Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy. Methods: Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (Kras^LSL-G12D/+; Trp53^LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A ⁶⁴Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs. Results: Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane® when evaluated at a concentration of 10 µM. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution. Conclusion: We present a scalable technique for fabricating efficacious squalenoylated-gemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional ⁶⁴Cu-SqNOTA-SqGem NP.

Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper (II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 wt% loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine‑copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42 °C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis.

Focal therapies play an important role in the treatment of cancers where palliation is desired, local control is needed, or surgical resection is not feasible. Pairing immunotherapy with such focal treatments is particularly attractive; however, there is emerging evidence that focal therapy can have a positive or negative impact on the efficacy of immunotherapy. Thermal ablation is an appealing modality to pair with such protocols, as tumors can be rapidly debulked (cell death occurring within minutes to hours), tumor antigens can be released locally, and treatment can be conducted and repeated without the concerns of radiation-based therapies. In a syngeneic model of epithelial cancer, we found that 7 days of immunotherapy (TLR9 agonist and checkpoint blockade), prior to thermal ablation, reduced macrophages and myeloid-derived suppressor cells and enhanced IFN-γ-producing CD8⁺ T cells, the M1 macrophage fraction, and PD-L1 expression on CD45⁺ cells. Continued treatment with immunotherapy alone or with immunotherapy combined with ablation (primed ablation) then resulted in a complete response in 80% of treated mice at day 90, and primed ablation expanded CD8⁺ T cells as compared with all control groups. When the tumor burden was increased by implantation of 3 orthotopic tumors, successive primed ablation of 2 discrete lesions resulted in survival of 60% of treated mice as compared with 25% of mice treated with immunotherapy alone. Alternatively, when immunotherapy was begun immediately after thermal ablation, the abscopal effect was diminished and none of the mice within the cohort exhibited a complete response. In summary, we found that immunotherapy begun before ablation can be curative and can enhance efficacy in the presence of a high tumor burden. Two mechanisms have potential to impact the efficacy of immunotherapy when begun immediately after thermal ablation: mechanical changes in the tumor microenvironment and inflammatory-mediated changes in immune phenotype.