 10 min after LPH administration. Plots show means ± SEM (N = 3). P values were determined by unpaired t tests. *P ≤ 0.05.

  • Fig. 3 Improved cationic LPH:siRNA extravasation, penetration, and cellular uptake in the GL261 glioma mouse tumors using MB-FUS.

    (A) In vivo experimental protocol for LPH:siRNA delivery in a GL261 glioma mice tumor model. LPH:siRNA distribution is analyzed at 8 hours after nanoparticle administration. (B) Representative fluorescent microscopy data of LPH:siRNA extravasation and penetration in tumor at 8 hours after LPH:siRNA administration. (C) Quantification of the LPH extravasation in tumor with and without FUS at 8 hours after treatment (13.7-fold, P = 0.044). (D) Quantification of the siRNA penetration in tumor with and without FUS at 8 hours after treatment (5.4-fold, P = 0.0045). (E) Quantification of siRNA delivery to cancer cells with and without FUS at 8 hours after treatment (9.5-fold, P = 0.0364). (F) Quantification of the ratio of LPH (blue) and Cy5-siRNA (red) delivery to cancer cells to total cell uptake at 8 hours after FUS treatment. (G) Representative fluorescent microscopy data of LPH:Cy5-siRNA cellular uptake in tumor at 8 hours after LPH:siRNA administration. Green arrows show the LPH:siRNA uptake by cancer cells, and white arrows show the LPH:siRNA uptake by brain cells. Plots show means ± SEM (N = 3). P values were determined by unpaired t tests. *P ≤ 0.05 and **P ≤ 0.01

  • Fig. 4 Enhanced delivery of siRNA into the SHH-activated medulloblastoma tumor using MB-FUS and cationic LPH:SMO-siRNA.

    (A) In vivo experiment setup using a custom-built USgFUS system (left). Schematic graph is created with BioRender.com. Representative contrast-enhanced T2-weighted MR images of medulloblastoma tumor–bearing mice (right, top) and contrast-enhanced T1-weighted MR images of healthy mice after FUS BBB disruption (BBBD) targeted at the location of medulloblastoma tumor (right, bottom). (B) Harmonic emission from the MB-mediated BTB disruption using FUS. (C) Quantification of the acoustic emissions. (D) In vivo experimental protocol for the delivery of cationic LPH:SMO-siRNA in a medulloblastoma mouse tumor model. (E) Representative fluorescent microscopy data of LPH accumulation in tumor for non–FUS-treated group (left) and FUS-treated group (right). (F) Quantification of the LPH accumulation in tumor with and without FUS at 30 hours after treatment (9.4-fold, P = 0.0472). (G) Representative fluorescent data of FISH assay on non–MB-FUS group (top) and MB-FUS group (bottom). Plots show means ± SEM (N = 3). P values were determined by unpaired t tests. n.s., no statistical significance; *P ≤ 0.05; ****P ≤ 0.0001.

  • Fig. 5 MB-FUS, in combination with cationic LPH:SMO-siRNA, induces cell apoptosis in SHH-activated subgroup of medulloblastoma.

    (A) Schematic illustration of LPH:siRNA-induced cell apoptosis. Schematic graph is created with BioRender.com. (B) Representative microscopy data of SMO protein immunostaining non–FUS-treated group (left) and FUS-treated group (right) demonstrating substantial SMO protein knockdown. SMO protein is shown in brown, and nucleus staining is shown in purple. (C) Quantification of SMO protein level in medulloblastoma tumor (fivefold, P = 0.0483). (D) Representative fluorescent microscopy data of SMO-siRNA–induced cell apoptosis in tumor at 30 hours after LPH:SMO-siRNA administration for non–FUS-treated group (top) and FUS-treated group (bottom) using TUNEL assay. (E) Quantification of tumor cell apoptosis (TUNEL-positive signal) that is LPH positive with and without FUS at 30 hours after treatment (16.6-fold, P = 0.0013). (F) Representative fluorescent microscopy data of SMO-siRNA–induced cell apoptosis in tumor at 30 hours after LPH:SMO-siRNA administration for non–FUS-treated group (top) and FUS-treated group (bottom) using CC3 assay. (G) Quantification of tumor cell apoptosis (CC3-positive signal) that is LPH positive 8 hours after nontherapeutic LPH:Cy5-siRNA delivery administration (n.s.) and 30 hours after LPH:SMO-siRNA delivery administration with and without FUS (34-fold, P = 0.0017). Plots show means ± SEM (N = 3). P values were determined by unpaired t tests. n.s., P > 0.05; *P ≤ 0.05; **P ≤ 0.01.

  • Fig. 6 Integrated quantitative microscopy and PBPK modeling guides the integration of LPH nanoparticles and MB-FUS technologies.

    (A) Parameter identification procedures to recover LPH pharmacokinetics from the experimentally determined RhoB-LPH penetration (line profile perpendicular to vessel wall, left) in the GL261 glioma tumor model using 2D tumor cord geometry. The model output and the reference solutions agreed (right). (B) Normalized parameter fit for non–FUS-treated and FUS-treated groups using 2D tumor cord PBPK model. (C) Structurally heterogeneous modeling of LPH transport in TME. (D) Sensitivity analysis of the model parameters. (E) Cellular uptake of LPH with different sizes for non-FUS and FUS. (F) Transvascular flux with different surface charge LPH for non-FUS and FUS. Difference between different LPH sizes for non-FUS and FUS (one-way ANOVA). *Difference between non-FUS and FUS for each LPH size or surface charge (unpaired t tests). Extracellular LPH concentration (Ce) and intracellular LPH concentration (Ci) normalized to maximum LPH concentration inside the vessel (Cv). Dv, vessel diffusion coefficient; Di, interstitium diffusion coefficient; Kv, vessel hydraulic conductivity; Ki, interstitium hydraulic conductivity; V, rate of endocytosis. The plots show means ± SEM (N = 3). In (B) and (C), the P values were determined by unpaired t tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ††P ≤ 0.01, †††P ≤ 0.001, ††††P ≤ 0.0001.

Supplementary Materials

  • Supplementary Materials

    Single-cell analysis reveals effective siRNA delivery in brain tumors with microbubble-enhanced ultrasound and cationic nanoparticles

    Yutong Guo, Hohyun Lee, Zhou Fang, Anastasia Velalopoulou, Jinhwan Kim, Midhun Ben Thomas, Jingbo Liu, Ryan G. Abramowitz, YongTae Kim, Ahmet F. Coskun, Daniel Pomeranz Krummel, Soma Sengupta, Tobey J. MacDonald, Costas Arvanitis

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