(T1030-10-54) Development of Dual-Ligand Nanoparticles for Systemic Chemoimmunotherapy of Cancer

Purpose: The premise of delivering immune-activating agents is to overcome the immunosuppressive environment of tumor and help the immune system to effectively attack cancer cells. The objective of this study is to enable systemic delivery of immune-activating agents to tumors. Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death (ICD). The surface of NPs was decorated with quinic acid (QA) derivative, a synthetic mimic of sialyl Lewis-x, as well as adenosine triphosphate (ATP), a damage-associated molecular pattern. The underlying hypothesis is that the dual ligands will facilitate the delivery of NPs to solid tumors by systemic administration to activate antitumor immune activity. First, QA selectively interacts with the peritumoral endothelium via E-/P-selectins, thereby transiently increasing the endothelial permeability of NPs, which deliver PTX to induce ICD and generate tumor antigens. Second, ATP recruits antigen-presenting cells (APCs) to facilitate cancer immunotherapy. The purpose of study is to develop dual ligand NPs and evaluate the contribution of QA and ATP ligands to PTX delivery and immune cell activation.

Methods: PTX-loaded PLGA NPs were prepared with the single emulsion method. The NPs were then coated with polydopamine by incubation in dopamine solution at pH 8.5 for 4h. Polydopamine-coated PLGA NPs (NP_pD) was further incubated with QA and ATP to produce a dual ligand NP: i.e., QA/ATP-coated NP (NP_pD-QA/ATP). The NP_pD-QA/ATP was tested for the functionality of each ligand. The ability of QA to increase the endothelial transport of NPs was tested with a Transwell laid with TNF-α activated human umbilical vein endothelial cells (HUVEC) [YY1] [HAT2] [YY3]. The ability of ATP to attract dendritic cells was tested by quantifying dendritic cells migrating from the upper chamber of Transwell to the lower chamber in response to ATP or ATP-bound NPs. Cytotoxicity of PTX loaded in NP_pD-QA/ATP (PTX@NP_pD-QA/ATP) was tested with murine CT26 colorectal carcinoma cells. Tumor accumulation of NP_pD-QA/ATP was assessed with PTX and DiR as payloads in B16F10 tumor-bearing immune-competent C57BL/6 male mice.

Results: The z-average of PTX-loaded NP_pD-QA/ATP was 206 ± 7.2 nm. NP_pD-QA/ATP was as effective as free ATP in attracting monocytes and dendritic cells across a Transwell insert with a pore size of 8 µm. With confocal imaging of JAWSII cells, NP_pD-QA/ATP was as effective as NP_pD-ATP and free ATP in activating JAWSII cells as they showed rougher cell surfaces which was due to activated purinergic receptors which cause actin rearrangement and morphological change, consistent with the expected role of ATP. (Fig.1). Also, NP_pD-QA/ATP was as effective as NP_pD-QA in increasing transendothelial transport of NPs across TNF-α activated HUVEC layer. PTX@NP_pDQA/ATP was showed similar cytotoxicity to CT26 cells as free PTX. Tumor accumulation of NP_pD-QA/ATP was assessed in B16f10 tumor-bearing C57BL/6 mice with PTX as a payload at a dose equivalent to 20 mg/kg PTX, compared with NP_pD-ATP, NP_pD-QA, and Taxol. PTX@NP_pD-QA/ATP and PTX@NP_pD-QA significantly increased PTX accumulation in tumor, compared to Taxol and PTX@NP_pD-ATP (Fig 2 a). NP_pD-QA/ATP loaded with DiR were administered to B16f10 tumor-bearing C57BL/6 mice at a dose equivalent to 8 mg NPs per mouse and compared with NP_pD, NP_pD-ATP, and NP_pD-QA. Both DiR@NP_pD-QA/ATP and DiR@NP_pD-QA significantly increased DiR tumor accumulation compared to DiR@NP_pD and DiR@NP_pD-ATP (Fig. 2 b, c)

Conclusion: The z-average of PTX-loaded NP_pD-QA/ATP [YY1] [HAT2] was 206 ± 7.2 nm in diameter. NP_pD-QA/ATP was as effective as free ATP in attracting monocytes and dendritic cells across a Transwell insert with a pore size of 8 µm. JAWSII dendritic cells incubated with NP_pD-QA/ATP showed rough surfaces indicating the activation of purinergic receptors, which cause actin rearrangement and morphological change, consistent with the expected role of ATP (Fig.1). NP_pD-QA/ATP was as effective as NP_pD-QA in increasing the transendothelial transport of NPs across TNF-α activated HUVEC layer. PTX@NP_pD-QA/ATP showed similar cytotoxicity to CT26 cells as free PTX. Tumor accumulation of NP_pD-QA/ATP was assessed in B16F10 tumor-bearing C57BL/6 mice with PTX as a payload at a dose equivalent to 20 mg/kg PTX and compared with NP_pD-ATP, NP_pD-QA, and Taxol. PTX@NP_pD-QA/ATP and PTX@NP_pD-QA significantly increased PTX accumulation in tumor, compared to Taxol and PTX@NP_pD-ATP (Fig 2 a). NP_pD-QA/ATP loaded with DiR were administered to B16f10 tumor-bearing C57BL/6 mice at a dose equivalent to 8 mg NPs per mouse and compared with DiR-loaded NP_pD, NP_pD-ATP, and NP_pD-QA. Both DiR@NP_pD-QA/ATP and DiR@NP_pD-QA significantly increased DiR tumor accumulation compared to DiR@NP_pD and DiR@NP_pD-ATP (Fig. 2 b, c).

References: 1- Amoozgar, Z., et al. (2013). "Development of quinic acid-conjugated nanoparticles as a drug carrier to solid tumors. Amoozgar, Z., et al. (2013). "Development of quinic acid-conjugated nanoparticles as a drug carrier to solid tumors." Biomacromolecules 14(7): 2389-2395.
2- Xu, J., et al. (2018). "Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins." Small 14(50): e1803601.
3- Kwon, S., et al. (2024). "Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors." ACS Nano.

Acknowledgements: We appreciate the financial support from NIH R01 CA232419 and technical support of Shrinidhi Annadka, Dr. Daniel Flaherty, Kyle Timothy, Dr. Severin Schneebeli for the purification of QA-NH2

Fig.1. Free ATP, NPpD-ATP and NPpD-QA/ATP activate dendritic cells causing their morphology change.
Morphology of JAWSII cells untreated and treated with NPpD, ATP, NPpD-ATP, or NPpD-QA/ATP for 6h. NPpD-QA/ATP showed comparable activity to NPpD-ATP and free ATP in activating JAWSII cells.



Fig.2. NPpD-QA and NPpD-QA/ATP increase tumor accumulation of the payloads (PTX or DiR dye). (a) PTX content in B16F10 tumors at 24 h after a single intravenous injection of NPs. PTX equivalent to 20 mg/kg. (b) DiR content in B16F10 tumors at 24 h after a single intravenous injection of NPs. (c) Ex vivo imaging of B16F10 tumor at 24 h after single intravenous injection of DiR-loaded NPs.