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Nanoparticle accumulation and penetration in 3D tumor models: the effect of size, shape, and surface charge

Cybulski, Pierre, Bravo, Maria, Chen, Jim Jui-Kai, Van Zundert, Indra, Krzyzowska, Sandra, Taemaitree, Farsai, Uji-i, Hiroshi, Hofkens, Johan, Rocha, Susana, Fortuni, Beatrice·2025·Frontiers in Cell and Developmental Biology
DOI 10.3389/fcell.2024.1520078PMC PMC11802510

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Abstract

Preclinical studies have demonstrated that nanoparticles (NPs) hold significant potential for advancing cancer therapy by enhancing therapeutic efficacy while reducing side effects. Their effectiveness in solid tumors is, however, often constrained by insufficient accumulation and penetration. Understanding how the physicochemical properties of NPs – such as size, shape, and surface charge – influence their interaction with cells within the tumor is critical for optimizing NP design. In this study, we addressed the challenge of inconsistent NP behavior by systematically evaluating NP uptake in both 2D and 3D tumor models, and NP penetration in spheroids. Our results showed that larger NPs exhibited higher internalization rates in 2D models but limited penetration in 3D spheroids. Furthermore, negatively charged NPs consistently achieved superior accumulation and deeper penetration than neutral and positively charged NPs. Spherical NPs outperformed rod-shaped NPs in tumor accumulation and penetration. These findings underscore the importance of carefully tailoring NP properties to the complex tumor microenvironment for improved therapeutic outcomes in real tumors.

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FIGURE 1 Nanoparticle characterization. Electron microscopy images of (A) AuNS10, (B) AuNS30, (C) AuNS50, (D) AuNS65, (E) AuNR(−); since no differences were observed between the AuNR samples in EM images, AuNR(−) is shown as a representative, while AuNRØ and AuNR(+) are displayed in Supplementary Figure S1A, B. Scale bars: 100 nm. (F) AuNSs size distribution based on electron microscopy images. (G) Zeta potential of AuNR(−), AuNRØ, and AuNR(+). (H) Extinction spectra in water. (I) Summary table with values shown as mean ± standard deviation.
FIGURE 1 Nanoparticle characterization. Electron microscopy images of (A) AuNS10, (B) AuNS30, (C) AuNS50, (D) AuNS65, (E) AuNR(−); since no differences were observed between the AuNR samples in EM images, AuNR(−) is shown as a representative, while AuNRØ and AuNR(+) are displayed in Supplementary Figure S1A, B. Scale bars: 100 nm. (F) AuNSs size distribution based on electron microscopy images. (G) Zeta potential of AuNR(−), AuNRØ, and AuNR(+). (H) Extinction spectra in water. (I) Summary table with values shown as mean ± standard deviation.CC BY · PubMed Central
FIGURE 2 The effect of NP size on cellular uptake and spheroid penetration. Representative confocal fluorescence images of AuNS10, AuNS30, AuNS50, and AuNS65 in (A-D) 2D monolayers and (E-P) 3D spheroids of A549 cells. Cells were incubated with ∼8×101⁰ NPs/mL for 3 h or ∼8×109 NPs/mL for 24 h, for 2D or 3D models, respectively. NP PL is shown in green while cellular staining appears in magenta (CellMask™ Deep Red plasma membrane staining for 2D models and Phalloidin CruzFluor™ 647 for cytoskeleton staining in 3D models). Scale bars: 50 µm. (A-D) xy images and orthogonal section of 2D monolayers. (E-H) Maximal intensity z-projection images of A549 spheroids (I-L), xy images and orthogonal section of 3D spheroids. (M-P) Zoomed-in xy images of the top left ¼ area of the spheroids shown in (I–L) with white arrows indicating NPs. Additional images are provided in Supplementary Figure S2 (for 2D models) and Supplementary Figure S3 (for 3D models). (Q) Normalized mean PL intensity of NPs versus spheroid depth. Curves for individual spheroids are shown in Supplementary Figure S4.
FIGURE 2 The effect of NP size on cellular uptake and spheroid penetration. Representative confocal fluorescence images of AuNS10, AuNS30, AuNS50, and AuNS65 in (A-D) 2D monolayers and (E-P) 3D spheroids of A549 cells. Cells were incubated with ∼8×101⁰ NPs/mL for 3 h or ∼8×109 NPs/mL for 24 h, for 2D or 3D models, respectively. NP PL is shown in green while cellular staining appears in magenta (CellMask™ Deep Red plasma membrane staining for 2D models and Phalloidin CruzFluor™ 647 for cytoskeleton staining in 3D models). Scale bars: 50 µm. (A-D) xy images and orthogonal section of 2D monolayers. (E-H) Maximal intensity z-projection images of A549 spheroids (I-L), xy images and orthogonal section of 3D spheroids. (M-P) Zoomed-in xy images of the top left ¼ area of the spheroids shown in (I–L) with white arrows indicating NPs. Additional images are provided in Supplementary Figure S2 (for 2D models) and Supplementary Figure S3 (for 3D models). (Q) Normalized mean PL intensity of NPs versus spheroid depth. Curves for individual spheroids are shown in Supplementary Figure S4.CC BY · PubMed Central
FIGURE 3 The effect of NP charge on cellular uptake and spheroid penetration. Representative confocal fluorescence images of AuNR(−), AuNRØ, and AuNR(+) in (A-C) 2D monolayers and (D-L) 3D spheroids of A549 cells. Cells were incubated with ∼4×1011 NPs/mL for 3 h or 24 h, for 2D or 3D models, respectively. NP PL is shown in green while cellular staining appears in magenta (CellMask™ Green plasma membrane staining for 2D models and Phalloidin CruzFluor™ 488 for cytoskeleton staining in 3D models). Scale bars: 50 µm. (A-C) xy images and orthogonal section of 2D monolayers. (D-F) Maximal intensity z-projection images of A549 spheroids (G-I) xy images and orthogonal section of 3D spheroids. The intensity range is adjusted for each image to highlight NP penetration. Images with the same intensity range are shown in Supplementary Figure S7. (J-L) Zoomed-in xy images of the top left ¼ area of the spheroids reported in (G–I). Additional images are provided in Supplementary Figure S5 (for 2D models) and Supplementary Figure S6 (for 3D models). (M) Normalized mean PL intensity of NPs versus spheroid depth. Curves for individual spheroids are shown in Supplementary Figure S7.
FIGURE 3 The effect of NP charge on cellular uptake and spheroid penetration. Representative confocal fluorescence images of AuNR(−), AuNRØ, and AuNR(+) in (A-C) 2D monolayers and (D-L) 3D spheroids of A549 cells. Cells were incubated with ∼4×1011 NPs/mL for 3 h or 24 h, for 2D or 3D models, respectively. NP PL is shown in green while cellular staining appears in magenta (CellMask™ Green plasma membrane staining for 2D models and Phalloidin CruzFluor™ 488 for cytoskeleton staining in 3D models). Scale bars: 50 µm. (A-C) xy images and orthogonal section of 2D monolayers. (D-F) Maximal intensity z-projection images of A549 spheroids (G-I) xy images and orthogonal section of 3D spheroids. The intensity range is adjusted for each image to highlight NP penetration. Images with the same intensity range are shown in Supplementary Figure S7. (J-L) Zoomed-in xy images of the top left ¼ area of the spheroids reported in (G–I). Additional images are provided in Supplementary Figure S5 (for 2D models) and Supplementary Figure S6 (for 3D models). (M) Normalized mean PL intensity of NPs versus spheroid depth. Curves for individual spheroids are shown in Supplementary Figure S7.CC BY · PubMed Central
FIGURE 4 The effect of NP shape on cellular uptake and spheroid penetration. Representative confocal fluorescence images of AuNS10, AuNS50, and AuNR(−) in (A-C) 2D monolayers and (D-L) 3D spheroids of A549. Cells were incubated with ∼8×101⁰ NPs/mL for 3 h or ∼5×101⁰ NPs/mL for 24 h, for 2D or 3D models, respectively. NP PL is shown in green while cellular staining appears in magenta (CellMask™ Deep Red and Green plasma membrane staining for 2D models incubated with AuNSs and AuNRs, respectively, and Phalloidin CruzFluor™ 647 and 488 for cytoskeleton staining in 3D models incubated with AuNSs and AuNRs, respectively). Scale bars: 50 µm. (A-C) xy images and orthogonal section of 2D monolayers. (D-F) Maximal intensity z-projection images of A549 spheroids (G-I) xy images and orthogonal section of 3D spheroids. The intensity range is adjusted for each image to highlight NP penetration. Images with the same intensity range are shown in Supplementary Figure S11. (J-L) Zoomed-in xy images of the top left ¼ area of the spheroids reported in (G–I). Additional images are provided in Supplementary Figure S9 (for 2D models) and Supplementary Figure S10 (for 3D models). (M) Normalized mean PL intensity of NPs versus spheroid depth. Curves for individual spheroids are shown in Supplementary Figure S12.
FIGURE 4 The effect of NP shape on cellular uptake and spheroid penetration. Representative confocal fluorescence images of AuNS10, AuNS50, and AuNR(−) in (A-C) 2D monolayers and (D-L) 3D spheroids of A549. Cells were incubated with ∼8×101⁰ NPs/mL for 3 h or ∼5×101⁰ NPs/mL for 24 h, for 2D or 3D models, respectively. NP PL is shown in green while cellular staining appears in magenta (CellMask™ Deep Red and Green plasma membrane staining for 2D models incubated with AuNSs and AuNRs, respectively, and Phalloidin CruzFluor™ 647 and 488 for cytoskeleton staining in 3D models incubated with AuNSs and AuNRs, respectively). Scale bars: 50 µm. (A-C) xy images and orthogonal section of 2D monolayers. (D-F) Maximal intensity z-projection images of A549 spheroids (G-I) xy images and orthogonal section of 3D spheroids. The intensity range is adjusted for each image to highlight NP penetration. Images with the same intensity range are shown in Supplementary Figure S11. (J-L) Zoomed-in xy images of the top left ¼ area of the spheroids reported in (G–I). Additional images are provided in Supplementary Figure S9 (for 2D models) and Supplementary Figure S10 (for 3D models). (M) Normalized mean PL intensity of NPs versus spheroid depth. Curves for individual spheroids are shown in Supplementary Figure S12.CC BY · PubMed Central

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Metadata

Authors
Cybulski, P., Bravo, M., Chen, J. J.-K., Van Zundert, I., Krzyzowska, S., Taemaitree, F., Uji-i, H., Hofkens, J., Rocha, S., & Fortuni, B.
Venue
Frontiers in Cell and Developmental Biology
Publisher
Frontiers Media SA
Published
2025-01-24
Volume
12
ISSN
2296-634X
DOI
10.3389/fcell.2024.1520078
PMC
PMC11802510
Cited by
51
References
4
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • H2020 Marie Skłodowska-Curie Actions (860914)
  • Fonds Wetenschappelijk Onderzoek (G0D4519N G081916N VS08523N G0C1821N G022724N 12X1419N 12X1423N 12A6N25N)
  • KU Leuven (IDN/20/021 C14/15/053 C14/19/079 C14/22/085 GPUM/21/025)

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Cybulski, P., Bravo, M., Chen, J. J.-K., Van Zundert, I., Krzyzowska, S., Taemaitree, F., Uji-i, H., Hofkens, J., Rocha, S., & Fortuni, B. (2025). Nanoparticle accumulation and penetration in 3D tumor models: the effect of size, shape, and surface charge. Frontiers in Cell and Developmental Biology. https://doi.org/10.3389/fcell.2024.1520078