Nanoparticle formulation of mycophenolate mofetil achieves enhanced efficacy against hepatocellular carcinoma by targeting tumour-associated fibroblast

Abstract

Hepatocellular carcinoma (HCC) is one of the most aggressive tumours with marked fibrosis. Mycophenolate mofetil (MMF) was well-established to have antitumour and anti-fibrotic properties. To overcome the poor bioavailability of MMF, this study constructed two MMF nanosystems, MMF-LA@DSPE-PEG and MMF-LA@PEG-PLA, by covalently conjugating linoleic acid (LA) to MMF and then loading the conjugate into polymer materials, PEG_5k -PLA_8k and DSPE- PEG_2k , respectively. Hepatocellular carcinoma cell lines and C57BL/6 xenograft model were used to examine the anti-HCC efficacy of nanoparticles (NPs), whereas NIH-3T3 fibroblasts and highly-fibrotic HCC models were used to explore the anti-fibrotic efficacy. Administration of NPs dramatically inhibited the proliferation of HCC cells and fibroblasts in vitro. Animal experiments revealed that MMF-LA@DSPE-PEG achieved significantly higher anti-HCC efficacy than free MMF and MMF-LA@PEG-PLA both in C57BL/6 HCC model and highly-fibrotic HCC models. Immunohistochemistry further confirmed that MMF-LA@DSPE-PEG dramatically reduced cancer-associated fibroblast (CAF) density in tumours, as the expression levels of alpha-smooth muscle actin (α-SMA), fibroblast activation protein (FAP) and collagen IV were significantly downregulated. In addition, we found the presence of CAF strongly correlated with increased HCC recurrence risk after liver transplantation. MMF-LA@DSPE-PEG might act as a rational therapeutic strategy in treating HCC and preventing post-transplant HCC recurrence.

Keywords: cancer-associated fibroblast; hepatocellular carcinoma; mycophenolate mofetil; nanoparticles.

FIGURE 1

Construction and characteristics of MMF‐LA NPs. A, Schematic illustration of the construction of MMF‐LA@DSPE‐PEG nanosystem. MMF‐LA@DSPE‐PEG was constructed by covalently conjugating linoleic acid (LA) to mycophenolate mofetil (MMF) and then loading the conjugate into polymer material, DSPE‐PEG₂₀₀₀. B, The morphology of MMF‐LA@DSPE‐PEG and MMF‐LA@PEG‐PLA characterized by transmission electron microscopy (TEM). The scale bars: 100 nm. C and D, The diameters and zeta potentials of MMF‐LA@DSPE‐PEG and MMF‐LA@PEG‐PLA characterized by Dynamic light scattering (DLS)

FIGURE 2

Anti‐HCC efficiency of MMF‐LA NPs in vitro. A, Cell viabilities of Huh7 cells, SUN‐449 cells, LM3 cells and Hep1‐6 cells treated with free MMF, MMF‐LA@DSPE‐PEG and MMF‐LA@PEG‐PLA for 48 h. B, Cell cycles determined by flow cytometry. Cells were treated with free MMF (2 µg/mL) or MMF‐LA NPs (at 2 µg/mL MMF‐equivalent dose) for 48 h. C, Quantitative analysis of the results in panel B. Data are shown as the mean ± SD (n = 3), *P < .05, **P < .01. D, Expression levels of cyclin D and cyclin E determined using Western blot. E, Colony Formation of Hep1‐6 cells, SUN‐449 cells and Huh7 cells. Hep1‐6 cells, SUN‐449 cells and Huh7 cells were treated with drugs at 0.5, 2 and 4 µg/mL (MMF‐equivalent concentration), respectively, for 96 h. All the results are representative of at least three independent experiments

FIGURE 3

Anti‐HCC efficiency of MMF‐LA NPs in vivo. A, Images of Hep1‐6 tumours after treatment with drugs at 20 mg/kg (MMF‐equivalent concentration), (n = 6). B, Tumour volumes of different groups (n = 6), *P < .05, ***P < .001. C, Tumour inhibition rates (R) of different treatments. $R=(\overline{v}nc‐v/\overline{v}nc)$, $\overline{v}nc$ represents the mean tumour volume of NC group, v represents the tumour volume of free MMF, MMF‐LA@DSPE‐PEG or MMF‐LA@PEG‐PLA groups, (n = 6), *P < .05, ***P < .001. D, Bodyweights (mean ± SD, n = 6) of mice in different groups. E, Expression levels of PCNA in different groups determined by Immunohistochemistry. The scale bars: 100 µm. F, Quantitative analysis of PCNA expression status in panel E (Image J software), data are shown as the mean ± SD, (n = 3), **P < .01, ***P < .001

FIGURE 4

Anti‐fibrotic efficiency of MMF‐LA NPs in vitro. A, Cell viability of NIH‐3T3 cells treated with free MMF, MMF‐LA@DSPE‐PEG and MMF‐LA@PEG‐PLA for 48 h. B, Expression levels of tubulin determined by Western blot. C, Expression levels of tubulin and cell morphology of fibroblasts determined by Immunofluorescence. Green: tubulin; red: vimentin, blue: nuclei. The scale bars: 50 µm. D, Quantitative analysis of tubulin expression status in panel C (Image J software), data are shown as the mean ± SD, (n = 3), **P < .01. E, NIH‐3T3 cells were seeded in the top chamber of transwell with serum‐free medium and treated with free MMF, MMF‐LA@DSPE‐PEG and MMF‐LA@PEG‐PLA. After 24 h, migrated cells were fixed, stained and photographed. The scale bars: 200 µm (F) Quantitative analysis of the migrated cell number, data are shown as the mean ± SD, (n = 3), ***P < .001

FIGURE 5

CAF significantly enhanced HCC growth in vivo. A, Expression levels of α‐SMA determined by Immunofluorescence. NIH‐3T3 fibroblasts were co‐cultured with TGF‐β1 or cultured alone for 96 h. B, Tumour images of different groups. Hep1‐6 cells were injected with activated fibroblasts or injected alone into the nude mice, (n = 6). C, Tumour growth curves of different groups, ***P < .001. D, Expression levels of α‐SMA, FAP and collagen IV determined by Immunohistochemistry. The scale bars: 50, 100 or 200 µm. E, Quantitative analysis of panel D (Image J software), data are shown as the mean ± SD, (n = 3), ***P < .001. F, Representative images showing low α‐SMA expression (CAF density = 1), median α‐SMA expression (CAF density = 2) and high α‐SMA expression (CAF density = 3) in HCC samples obtained from HCC patients underwent liver transplantation. G, Kaplan‐Meier analysis of patients with moderate CAF density (group 1 + 2) and high CAF density (group 3)

FIGURE 6

MMF‐LA@DSPE‐PEG inhibited HCC growth by depleting CAF. Mice were orally administrated with free MMF (20 mg/kg) or intravenously injected with MMF‐LA NPs (at 20 mg/kg MMF‐equivalent dose) every other day for four times. A, Tumour images of different groups, (n = 6). B, Tumour growth curves of different groups, **P < .01. C, Tumour inhibition rates of different treatments. (n = 6), **P < .01, ***P < .001. D, Bodyweights (mean ± SD, n = 6) of mice in different groups. E, Expression levels of α‐SMA, FAP, collagen IV and CD31 determined by Immunohistochemistry. The scale bars: 200 µm. F, Quantitative analysis of panel E (Image J software), data are shown as the mean ± SD, (n = 3), **P < .01, ***P < .001

FIGURE 7

CAF‐targeting capacity of MMF‐LA@DSPE‐PEG. A, Accumulation of Free‐DIR and MMF‐LA@DSPE‐PEG‐DIR within tumours. B, Quantitative analysis of fluorescence intensity, n = 3, ***P < .001. C, Colocalization of MMF‐LA@DSPE‐PEG‐DIR and CAF. Green: α‐SMA; red: MMF‐LA@DSPE‐PEG‐DIR, blue: nuclei. The scale bars: 50 µm. D, Schematic illustration of the working mechanism of MMF‐LA@DSPE‐PEG. Cancer‐associated fibroblast (CAF) greatly enhance tumour growth. The established MMF‐LA@DSPE‐PEG nanoparticles effectively target CAF and then are internalized by these cells. As a consequence, CAF are dramatically suppressed and CAF‐related tumour growth is inhibited