(T1030-09-56) Cellular Uptake Mechanism of Hollow Polydopamine Nanoparticles, Nanosac

Purpose: Recently polydopamine nanoparticles or polydopamine-coated nanoparticles have been applied to various biological applications, such as cancer therapy, bioimaging, and tissue engineering. We have developed polydopamine nanocapsules for systemic delivery of siRNA, called Nanosac. Nanosac is prepared by coating of amine-modified mesoporous silica nanoparticles (MSN^a) with siRNA and polydopamine, followed by removal of the MSN core. Previously, the cellular uptake mechanism of Nanosac was tested using inhibitors of endocytosis pathways (chlorpromazine, methyl-β-cyclodextrin, and amiloride hydrochloride). Cellular uptake of Nanosac was hindered by methyl-β-cyclodextrin, suggesting Nanosac was internalized via caveolae-mediated endocytosis. To verify the mechanism of Nanosac entry to cancer cells, endocytosed Nanosac was visualized by confocal microscopy and compared with other nanoparticles in the cells.

Methods: 2x10⁴ B16F10 cells were seeded in a 30 mm³ confocal dish for 24 hours. The medium was replaced with a suspension of Cy3-siRNA-containing nanoparticles (Lipofectamine 2000, MSN^a, and Nanosac) and incubated for 4 hours at 37°C. The cells were rinsed with PBS twice and incubated with LysoTracker Red DND-99 for 1 hour. After rinsing twice with PBS, cells were fixed in 4% paraformaldehyde for 10 mins. The caveolin-1 receptor was stained with caveolin-1 antibody (Alexa 488), and the cell nucleus was stained with DAPI. Confocal microscopy was performed by the Nikon A1R confocal microscope (Melville, NT, USA).

Results: The signal of Cy3-siRNA containing Lipofectamine 2000 (Cy3-siRNA@L2K) was colocalized with LysoTracker and Caveolin-1 signals, indicating that Cy3-siRNA@L2K was internalized via both caveolae and lysosome. The majority of Cy3-siRNA loaded MSN (Cy3-siRNA@MSN^a) signal was colocalized with LysoTracker, suggesting that the endocytosed Cy3-siRNA@MSN^a was transported to the lysosomes. In contrast, Cy3-siRNA@Nanosac signal colocalized more with Caveolin-1 than lysotracker, which indicates Nanosac entered the cells through caveolae. The polydopamine surface of Nanosac recruits albumin, which can interact with cancer cells. The caveolae-mediated endocytosis of Nanosac may be explained by the surface-bound albumin entering the cells through caveolae.

Conclusion: Nanosac showed a different cellular uptake pattern than bare MSN^a without polydopamine coating: siRNA delivered by Nanosac mainly appeared in the caveolae, whereas those delivered by bare MSN^a or L2K. Serum albumin recruited by the polydopamine surface of Nanosac may be responsible for the difference in cellular uptake.

References: Kim, H., et al. (2021). Nanosac, a Noncationic and Soft Polyphenol Nanocapsule, Enables Systemic Delivery of siRNA to Solid Tumors. ACS Nano 15(3): 4576-4593.
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Chatterjee, M.; Ben-Josef, E.; Robb, R.; Vedaie, M.; Seum, S.; Thirumoorthy, K.; Palanichamy, K.; Harbrecht, M.; Chakravarti, A.; Williams, T. M. Caveolae-Mediated Endocytosis Is Critical for Albumin Cellular Uptake and Response to Albumin-Bound Chemotherapy. Cancer Res. 2017, 77 (21), 5925.

Acknowledgements: This work was supported by the National Institutes of HealthR01 CA232419, R01 CA199663, and R01 CA258737), National Science Foundation (CBET-1705560), Indiana Clinical and Translational, and funded in part by grant National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.

Figure 1. Schematic illustration (a) Schematics diagram of the preparation of fluorescence-labeled Nanosac. MSN: mesoporous silica nanoparticles; MSNa: MSN conjugated with (3-Aminopropyl) triethoxysilane (APTES); MSNa /siRNA: MSNa with siRNA loaded on the surface; MSNa /siRNA/pD: MSNa /siRNA covered with pD; and O/siRNA/pD (Nanosac): siRNA-loaded nanocapsules after MSN core removal. (B) Schematic of Cy5-labeled MSNa synthesis.

Figure 2. Characterization of the fluorescence-labeled Nanosac. (a) The ζ potential / z-average and (b) TEM images of Cy3-siRNA-loaded or Cy5-labeled Nanosac. (c) Confocal images of fluorescence-labeled Nanosac, scale bar; 10 µm.

Figure 3. (a) Confocal microscopic images of Cy3-siRNA@L2K, Cy3-siRNA@MSNa, and Cy3-siRNA@Nanosac in B16F10 cells. Scale bar: 20 µm. (b) Magnified images to visualize the colocalization of Cy3-siRNA and caveolin-1 antibody (Alexa 488). Red: Cy3-siRNA; Green: Cavelin-1 antibody; and Blue: nuclei. Scale bar: 10 µm.