(T1130-03-17) Transcytosis Mediated Deep Tumor Penetration for Enhanced Chemotherapy and Immune Activation of Pancreatic Cancer

Purpose: A versatile drug-polymer conjugate PODEA-Gem-HMI is prepared and assembled into nanoparticles for the treatment of pancreatic ductal adenocarcinoma (PDAC) through the codelivery of gemcitabine and FAK inhibitor defactinib. While sensing the mild acidity in the tumor microenvironment, nanoparticles will disintegrate and release defactinib to modulate the tumor stroma. The PODEA block of the conjugate can bind with cell membranes reversibly and trigger adsorption-mediated transcytosis (AMT) for promoted tumor penetration and cellular uptake. The internalized conjugates will release gemcitabine responding to the overexpressed glutathione (GSH) for enhanced chemotherapy, and PHMI can condensate the STING monomers for prolonged spontaneous immune stimulation.

Methods: The size and distribution of nanoparticles were well characterized with DLS and TEM. HPLC was used to evaluate the release profile of gemcitabine and defactinib. Stained cells or tumor sections were observed with CLSM. WB was applied to evaluate the protein expression level. The portion of cytotoxic T cells and DC maturation was measured by FACS. The expression of anti-/pro-tumor cytokines was investigated with RT-qPCR or ELISA.

Results: The PHMI block in nanoparticles can capture protons in a mild acidic microenvironment and achieve hydrophobic to hydrophilic transition. Apparent transformations could be observed in both particle size and morphology. The increased ζ potential confirmed that the nanoparticles could protonate and attain charge reversal for promoted cellular uptake. The mild acidity triggered drug release kinetics profile was also estimated, showing that approximately 80% of the encapsulated defactinib could be released within 8 hours. And the quantitative analysis showed that the release of conjugated gemcitabine presented reducing substance concentration and time dual dependent release kinetics. The in vitro transcytosis procedure was visualized with the coverslip transfer experiment. Stronger fluorescence could be found in all three coverslips treated with PODEA nanoparticles, indicating nanoparticles were transmitted among cells on different coverslips, and verified the transcytosis-inducing ability of PODEA. In contrast, weak fluorescence could be observed only on the first and second coverslip of the PEG group, while the signal could hardly be detected on the third coverslip. To verify the enhanced STING stimulation profile, the activation kinetics and the phosphorylation of downstream protein TBK were investigated by Western blot with bone marrow derived macrophages. Compared to other traditional STING agonists, PHMI exhibited delayed and sustained STING activation profiles, as the increased STING phosphorylation can be detected from 6 h and can still be found at 48 h. Inspired by the in vitro results, we further evaluated the tumor penetration capabilities of the nanoparticles. The fluorescence probe labeled nanoparticles were intravenously injected into orthotopic PDAC bearing mice. Tumor tissues were harvested at 12 h after injection and sliced into frozen sections. As the immunofluorescence staining indicated, much more PODEA nanoparticles crossed the hindrance of tumor stroma and accumulated in the core of tumor tissue than PEG nanoparticles. In vivo tumor killing, stromal modulation, and immune activation ability were also investigated with orthotopic PDAC model. The minor tumors were found in PODEA-Gem-HMI nanoparticles-treated mice, and the longest median survival time was also observed in this group. And no noticeable body weight changes in the treated mice suggested favorable biosafety. The FACS analysis confirmed the elevated portion of infiltrated cytotoxic T cells and enlarged portion of matured DCs. Also, the promoted expression of anti-tumor cytokines like IFN-α and lessened secretion of pro-tumor cytokines like TGF-β1 were also measured to evaluate the immune activation efficacy.

Conclusion: In this work, distinct from the common strategy of modifying nanoparticles' particle size or electrical properties, a permeable drug delivery system that can trigger transcytosis to promote deep penetration against PDAC was developed. This strategy exquisitely complies with the pathological characteristics of the excessive proliferation of stromal cells in the PDAC. Defactinib can be specifically released in the microenvironment to soften the tumor stroma. With the implementation of transcytosis, the stromal cells are transformed from janitors to porters, and transport the drug-polymer conjugate to the deep PDAC autonomously. The conjugated gemcitabine would be responsively released and selectively restrain the proliferation of PDAC cells. Besides, the conjugate would also induce the STING condensation and stimulate the anti-PDAC immune responses. Overall, with the exclusive design, the delivery system exhibited enhanced tumor penetration capacity, displayed promoted anti-tumor efficacy and offered a new strategy for PDAC treatment.

References: Chen, H. et al. Transcytosis Mediated Deep Tumor Penetration for Enhanced Chemotherapy and Immune Activation of Pancreatic Cancer. Adv. Funct. Mater., doi: 10.1002/adfm.202214937 (2023).

Acknowledgements: This study was supported by the National Natural Science Foundation of China (Grant No. 32030059 and 82121002), Key Projects of Shanghai Science Foundation (Grant No. 19JC1410800), Shanghai Municipal Science and Technology Major Project (Grant No. 2018SHZDZX01) and ZJLab. All animal experiments described in this study were performed according to the protocols approved by Fudan University Institutional Animal Care and Use Committee.

Figure 1. Schematic illustration of the transcytosis mediated tumor penetration, microenvironment acidity and intracellular GSH dual-responsive drug release profile of nanoparticles with the stroma modulation and combined chemo/immunotherapy promotion capacity.

Figure 2. The profiles of nanoparticles in vitro. a) Illustration of mild acidity triggered drug release and charge reversal of the PODEA-Gem-HMI nanoparticles. The TEM images of the nanoparticles at b) pH 7.4 and c) pH 6.8. Scale bars = 100 nm. The change of the d) size and e) ζ potential of the nanoparticles at pH 7.4 and 6.8. f) Release profile of defactinib in PBS 7.4 and 6.8 at 37 °C. g) The release profile of gemcitabine triggered by different concentrations of dithiothreitol in PBS 7.4 at 37 °C. h) Illustration showing the protocol of visualizing transcytosis with coverslip coincubation. i) CLSM images of the cells on different coverslips. Scale bar = 30 µm. j) Western blot of STING, pSTING, and pTBK expression in primary macrophages at different time points after being treated with PODEA-HMI. β-Tubulin was used as the loading control. The data are represented as the means ± SD (n = 3).

Figure 3. The profiles of nanoparticles in vivo. a) Representative fluorescent images of frozen tumor slices from PDAC-bearing C57 mice. Blue: DAPI for staining cell nucleus; green: Alexa Fluor 488 labeled α-SMA for staining tumor stroma; red: BODIPY 630/650 labeled nanoparticles. Scale bars = 100 µm. b) Tumor volume changes measured by the bioluminescence signal. c) Survival curves of different groups of treatment. d) Body weight recorded 20 days after drug administration. Statistical results of the FACS analysis of e) cytotoxic T cells (CD45+CD4−CD8+) and f) matured DCs (CD11c+CD80+CD86+). g) The expression of IFN-α in the tumor tissue detected by RT-qPCR. h) Expression of TGF-β1 in tumors. The data are represented as means ± SD (n = 4). *, **, and *** denote p < 0.01, p < 0.005, and p < 0.001.