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.

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.

Heterogeneous computing for epidemiological model fitting and simulation.

BACKGROUND: Over the last years, substantial effort has been put into enhancing our arsenal in fighting epidemics from both technological and theoretical perspectives with scientists from different fields teaming up for rapid assessment of potentially urgent situations. This paper focusses on the computational aspects of infectious disease models and applies commonly available graphics processing units (GPUs) for the simulation of these models. However, fully utilizing the resources of both CPUs and GPUs requires a carefully balanced heterogeneous approach. RESULTS: The contribution of this paper is twofold. First, an efficient GPU implementation for evaluating a small-scale ODE model; here, the basic S(usceptible)-I(nfected)-R(ecovered) model, is discussed. Second, an asynchronous particle swarm optimization (PSO) implementation is proposed where batches of particles are sent asynchronously from the host (CPU) to the GPU for evaluation. The ultimate goal is to infer model parameters that enable the model to correctly describe observed data. The particles of the PSO algorithm are candidate parameters of the model; finding the right one is a matter of optimizing the likelihood function which quantifies how well the model describes the observed data. By employing a heterogeneous approach, in which both CPU and GPU are kept busy with useful work, speedups of 10 to 12 times can be achieved on a moderate machine with a high-end consumer GPU as compared to a high-end system with 32 CPU cores. CONCLUSIONS: Utilizing GPUs for parameter inference can bring considerable increases in performance using average host systems with high-end consumer GPUs. Future studies should evaluate the benefit of using newer CPU and GPU architectures as well as applying this method to more complex epidemiological scenarios.

Delivery of the gene encoding the tumor suppressor Sef into prostate tumors by therapeutic-ultrasound inhibits both tumor angiogenesis and growth.

Carcinomas constitute over 80% of all human cancer types with no effective therapy for metastatic disease. Here, we demonstrate, for the first time, the efficacy of therapeutic-ultrasound (TUS) to deliver a human tumor suppressor gene, hSef-b, to prostate tumors in vivo. Sef is downregulated in various human carcinomas, in a manner correlating with tumor aggressiveness. In vitro, hSef-b inhibited proliferation of TRAMP C2 cells and attenuated activation of ERK/MAPK and the master transcription factor NF-κB in response to FGF and IL-1/TNF, respectively. In vivo, transfection efficiency of a plasmid co-expressing hSef-b/eGFP into TRAMP C2 tumors was 14.7 ± 2.5% following a single TUS application. Repeated TUS treatments with hSef-b plasmid, significantly suppressed prostate tumor growth (60%) through inhibition of cell proliferation (60%), and reduction in blood vessel density (56%). In accordance, repeated TUS-treatments with hSef-b significantly inhibited in vivo expression of FGF2 and MMP-9. FGF2 is a known mitogen, and both FGF2/MMP-9 are proangiogenic factors. Taken together our results strongly suggest that hSef-b acts in a cell autonomous as well as non-cell autonomous manner. Moreover, the study demonstrates the efficacy of non-viral TUS-based hSef-b gene delivery approach for the treatment of prostate cancer tumors, and possibly other carcinomas where Sef is downregulated.

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.

Ultrasound-Mediated Mesenchymal Stem Cells Transfection as a Targeted Cancer Therapy Platform.

Mesenchymal stem cells (MSCs) hold tremendous potential as a targeted cell-based delivery platform for inflammatory and cancer therapy. Genetic manipulation of MSCs, however, is challenging, and therefore, most studies using MSCs as therapeutic cell carriers have utilized viral vectors to transduce the cells. Here, we demonstrate, for the first time, an alternative approach for the efficient transfection of MSCs; therapeutic ultrasound (TUS). Using TUS with low intensities and moderate frequencies, MSCs were transfected with a pDNA encoding for PEX, a protein that inhibits tumor angiogenesis, and studied as a cell vehicle for in vivo tumor therapy. TUS application did not alter the MSCs' stemness or their homing capabilities, and the transfected MSCs transcribed biologically active PEX. Additionally, in a mouse model, 70% inhibition of prostate tumor growth was achieved following a single I.V. administration of MSCs that were TUS-transfected with pPEX. Further, the repeated I.V. administration of TUS-pPEX transfected-MSCs enhanced tumor inhibition up to 84%. Altogether, these results provide a proof of concept that TUS-transfected MSCs can be effectively used as a cell-based delivery approach for the prospective treatment of cancer.

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.

Spatiotemporal Evolution of Ebola Virus Disease at Sub-National Level during the 2014 West Africa Epidemic: Model Scrutiny and Data Meagreness.

BACKGROUND: The Ebola outbreak in West Africa has infected at least 27,443 individuals and killed 11,207, based on data until 24 June, 2015, released by the World Health Organization (WHO). This outbreak has been characterised by extensive geographic spread across the affected countries Guinea, Liberia and Sierra Leone, and by localized hotspots within these countries. The rapid recognition and quantitative assessment of localised areas of higher transmission can inform the optimal deployment of public health resources. METHODS: A variety of mathematical models have been used to estimate the evolution of this epidemic, and some have pointed out the importance of the spatial heterogeneity apparent from incidence maps. However, little is known about the district-level transmission. Given that many response decisions are taken at sub-national level, the current study aimed to investigate the spatial heterogeneity by using a different modelling framework, built on publicly available data at district level. Furthermore, we assessed whether this model could quantify the effect of intervention measures and provide predictions at a local level to guide public health action. We used a two-stage modelling approach: a) a flexible spatiotemporal growth model across all affected districts and b) a deterministic SEIR compartmental model per district whenever deemed appropriate. FINDINGS: Our estimates show substantial differences in the evolution of the outbreak in the various regions of Guinea, Liberia and Sierra Leone, illustrating the importance of monitoring the outbreak at district level. We also provide an estimate of the time-dependent district-specific effective reproduction number, as a quantitative measure to compare transmission between different districts and give input for informed decisions on control measures and resource allocation. Prediction and assessing the impact of control measures proved to be difficult without more accurate data. In conclusion, this study provides us a useful tool at district level for public health, and illustrates the importance of collecting and sharing data.

The effect of AZD2171- or sTRAIL/Apo2L-loaded polylactic-co-glycolic acid microspheres on a subcutaneous glioblastoma model.

Studies with AZD2171-a new anti-angiogenic inhibitor of tyrosine kinases associated with VEGF signaling-have shown great promise for treating glioblastoma. Unfortunately, AZD2171 success is limited by low permeability through the blood-brain barrier. Due to AZD2171's short half-life and high toxicity, its local administration will require multiple intracranial procedures, making this approach clinically unfeasible. In this study, we investigated the potential of the highly hydrophobic AZD2171, released from modified polylactic-co-glycolic acid microspheres (PLGA-MS), to treat glioblastoma. To further demonstrate the versatile loading capacity of this system, the same PLGA formulation, which was found optimal for the loading and release of AZD2171, was tested with sTRAIL/Apo2L-a biologic drug that is very different than AZD2171 in its molecular weight, solubility, and charge. AZD2171 released from PLGA-MS was at least effective as the free drug in inhibiting endothelial growth and proliferation (in vitro), and, surprisingly, had a profound cytotoxic effect also towards in vitro cultured glioblastoma cell-lines (U87 and A172). Complete tumor inhibition was achieved following a single treatment with AZD2171-loaded PLGA-MS (6 (mg)/kg) administered locally adjacent to human U87 glioma tumors inoculated subcutaneously in nude mice. This improved effect, compared to other therapeutic approaches involving AZD2171, was shown to affect both tumor vasculature and the glioma cells. sTRAIL-loaded microspheres, administered at very low doses (0.3 (mg)/kg), led to 35 % inhibition of tumor growth in 2 weeks. Collectively, our results provide pre-clinical evidence for the potential of PLGA formulations of AZD2171 and sTRAIL to serve as an effective treatment for glioblastoma.

Insight concerning the mechanism of therapeutic ultrasound facilitating gene delivery: increasing cell membrane permeability or interfering with intracellular pathways?

Nonviral gene delivery methods encounter major barriers in plasmid DNA (pDNA) trafficking toward the nucleus. The present study aims to understand the role and contribution of therapeutic ultrasound (TUS), if any, in pDNA trafficking in primary cells such as fibroblasts and cell lines (e.g., baby hamster kidney [BHK]) during the transfection process. Using compounds that alter the endocytic pathways and the cytoskeletal network, we show that after TUS application, pDNA trafficking in the cytoplasm is not mediated by endocytosis or by the cytoskeletal network. Transfection studies and confocal analyses showed that the actin fibers impeded TUS-mediated transfection in BHK cells, but not in fibroblasts. Flow cytometric analyses indicated that pDNA uptake by cells occurs primarily when the pDNA is added before and not after TUS application. Taken together, these results suggest that TUS by itself operates as a mechanical force driving the pDNA through the cell membrane, traversing the cytoplasmic network and into the nucleus.

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro.

BACKGROUND: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. DESIGN AND METHODS: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. RESULTS: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. CONCLUSIONS: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.