(W1130-08-48) Albumin Corona Improves Colloidal Stability of Nanosac: A Non-cationic Polyphenol Gene Carrier

Purpose: To avoid toxicity of cationic carriers and improve extrahepatic delivery of siRNA, we have developed a polyphenol-based nanocapsule system called Nanosac. Nanosac is negatively charged, thus avoiding positive charge-associated toxicity [1]. The softness of Nanosac is favorable for evading the reticuloendothelial system sequestration. With these unique features, Nanosac delivered siRNA targeting PD-L1 (siPD-L1) to tumors systemically, proving superior to the hard counterpart and anti-PD-L1 antibody in suppressing tumor growth via immune checkpoint blockade [1]. Another relevant feature is the tendency of Nanosac to co-opt albumin corona in serum. We hypothesize that albumin binding plays a critical role in preventing Nanosac aggregation in electrolyte solution and mediating interactions with peritumoral endothelial cells and tumor cells. To test this hypothesis, we coat Nanosac with albumin a priori (pre-albuminylate) and evaluate its colloidal stability, transendothelial transport, and interaction with SPARC-positive NCI/ADR-RES tumor cells.

Methods: Nanosac was produced by sequential coating of mesoporous silica nanoparticles (MSN) with siRNA and polydopamine (pD), followed by removal of the sacrificial MSN core by a dilute fluoride buffer. Fresh Nanosac was suspended in electrolyte solutions, albumin solution, or a solution of albumin and electrolyte mixture. Alternatively, Nanosac was mixed with albumin first and then with electrolytes. The size of Nanosac were measured by dynamic light scattering. For pre-albuminylation, Nanosac was incubated in albumin solutions overnight at room temperature and used as is. Transendothelial transport of Nanosac was evaluated with human umbilical vein endothelial cells (HUVEC) and human cerebral microvascular endothelial cells (hCMEC/D3) laid on the Transwell insert with 3 µm pores. 0.1 mg/mL of Cy3-labeled Nanosac or Alb-Nanosac (or Cy5-siRNA loaded Nanosac or Alb-Nanosac) was added to the donor side of the Transwell with a confluent layer of endothelial cells, either resting or activated by 10 ng/mL of TNF-α (simulating peritumoral endothelium). After 6 h, fluorescence intensity of the medium in the receptor side was measured to quantify the labeled Nanosac transported across the endothelial layer.To identify endothelial membrane proteins responsible for Alb-Nanosac transport, cell membrane fragments from lysed cells were isolated by differential centrifugation. Proteins upregulated due to TNF-α challenge are identified based on spectral counts in LC-MS/MS analysis. NCI/ADR-RES tumor cell uptake of Nanosac or Alb-Nanosac was visualized by confocal microscopy. Nanosacs were labeled with Alexa Fluor 594 (AF594), nuclei were stained with DAPI, and cell membranes were labeled with Wheat Germ Agglutinin-Alexa Fluor 488. For testing RNA delivery efficiency, siRNA targeting P-glycoprotein (P-gp) was encapsulated in Alb-Nanosac and applied to NCI/ADR-RES cells. P-gp expression was quantified by Western blotting. Lipid nanoparticles (LNPs) containing D-Lin-MC3-DMA (MC3-LNP) was used as a benchmark carrier.

Results: Bare Nanosac was prone to aggregation in the presence of electrolytes due to the neutralization of the negative charge of Nanosac (Fig.1). However, Nanosac was protected from electrolyte-induced aggregation in PBS in the presence of relatively concentrated albumin (10-80 mg/mL) (Fig.1C). This result suggests that albumin binding to Nanosac occurs prior to the surface charge neutralization. To ensure protective effect of albumin, we bathed Nanosac in albumin solution, making albumin-coated Nanosac (Alb-Nanosac). Pre-albuminylation did not affect the Nanosac structure (Fig.1D). We examined the effect of surface-bound albumin on Nanosac-cell interactions using cell models relevant to the delivery of circulating nanoparticles to solid tumors. We used HUVEC and hCMEC/D3 cells to examine the interactions with endothelial cells. We confirmed the integrity of the endothelial monolayer based on the Trans-Epithelial Electrical Resistance (TEER) value reaching the plateau (Fig.2B) and the limited transport of 70 kDa FITC-dextran across the cell layer (Fig.2C). Despite the larger size, Nanosac and Alb-Nanosac exhibited significantly higher transendothelial transport across HUVEC and hCMEC/D3 monolayers than FITC-dextran, indicating that its passage involved active transport mechanisms. Alb-Nanosac passed TNF-α-activated HUVEC better than bare Nanosac. In contrast, Alb-Nanosac showed no difference from bare Nanosac in resting (non-TNF-α challenged) HUVEC or hCMEC/D3 cells irrespective TNF-α challenge (Fig.2D).To identify proteins mediating the transport of Alb-Nanosac across the activated HUVEC, we analyzed protein expressed on HUVEC and hCMEC/D3 cells. TNFα-activated HUVECs showed upregulated expression of 99 proteins compared to resting HUVECs (Figs.2E and 2F). The expression of endothelial proteins responsible for albumin transport, such as SPARC, FcRN, and TGF-ꞵ, were more pronounced in HUVEC than in hCMEC/D3 cells, irrespective of TNFα stimulation (Fig.2G), and may also have contributed to Alb-Nanosac transport across HUVEC.We tested if Alb-Nanosac delivers siRNA to SPARC⁺ tumor cells, using siRNA that downregulates ABCB1 expression, thereby inhibiting P-gp. We chose NCI/ADR-RES cells, a multidrug-resistant cancer cell line, due to the overexpression of P-gp and SPARC. Confocal microscopy demonstrated cellular uptake of siRNA-loaded Nanosac (Fig.3A). Western blot analysis showed effective P-gp silencing in Alb-Nanosac-treated cells compared to controls (Figs.3B and 3C).

Conclusion: Albumin binding was critical to the colloidal stability of Nanosac. Pre-albuminylation enhanced the transport of Nanosac across the TNFα-activated endothelial cells. Pre-albuminylated Nanosac silenced P-gp expression in SPARC-expressing NCI/ADR cells to the extent comparable to LNP.

References: 1. Kim, H., et al., Nanosac, a noncationic and soft polyphenol nanocapsule, enables systemic delivery of siRNA to solid tumors. ACS nano, 2021. 15(3): p. 4576-4593.

Acknowledgements: We acknowledge the financial support from NIH R01 CA258737, Purdue Center for Cancer Research, and Purdue Institute for Drug Discovery. We also acknowledge the Fellowship support from the Egyptian Government Ministry of Higher Education Missions Sector to M.E.

Fig.1. Albumin corona improves colloidal stability of Nanosac.
A) Summary on colloidal stability of Nanosac
B) Nanosac colloidal stability. Effect of electrolyte on Nanosac.
C) Effect of pre-albuminylation on colloidal stability of Nanosac in PBS.
D) TEM images of Nanosac and Alb-Nanosac.



Fig.2. Transendothelial transport of Nanosac or Alb-Nanosac through endothelial cells.
A) Nanosac or Alb-Nanosac have no effect on the viability of HUVEC or hCMEC/D3.
B) TEER values of endothelial cells indicate the integrity of the confluent endothelial layer and the establishment of tight junctions.
C) The low transport of 70 kDa FITC-dextran across the endothelial cell layer ( <20%) further supports the continuity of endothelial layer.
D) Pre-albuminylation increased Nanosac transport across activated HUVEC but not hCMEC/D3.
E) Venn diagram highlighting proteins upregulated in TNFα-activated endothelial cells compared to resting cells.
F) Heatmap of Z-score normalized log2-transformed protein signal intensities from LC-MS/MS analysis of endothelial cell membranes (hCMEC/D3 and HUVEC) with and without TNFα stimulation. Each row represents a protein, and each column represents a biological replicate. Color intensity reflects the relative abundance of each protein across samples.
G) Endothelial proteins more expressed in activated HUVEC than hCMEC/D3 cells and responsible for albumin transport.

Fig.3. Alb-Nanosac-mediated siPgp delivery to NCI/ADR-RES cells
A) Uptake of AF594-*Nanosac and AF594-Alb-*Nanosac by NCI/ADR-RES cells.
B) Western blot analysis of P-gp expression in NCI/ADR cells. Representative blots showing the expression levels of P-gp in NCI/ADR cells treated with blank Alb-Nanosac, siP-gp@Alb-Nanosac, or siP-gp@MC3-LNP. GAPDH was used to confirm equal protein loading.
C)P-gp band intensity normalized to GAPDH.