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Formulation and Delivery - Biomolecular

(T1130-07-37) Albumin Corona Improves Colloidal Stability of Nanosac, a Non-Cationic Polyphenol Gene Carrier

Tuesday, October 22, 2024
11:30 AM – 12:30 PM MT
Purpose: To avoid toxicity of cationic carriers and improve extrahepatic delivery of siRNA, we have developed a nanocapsule system called “Nanosac.” Nanosac is a soft, non-cationic nanocapsule encapsulating siRNA inside [1]. Nanosac does not depend on cationic charges to load nucleic acids, thus avoiding charge-associated toxicity. The softness of Nanosac is favorable for evading the reticuloendothelial system sequestration [2-5]. 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]. Given the effectiveness of Nanosac in vivo, it is likely that circulating Nanosac can reach solid tumors. However, it is also observed that Nanosac interacts with components in physiological fluids and undergoes aggregation. We improve the colloidal stability of Nanosac by coating the surface with albumin (pre-albuminylation). Additionally, we expect that the interaction of surface-bound albumin with 60-kDa sialoglycoprotein (gp60) on endothelial cells and secreted protein acidic and rich in cysteine (SPARC) of tumor cells as well as their extracellular matrices facilitate the transport of Nanosac to tumors. To test this, we evaluate transendothelial transport of the pre-albuminylated Nanosac and the silencing effect using PANC-1 pancreatic cancer cells and siRNA targeting k-ras, a mutant oncogene that triggers a cascade of events promoting tumor growth.

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 in these solutions were measured by dynamic light scattering. For prealbuminylation, Nanosac was incubated in albumin solutions overnight at room temperature. The prealbuminylated Nanosac was added to PBS, and the size was measured. For the evaluation of transendothelial transport of Nanosac, human umbilical vein endothelial cells (HUVEC) and human cerebral microvascular endothelial cells (hCMEC/D3) were seeded at a density of 80,000 cells per well in the Transwell insert with a polycarbonate membrane filter (3 µm pore), pre-coated with collagen-I. Trans-Epithelial Electrical Resistance (TEER) across the Transwell was monitored every day to confirm the integrity of cell layer throughout the assay. When the TEER value reached a plateau, 0.1 mg/mL of Cy3-labeled Nanosac or Cy3-labeled Nanosac/Alb (or Cy5-siRNA loaded Nanosac or Cy5-siRNA loaded Nanosac/Alb) was added to the apical 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, the fluorescence intensity of the medium in the basolateral side was measured to quantify the labeled Nanosac transported across the endothelial layer.

Results: Nanosac was prone to aggregation in the presence of electrolytes such as NaCl, KCl, or PBS, 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). This result suggests that albumin binding to Nanosac occurs prior to the surface charge neutralization. To reduce the risk of electrolyte-induced aggregation, Nanosac was prealbuminylated prior to the following cell studies. The effect of surface-bound albumin on Nanosac-cell interactions was examined using different cell models relevant to the delivery of circulating nanoparticles to solid tumors. HUVEC and hCMEC/D3 cells were chosen as endothelial cell models to examine the interactions with gp60 of peritumoral endothelium. HUVEC and hCMEC/D3 represent a gp60-expressing (gp60+) and a non-gp60-expressing (gp60-) cell line, respectively [6]. Pre-albuminylated Nanosac was transported across TNF-α-activated HUVEC better than the unmodified Nanosac. In contrast, there was no difference due to pre-albuminylation in resting HUVEC (no TNF-α challenge) or HCMEC/D3 cells (with or without TNF-α challenge) (Fig. 2). Since the prominent difference between HCMEC/D3 cells (or resting HUVEC) and TNF-α-stimulated HUVEC is the level of gp60 expression [6, 7], we speculate that the differential transendothelial transport is mediated by gp60.

Conclusion: Pre-exposure to albumin was critical to the colloidal stability of Nanosac. Prealbuminylation reduces the risk of Nanosac aggregation in serum solution and enhances the transport of Nanosac across the TNFα-activated endothelial cells.

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.
2. Liang, Q., et al., The softness of tumour-cell-derived microparticles regulates their drug-delivery efficiency. Nature biomedical engineering, 2019. 3(9): p. 729-740.
3. Anselmo, A.C. and S. Mitragotri, Impact of particle elasticity on particle-based drug delivery systems. Advanced drug delivery reviews, 2017. 108: p. 51-67.
4. Anselmo, A.C., et al., Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting. ACS nano, 2015. 9(3): p. 3169-3177.
5. Key, J., et al., Soft discoidal polymeric nanoconstructs resist macrophage uptake and enhance vascular targeting in tumors. ACS nano, 2015. 9(12): p. 11628-11641.
6. Hyun, H., et al., Surface modification of polymer nanoparticles with native albumin for enhancing drug delivery to solid tumors. Biomaterials, 2018. 180: p. 206-224.
7. Tao, H.-y., et al., The development of human serum albumin-based drugs and relevant fusion proteins for cancer therapy. International journal of biological macromolecules, 2021. 187: p. 24-34.

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 improves colloidal stability of Nanosac
A) Schematic summary of colloidal stability of Nanosac.
B) Effect of electrolyte on Nanosac.
Nanosac was prone to aggregation in the presence of electrolytes such as NaCl, KCl, or PBS, due to the neutralization of the negative charge of Nanosac.
C,D) Effect of pre-albuminylation on colloidal stability of Nanosac in PBS.
Pre-albuminylated Nanosac, especially in relatively concentrated albumin (10-80 mg/mL), was protected from electrolyte-induced aggregation in PBS.


Fig. 2.: Fold change in the transendothelial transport of Nanosac due to albumin modification
The fold change of Nanosac crossing HUVEC and HCMEC/D3 monolayer, resting or TNF-α-activated (n = 8 tests of a representative batch, mean ± s.d.). Nanosac or pre-albuminylated Nanosac (Alb-Nanosac) were passed through the endothelial monolayers for 6 h. Alb-Nanosac was transported across TNFα-activated HUVEC better than the unmodified Nanosac.