Large, Anionic Liposomes Enable Targeted Intraperitoneal Delivery of a TLR 7/8 Agonist To Repolarize Ovarian Tumors' Microenvironment.

Ovarian cancer is the most lethal gynecological malignancy in the United States. Current standard of treatment includes surgical debulking and chemotherapy, such as cisplatin and paclitaxel. However, the patients' response rate for chemotherapy in ovarian cancer is not optimal, and they often develop chemoresistance and suffer from side effects. Current clinical trials make extensive use of immune checkpoint blockade (ICB) as a novel cancer immunotherapeutic strategy against ovarian tumors. However, the response rates for ICB antibodies remain limited to 10-20% of treated ovarian cancer patients despite the success of this approach in melanoma, renal, head and neck, and nonsmall cell lung cancers. This lack of efficacy is often attributed to the "cold" immune status of ovarian tumors, as these tumors often have a low number of tumor-infiltrating lymphocytes (TILs) but a high number of suppressive immune cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), or regulatory T cells (Tregs). Repolarizing TAMs could be a promising strategy to reshape the tumor immune microenvironment and promote antitumor activity when combined with ICBs. Toll-like receptor (TLR) 7 and 8 agonists, such as imiquimod and resiquimod, are potent immunostimulatory molecules with potential to repolarize macrophages. However, these small molecules have poor pharmacokinetic profiles and can induce severe side effects when administered systemically. Previously, our group demonstrated that various large, anionic nanomaterials (silica, PLGA, and polystyrene) specifically target TAMs when administered intraperitoneally (IP) to ovarian tumor-bearing mice. In the present study, we demonstrate that large, anionic liposomes administered IP also efficiently localize to TAMs and can be used to target the delivery of resiquimod. Resiquimod delivered in this targeted fashion promoted activation of M1 macrophages and T cell infiltration, while reducing the percentage of Tregs in the tumor microenvironment. Finally, liposome-formulated resiquimod significantly enhanced the efficacy of PD1 blockade against syngeneic ovarian tumors. We anticipate that further optimization of our liposomal delivery strategy can generate a clinically relevant strategy for more effective and safer immunotherapy for ovarian cancer patients.

Thermal analysis of laser irradiation-gold nanorod combinations at 808 nm, 940 nm, 975 nm and 1064 nm wavelengths in breast cancer model.

BACKGROUND: Photothermal therapy is currently under the spotlight to improve the efficacy of minimally invasive thermal treatment of solid tumors. The interplay of several factors including the radiation wavelengths and the nanoparticle characteristics underlie the thermal outcome. However, a quantitative thermal analysis in in vivo models embedding nanoparticles and under different near-infrared (NIR) wavelengths is missing. PURPOSE: We evaluate the thermal effects induced by different combinations of NIR laser wavelengths and gold nanorods (GNRs) in breast cancer tumor models in mice. MATERIALS AND METHODS: Four laser wavelengths within the therapeutic window, i.e., 808, 940, 975, and 1064 nm were employed, and corresponding GNRs were intratumorally injected. The tissue thermal response was evaluated in terms of temperature profile and time constants, considering the step response of a first-order system as a model. RESULTS: The 808 nm and 1064 nm lasers experienced the highest temperature enhancements (>24%) in presence of GNRs compared to controls; conversely, 975 nm and 940 nm lasers showed high temperatures in controls due to significant tissue absorption and the lowest temperature difference with and without GNRs (temperature enhancement <10%). The presence of GNRs resulted in small time constants, thus quicker laser-induced thermal response (from 67 s to 33 s at 808 nm). CONCLUSIONS: The thermal responses of different GNR-laser wavelength combinations quantitatively validate the widespread usage of 808 nm laser for nanoparticle-assisted photothermal procedures. Moreover, our results provide insights on other usable wavelengths, toward the identification of an effective photothermal treatment strategy for the removal of focal malignancies.

Neural Stem Cells Improve the Delivery of Oncolytic Chimeric Orthopoxvirus in a Metastatic Ovarian Cancer Model.

Oncolytic virotherapy represents a promising approach for treating recurrent and/or drug-resistant ovarian cancer. However, its successful application in the clinic has been hampered by rapid immune-mediated clearance, which reduces viral delivery to the tumor. Patient-derived mesenchymal stem cells that home to tumors have been used as viral delivery tools, but variability associated with autologous cell isolations limits the clinical applicability of this approach. We previously developed an allogeneic, clonal neural stem cell (NSC) line (HB1.F3.CD21) that can be used to deliver viral cargo. Here, we demonstrate that this NSC line can improve the delivery of a thymidine kinase gene-deficient conditionally replication-competent orthopoxvirus, CF33, in a preclinical cisplatin-resistant peritoneal ovarian metastases model. Overall, our findings provide the basis for using off-the-shelf allogeneic cell-based delivery platforms for oncolytic viruses, thus providing a more efficient delivery alternative compared with the free virus administration approach.

Specific targeting of ovarian tumor-associated macrophages by large, anionic nanoparticles.

Immunotherapy is emerging as one of the most effective methods for treating many cancers. However, immunotherapy can still introduce significant off-target toxicity, and methods are sought to enable targeted immunotherapy at tumor sites. Here, we show that relatively large (>100-nm) anionic nanoparticles administered intraperitoneally (i.p.) selectively accumulate in tumor-associated macrophages (TAMs). In a mouse model of metastatic ovarian cancer, fluorescently labeled silica, poly(lactic-co-glycolic acid), and polystyrene nanoparticles administered i.p. were all found to selectively accumulate in TAMs. Quantifying silica particle uptake indicated that >80% of the injected dose was in TAMs. Particles that were smaller than 100 nm or cationic or administered intravenously (i.v.) showed no TAM targeting. Moreover, this phenomenon is likely to occur in humans because when freshly excised human surgical samples were treated with the fluorescent silica nanoparticles no interaction with healthy tissue was seen but selective uptake by TAMs was seen in 13 different patient samples. Ovarian cancer is a deadly disease that afflicts ∼22,000 women per year in the United States, and the presence of immunosuppressive TAMs at tumors is correlated with decreased survival. The ability to selectively target TAMs opens the door to targeted immunotherapy for ovarian cancer.

A Systematic comparison of in vitro cell uptake and in vivo biodistribution for three classes of gold nanoparticles with saturated PEG coatings.

A great deal of attention has been focused on nanoparticles for cancer therapy, with the promise of tumor-selective delivery. However, despite intense work in the field over many years, the biggest obstacle to this vision remains extremely low delivery efficiency of nanoparticles into tumors. Due to the cost, time, and impact on the animals for in vivo studies, the nanoparticle field predominantly uses cellular uptake assays as a proxy to predict in vivo outcomes. Extensive research has focused on decreasing macrophage uptake in vitro as a proxy to delay nanoparticle accumulation in the mononuclear phagocytic system (MPS), mainly the liver and spleen, and thereby increase tumor accumulation. We have recently reported novel synthetic methods employing small molecule crosslinkers for the controlled assembly of small nanoparticles into larger aggregates and found that these nanoaggregates had remarkably high surface coverage and low cell uptake, even in macrophages. We further found that this extremely low cellular uptake could be recapitulated on solid gold nanoparticles by densely coating their surface with small molecules. Here we report our studies on the biodistribution and clearance of these materials in comparison to more conventional PEGylated gold nanoparticles. It was expected that the remarkably low macrophage uptake in vitro would translate to extended blood circulation time in vivo, but instead we found no correlation between either surface coverage or in vitro macrophage cell uptake and in vivo blood circulation. Gold nanoaggregates accumulate more rapidly and to a higher level in the liver compared to control gold nanoparticles. The lack of correlation between in vitro macrophage uptake and in vivo blood circulation suggests that the field must find other in vitro assays to use as a primary proxy for in vivo outcomes or use direct in vivo experimentation as a primary assay.

Silica Coated Paclitaxel Nanocrystals Enable Neural Stem Cell Loading For Treatment of Ovarian Cancer.

Ovarian cancer is commonly diagnosed only after it has metastasized to the abdominal cavity (stage III). While the current standard of care of intraperitoneal (IP) administration of cisplatin and paclitaxel (PTX) combination chemotherapy has benefit, patient 5-year survival rates are low and have not significantly improved in the past decade. The ability to target chemotherapy selectively to ovarian tumors while sparing normal tissue would improve efficacy and decrease toxicities. We have previously shown that cisplatin-loaded nanoparticles (NPs) loaded within neural stem cells (NSCs) are selectively delivered to ovarian tumors in the abdominal cavity following IP injection, with no evidence of localization to normal tissue. Here we extended the capabilities of this system to also include PTX delivery. NPs that will be loaded into NSCs must contain a high amount of drug by weight but constrain the release of the drug such that the NSCs are viable after loading and can successfully migrate to tumors. We developed silica coated PTX nanocrystals (Si[PTX-NC]) meeting these requirements. Si[PTX-NC] were more effective than uncoated PTX-NC or Abraxane for loading NSCs with PTX. NSCs loaded with Si[PTX-NC] maintained their migratory ability and, for low dose PTX, were more effective than free PTX-NC or Si[PTX-NC] at killing ovarian tumors in vivo. This work demonstrates that NSC/NP delivery is a platform technology amenable to delivering different therapeutics and enables the pursuit of NSC/NP targeted delivery of the entire preferred chemotherapy regimen for ovarian cancer. It also describes efficient silica coating chemistry for PTX nanocrystals that may have applications beyond our focus on NSC transport.