Conor P. Foley

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.

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.

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.

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.

Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors

Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

501. Radioiodinated Adeno-Associated Virus: A Promising New Approach for Monitoring Gene Therapy

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a form of Batten disease that is caused by mutations in the CLN2 gene. These defects cause widespread neurodegeneration resulting in death by the age of 10-12 years. One treatment for LINCL that has shown promise in animal and clinical studies is gene therapy using adeno-associated virus (AAV) as a vehicle to deliver the CLN2 gene throughout the brain. This is currently accomplished by direct infusion, but there is no way to measure the spatial distribution of administered vector. Iodine-124 labeling of the viral capsid may offer a means for non-invasive determination of spatial distribution using MicroPET imaging. The production of AAVrh.10CLN2 under GMP conditions met endotoxin, mycoplasma, sterility and transgene expression release criteria. Purified AAVrh.10CLN2 was concentrated to 1013 gene copies/ml. Labeling with Na124I was carried out at 2-5 0C under mild oxidizing conditions in pH 7.5 buffer. Following radiolabeling, the product mixture was purified using an anion exchange cartridge and centrifugal filtration. Purified I124-AAVrh.10CLN2 was formulated in a pH 7.4 PBS buffer. The sterile formulation was injected (2 ml at 2.5 μCi/ml) intraparenchymally to the striatum in the murine brain and imaged on a Siemens Inveon MicroPET scanner. Thirty minute PET scans were acquired for each mouse (n=3). For the control group, we performed the same procedure using free Na 124I alone (n=3). The radiolabeling efficiency of AAV was in the range of 12-14%. PET/CT imaging clearly demonstrated the spatial distribution of vector over a ten day period, with minimal 124I uptake in the unblocked thyroid. In contrast, free iodide was rapidly cleared from the brain within 2 days (Figure 1). In conclusion, adeno-associated virus was successfully labeled with 124I and its distribution in the mouse brain was observed. This radiolabeling approach has the potential for wide application in gene therapy trials. View Large Image | Download PowerPoint Slide