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.

Surgery-Guided Removal of Ovarian Cancer Using Up-Converting Nanoparticles.

Ovarian cancer survival and the recurrence rate are drastically affected by the amount of tumor that can be surgically removed prior to chemotherapy. Surgeons are currently limited to visual inspection, making smaller tumors difficult to be removed surgically. Enhancing the surgeon's ability to selectively remove cancerous tissue would have a positive effect on a patient's prognosis. One approach to aid in surgical tumor removal involves using targeted fluorescent probes to selectively label cancerous tissue. To date, there has been a trade-off in balancing two requirements for the surgeon: the ability to see maximal tumors and the ability to identify these tumors by eye while performing the surgery. The ability to see maximal tumors has been prioritized and this has led to the use of fluorophores activated by near-infrared (NIR) light as NIR penetrates most deeply in this surgical setting, but the light emitted by traditional NIR fluorophores is invisible to the naked eye. This has necessitated the use of specialty detectors and monitors that the surgeon must consult while performing the surgery. In this study, we develop nanoparticles that selectively label ovarian tumors and are activated by NIR light but emit visible light. This potentially allows for maximal tumor observation and real-time detection by eye during surgery. We designed two generations of up-converting nanoparticles that emit green light when illuminated with NIR light. These particles specifically label ovarian tumors most likely via tumor-associated macrophages, which are prominent in the tumor microenvironment. Our results demonstrate that this approach is a viable means of visualizing tumors during surgery without the need for complicated, expensive, and bulky detection equipment. Continued improvement and experimentation could expand our approach into a much needed surgical technique to aid ovarian tumor removal.

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.

Use of a bioengineered antioxidant in mouse models of metabolic syndrome.

Background: Oxidative stress has been implicated in metabolic syndrome (MetS); however, antioxidants such as vitamin E have had limited success in the clinic. This prompts the question of what effects amore potent antioxidant might produce. A prime candidate is the recently developed bioengineered antioxidant, poly(ethylene glycol)-functionalizedhydrophilic carbon clusters (PEG-HCCs), which are capable of neutralizing the reactive oxygen species (ROS) superoxide anion and hydroxyl radical at106/molecule of PEG-HCC. In this project, we tested the potential of PEG-HCCs as a possible therapeutic for MetS.Results: PEG-HCC treatment lessened lipid peroxidation, aspartate aminotransferase levels, non-fastingblood glucose levels, and JNK phosphorylation inob/ob mice. PEG-HCC-treated WT mice had an increased response to insulin by insulin tolerance tests and adecrease in blood glucose by glucose tolerance tests. These effects were not observed in HFD-fed mice, regardless of treatment. PEG-HCCs were observed in the interstitial space of liver, spleen, skeletal muscle, and adipose tissue. No significant difference was shown in gluconeogenesis or inflammatory gene expression between treatment and dietary groups.Expert Opinion: PEG-HCCs improved some parameters of disease possibly due to a resulting increase in peripheral insulin sensitivity. However, additional studies are needed to elucidate how PEG-HCCsare producing these effects.

Dynamically Programmable Magnetic Fields for Controlled Movement of Cells Loaded with Iron Oxide Nanoparticles.

Cell-based therapies are becoming increasingly prominent in numerous medical contexts, particularly in regenerative medicine and the treatment of cancer. However, since the efficacy of the therapy is largely dependent on the concentration of therapeutic cells at the treatment area, a major challenge associated with cell-based therapies is the ability to move and localize therapeutic cells within the body. In this article, a technique based on dynamically programmable magnetic fields is successfully demonstrated to noninvasively aggregate therapeutic cells at a desired location. Various types of therapeutically relevant cells (neural stem cells, monocytes/macrophages, and chimeric antigen receptor T cells) are loaded with iron oxide nanoparticles and then focused at a particular site using externally controlled electromagnets. These experimental results serve as a readily scalable prototype for designing an apparatus that patients can wear to focus therapeutic cells at the anatomical sites needed for treatment.

Colloidal Capsules Assembled from Gold Nanoparticles Using Small-Molecule Hydrophobic Cross-linkers.

Colloidal capsules (or colloidosomes) have been studied for various applications such as therapeutic agent encapsulation, photothermal therapy, imaging, and energy storage. Emulsion-based synthesis is the most common approach for preparing colloidal capsules as it is relatively straightforward and scalable. However, while the initial formation requires only introducing the colloidal subunits into an emulsion and letting them assemble at the interface, a second step is required in order to prepare stable, covalently linked colloidal capsules, and preparing submicron colloidal capsules is quite challenging. Here, we describe a simple and quick one-step method to synthesize covalently linked, stable nanoscale colloidal capsules consisting of gold nanoparticles (NPs) (AuNP) and thiol-containing cross-linkers. Gold nanoparticle capsules (AuNCs) were formed by coating emulsion droplets containing thiol-containing cross-linkers with citrate-stabilized AuNPs. The physicochemical properties of the colloidal capsules can be tailored by changing the building blocks. In order to demonstrate this, colloidal capsules were assembled from AuNPs ranging from 5 to 20 nm in size. The use of the larger 20 nm starting particles resulted in AuNCs with a sufficiently pronounced red shift for λmax to be suitable for biological photothermal applications, where use of a near-infrared laser is strongly preferred. The AuNCs were found to be biocompatible and stable in cell culture conditions and to provide moderate heating. This demonstrates the modularity of the synthesis and the potential advantages of a one-step synthesis to prepare nanoscale gold colloidal capsules.

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.

Immunostimulatory CpG on Carbon Nanotubes Selectively Inhibits Migration of Brain Tumor Cells.

Even when treated with aggressive current therapies, patients with glioblastoma usually survive less than two years and exhibit a high rate of recurrence. CpG is an oligonucleotide that activates the innate immune system via Toll-like receptor 9 (TLR9) activation. Injection of CpG into glioblastoma tumors showed promise as an immunotherapy in mouse models but proved disappointing in human trials. One aspect of glioma that is not addressed by CpG therapy alone is the highly invasive nature of glioma cells, which is associated with resistance to radiation and chemotherapy. Here, we demonstrate that single-walled carbon nanotubes noncovalently functionalized with CpG (SWNT/CpG), which retain the immunostimulatory property of the CpG, selectively inhibit the migration of glioma cells and not macrophages without affecting cell viability or proliferation. SWNT/CpG also selectively decreased NF-κB activation in glioma cells, while activating macrophages by induction of the TLR9/NF-κB pathway, as we have previously reported. The migration inhibition of glioma cells was correlated with selective reduction of intracellular levels of reactive oxygen species (ROS), suggesting that an antioxidant-based mechanism mediates the observed effects. To the best of our knowledge, SWNT/CpG is the first nanomaterial that inhibits the migration of cancer cells while stimulating the immune system.

Coating Metal Nanoparticle Surfaces with Small Organic Molecules Can Reduce Nonspecific Cell Uptake.

Elucidation of mechanisms of uptake of nanoparticles by cells and methods to prevent this uptake is essential for many applications of nanoparticles. Most recent studies have focused on the role of proteins that coat nanoparticles and have employed PEGylation, particularly dense coatings of PEG, to reduce protein opsonization and cell uptake. Here we show that small molecule coatings on metallic nanoparticles can markedly reduce cell uptake for very sparsely PEGylated nanoparticles. Similar results were obtained in media with and without proteins, suggesting that protein opsonization is not the primary driver of this phenomenon. The reduction in cell uptake is proportional to the degree of surface coverage by the small molecules. Probing cell uptake pathways using inhibitors suggested that the primary role of increased surface coverage is to reduce nanoparticles' interactions with the scavenger receptors. This work highlights an under-investigated mechanism of cell uptake that may have played a role in many other studies and also suggests that a wide variety of molecules can be used alongside PEGylation to stably passivate nanoparticle surfaces for low cell uptake.

Impact of Cross-Linker Valency on Gold Nanoparticle Aggregate Formation and Cellular Uptake.

Synthesis of spherical, biocompatible nanoparticle aggregates using a small molecular cross-linker is a simple and flexible approach for the controlled assembly of gold nanoparticles. This strategy can be extended to a variety of cross-linkers, making it possible to the test the effect of cross-linker properties on aggregate formation and physicochemical properties. Here, we synthesized aggregates using a series of structurally homologous cross-linkers with differing valencies. These aggregates have the same size, morphology, surface charge, surface coating, and stability in salt, media, and low pH conditions, but they differ in their stability to cyanide etching and uptake by cells. This highlights the fine-tuning of nanoparticle aggregate properties that can be achieved by using small-molecule cross-linkers.

Exploiting homing abilities of cell carriers: Targeted delivery of nanoparticles for cancer therapy.

Off target toxicities is one of the hallmarks of conventional chemotherapy as only a tiny percentage of the injected dose actually reaches the tumor(s). Numerous strategies have been employed in attempts to achieve targeted therapeutic delivery to tumors. One strategy that has received immense attention has been the packaging of these chemotherapeutics into nanoparticles and relying on the enhanced permeation and retention (EPR) effect for targeting. However, few, if any, nanoformulations have been used clinically that actually show enhanced drug delivery to tumors. There are a number of biological barriers to successful targeted delivery and nanoparticles large enough to theoretically benefit from the EPR effect predominantly accumulate in the liver and spleen after systemic administration. Nanoparticles that do reach the tumor will experience challenges such as difficulty penetrating deeply into tumors and rapid uptake by macrophages rather than tumor cells. In order to overcome this, researchers are investigating a new drug delivery system by utilizing T-cells, macrophages, or stem cells (Mesenchymal/Neural Stem Cells) and loading them with therapeutic nanoparticles for targeted delivery due to either their organotropic or tumor tropic migratory capabilities. By exploiting the migration and motility of these particular cells, researchers have delivered drug-loaded nanoparticles as well as nanoparticles for use in thermal ablation and magnetic field treatments, with the goals of decreasing off-target toxicities and increasing intratumoral distribution of the therapeutic payload. This is an inherently complex drug delivery system that requires optimization of multiple parameters - including cell type, payload, cell loading, release rate from nanoparticle and more - for success. Here we review recent advances and upcoming challenges for the field.

Challenges in realizing selectivity for nanoparticle biodistribution and clearance: lessons from gold nanoparticles.

The field of nanomedicine has received much attention for its potential to allow for targeted identification and treatment of tumors, while sparing healthy tissue. This promise has yet to be clinically realized; instead nanomedicine has translated into clinical benefit via formulations that improve the pharmacokinetics and toxicity profiles of toxic chemotherapeutic agents. In this perspective, we highlight that several of the defining strategies for using nanoparticles intravenously to target solid tumors have limited supporting data in animal studies. Namely, it does not appear that reducing macrophage (and other cell type) uptake in vitro leads to better biodistribution in vivo, nor does increasing blood circulation time nor active targeting. We suggest instead that the coming decade will primarily see nanoparticles impact immunotherapy and local/pseudolocal cancer therapy.

Intraperitoneal Administration of Neural Stem Cell-Nanoparticle Conjugates Targets Chemotherapy to Ovarian Tumors.

Ovarian cancer is particularly aggressive once it has metastasized to the abdominal cavity (stage III). Intraperitoneal (IP) as compared to intravenous (IV) administration of chemotherapy improves survival for stage III ovarian cancer, demonstrating that concentrating chemotherapy at tumor sites has therapeutic benefit; unfortunately, IP therapy also increases toxic side effects, thus preventing its completion in many patients. The ability to target chemotherapy selectively to ovarian tumors while sparing normal tissue would improve efficacy and decrease toxicities. We have previously shown that tumor-tropic neural stem cells (NSCs) dramatically improve the intratumoral distribution of nanoparticles (NPs) when given intracerebrally near an orthotopic brain tumor or into a flank xenograft tumor. Here, we show that NPs either conjugated to the surface of NSCs or loaded within the cells are selectively delivered to and distributed within ovarian tumors in the abdominal cavity following IP injection, with no evidence of localization to normal tissue. IP administration is significantly more effective than IV administration, and NPs carried by NSCs show substantially deeper penetration into tumors than free NPs. The NSCs and NPs target and localize to ovarian tumors within 1 h of administration. Pt-loaded silica NPs (SiNP[Pt]) were developed that can be transported in NSCs, and it was found that the NSC delivery of SiNP[Pt] (NSC-SiNP[Pt]) results in higher levels of Pt in tumors as compared to free drug or SiNP[Pt]. To the best of our knowledge, this work represents the first demonstration that cells given IP can target the delivery of drug-loaded NPs.

Colorimetric Detection of Staphylococcus aureus Contaminated Solutions without Purification.

Current water quality monitoring methods rely on growth-based measurements to detect fecal indicator bacteria, such as Escherichia coli and enterococci, and Staphylococcus aureus (S. aureus). These growth-based measurements, however, can take days to complete. This is a significant limitation in the evaluation of contaminated food and water sources. Various methods for selective in vitro detection of S. aureus have also been reported; however, these strategies, such as ELISA, agar-diffusion, PCR, or liquid chromatography-tandem mass spectrometry, all require overnight culturing or sophisticated instrumentation. There is a pressing need for a portable, simple diagnostic for S. aureus. Here, we demonstrate that oligonucleotide-functionalized gold nanoparticles (Oligo-AuNPs) can be designed to rapidly and selectively detect S. aureus with a colorimetric readout. We have functionalized a chemically modified 11-mer sequence onto AuNPs and have found that aggregation occurs in the presence of S. aureus supernantants. The particles can be stored as a lyophilized powder and reconstituted at time of use, and this has been tested in biologically relevant samples such as creek and ocean water. This approach requires minimal sample preparation and requires no extraneous instrumentation, leading to a rapid and simple diagnostic read-out that could be used in field tests to monitor food and water sources.

Focusing light inside scattering media with magnetic-particle-guided wavefront shaping.

Optical scattering has traditionally limited the ability to focus light inside scattering media such as biological tissue. Recently developed wavefront shaping techniques promise to overcome this limit by tailoring an optical wavefront to constructively interfere at a target location deep inside scattering media. To find such a wavefront solution, a "guide-star" mechanism is required to identify the target location. However, developing guidestars of practical usefulness is challenging, especially in biological tissue, which hinders the translation of wavefront shaping techniques. Here, we demonstrate a guidestar mechanism that relies on magnetic modulation of small particles. This guidestar method features an optical modulation efficiency of 29% and enables micrometer-scale focusing inside biological tissue with a peak intensity-to-background ratio (PBR) of 140; both numbers are one order of magnitude higher than those achieved with the ultrasound guidestar, a popular guidestar method. We also demonstrate that light can be focused on cells labeled with magnetic particles, and to different target locations by magnetically controlling the position of a particle. Since magnetic fields have a large penetration depth even through bone structures like the skull, this optical focusing method holds great promise for deep-tissue applications such as optogenetic modulation of neurons, targeted light-based therapy, and imaging.

Gold nanorod-mediated near-infrared laser ablation: in vivo experiments on mice and theoretical analysis at different settings.

PURPOSE: The aim of the present study was the in vivo assessment of the effects of gold nanorod (AuNR)-mediated laser ablation (LA) of flank xenograft tumours. We investigated: the differences between intra-tumoural (TIT) and surface tumoural temperature (TS); the influence of AuNRs concentration and laser power (P) on both these temperatures and on tumour regression. Lastly, experimental data were used to validate a theoretical model developed to predict the effects of AuNR-mediated LA. MATERIALS AND METHODS: Thirty-two nude mice were treated using near-infra-red light at two P, 3 d after injecting increasing AuNR doses. TIT and TS were recorded during the procedure by two thermocouples, one located within the tumour and the other one on the skin adjacent to the tumour. Tumour regression was assessed 2 d after near-infra-red exposure via Xenogen imaging. A three-dimensional temperature map was obtained by finite element modelling. RESULTS: TIT and TS difference is substantial when AuNRs are injected. Moreover, the maximum temperature reached is strongly influenced by both P and AuNR concentration. Tumours heated above 55 °C experienced regression. Good agreement between experimental and theoretical TIT was found (maximum difference of 4 °C). CONCLUSIONS: Data show significant influence of P and AuNR concentration on the temperatures reached during AuNR-mediated LA of solid tumours. TS and TIT difference increases with AuNRs concentration. Simulated temperatures agree quite well with experimental data. Together, these results represent the first step towards a rationally designed strategy to select the most promising laser settings and AuNRs concentration to improve solid tumour treatment outcomes.

Metronomic Doses of Temozolomide Enhance the Efficacy of Carbon Nanotube CpG Immunotherapy in an Invasive Glioma Model.

Even when treated with aggressive current therapies, most patients with glioblastoma survive less than two years. Rapid tumor growth, an invasive nature, and the blood-brain barrier, which limits the penetration of large molecules into the brain, all contribute to the poor tumor response associated with conventional therapies. Immunotherapy has emerged as a therapeutic approach that may overcome these challenges. We recently reported that single-walled carbon nanotubes (SWCNTs) can be used to dramatically increase the immunotherapeutic efficacy of CpG oligonucleotides in a mouse model of glioma. Following implantation in the mouse brain, the tumor cell line used in these previous studies (GL261) tends to form a spherical tumor with limited invasion into healthy brain. In order to evaluate SWCNT/CpG therapy under more clinically-relevant conditions, here we report the treatment of a more invasive mouse glioma model (K-Luc) that better recapitulates human disease. In addition, a CpG sequence previously tested in humans was used to formulate the SWCNT/CpG which was combined with temozolomide, the standard of care chemotherapy for glioblastoma patients. We found that, following two intracranial administrations, SWCNT/CpG is well-tolerated and improves the survival of mice bearing invasive gliomas. Interestingly, the efficacy of SWCNT/CpG was enhanced when combined with temozolomide. This enhanced anti-tumor efficacy was correlated to an increase of tumor-specific cytotoxic activity in splenocytes. These results reinforce the emerging understanding that immunotherapy can be enhanced by combining it with chemotherapy and support the continued development of SWCNT/CpG.