Convection-enhanced Delivery Method Research Articles

Intracellular trafficking and functional monitoring of miRNA delivery in glioblastoma using lipopolyplexes and the miRNA-ON RILES reporter system

MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.

Convection-enhanced Drug Delivery for Glioblastoma: A Systematic Review Focused on Methodological Differences in the Use of the Convection-enhanced Delivery Method.

Glioblastoma (GBM) is a leading cause of brain cancer-related death. The blood–brain barrier (BBB) prevents the transport of most systemic delivered molecules to the brain. This constitutes a major problem in the therapy of brain tumors. In the last decade, numerous different drug-delivery approaches have been developed to overcome the BBB. The objective of this study is to provide an overview of the methodological aspects used in all preclinical and clinical studies published from 2011 to 2016 where convection-enhanced delivery (CED) was used for drug delivery in the treatment of GBM. A systematic review of English articles published in the past 5 years was undertaken using PubMed and Embase. The search terms (brain tumor [MeSH Terms]) AND (CED OR convection enhanced delivery) were used in PubMed and a similar search was carried out in Embase using their “multi-field search.” All studies using CED on an intracranial GBM model were included. The search resulted in 151 hits after duplicates were removed. In total, 30 studies were included in the review. Of these, two publications studied the technical aspects of the CED method. Furthermore, only one study was a clinical study. The research field is focused on preclinical drug development trials and less emphasis is placed on the CED technique itself. However, it is important that future studies focus on establishing optimal protocols for the use of CED in rodents as well as for big brain models to be able to use the CED method in patients with GBM.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood-brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.

Recombinant adeno-associated virus (rAAV) has become an extremely useful tool for the study of gene over expression or knockdown in the central nervous system of experimental animals. One disadvantage of intracranial injections of rAAV vectors into the brain parenchyma has been restricted distribution to relatively small volumes of the brain. Convection enhanced delivery (CED) is a method for delivery of clinically relevant amounts of therapeutic agents to large areas of the brain in a direct intracranial injection procedure. CED uses bulk flow to increase the hydrostatic pressure and thus improve volume distribution. The CED method has shown robust gene transfer and increased distribution within the CNS and can be successfully used for different serotypes of rAAV for increased transduction of the mouse CNS. This chapter details the surgical injection of rAAV by CED into a mouse brain.

Neurotrophic factors: promising candidates in tissue regeneration.

Neurotrophic factors, also referred as neurotrophins, are growth factors originally identified in the nervous system. As indicated by the name, neurotrophic factors are essential for the survival and development of neurons. Nerve growth factor (NGF) is the first identified neurotrophic factor, and is necessary for the development of sensory neurons in the dorsal root ganglia, and cholinergic neurons (Levi-Montalcini and Hamburger, 1951). Brain derived neurotrophic factor (BDNF) is required for survival of sensory neuron in the dorsal root ganglia, hippocampus and cerebral cortex, but not for motor neurons (Conover et al., 1995). On the other hand, Neurotrophin-3 (NT-3) is critical for the survival and proliferation of both sensory neurons, and motor neurons (Woolley et al., 2005). Glial cell derived neurotrophic factors (GDNF) family of ligands (GFLs) include GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). GFLs enhance the survival of dopaminergic neurons in the midbrain (Lin et al., 1993), as well as the survival of enteric neurons in the gastrointestinal tract (Sanchez et al., 1996). Neuropoietic cytokines, such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are also considered members of neurotrophic factor family, and promote survival of multiple types of neurons and glial cells (Stolp, 2013). There are numerous reports on the role of neurotrophic factors in healthy and damaged neurons. Neurotrophic factors promote the development, survival, proliferation, and differentiation of healthy neurons. They also exhibit a role in anti-inflammation, anti-apoptosis, re-myelination and axon regeneration, thereby facilitating neuronal tissue regeneration. More recent discoveries indicate that neurotrophic factors are expressed in non-neuronal tissues, and may play a role in the tissue repair as well. NGF, BDNF, and NT3 have been noted to promote proliferation, vascularization, and neural differentiation in mesenchymal cells such as bone marrow stem cells (BMSCs) and fibroblasts. NT3 and GDNF are reported to promote ovarian follicle differentiation (Nilsson et al., 2009) and spermatogenesis respectively (Meng et al., 2000). GDNF is also critical in the ureteric bud origination during kidney development (Sanchez et al., 1996). In addition, increased level of NGF is linked with improved regeneration of cardiomyocytes (Caporali et al., 2008) and pancreatic islet (Hata et al., 2015)(Figure 1). Figure 1 Schematic map of neurotrophic factors and their functions. In a recent study, we reported that GDNF is enriched in the salivary gland stem cells (SSCs), but not in the acinar cell. More interestingly, the expression of GDNF in the SSCs is elevated post radiation to the head and neck region in both mice and humans. This neurotrophic factor is able to increase the proliferation of SSCs in a dose dependent manner in vitro. We also found that one time delivery of GDNF to the salivary gland either before or post radiation to the head and neck region of the mice would significantly increase the number of surviving SSCs, improve the morphology of the salivary gland and rescue the saliva secretion. The data suggested that GDNF protects the salivary gland from radiation induced damage by promoting salivary gland stem cell regeneration and proliferation in vivo (Xiao et al., 2014). Besides the healthy tissues, neurotrophic factors are also reported to regulate tumor cell growth, invasion, metastasis along nerve, and resistance to therapy. BDNF is found to enhance the proliferation of malignant gliomas, breast cancer and lung cancer. GDNF pathways have been associated with growth and metastasis of neuroblastoma, glioma, breast cancer, small cell lung cancer, thyroid cancer, pancreatic cancer, colon cancer and testicular cancer. It would be necessary to test whether GDNF promotes head and cancer (HNC) survival, proliferation and migration, before applying it to the post radiation HNC patients. The mechanism through which neurotrophic factors mediate tissue regeneration and tumor cell behaviorstill needs further investigation. NGF, BDNF and NTs (Neurotrophins) bind to the neurotrophin receptor p75 at low affinity. The binding between NGF, BDNF, NTs and the receptor tyrosine kinases (Trk) are stronger and more specific. NGF specifically binds to TrkA while BDNF preferentially binds to TrkB. NT-3 preferentially binds to TrkC, but can also activate TrkA and TrkB, while NT-4/5 preferentially binds to TrkB. The PI3K/AKT, MEK/ERK are reported to be downstream of the neurotrophin activation (Skaper, 2012). GDNF is the most well studied member of the GFL family. It preferentially binds to the receptor GFRα1, which then activates the receptor tyrosine kinase RET or the neural cell adhesion molecule (NCAM) (Zhou et al., 2003). The PI3K/AKT, MEK/ERK, SRC/c-Jun kinase (JNK), FYN/focal adhesion kinase (FAK) pathways have all been reported to be downstream of the GDNF signal. In our study, elevated GDNF were found co-localized with NCAM and phosphorylated FAK signal in the SSC of post radiation HNC patients. Although phosphorylated AKT and phosphorylated ERK level both increased in the post radiation salivary gland, the signal was neither enriched in SSCs nor overlay with GDNF. The results indicate that GDNF mainly work through the NCAM and FAK pathway in promoting salivary gland regeneration (Xiao et al., 2014). These results indicate that neurotrophic factors could be promising drug candidates for tissue regeneration in the future. Neurotrophic factors and their modulators are tested in clinical trials already for treating neurodegenerative diseases, such as Alzheimer's disease ({type:clinical-trial,attrs:{text:NCT01163825,term_id:NCT01163825}}NCT01163825, {type:clinical-trial,attrs:{text:NCT00876863,term_id:NCT00876863}}NCT00876863, {type:clinical-trial,attrs:{text:NCT02271750,term_id:NCT02271750}}NCT02271750) and Parkinson's disease ({type:clinical-trial,attrs:{text:NCT00985517,term_id:NCT00985517}}NCT00985517, {type:clinical-trial,attrs:{text:NCT01621581,term_id:NCT01621581}}NCT01621581), progressive supranuclear palsy ({type:clinical-trial,attrs:{text:NC5903,term_id:NC5903}}NC5903), traumatic brain injury ({type:clinical-trial,attrs:{text:NCT01212679,term_id:NCT01212679}}NCT01212679, {type:clinical-trial,attrs:{text:NCT02276079,term_id:NCT02276079}}NCT02276079), and cerebral radiation necrosis ({type:clinical-trial,attrs:{text:NCT02032147,term_id:NCT02032147}}NCT02032147). Neurotrophic factors are delivered to patients through direct injection, infusion pumps, encapsulated particles, virus mediated infection, etc. A clinical study on the safety and efficacy of a recombinant human NGF eye drop solution is ongoing recruiting patients with persistent epithelial defect of cornea or keratitis of the cornea ({type:clinical-trial,attrs:{text:NCT01756456,term_id:NCT01756456}}NCT01756456, {type:clinical-trial,attrs:{text:NCT01411657,term_id:NCT01411657}}NCT01411657). Neuropoietic cytokine CNTF has been shown in several clinical models to enhance survival and regeneration of the retinal ganglion neurons ({type:clinical-trial,attrs:{text:NCT01408472,term_id:NCT01408472}}NCT01408472). There are controversies about applying the neurotrophic factors. Amgen withheld the clinical trial of GDNF on patients with Parkinson's in 2004 concerning the efficacy and safety issues. With the increasing evidences indicating the drug delivery method may play critical role in the effect, MedGenesis Therapeutix Inc. reopened the trial using convection enhanced delivery method to deliver GDNF. It is promising to see more bedside studies on the potential therapeutic effect of neurotrophic factors, as more and more benchside studies demonstrate that neurotrophic factors play important roles in regulating stem cell behavior and promotes tissue regeneration.

Convection-enhanced delivery of nanodiamond drug delivery platforms for intracranial tumor treatment

This study examined a novel drug delivery system for treatment of malignant brain gliomas: DOX complexed with nanodiamonds (ND-Dox), and administered via convection-enhanced delivery (CED). Drug retention and toxicity were examined in glioma cell lines, and distribution, retention and toxicity were examined in normal rat parenchyma. Efficacy was assessed in a bioluminescence rodent tumor model. NDs markedly enhanced DOX uptake and retention in glioma cells. ND-Dox delivered via CED extended DOX retention and localized DOX toxicity in normal rodent parenchyma, and was significantly more efficient at killing tumor cells than uncomplexed DOX. Outcomes from this work suggest that CED of ND-Dox is a promising approach for brain tumor treatment. From the Clinical EditorIn this paper, nanodiamonds were utilized to enhance delivery of DOX in a preclinical glioma model using a convection-enhanced delivery method, demonstrating remarkably enhanced efficacy.

Convection-enhanced delivery of M13 bacteriophage to the brain

Recent studies indicate that M13 bacteriophage, a very large nanoparticle, binds to β-amyloid and α-synuclein proteins, leading to plaque disaggregation in models of Alzheimer and Parkinson disease. To determine the feasibility, safety, and characteristics of convection-enhanced delivery (CED) of M13 bacteriophage to the brain, the authors perfused primate brains with bacteriophage. Four nonhuman primates underwent CED of M13 bacteriophage (900 nm) to thalamic gray matter (4 infusions) and frontal white matter (3 infusions). Bacteriophage was coinfused with Gd-DTPA (1 mM), and serial MRI studies were performed during infusion. Animals were monitored for neurological deficits and were killed 3 days after infusion. Tissues were analyzed for bacteriophage distribution. Real-time T1-weighted MRI studies of coinfused Gd-DTPA during infusion demonstrated a discrete region of perfusion in both thalamic gray and frontal white matter. An MRI-volumetric analysis revealed that the mean volume of distribution (Vd) to volume of infusion (Vi) ratio of M13 bacteriophage was 2.3 ± 0.2 in gray matter and 1.9 ± 0.3 in white matter. The mean values are expressed ± SD. Immunohistochemical analysis demonstrated mean Vd:Vi ratios of 2.9 ± 0.2 in gray matter and 2.1 ± 0.3 in white matter. The Gd-DTPA accurately tracked M13 bacteriophage distribution (the mean difference between imaging and actual bacteriophage Vd was insignificant [p > 0.05], and was -2.2% ± 9.9% in thalamic gray matter and 9.1% ± 9.5% in frontal white matter). Immunohistochemical analysis revealed evidence of additional spread from the initial delivery site in white matter (mean Vd:Vi, 16.1 ± 9.1). All animals remained neurologically intact after infusion during the observation period, and histological studies revealed no evidence of toxicity. The CED method can be used successfully and safely to distribute M13 bacteriophage in the brain. Furthermore, additional white matter spread after infusion cessation enhances distribution of this large nanoparticle. Real-time MRI studies of coinfused Gd-DTPA (1 mM) can be used for accurate tracking of distribution during infusion of M13 bacteriophage.

Convection-enhanced delivery and systemic mannitol increase gene product distribution of AAV vectors 5, 8, and 9 and increase gene product in the adult mouse brain

The use of recombinant adeno-associated viral (rAAV) vectors as a means of gene delivery to the central nervous system has emerged as a potentially viable method for the treatment of several types of degenerative brain diseases. However, a limitation of typical intracranial injections into the adult brain parenchyma is the relatively restricted distribution of the delivered gene to large brain regions such as the cortex, presumably due to confined dispersion of the injected particles. Optimizing the administration techniques to maximize gene distribution and gene expression is an important step in developing gene therapy studies. Here, we have found additive increases in distribution when 3 methods to increase brain distribution of rAAV were combined. The convection enhanced delivery (CED) method with the step-design cannula was used to deliver rAAV vector serotypes 5, 8 and 9 encoding GFP into the hippocampus of the mouse brain. While the CED method improved distribution of all 3 serotypes, the combination of rAAV9 and CED was particularly effective. Systemic mannitol administration, which reduces intracranial pressure, also further expanded distribution of GFP expression, in particular, increased expression on the contralateral hippocampi. These data suggest that combining advanced injection techniques with newer rAAV serotypes greatly improves viral vector distribution, which could have significant benefits for implementation of gene therapy strategies.

Clinically Relevant Effects of Convection-Enhanced Delivery of AAV2-GDNF on the Dopaminergic Nigrostriatal Pathway in Aged Rhesus Monkeys

Growth factor therapy for Parkinson's disease offers the prospect of restoration of dopaminergic innervation and/or prevention of neurodegeneration. Safety and efficacy of an adeno-associated virus (AAV2) encoding human glial cell-derived neurotrophic factor (GDNF) was investigated in aged nonhuman primates. Positron emission tomography with 6-[(18)F]-fluoro-l-m-tyrosine (FMT-PET) in putamen was assessed 3 months before and after AAV2 infusion. In the right putamen, monkeys received either phosphate-buffered saline or low-dose (LD) or high-dose (HD) AAV2-GDNF. Monkeys that had received putaminal phosphate-buffered saline (PBS) infusions additionally received either PBS or HD AAV2-GDNF in the right substantia nigra (SN). The convection-enhanced delivery method used for infusion of AAV2-GDNF vector resulted in robust volume of GDNF distribution within the putamen. AAV2-GDNF increased FMT-PET uptake in the ipsilateral putamen as well as enhancing locomotor activity. Within the putamen and caudate, the HD gene transfer mediated intense GDNF fiber and extracellular immunoreactivity (IR). Retrograde and anterograde transport of GDNF to other brain regions was observed. AAV2-GDNF did not significantly affect dopamine in the ipsilateral putamen or caudate, but increased dopamine turnover in HD groups. HD putamen treatment increased the density of dopaminergic terminals in these regions. HD treatments, irrespective of the site of infusion, increased the number of nonpigmented TH-IR neurons in the SN. AAV2-GDNF gene transfer does not appear to elicit adverse effects, delivers therapeutic levels of GDNF within target brain areas, and enhances utilization of striatal dopamine and dopaminergic nigrostriatal innervation.

Real-time imaging of convection-enhanced delivery of viruses and virus-sized particles

Despite recent evidence showing that convection-enhanced delivery (CED) of viruses and virus-sized particles to the central nervous system (CNS) is possible, little is known about the factors influencing distribution of these vectors with convection. To better define the delivery of viruses and virus-sized particles in the CNS, and to determine optimal parameters for infusion, the authors coinfused adeno-associated virus ([AAV], 24-nm diameter) and/or ferumoxtran-10 (24 nm) by using CED during real-time magnetic resonance (MR) imaging. Sixteen rats underwent intrastriatal convective coinfusion with 4 microl of 35S-AAV capsids (0.5-1.0 x 10(14) viral particles/ml) and increasing concentrations (0.1, 0.5, 1, and 5 mg/ml) of a similar sized iron oxide MR imaging agent (ferumoxtran-10). Five nonhuman primates underwent either convective coinfusion of 35S-AAV capsids and 1 mg/ml ferumoxtran-10 (striatum, one animal) or infusion of 1 mg/ml ferumoxtran-10 alone (striatum in two animals; frontal white matter in two). Clinical effects, MR imaging studies, quantitative autoradiography, and histological data were analyzed. Real-time, T2-weighted MR imaging of ferumoxtran-10 during infusion revealed a clearly defined hypointense region of perfusion. Quantitative autoradiography confirmed that MR imaging of ferumoxtran-10 at a concentration of 1 mg/ml accurately tracked viral capsid distribution in the rat and primate brain (the mean difference in volume of distribution [Vd] was 7 and 15% in rats and primates, respectively). The Vd increased linearly with increasing volume of infusion (Vi) (R2 = 0.98). The mean Vd/Vi ratio was 4.1 +/- 0.2 (mean +/- standard error of the mean) in gray and 2.3 +/- 0.1 in white matter (p < 0.01). The distribution of infusate was homogeneous. Postinfusion MR imaging revealed leakback along the cannula track at infusion rates greater than 1.5 microl/minute in primate gray and white matter. No animal had clinical or histological evidence of toxicity. The CED method can be used to deliver AAV capsids and similar sized particles to the CNS safely and effectively over clinically relevant volumes. Moreover, real-time MR imaging of ferumoxtran-10 during infusion reveals that AAV capsids and similar sized particles have different convective delivery properties than smaller proteins and other compounds.

Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents

Clinical application of the convection-enhanced delivery (CED) technique is currently limited by low infusion speed and reflux of the delivered agent. The authors developed and evaluated a new step-design cannula to overcome present limitations and to introduce a rapid, reflux-free CED method for future clinical trials. The CED of 0.4% trypan blue dye was performed in agarose gel to test cannula needles for distribution and reflux. Infusion rates ranging from 0.5 to 50 microl/minute were used. Agarose gel findings were translated into a study in rats and then in cynomolgus monkeys (Macacafascicularis) by using trypan blue and liposomes to confirm the efficacy of the reflux-free step-design cannula in vivo. Results of agarose gel studies showed reflux-free infusion with high flow rates using the step-design cannula. Data from the study in rats confirmed the agarose gel findings and also revealed increasing tissue damage at a flow rate above 5-microl/minute. Robust reflux-free delivery and distribution of liposomes was achieved using the step-design cannula in brains in both rats and nonhuman primates. The authors developed a new step-design cannula for CED that effectively prevents reflux in vivo and maximizes the distribution of agents delivered in the brain. Data in the present study show reflux-free infusion with a constant volume of distribution in the rat brain over a broad range of flow rates. Reflux-free delivery of liposomes into nonhuman primate brain was also established using the cannula. This step-design cannula may allow reflux-free distribution and shorten the duration of infusion in future clinical applications of CED in humans.

Progressive and extensive dopaminergic degeneration induced by convection-enhanced delivery of 6-hydroxydopamine into the rat striatum: a novel rodent model of Parkinson disease

A striatal dopamine lesion induces progressive nigral degeneration in rodents; however, intrastriatal injection of 6-hydroxydopamine (6-OHDA) causes only limited lesions due to spontaneous regeneration of the neurons that survive. To make an extensive lesion, the authors used a convection-enhanced delivery (CED) method for intrastriatal infusion of 6-OHDA and evaluated the animals for a model of Parkinson disease (PD). Different doses of 6-OHDA were infused into the unilateral striatum in rats by using the CED method. The dopaminergic neuronal degeneration was evaluated based on morphological, biochemical, and behavioral measurements until 8 weeks postlesion. Due to the wide distribution of the drug, CED of 20 microg of 6-OHDA into the striatum was sufficient to obtain a progressive and extensive nigrostriatal lesion as defined by morphological (> 80% cell loss in the substantia nigra [SN]) and biochemical (> 95% decrease in striatal dopamine) criteria. The extent of the lesion manifested as a stable turning behavior with amphetamine (> 6 turns/minute) and apomorphine (> 4 turns/minute). It also appeared that at I week postlesion the apoptotic markers were maximal in neurons of the SN. A rat model of PD with a progressive and extensive dopamine lesion was successfully made by intrastriatal CED of 6-OHDA. In this model, the therapeutic value can be assessed using behavioral, biochemical, and histochemical measurements. The delay of nigral neuronal death with respect to the time of 6-OHDA administration may provide a therapeutic window for testing neuroprotective strategies.