(T1130-08-54) Quinic Acid and ATP-Dual Ligand Nanoparticles for Systemic Cancer Chemoimmunotherapy

Purpose: Chemoimmunotherapy combines chemotherapy inducing immunogenic cell death (ICD) and immunostimulants activating immune cells in tumors. We previously identified two ligands that can help systemic delivery of chemotherapy and immunotherapy, respectively. First, quinic acid (QA) derivative (QA-NH₂), a synthetic mimic of sialyl Lewis-x, selectively interacts with the peritumoral endothelium via E-/P-selectins, thereby increasing the endothelial transport of drug carriers¹. Second, adenosine triphosphate (ATP), a damage-associated molecular pattern, recruits antigen-presenting cells (APCs) to the treated tumors². Using polydopamine-based surface modification, we decorate PTX-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) with QA-NH₂ and ATP. We hypothesize that dual ligands will facilitate NP delivery to solid tumors by systemic administration and activation of antitumor immune responses. We evaluate the contribution of QA and ATP ligands to PTX delivery and immune cell activation and their translation to antitumor activity.

Methods: PTX-loaded PLGA NPs were prepared by the single emulsion method and coated with polydopamine by incubation in dopamine solution at pH 8.5 for 4 h. Polydopamine-coated PLGA NPs (NP_pD) was further incubated with QA-NH₂ 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. QA activity was evaluated based on the NP transport across TNF-α-activated human umbilical vein endothelial cells (HUVEC) laid on a Transwell. ATP activity was evaluated based on the ability of ATP-bound NPs to activate JAWSII dendritic cells with and without apyrase. Tumor accumulation of NP_pD-QA/ATP was assessed in subcutaneous B16F10 tumor-bearing C57BL/6 male mice with PTX and DiR as payloads. Antitumor efficacy of PTX@NP_pD-QA/ATP was evaluated in B16F10 tumor-bearing mice by intravenous injection at a dose equivalent to PTX 20 mg/kg, administered every 3 days for a total of 4 doses.

Results: NP_pD-QA/ATP was as effective as free ATP or NP_pD-ATP in activating JAWSII dendritic cells, which manifested as rough cell surfaces (Fig.1 a). To evaluate the ability of NPs to protect ATP from enzymatic degradation, we exposed JAWSII dendritic cells to NP_pD-QA/ATP, NP_pD-ATP, or free ATP for 6 h in the presence of potato apyrase, a recombinant enzyme that inactivates ATP. Both NP_pD-QA/ATP and NP_pD-ATP were significantly more resistant to apyrase than free ATP (Fig. 1 b). NP_pD-QA/ATP was as effective as NP_pD-QA in crossing TNF-α activated HUVEC layer, compared to NP_pD. These results indicate that NP_pD-QA/ATP have functions of both ATP and QA. We assessed tumor accumulation of NP_pD-QA/ATP 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_p®D-ATP (Fig. 2 b, c). These results indicate that QA ligand improved the delivery of NPs and its payload to tumors. PTX@NP_pD -QA/ATP showed greater antitumor efficacy and the survival rate in the B16F10 model than Taxol^®, PTX@NP_pD-QA, PTX@NP_pD -ATP, and PTX@NP_pD at dose equivalent to 20 mg/kg PTX given every 3 days for a total of 4 doses (Fig. 3a). This result shows that dual modification of NPs with QA ligand and ATP can enhance the antitumor activity of PTX significantly. PTX@NP_pD-QA/ATP NPs also outperformed a mixture of (PTX@ NP_pD-QA and PTX@ NP_pD -ATP) or a clinical benchmark of chemoimmunotherapy (Abraxane^® co-administered with anti-PD-1 antibody)³ (Fig. 3b). These results indicate that co-presence of QA and ATP ligands on the same NPs is critical to their cooperative functions – QA to increase PTX delivery for ICD induction and ATP to recruit antigen presenting cells to tumor antigens generated by ICD.

Conclusion: Dual-ligand nanoparticles (NP_pD-QA/ATP) recruited dendritic cells and enhanced trans-endothelial transport in vitro to a level comparable with NPs containing each ligand individually. Additionally, NP_pD-QA/ATP, similar to NP_pD-QA, significantly improved the delivery of payloads (PTX or DiR) to B16F10 tumors compared to control groups without QA. PTX@ NP_pD-QA/ATP improved antitumor efficacy in B16F10 tumor model compared to Taxol^®, PTX@ NP_pD, PTX@ NP_pD-QA and PTX@ NP_pD-ATP, a mixture of PTX@ NP_pD-QA and PTX@ NP_pD-ATP, or Abraxane^® administered with anti PD-1 antibody. These results show that simple modification of NP surface with two functional ligands (QA and ATP) improves the efficacy of chemotherapy far beyond benchmark treatments, such as Taxol or the combination of Abraxane and anti-PD-1 antibodies, due to the improved drug delivery and likely antitumor immune activation. <

References: 1- Xu, J., et al. (2018). "Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins." Small 14(50): e1803601.
2- Kwon, S., et al. (2024). "Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors." ACS Nano. 18, 3681−3698
3- Li, J. J. et al. (2022). "Efficacy and safety of anti-PD-1 inhibitor combined with nab-paclitaxel in Chinese patients with refractory melanoma". J Cancer Res Clin Oncol 148(5): 1159-1169.

Acknowledgements: This work was supported by the National Institutes of Health (R01 CA232419, R01 CA258737).

Fig.1. ATP-modified NPs (NPpD-ATP and NPpD-QA/ATP) activate dendritic cells. (a) Free ATP, NPpD-ATP and NPpD-QA/ATP activate JAWSII dendritic cells causing their morphology change. NPpD-QA/ATP showed comparable activity to NPpD-ATP and free ATP in activating JAWSII cells. (b) Morphology of JAWSII dendritic cells treated with free ATP, NPpD-ATP and NPpD-QA/ATP in the presence of apyrase (ATP degrading enzyme) for 6 h. (c) Graph shows perimeters of cells, an indication of dendritic cell activation.

Fig.2. QA-modified NPs (NPpD-QA and NPpD-QA/ATP) increase tumor accumulation of payloads (PTX or DiR dye). (a) PTX content in B16F10 tumors at 24 h after a single intravenous injection of NPs (Unmodified NPs, NPpD-ATP, NPpD-QA, or NPpD-QA/ATP) delivering PTX 20 mg/kg. (b) DiR content in B16F10 tumors at 24 h after a single intravenous injection of DiR-loaded NPs. (c) Ex vivo imaging of B16F10 tumor at 24 h after single intravenous injection of DiR-loaded NPs. % ID: % of the injected dose.

Fig.3. Dual modified NPs (PTX@ NPpD-QA/ATP) improve antitumor efficacy of PTX against B16f10 melanoma. (a) PTX@ NPpD-QA/ATP compared with Taxol®, PTX@ NPpD-QA, PTX@ NPpD -ATP and PTX@ NPpD, (b) PTX@ NPpD-QA/ATP compared with a mixture of PTX@ NPpD-QA and PTX@ NPpD -ATP or a combination of Abraxane and anti-PD1 antibody (PD-1). All groups (except for PBS) received treatments equivalent to PTX 20 mg/kg/dose. Tumor specific growth rate= ln(V2/V1) / (t2 - t1), where V1 and V2 are tumor volumes at time t1 and t2, respectively. PTX@ NPpD-Q/T: PTX@ NPpD-QA/ATP. PTX@NP Mix: mixture of PTX@ NPpD-QA and PTX@ NPpD -ATP.