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. 2024 Dec;36(50):e2404605.
doi: 10.1002/adma.202404605. Epub 2024 Oct 30.

Engineering Radiocatalytic Nanoliposomes with Hydrophobic Gold Nanoclusters for Radiotherapy Enhancement

Affiliations

Engineering Radiocatalytic Nanoliposomes with Hydrophobic Gold Nanoclusters for Radiotherapy Enhancement

Nazareth Milagros Carigga Gutierrez et al. Adv Mater. 2024 Dec.

Abstract

Chemoradiation therapy is on the forefront of pancreatic cancer care, and there is a continued effort to improve its safety and efficacy. Liposomes are widely used to improve chemotherapy safety, and may accurately deliver high-Z element- radiocatalytic nanomaterials to cancer tissues. In this study, the interaction between X-rays and long-circulating nanoliposome formulations loaded with gold nanoclusters is explored in the context of oxaliplatin chemotherapy for desmoplastic pancreatic cancer. Hydrophobic gold nanoclusters stabilized with dodecanethiol (AuDDT) are efficiently incorporated in nanoliposomal bilayers. AuDDT-nanoliposomes significantly augmented radiation-induced OH production, which is most effective with monochromatic X-rays at energies that exceed the K-shell electron binding energy of Au (81.7 keV). Cargo release assays reveal that AuDDT-nanoliposomes can permeabilize lipid bilayers in an X-ray dose- and formulation-dependent manner. The radiocatalytic effect of AuDDT-nanoliposomes significantly augments radiotherapy and oxaliplatin-chemoradiotherapy outcomes in 3D pancreatic microtumors. The PEGylated AuDDT-nanoliposomes display high tumor accumulation in an orthotopic mouse model of pancreatic cancer, showing promise for nanoliposomes as carriers for radiocatalytic nanomaterials. Altogether, compelling proof for chemo-radiation dose-enhancement using AuDDT-nanoliposomes is presented. Further improving the nanoliposomal loading of high-Z elements will advance the safety, efficacy, and translatability of such chemoradiation dose-enhancement approaches.

Keywords: 3D culture models; chemoradiotherapy; lipid nanotechnology; monochromatic synchrotron radiation; oxaliplatin; pancreatic cancer; radiation therapy; radiotherapy‐controlled drug delivery; tumor permeability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of AuDDT‐OXILs. A) AuDDT morphology imaged by TEM microscopy and B) hydrodynamic diameters measured by dynamic light‐scattering. C) Absorption (black line) and fluorescence emission spectra of AuDDT (100 µm Au in CHCl3). D) AuDDT‐NLs morphology imaged by cryo‐TEM and E) hydrodynamic diameter measured by dynamic light scattering spectroscopy. F) Absorption (black line) and fluorescence emission spectra of AuDDT‐NLs (100 µm Au) in PBS. G) Size and polydispersity of AuDDT‐NLs at different Au: lipid ratios. H) Semi‐quantitative encapsulation efficiencies (EE%) of AuDDT at varying molar ratios in NLs (box whisker plots depict the mean, 25th and 75th percentile, and the 95% CI). I) Effect of AuDDT on NLs ζ‐potentials. All data was obtained from N ≥ 9 from ≥3 technical repeats.
Figure 2
Figure 2
Cryo‐electron microscopy analysis of AuDDT‐NLs reveals single nanoclusters and aggregate formation within lipid bilayers. Depicted images were obtained of empty NLs (A), NLs containing 0.5 moL% AuDDT (B), or NLs containing 2 moL% AuDDT (C). Four types of liposomes were observed, containing either no nanoclusters (D), NLs with a single AuDDT that pushed apart the inner and outer membrane leaflet (E), NLs containing a single AuDDT or aggregated AuDDT that caused lipid bilayer fusion (F), and, G) NLs containing aggregates of nanoclusters between the membrane leaflets.
Figure 3
Figure 3
X‐ray interactions with Au increases the generation of secondary electrons and elevates hydroxyl radical formation. A) The X‐ray mass energy absorption coefficient was plotted as a function of X‐ray energy for Au and soft tissue. Simulated secondary electron spectra obtained when 79.7 keV X‐rays (B) and 81.7 keV X‐rays (C) interact with Au. D) Ratio of the mass energy absorption coefficients of Au and soft tissue, plotted as a function of the X‐ray energy. Indicated in gray are the X‐ray energies selected for the APF oxidation assays. Simulated secondary electron spectra obtained when 79.7 keV X‐rays (E) and 81.7 keV X‐rays (F) interact with water. G) APF oxidation by monochromatic synchrotron radiation in the presence of NL containing only BPD (BPD‐NLs, dark green, control), or AuDDT‐BPD‐NL (red). Data was analyzed using a One‐way ANOVA and Sidak's multiple comparisons test. APF oxidation as a function of X‐ray dose in the presence of BPD‐NLs (green), AuDDT‐NLs (gold), and AuDDT‐BPD‐NLs (red), under X‐ray energy of either 79.7 keV (H) or 81.7 keV (I). Data was fitted using an agonist versus response (three‐parameter) fit.
Figure 4
Figure 4
Cargo‐release from AuDDT‐BPD‐OXILs occurs in a formulation‐dependent manner. A) X‐ray‐triggered cargo release from AuDDT‐BPD‐NLs composed of DOPC: DSPE‐PEG (98:2 mol%) upon irradiation with 81.7 keV monochromatic synchrotron radiation. Data represents N = 9 from three technical repeats. B) X‐ray‐triggered cargo release from AuDDT(‐BPD)‐OXILs composed of increasing DOPE content (0–48 mol% at the expense of DSPC (48–0 mol%), and further supplemented with cholesterol (48 mol%) and DSPE‐PEG (4 mol%). Cargo release was induced upon irradiation with 81.7 keV monochromatic synchrotron radiation. Data represents N = 12–20 from four technical repeats. C) X‐ray‐triggered cargo release from AuDDT(‐BPD)‐OXILs composed of increasing DLinPE content (0–48 mol% at the expense of DSPC (48–0 mol%), and further supplemented with cholesterol (48 mol%) and DSPE‐PEG (4 mol%). Cargo release was induced upon irradiation with 81.7 keV monochromatic synchrotron radiation. Data represents N = 8–10 from three technical repeats. All data was fitted using agonist versus normalized response fits, and curves were statistically compared using an extra sum‐of‐squares F‐test.
Figure 5
Figure 5
The radiocatalytic effects of AuDDT‐BPD‐OXILs enhance radiotherapy outcomes on pancreatic microtumors composed of MIA PaCa‐2 and HPSC cells. A) Representative brightfield images and viability heatmaps of pancreatic microtumors following exposure to monochromatic synchrotron radiation (81.7 keV) in the presence/absence of oxaliplatin (scalebar = 500 µm). B,C) The efficacy of radiotherapy in the absence (black) and presence of AuDDT‐BPD‐OXILs (blue) based on microtumor viability (B) and microtumor size (C). D,E) Radiosensitization by AuDDT‐BPD‐OXILs was confirmed by viability assessment (D) and microtumor size quantification (E) following monochromatic X‐rays tuned below (79.7 keV) and above (81.7 keV) the K‐edge of Au (80.7 keV). F–H) Integrated comparison of microtumor size and viability of following chemoradiotherapy at 0 Gy (F), 4 Gy (G), and 16 Gy (H), given with 81.7 keV X‐rays. Treatment groups were radiotherapy alone (black), AuDDT‐BPD‐OXILs + radiotherapy (blue), oxaliplatin + radiotherapy (red), and AuDD‐BPD‐OXILs + oxaliplatin + radiotherapy. All box‐whisker plots depict the mean, 25th, and 75th percentile, and the 95% confidence interval from N = 8. Data passed normality tests (Pearson‐Omnibus test), and statistical analyses were performed using a One‐way ANOVA and Sidak's post‐hoc test for multiple comparisons (asterisks), or using a student's t‐tests between two defined groups (hashtags).
Figure 6
Figure 6
AuDDT‐OXILs improve oxaliplatin‐chemoradiotherapy outcomes in MIA PaCa‐2+HPSC pancreatic microtumors. A) Mean calcein emission from MIA PaCa‐2+HPSC microtumors following radiotherapy. B) Cross sections of MIA PaCa‐2+HPSC microtumors, displaying the calcein fluorescence emission, after 0 Gy (B) and 4 Gy (C) monochromatic irradiation (81.7 keV), acquired using confocal fluorescence microscopy (22.5 µm focal plane). Scalebar = 500 µm. C) Mean calcein emission from PANC‐1+HPSC microtumors following radiotherapy. D) Cross sections of PANC‐1+HPSC microtumors, displaying the calcein fluorescence emission, after 0 Gy (B), 4 Gy (C), and 16 Gy (D) radiotherapy delivered using monochromatic X‐rays (81.7 keV), acquired using confocal fluorescence microscopy (22.5 µm focal plane). Scalebar = 250 µm. Data passed normality tests (panel A: Kolmogorov–Smirnov test, Panel C: Pearson–Omnibus test), and statistical analyses were performed using a One‐way ANOVA and Sidak's post‐hoc test for multiple comparisons.
Figure 7
Figure 7
Differential biodistribution of lipid‐anchored BPD and AuDDT when administered as AuDDT‐BPD‐OXILs. A) Side views of a representative mouse that received AUDDT‐BPD‐OXILs via i.v. injection, obtained by the whole body in vivo fluorescence imaging at different timepoints. B) The accumulation of AuDDT‐BPD‐OXILs in cancer tissues was imaged using 3D fluorescence tomography zoomed into the area containing the spleen, pancreas, and tumor. C) Distribution of OXILS based on the fluorescence of BPD‐PC determined at 5 h post‐administration (dark red) or 24 h post‐administration (bright red). D) Distribution of AuDDT determined by SWIR fluorescence imaging at 5 h post‐administration (dark blue) or 24 h post‐administration (light blue). Data from panels C and D are from N = 3 per timepoint, and statistical analysis was performed with a One‐way ANOVA and Sidak's multiple comparison's test (asterisks) or a student's t‐test (hashtags).

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