Therapeutic strategies to improve drug delivery across the blood-brain barrier.

Resection of brain tumors is followed by chemotherapy and radiation to ablate remaining malignant cell populations. Targeting these populations stands to reduce tumor recurrence and offer the promise of more complete therapy. Thus, improving access to the tumor, while leaving normal brain tissue unscathed, is a critical pursuit. A central challenge in this endeavor lies in the limited delivery of therapeutics to the tumor itself. The blood-brain barrier (BBB) is responsible for much of this difficulty but also provides an essential separation from systemic circulation. Due to the BBB's physical and chemical constraints, many current therapies, from cytotoxic drugs to antibody-based proteins, cannot gain access to the tumor. This review describes the characteristics of the BBB and associated changes wrought by the presence of a tumor. Current strategies for enhancing the delivery of therapies across the BBB to the tumor will be discussed, with a distinction made between strategies that seek to disrupt the BBB and those that aim to circumvent it.

Quinic acid derivative KZ-41 exhibits radiomitigating activity in preclinical models of radiation injury.

Acute radiation syndrome is induced when a significant portion of the body receives high-dose, as well as high-dose rate, radiation. We have previously identified a quinic acid-based derivative, KZ-41, that protects from radiation injury. Further preclinical efficacy studies were conducted to determine the radiomitigating activity of KZ-41. C57BL/6 mice received total body irradiation (TBI-LD80/30, ¹³7Cs; ∼2 min) followed by either normal saline or KZ-41 (100 mg/kg sc ∼26 h post-TBI). KZ-41 increased 30-day survival by approximately 45% compared with vehicle controls (P < 0.05). To further investigate the potential radiomodulating mechanisms of KZ-41, we developed a combined radiation and vascular injury model. C57BL/6 mice surgically fixed with dorsal windows for dermal vasculature imaging received either sham or TBI (¹³7Cs; 6 Gray). Postcapillary venule injury was induced (24, 48, 72, and 96 h post-TBI) followed by imaging at 5 min and 24 h to assess clot formation and blood flow. Impairment in flow (P < 0.05) and clot formation (P < 0.05) were observed as early as 48 and 72 h, respectively. Thus, vascular injury 72 h post-TBI was used to evaluate intervention (KZ-41; 100 mg/kg i.p. at 12, 36, and 60 h post-TBI) on radiation-induced changes in both flow and clot formation. KZ-41, although not improving flow, increased clot formation (P < 0.05). Platelet counts were lower in both irradiated groups compared with sham controls (P < 0.05). In summary, KZ-41 exerts radiomitigating activity in lethally irradiated mice. Imaging results suggest KZ-41 exerts radiomitigating activity through mechanisms involving promotion of initial clot formation and vascular flow restoration. The imaging model described herein is useful for further examination of radiation-induced vascular injury repair mechanisms.

Gold nanostars: surfactant-free synthesis, 3D modelling, and two-photon photoluminescence imaging.

Understanding the control of the optical and plasmonic properties of unique nanosystems--gold nanostars--both experimentally and theoretically permits superior design and fabrication for biomedical applications. Here, we present a new, surfactant-free synthesis method of biocompatible gold nanostars with adjustable geometry such that the plasmon band can be tuned into the near-infrared region 'tissue diagnostic window', which is most suitable for in vivo imaging. Theoretical modelling was performed for multiple-branched 3D nanostars and yielded absorption spectra in good agreement with experimental results. The plasmon band shift was attributed to variations in branch aspect ratio, and the plasmon band intensifies with increasing branch number, branch length, and overall star size. Nanostars showed an extremely strong two-photon photoluminescence (TPL) process. The TPL imaging of wheat-germ agglutinin (WGA) functionalized nanostars on BT549 breast cancer cells and of PEGylated nanostars circulating in the vasculature, examined through a dorsal window chamber in vivo in laboratory mouse studies, demonstrated that gold nanostars can serve as an efficient contrast agent for biological imaging applications.

In vivo particle tracking and photothermal ablation using plasmon-resonant gold nanostars.

Gold nanostars offer unique plasmon properties that efficiently transduce photon energy into heat for photothermal therapy. Nanostars, with their small core size and multiple long thin branches, exhibit high absorption cross-sections that are tunable in the near-infrared region with relatively low scattering effect, making them efficient photothermal transducers. Here, we demonstrate particle tracking and photothermal ablation both in vitro and in vivo. Using SKBR3 breast cancer cells incubated with bare nanostars, we observed photothermal ablation within 5 minutes of irradiation (980-nm continuous-wave laser, 15 W/cm2). On a mouse injected systemically with PEGylated nanostars for 2 days, extravasation of nanostars was observed and localized photothermal ablation was demonstrated on a dorsal window chamber within 10 minutes of irradiation (785-nm continuous-wave laser, 1.1 W/cm2). These preliminary results of plasmon-enhanced localized hyperthermia are encouraging and have illustrated the potential of gold nanostars as efficient photothermal agents in cancer therapy. FROM THE CLINICAL EDITOR: Gold nanostars are tunable in the near-infrared region with low scattering, thus enable photothermal therapy. Encouraging preliminary results of plasmon-enhanced localized hyperthermia both in vitro and in vivo demonstrate that Au nanostars may be efficient photothermal agents for cancer therapy.

Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion.

Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

Continuous delivery of IFN-beta promotes sustained maturation of intratumoral vasculature.

IFNs have pleiotropic antitumor mechanisms of action. The purpose of this study was to further investigate the effects of IFN-beta on the vasculature of human xenografts in immunodeficient mice. We found that continuous, systemic IFN-beta delivery, established with liver-targeted adeno-associated virus vectors, led to sustained morphologic and functional changes of the tumor vasculature that were consistent with vessel maturation. These changes included increased smooth muscle cell coverage of tumor vessels, improved intratumoral blood flow, and decreased vessel permeability, tumor interstitial pressure, and intratumoral hypoxia. Although these changes in the tumor vasculature resulted in more efficient tumor perfusion, further tumor growth was restricted, as the mature vasculature seemed to be unable to expand to support further tumor growth. In addition, maturation of the intratumoral vasculature resulted in increased intratumoral penetration of systemically administered chemotherapy. Finally, molecular analysis revealed increased expression by treated tumors of angiopoietin-1, a cytokine known to promote vessel stabilization. Induction of angiopoietin-1 expression in response to IFN-beta was broadly observed in different tumor lines but not in those with defects in IFN signaling. In addition, IFN-beta-mediated vascular changes were prevented when angiopoietin signaling was blocked with a decoy receptor. Thus, we have identified an alternative approach for achieving sustained vascular remodeling-continuous delivery of IFN-beta. In addition to restricting tumor growth by inhibiting further angiogenesis, maturation of the tumor vasculature also improved the efficiency of delivery of adjuvant therapy. These results have significant implications for the planning of combination anticancer therapy.

Effects of fractionated radiation on the brain vasculature in a murine model: blood-brain barrier permeability, astrocyte proliferation, and ultrastructural changes.

PURPOSE: Radiation therapy of CNS tumors damages the blood-brain barrier (BBB) and normal brain tissue. Our aims were to characterize the short- and long-term effects of fractionated radiotherapy (FRT) on cerebral microvasculature in mice and to investigate the mechanism of change in BBB permeability in mice. METHODS AND MATERIALS: Intravital microscopy and a cranial window technique were used to measure BBB permeability to fluorescein isothiocyanate (FITC)-dextran and leukocyte endothelial interactions before and after cranial irradiation. Daily doses of 2 Gy were delivered 5 days/week (total, 40 Gy). We immunostained the molecules to detect the expression of glial fibrillary acidic protein and to demonstrate astrocyte activity in brain parenchyma. To relate the permeability changes to endothelial ultrastructural changes, we used electron microscopy. RESULTS: Blood-brain barrier permeability did not increase significantly until 90 days after FRT, at which point it increased continuously until 180 days post-FRT. The number of adherent leukocytes did not increase during the study. The number of astrocytes in the cerebral cortex increased significantly; vesicular activity in endothelial cells increased beginning 90 days after irradiation, and most tight junctions stayed intact, although some were shorter and less dense at 120 and 180 days. CONCLUSIONS: The cellular and microvasculature response of the brain to FRT is mediated through astrogliosis and ultrastructural changes, accompanied by an increase in BBB permeability. The response to FRT is delayed as compared with single-dose irradiation treatment, and does not involve leukocyte adhesion. However, FRT induces an increase in the BBB permeability, as in the case of single-dose irradiation.

Tracking mesenchymal stromal cells using an ultra-bright TAT-functionalized plasmonic-active nanoplatform.

High-resolution tracking of stem cells remains a challenging task. An ultra-bright contrast agent with extended intracellular retention is suitable for in vivo high-resolution tracking of stem cells following the implantation. Here, a plasmonic-active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide-functionalized gold nanostars (TAT-GNS) that emit ultra-bright two-photon photoluminescence capable of tracking MSCs under high-resolution optical imaging. In vitro experiment showed TAT-GNS-labeled MSCs retained a similar differentiability to that of non-labeled MSCs controls. Due to their star shape, TAT-GNS exhibited greater intracellular retention than that of commercial Q-Tracker. In vivo imaging of TAT-GNS-labeled MSCs five days following intra-arterial injections in mice kidneys showed possible MSCs implantation in juxta-glomerular (JG) regions, but non-specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic-active nanoplatforms may be a useful intracellular tracking tool for stem cell research. An ultra-bright intracellular contrast agent is developed using TAT peptide-functionalized gold nanostars (TAT-GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two-photon photoluminescence and superior labeling efficiency than commercial Q-Tracker. Following renal implantation, some TAT-GNS-labeled MSCs permeate blood vessels and migrate to the juxta-glomerular region.

A Bioengineered Peptide that Localizes to and Illuminates Medulloblastoma: A New Tool with Potential for Fluorescence-Guided Surgical Resection.

Tumors of the central nervous system are challenging to treat due to the limited effectiveness and associated toxicities of chemotherapy and radiation therapy. For tumors that can be removed surgically, extent of malignant tissue resection has been shown to correlate with disease progression, recurrence, and survival. Thus, improved technologies for real-time brain tumor imaging are critically needed as tools for guided surgical resection. We previously engineered a novel peptide that binds with high affinity and unique specificity to αVß3, αVß5, and α5ß1 integrins, which are present on tumor cells, and the vasculature of many cancers, including brain tumors. In the current study, we conjugated this engineered peptide to a near infrared fluorescent dye (Alexa Fluor 680), and used the resulting molecular probe for non-invasive whole body imaging of patient-derived medulloblastoma xenograft tumors implanted in the cerebellum of mice. The engineered peptide exhibited robust targeting and illumination of intracranial medulloblastoma following both intravenous and intraperitoneal injection routes. In contrast, a variant of the engineered peptide containing a scrambled integrin-binding sequence did not localize to brain tumors, demonstrating that tumor-targeting is driven by specific integrin interactions. Ex vivo imaging was used to confirm the presence of tumor and molecular probe localization to the cerebellar region. These results warrant further clinical development of the engineered peptide as a tool for image-guided resection of central nervous system tumors.

Plasmonics-enhanced and optically modulated delivery of gold nanostars into brain tumor.

Plasmonics-active gold nanostars exhibiting strong imaging contrast and efficient photothermal transduction were synthesized for a novel pulsed laser-modulated plasmonics-enhanced brain tumor microvascular permeabilization. We demonstrate a selective, optically modulated delivery of nanoprobes into the tumor parenchyma with minimal off-target distribution.

Effects of irradiation on brain vasculature using an in situ tumor model.

PURPOSE: Damage to normal tissue is a limiting factor in clinical radiotherapy (RT). We tested the hypothesis that the presence of tumor alters the response of normal tissues to irradiation using a rat in situ brain tumor model. METHODS AND MATERIALS: Intravital microscopy was used with a rat cranial window to assess the in situ effect of rat C6 glioma on peritumoral tissue with and without RT. The RT regimen included 40 Gy at 8 Gy/day starting Day 5 after tumor implant. Endpoints included blood-brain barrier permeability, clearance index, leukocyte-endothelial interactions and staining for vascular endothelial growth factor (VEGF) glial fibrillary acidic protein, and apoptosis. To characterize the system response to RT, animal survival and tumor surface area and volume were measured. Sham experiments were performed on similar animals implanted with basement membrane matrix absent of tumor cells. RESULTS: The presence of tumor alone increases permeability but has little effect on leukocyte-endothelial interactions and astrogliosis. Radiation alone increases tissue permeability, leukocyte-endothelial interactions, and astrogliosis. The highest levels of permeability and cell adhesion were seen in the model that combined tumor and irradiation; however, the presence of tumor appeared to reduce the volume of rolling leukocytes. Unirradiated tumor and peritumoral tissue had poor clearance. Irradiated tumor and peritumoral tissue had a similar clearance index to irradiated and unirradiated sham-implanted animals. Radiation reduces the presence of VEGF in peritumoral normal tissues but did not affect the amount of apoptosis in the normal tissue. Apoptosis was identified in the tumor tissue with and without radiation. CONCLUSIONS: We developed a novel approach to demonstrate that the presence of the tumor in a rat intracranial model alters the response of normal tissues to irradiation.

Dual-mode IVUS transducer for image-guided brain therapy: preliminary experiments.

In this study, we investigated the feasibility of using 3.5-Fr intravascular ultrasound (IVUS) catheters for minimally-invasive, image-guided hyperthermia treatment of tumors in the brain. Feasibility was demonstrated by: (1) retro-fitting a commercial 3.5-Fr IVUS catheter with a 5 × 0.5 × 0.22 mm PZT-4 transducer for 9-MHz imaging and (2) testing an identical transducer for therapy potential with 3.3-MHz continuous-wave excitation. The imaging transducer was compared with a 9-Fr, 9-MHz ICE catheter when visualizing the post-mortem ovine brain and was also used to attempt vascular access to an in vivo porcine brain. A net average electrical power input of 700 mW was applied to the therapy transducer, producing a temperature rise of +13.5°C at a depth of 1.5 mm in live brain tumor tissue in the mouse model. These results suggest that it may be feasible to combine the imaging and therapeutic capabilities into a single device as a clinically-viable instrument.

Radiation-induced astrogliosis and blood-brain barrier damage can be abrogated using anti-TNF treatment.

PURPOSE: In this article, we investigate the role of tumor necrosis factor-alpha (TNF) in the initiation of acute damage to the blood-brain barrier (BBB) and brain tissue following radiotherapy (RT) for CNS tumors. METHODS AND MATERIALS: Intravital microscopy and a closed cranial window technique were used to measure quantitatively BBB permeability to FITC-dextran 4.4-kDa molecules, leukocyte adhesion (Rhodamine-6G) and vessel diameters before and after 20-Gy cranial radiation with and without treatment with anti-TNF. Immunohistochemistry was used to quantify astrogliosis post-RT and immunofluorescence was used to visualize protein expression of TNF and ICAM-1 post-RT. Recombinant TNF (rTNF) was used to elucidate the role of TNF in leukocyte adhesion and vessel diameter. RESULTS: Mice treated with anti-TNF showed significantly lower permeability and leukocyte adhesion at 24 and 48 h post-RT vs. RT-only animals. We observed a significant decrease in arteriole diameters at 48 h post-RT that was inhibited in TNF-treated animals. We also saw a significant increase in activated astrocytes following RT that was significantly lower in the anti-TNF-treated group. In addition, immunofluorescence showed protein expression of TNF and ICAM-1 in the cerebral cortex that was inhibited with anti-TNF treatment. Finally, administration of rTNF induced a decrease in arteriole diameter and a significant increase in leukocyte adhesion in venules and arterioles. CONCLUSIONS: TNF plays a significant role in acute changes in BBB permeability, leukocyte adhesion, arteriole diameter, and astrocyte activation following cranial radiation. Treatment with anti-TNF protects the brain's microvascular network from the acute damage following RT.