Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption.

The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption that we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single administration.

Cannulation of the internal carotid artery in mice: a novel technique for intra-arterial delivery of therapeutics.

We have developed a novel minimally invasive technique for the intra-arterial delivery of therapeutics to the mouse brain. CD-1 mice were anesthetized and placed in a lateral decubitus position. A 10mm midline longitudinal incision was made over the thyroid bone. The omohyoid and sternomastoid muscles were retracted to expose the common carotid artery and external carotid artery (ECA). To maximize delivery of administered agents, the superior thyroid artery was ligated or coagulated, and the occipital artery and the pterygopalatine artery (PPA) were temporarily occluded with 6-0 prolene suture. The ECA was carefully dissected and a permanent ligature was placed on its distal segment while a temporary 6-0 prolene ligature was placed on the proximal segment in order to obtain a flow-free segment of vessel. A sterilized 169 µm outer diameter polyimide microcatheter was introduced into the ECA and advanced in retrograde fashion toward the carotid bifurcation. The catheter was then secured and manually rotated so that the microcatheter tip was oriented cephalad in the internal carotid artery (ICA). We were able to achieve reproducible results for selective ipsilateral hemispheric carotid injections of mannitol mediated therapeutics and/or gadolinium-based MRI contrast agent. Survival rates were dependent on the administered agent and ranged from 78 to 90%. This technique allows for reproducible delivery of agents to the ipsilateral cerebral hemisphere by utilizing anterograde catheter placement and temporary ligation of the PPA. This method is cost-effective and associated with a low rate of morbimortality.

Orthotopic glioblastoma stem-like cell xenograft model in mice to evaluate intra-arterial delivery of bevacizumab: from bedside to bench.

Bevacizumab (BV), a humanized monocolonal antibody directed against vascular endothelial growth factor (VEGF), is a standard intravenous (IV) treatment for recurrent glioblastoma multiforme (GBM), that has been introduced recently as an intra-arterial (IA) treatment modality in humans. Since preclinical models have not been reported, we sought to develop a tumor stem cell (TSC) xenograft model to investigate IA BV delivery in vivo. Firefly luciferase transduced patient TSC were injected into the cortex of 35 nude mice. Tumor growth was monitored weekly using bioluminescence imaging. Mice were treated with either intraperitoneal (IP) or IA BV, with or without blood-brain barrier disruption (BBBD), or with IP saline injection (controls). Tumor tissue was analyzed using immunohistochemistry and western blot techniques. Tumor formation occurred in 31 of 35 (89%) mice with a significant signal increase over time (p=0.018). Post mortem histology revealed an infiltrative growth of TSC xenografts in a similar pattern compared to the primary human GBM. Tumor tissue analyzed at 24 hours after treatment revealed that IA BV treatment with BBBD led to a significantly higher intratumoral BV concentration compared to IA BV alone, IP BV or controls (p<0.05). Thus, we have developed a TSC-based xenograft mouse model that allows us to study IA chemotherapy. However, further studies are needed to analyze the treatment effects after IA BV to assess tumor progression and overall animal survival.

Intra-arterial bevacizumab with blood brain barrier disruption in a glioblastoma xenograft model.

PURPOSE: In this study we investigated the treatment response and survival of intra-arterial (IA) compared to intra-peritoneal (IP) delivery of bevacizumab (BV) in a glioblastoma (GBM) xenograft mouse model. METHODS: 3x10(5) U87-Luc cells were stereotactically implanted into the cortex of 35 nude mice and grouped for treatment (n = 7 in each group): IP saline (group 1), single IP BV (group 2), biweekly IP BV for 3 weeks (group 3), single intra-arterial (IA) BV alone (group 4) and single IA BV with blood brain barrier disruption (BBBD) (group 5). Tumor growth was monitored every 3 to 4 days using bioluminescence imaging (BLI) and survival was analyzed by the Kaplan Meier method. Tumor tissue was analyzed using H&E staining and immunohistochemistry. RESULTS: Based on BLI, BV treated mice showed a delayed tumor growth over time compared to control. Kaplan Meier analysis demonstrated a median survival time of 28 days for group 1,31 days for group 2, 34 days for group 3, 36 days for group 4 and 36 days for group 5 (p < 0.0001). Mice treated with repeated IP BV (p = 0.003) or single IA BV with (p = 0.015) or without (p = 0.005) BBBD showed a significant survival benefit compared to single IP BV treated mice. Post mortem analysis revealed a histological pattern with a more discontinuous border between tumor and mouse brain in the repeated IP BV and single IA BV with or without BBBD treated mice compared to the sharply defined edges of single IP BV treated and control mice. CONCLUSIONS: In this study we showed a significant survival benefit of repeated IP BV and single IA BV with or without BBBD treated mice compared to single IP BV treated and control mice in a U87 xenograft model.

Real-time imaging of perivascular transport of nanoparticles during convection-enhanced delivery in the rat cortex.

Convection-enhanced delivery (CED) is a promising technique for administering large therapeutics that do not readily cross the blood brain barrier to neural tissue. It is of vital importance to understand how large drug constructs move through neural tissue during CED to optimize construct and delivery parameters so that drugs are concentrated in the targeted tissue, with minimal leakage outside the targeted zone. Experiments have shown that liposomes, viral vectors, high molecular weight tracers, and nanoparticles infused into neural tissue localize in the perivascular spaces of blood vessels within the brain parenchyma. In this work, we used two-photon excited fluorescence microscopy to monitor the real-time distribution of nanoparticles infused in the cortex of live, anesthetized rats via CED. Fluorescent nanoparticles of 24 and 100 nm nominal diameters were infused into rat cortex through microfluidic probes. We found that perivascular spaces provide a high permeability path for rapid convective transport of large nanoparticles through tissue, and that the effects of perivascular spaces on transport are more significant for larger particles that undergo hindered transport through the extracellular matrix. This suggests that the vascular topology of the target tissue volume must be considered when delivering large therapeutic constructs via CED.

Microfluidic probes in the treatment of brain-related diseases.

Many new therapeutic compounds have been developed that target malignancies and other disorders of the brain. However, delivering these compounds to diseased tissue remains a difficult challenge. One option for local drug delivery in the brain is direct infusion of the compounds through a catheter into the brain parenchyma. Over the last decade, new infusion catheters have been developed to improve this delivery method. Some of these catheters are needles or cannulas that have been modified specifically to increase the infusion rate that can be achieved without leakage of the infusate out of the brain. Other new catheters have been fabricated using micromachining techniques adapted from electronics manufacturing. These microfabricated catheters can achieve comparable infusion rates as standard needles, but they also can incorporate features that would be difficult to build into needles or cannulas to improve drug delivery. This article reviews the development of these devices, their performance in preclinical studies and their potential benefits to neural drug delivery.

Flexible microfluidic devices supported by biodegradable insertion scaffolds for convection-enhanced neural drug delivery.

Convection enhanced delivery (CED) can improve the spatial distribution of drugs delivered directly to the brain. In CED, drugs are infused locally into tissue through a needle or catheter inserted into brain parenchyma. Transport of the infused material is dominated by convection, which enhances drug penetration into tissue compared with diffusion mediated delivery. We have fabricated and characterized an implantable microfluidic device for chronic convection enhanced delivery protocols. The device consists of a flexible parylene-C microfluidic channel that is supported during its insertion into tissue by a biodegradable poly(DL-lactide-co-glycolide) scaffold. The scaffold is designed to enable tissue penetration and then erode over time, leaving only the flexible channel implanted in the tissue. The device was able to reproducibly inject fluid into neural tissue in acute experiments with final infusate distributions that closely approximate delivery from an ideal point source. This system shows promise as a tool for chronic CED protocols.

Dilation and degradation of the brain extracellular matrix enhances penetration of infused polymer nanoparticles.

This study investigates methods of manipulating the brain extracellular matrix (ECM) to enhance the penetration of nanoparticle drug carriers in convection-enhanced delivery (CED). A probe was fabricated with two independent microfluidic channels to infuse, either simultaneously or sequentially, nanoparticles and ECM-modifying agents. Infusions were performed in the striatum of the normal rat brain. Monodisperse polystyrene particles with a diameter of 54 nm were used as a model nanoparticle system. Because the size of these particles is comparable to the effective pore size of the ECM, their transport may be significantly hindered compared with the transport of low molecular weight molecules. To enhance the transport of the infused nanoparticles, we attempted to increase the effective pore size of the ECM by two methods: dilating the extracellular space and degrading selected constituents of the ECM. Two methods of dilating the extracellular space were investigated: co-infusion of nanoparticles and a hyperosmolar solution of mannitol, and pre-infusion of an isotonic buffer solution followed by infusion of nanoparticles. These treatments resulted in an increase in the nanoparticle distribution volume of 51% and 123%, respectively. To degrade hyaluronan, a primary structural component of the brain ECM, a pre-infusion of hyaluronidase (20,000 U/mL) was followed after 30 min by infusion of nanoparticles. This treatment resulted in an increase in the nanoparticle distribution of 64%. Our results suggest that both dilation and enzymatic digestion can be incorporated into CED protocols to enhance nanoparticle penetration.