Ultrasound-Guided Microbubble-Mediated Locoregional Delivery of Multiple MicroRNAs Improves Chemotherapy in Hepatocellular Carcinoma

Abstract

Rationale: To assess treatment effects of 4 complementary miRNAs (miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21) encapsulated in a biodegradable PLGA-PEG nanoparticle, administered by an ultrasound-guided microbubble-mediated targeted delivery (UGMMTD) approach in mouse models of hepatocellular carcinoma (HCC). Methods:In vitro apoptotic index was measured in HepG2 and Hepa1-6 HCC cells treated with various combinations of the 4 miRNAs with doxorubicin. Three promising combinations were further tested in vivo by using UGMMTD. 63 HepG2 xenografts in mice were randomized into: group 1, miRNA-122/antimiRNA-10b/antimiRNA-21/US/doxorubicin; group 2, miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21/US/doxorubicin; group 3, miRNA-100/miRNA-122/antimiRNA-10b/US/doxorubicin; group 4, miRNA-122/anitmiRNA-10b/antimiRNA-21/doxorubicin; group 5, miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21/doxorubicin; group 6, miRNA-100/miRNA-122/antimiRNA-10b/doxorubicin; group 7, doxorubicin only treatment; and group 8, without any treatment. Tumor volumes were measured through 18 days. H&E staining, TUNEL assay, and qRT-PCR quantification for delivered miRNAs were performed. Results:In vivo results showed that UGMMTD of miRNAs with doxorubicin in groups 1-3 significantly (P<0.05) delayed tumor growth compared to control without any treatment, and doxorubicin only from day 7 to 18. On qRT-PCR, levels of delivered miRNAs were significantly (P<0.05) higher in groups 1-3 upon UGMMTD treatment compared to controls. TUNEL assay showed that upon UGMMTD, significantly higher levels of apoptotic cell populations were observed in groups 1-3 compared to controls. Toxicity was not observed in various organs of different groups. Conclusions: UGMMTD of miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21 combination improved therapeutic outcome of doxorubicin chemotherapy in mouse models of HCC by substantial inhibition of tumor growth and significant increase in apoptotic index.

Keywords: drug delivery; hepatocellular carcinoma (HCC); microRNA-100/microRNA-122/microRNA-10/microRNA-21; microbubble; ultrasound.

Figure 1

Schematic illustration of signaling pathways regulated by miRNA-100, miRNA-122, miRNA-10b, and miRNA-21 in hepatocellular carcinoma (HCC), and the mechanisms by which the microRNAs modulated regulation can achieve therapeutic enhancement of chemotherapy.

Figure 2

Schematic figure showing the overall in vitro, in vivo and ex vivo experimental design for evaluating the microRNA targeted HCC chemotherapy.

Figure 3

Size zeta potential and loading efficiency of miRNA-loaded nanoparticles.

Figure 4

In vitro treatment evaluation of HepG2 human HCC cells to various combination of microRNAs in the presence of doxorubicin chemotherapy and the downstream functional effect by immunoblot analysis. (a) Doxorubicin dose mediated apoptotic evaluation, (b) MicroRNAs combination therapy evaluated in the presence of 0.25 μM doxorubicin using PI staining based FACS analysis for nine different miRNA combinations delivered by PLGA-b-PEG nanoparticles, (c) Quantitative graph showing the results of apoptotic and live cell populations measured by the FACS data shown in 'b', (d) Western blot results showing strongest effect either on both downregulation of the target anti-apoptotic proteins HoxD10, IGF1R, CD320 and BCL2, or/and upregulation of the target pro-apoptotic proteins PDCD4 and Bax after various treatment conditions in HepG2 cells.

Figure 5

In vitro treatment evaluation of Hepa1-6 mouse HCC cells to various combination of microRNAs in the presence of doxorubicin chemotherapy and the downstream functional effect by immunoblot analysis. (a) Doxorubicin dose mediated apoptotic evaluation by PI staining based FACS analysis, (b) MicroRNAs combination therapy evaluated in the presence of 0.25 μM doxorubicin by PI staining based FACS analysis using nine different miRNA combinations delivered by PLGA-b-PEG nanoparticles, (c) Quantitative graph showing the results of apoptotic and live cell populations measured by the FACS data shown in 'b', (d) Western blot results showing strongest effect either on both downregulation of the target anti-apoptotic proteins HoxD10, IGF1R, CD320 and BCL2, or/and upregulation of the target pro-apoptotic proteins PDCD4 and Bax after various treatment conditions in Hepa1-6 cells.

Figure 6

Antitumor effect of complementary miRNAs and doxorubicin co-treatment in the presence and absence of US-MB delivery in HepG2 human HCC xenografts in vivo. (a). Schematic workflow summarizes the in vivo treatment conditions, therapy evaluation, and ex vivo analyses used for monitoring therapeutic outcome. Three repeated cycles of combination treatment were used on day 1, 7, and 12, for the delivery of miRNA-loaded NPs using an ultrasound-guided and microbubble-mediated delivery approach. On days 2, 3, 4, 8, 9, 10, 13, 14, and 15, the mice in groups 1-7 also received i.p. injection of low dose doxorubicin at 2.5 mg/kg. Tumor volume was calculated using the formula: volume = [length x width x height) × π /6. The tumor volumes were measured at the baseline (day 1) before treatment started, and 7, 10, 12, 15, and 18 days after treatment. To facilitate the comparisons between the tumor volumes in different groups, the tumor volumes measured at different time points in each animal were normalized to its own baseline value. Therefore, the normalized tumor volume value was 1.0 for each animal at the baseline. (b). The results of the treatment condition g with miRNAs (antimiRNA-21, antimiRNA-10b, and miRNA-122) and doxorubicin, with (group 1) and without (group 4) US-MB treatment along with doxorubicin only and untreated control group monitored for tumor growth over 18 days. (c). The results of the treatment condition h with miRNAs (antimiRNA-21, antimiRNA-10b, miRNA-100 and miRNA-122) and doxorubicin, with (group 2) and without (group 5) US-MB treatment along with doxorubicin only and untreated control group monitored for tumor growth over 18 days. (d). The results of the condition group i with miRNAs (miRNA-122, miRNA-100 and antimiRNA-10b) and doxorubicin, with (group 3) and without (group 6) US-MB treatment along with doxorubicin only and untreated control group monitored for tumor growth over 18 days. (e). The comparison between the three treatment groups (miRNA + doxorubicin + ultrasound) showing no statistical significance at day 7 to 18. Note that values were normalized and compared to day 1 value before the treatment. Each box in the plot represents the 25^th and 75^th quartiles, the line inside each box identifies the median and the whiskers indicate the 5^th and 95^th percentile of measurements excluding the outliers. ♦ represent outliers. * indicates P value < 0.05 between treated and control groups.

Figure 7

Quantitative RT-PCR results in human HepG2 HCC xenografts following intravenous injection of miR-100, miR-122, antimiR-10b, and antimiR-21-loaded PLGA-b-PEG-NPs and treated with ultrasound-guided and microbubble-mediated sonoporation. Note significant increases in the amount of delivered miRNAs were observed after 3 repeated treatment cycles in group 1-3 compared to controls.

Figure 8

Representative TUNEL stained sections obtained from the animals with combination treatment show increased apoptosis (brown color) in treated tumors after 3 repeated treatment cycles compared to control tumors. Scale bars = 250 µm.

Figure 9

Histological analysis of tumors and different organs assessed for toxicity and treatment effect using Hematoxylin and Eosin (H&E) staining. (a) H&E stained sections show large tumor cell populations characterized by dense nuclear distribution with higher instance of actively dividing nuclear morphology in a representative animal of group 8. On the other hand, H&E stained sections of treated tumor demonstrate the enhanced necrosis in groups 1-3 with miRNA, doxorubicin and ultrasound. (b) H&E stained sections of various organ, including heart, lung, liver, pancreas, spleen and kidneys do not show the toxicity in the tumors treated with miRNA, doxorubicin and ultrasound. Scale bars = 250 µm.