Tumor-specific delivery of gemcitabine with activatable liposomes.

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 gemcitabine­copper 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.

A Scalable Method for Squalenoylation and Assembly of Multifunctional 64Cu-Labeled Squalenoylated Gemcitabine Nanoparticles.

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 (KrasLSL-G12D/+; Trp53LSL-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.

Priming is key to effective incorporation of image-guided thermal ablation into immunotherapy protocols.

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.

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvß3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

On-command drug release from nanochains inhibits growth of breast tumors.

PURPOSE: To evaluate the ability of radiofrequency (RF)-triggered drug release from a multicomponent chain-shaped nanoparticle to inhibit the growth of an aggressive breast tumor. METHODS: A two-step solid phase chemistry was employed to synthesize doxorubicin-loaded nanochains, which were composed of three iron oxide nanospheres and one doxorubicin-loaded liposome assembled in a 100-nm-long linear nanochain. The nanochains were tested in the 4T1-LUC-GFP orthotopic mouse model, which is a highly aggressive breast cancer model. The 4T1-LUC-GFP cell line stably expresses firefly luciferase, which allowed the non-invasive in vivo imaging of tumor response to the treatment using bioluminescence imaging (BLI). RESULTS: Longitudinal BLI imaging showed that a single nanochain treatment followed by application of RF resulted in an at least 100-fold lower BLI signal compared to the groups treated with nanochains (without RF) or free doxorubicin followed by RF. A statistically significant increase in survival time of the nanochain-treated animals followed by RF (64.3 days) was observed when compared to the nanochain-treated group without RF (35.7 days), free doxorubicin-treated group followed by RF (38.5 days), and the untreated group (30.5 days; n=5 animals per group). CONCLUSIONS: These studies showed that the combination of RF and nanochains has the potential to effectively treat highly aggressive cancers and prolong survival.

Treatment of cancer micrometastasis using a multicomponent chain-like nanoparticle.

While potent cytotoxic agents are available to oncologists, the clinical utility of these agents is limited due to their non-specific distribution in the body and toxicity to normal tissues leading to use of suboptimal doses for eradication of metastatic disease. Furthermore, treatment of micrometastases is impeded by several biobarriers, including their small size and high dispersion to organs, making them nearly inaccessible to drugs. To circumvent these limitations in treating metastatic disease, we developed a multicomponent, flexible chain-like nanoparticle (termed nanochain) that possesses a unique ability to gain access to and be deposited at micrometastatic sites. Moreover, coupling nanochain particles to radiofrequency (RF)-triggered cargo delivery facilitated widespread delivery of drug into hard-to-reach cancer cells. Collectively, these features synergistically facilitate effective treatment and ultimately eradication of micrometastatic disease using a low dose of a cytotoxic drug.