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. 2021 Jul 20;22(14):7743.
doi: 10.3390/ijms22147743.

Liposomes Loaded with Everolimus and Coated with Hyaluronic Acid: A Promising Approach for Lung Fibrosis

Laura Pandolfi  1 , Alessandro Marengo  2 , Kamila Bohne Japiassu  2 , Vanessa Frangipane  1 , Nicolas Tsapis  2 , Valeria Bincoletto  3 , Veronica Codullo  4 , Sara Bozzini  1 , Monica Morosini  1 , Sara Lettieri  5 , Valentina Vertui  5 , Davide Piloni  5 , Silvia Arpicco  3 , Elias Fattal  2 , Federica Meloni  1   5
Affiliations

Liposomes Loaded with Everolimus and Coated with Hyaluronic Acid: A Promising Approach for Lung Fibrosis

Laura Pandolfi et al. Int J Mol Sci. .

Abstract

Chronic lung allograft dysfunction (CLAD) and interstitial lung disease associated with collagen tissue diseases (CTD-ILD) are two end-stage lung disorders in which different chronic triggers induce activation of myo-/fibroblasts (LFs). Everolimus, an mTOR inhibitor, can be adopted as a potential strategy for CLAD and CTD-ILD, however it exerts important side effects. This study aims to exploit nanomedicine to reduce everolimus side effects encapsulating it inside liposomes targeted against LFs, expressing a high rate of CD44. PEGylated liposomes were modified with high molecular weight hyaluronic acid and loaded with everolimus (PEG-LIP(ev)-HA400kDa). Liposomes were tested by in vitro experiments using LFs derived from broncholveolar lavage (BAL) of patients affected by CLAD and CTD-ILD, and on alveolar macrophages (AM) and lymphocytes isolated, respectively, from BAL and peripheral blood. PEG-LIP-HA400kDa demonstrated to be specific for LFs, but not for CD44-negative cells, and after loading everolimus, PEG-LIP(ev)-HA400kDa were able to arrest cell cycle arrest and to decrease phospho-mTOR level. PEG-LIP(ev)-HA400kDa showed anti-inflammatory effect on immune cells. This study opens the possibility to use everolimus in lung fibrotic diseases, demonstrating that our lipids-based vehicles can vehicle everolimus inside cells exerting the same drug molecular effect, not only in LFs, but also in immune cells.

Keywords: everolimus; hyaluronic acid; liposomes; lung diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
1H-NMR of DPPE, HA and HA-DPPE conjugates (left) and corresponding chemical structures (right). Methylene of DPPE group (black arrow). N-acetyl proton (methyl) of HA (orange arrow). Hydroxyl groups of HA (red arrow).
Figure 2
Figure 2
FTIR spectra of DPPE (green line), HA (blue line) and HA-DPPE (red line).
Figure 3
Figure 3
Variations of liposome size, PdI, and EE as a function of mg of everolimus added in the PEGylated liposome formulation.
Figure 4
Figure 4
Analysis of fluorescently labeled PEG-LIP and PEG-LIP-HA400kDa internalization in LFs derived from CLAD and ILD patients. (a,b) Flow cytometry analysis of (a) CLAD and (b) ILD LFs after 4 h of incubation with liposomes. Data are represented as mean ± SD. *, p < 0.05 vs. CTR; ***, p < 0.01 vs. CTR; #, p < 0.001 vs. LIP. (c,f) Representative confocal images of LFs CLAD incubated with (c) PEG-LIP or (d) PEG-LIP-HA400kDa or CTD-ILD LFs incubated with (e) PEG-LIP or (f) PEG-LIP-HA400kDa. Nuclei of cells = light blue (DAPI); liposomes = red signals. Scale bar = 100 μm.
Figure 5
Figure 5
Cell proliferation analysis by flow cytometry of LFs derived from (a) CLAD and (b) CTD-ILD treated with liposomes loaded with everolimus and everolimus alone (50 nM). Data are represented as mean ± SD of CFSE signals read by flow cytometer for each sample. ***, p < 0.001 vs. CTR; *, p < 0.05 vs. CTR; #, p < 0.001 vs. PEG-LIP(ev)-HA400kDa (One-way ANOVA followed by Dunnett post-hoc).
Figure 6
Figure 6
Cell cycle analysis of LFs derived from (a,c) CLAD and (b,d) CTD-ILD after incubation with liposomes and everolimus alone (50 nM) for (a,b) 24 h and (c,d) 48 h. Data are represented as mean of percentage of cells ± SD in each cell cycle phase. ***, p < 0.001 vs. CTR; *, p < 0.05 vs. CTR; ^, p < 0.05 vs. PEG-LIP(ev)-HA400kDa. (Two-way ANOVA followed by Tukey post-hoc).
Figure 7
Figure 7
Western blot analysis of mTOR and p-mTOR on LFs derived from (a) CLAD and (b) CTD-ILD after 24 h of treatment with liposomes and everolimus alone (50 nM). (a,b) Representative blot of immunedecoration using anti-mTOR, anti-p-mTOR, and β-actin. (c) Quantitative analysis of immunoblots representing the expression level of p-mTOR in CLAD and CTD-ILD normalized to CTR = 1.
Figure 8
Figure 8
Assessment of liposomes internalization in AM and CD3+ lymphocytes derived from CLAD patients. (a–c) Confocal microscopy analysis of Rhodamine-labeled (a) PEG-LIP and (c) PEG-LIP-HA400kDa internalized by AM after 1 h of incubation (liposomes = red signal; DAPI = light blue), with (b) quantification of mean fluorescence intensity of red signal present in three different ROI in (a) and (c). Data are represented as mean ± SD, ***, p < 0.001 vs. PEG-LIP. (d) Quantification of the interaction of Rhodamine-labeled PEG-LIP and PEG-LIP-HA400kDa with CD3+ lymphocytes after 1 h of incubation and analyzed by flow cytometry. Data are represented as mean ± SD. ***, p < 0.001 vs. CTR. Scale bar = 30 μm. ^, p < 0.05 vs. PEG-LIP(ev)-HA400kDa.
Figure 9
Figure 9
Effect on IL8 and TGF-β release by AM after 48 h of treatments (final concentration of everolimus = 50 nM). (a,b) IL8 quantification of AM derived from BAL of (a) CLAD or (b) CTD-ILD. (c,d) TGF-β quantification of AM derived from BAL of (c) CLAD or (d) CTD-ILD. Data have been normalized to control cells (100%) and are represented as mean ± SD. ***, p < 0.001 vs. CTR.
Figure 10
Figure 10
Evaluation of IFN-γ and IL17a release from CD3+ lymphocytes derived from peripheral blood of (a,b) CLAD and (c,d) CTD-ILD patients after treatment with PEG-LIP(ev), PEG-LIP(ev)-HA400kDa and Ev (50 nM). Data are represented as mean ± SD of number of spots normalized to CTR set at 100%. ***, p < 0.001 vs. CTR.
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