Catherine E. Kang

An injectable drug delivery platform for sustained combination therapy

We report the development of a series of physical hydrogel blends composed of hyaluronan (HA) and methyl cellulose (MC) designed for independent delivery of one or more drugs, from 1 to 28 days, for ultimate application in spinal cord injury repair strategies. To achieve a diversity of release profiles we exploit the combination of fast diffusion-controlled release of dissolved solutes from the HAMC itself and slow drug release from poly(lactide-co-glycolide) particles dispersed within the gel. Delivery from the composite hydrogels was demonstrated using the neuroprotective molecules NBQX and FGF-2, which were released for 1 and 4 days, respectively; the neuroregenerative molecules dbcAMP and EGF, and proteins alpha-chymotrypsin and IgG, which were released for 28 days. alpha-chymotrypsin and IgG were selected as model proteins for the clinically relevant neurotrophin-3 and anti-NogoA. Particle loaded hydrogels were significantly more stable than HAMC alone and drug release was longer and more linear than from particles alone. The composite hydrogels are minimally swelling and injectable through a 30 gauge/200 microm inner diameter needle at particle loads up to 15 wt.% and particle diameters up to 15 microm.

Intrathecal delivery of a polymeric nanocomposite hydrogel after spinal cord injury

Major traumatic spinal cord injury (SCI) results in permanent paralysis below the site of injury. The effectiveness of systemically delivered pharmacological therapies against SCI can be limited by the blood-spinal cord barrier and side effects. Local drug delivery to the injured spinal cord can be achieved using a minimally invasive biopolymer matrix of hyaluronan and methylcellulose injected into the intrathecal space, bypassing the blood-spinal cord barrier and overcoming limitations of existing strategies. Composite hydrogels of drug-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles dispersed in this biopolymer matrix meet the in vitro design criteria for prolonged local release. Using a blank (without drug) composite designed for 28-day sustained release, we presently explore the mechanism of particle-mediated hydrogel stabilization in vitro and aspects of biocompatibility and safety in vivo. The composite hydrogel is well tolerated in the intrathecal space of spinal cord injured rats, showing no increase in inflammation, scarring, or cavity volume relative to controls, and no significant effect on locomotor function up to 28 days. Furthermore, there was no effect on locomotor function in healthy animals which received the composite hydrogel, although a qualitative increase in ED-1 staining was apparent. These data support the further development of composite hydrogels of hyaluronan and methylcellulose containing PLGA nanoparticles for sustained local delivery to the injured spinal cord, an application for which there are no approved alternatives.

A New Paradigm for Local and Sustained Release of Therapeutic Molecules to the Injured Spinal Cord for Neuroprotection and Tissue Repair

After spinal cord injury (SCI), a complex cascade of events leads to tissue degeneration and a penumbra of cell death. Neuroprotective molecules to limit tissue loss are promising; however, intravenous delivery is limited by the blood-spinal cord barrier and short systemic half-life. Current local delivery strategies are flawed: bolus injection results in drug dispersion throughout the intrathecal (IT) space, and catheters/pumps are invasive and open to infection. Our laboratory previously developed a hydrogel of hyaluronan (HA) and methylcellulose (MC) (HAMC) that, when injected into the IT space, was safe and, remarkably, had some therapeutic benefit on its own. In order to test this new paradigm of local and sustained delivery, relative to conventional delivery strategies, we tested, for the first time, the in vivo efficacy of HAMC as an IT drug delivery system by delivering a known neuroprotective molecule, erythropoietin (EPO). In vitro studies showed that EPO was released from HAMC within 16 h, with 80% bioactivity maintained. When the material alone was injected in vivo, individual fluorescent labels on HA and MC showed that HA dissolved from the gel within 24 h, whereas the hydrophobically associated MC persisted in the IT space for 4-7 days. Using a clip compression injury model of moderate severity, HAMC with EPO was injected in the IT space and, in order to better understand the potential of this delivery system, compared to the therapeutic effect of both common delivery strategies-IT EPO and intraperitoneal EPO-and a control of IT HAMC alone. IT HAMC delivery of EPO resulted in both reduced cavitation after SCI and a greater number of neurons relative to the other delivery strategies. These data suggest that the localized and sustained release of EPO at the tissue site by HAMC delivery enhances neuroprotection. This new system of IT delivery holds great promise for the safe, efficacious, and local delivery of therapeutic molecules directly to the spinal cord.

Accelerated release of a sparingly soluble drug from an injectable hyaluronan-methylcellulose hydrogel.

An injectable hydrogel, comprised of hyaluronan and methylcellulose (HAMC), shows promise for localized, sustained delivery of growth factors for treatment of spinal cord injury (SCI). To better understand its potential for the delivery of small molecules, the release of sparingly soluble neuroprotectant, nimodipine, was investigated experimentally and via continuum modeling. This revealed that the MC in HAMC increased the solubility of sparingly soluble drug by over an order of magnitude, and enabled highly tunable release rates to be achieved by varying the method by which the drug was introduced into the scaffold. When nimodipine was introduced into HAMC in solubilized form, it was rapidly released from the scaffold within 8 h. Conversely, when nimodipine was blended into HAMC in particulate form, the release rates were greatly reduced, giving rise to complete release over 2-3 days for small, sub-micron particles, and longer times for large, 100 mum particles. The nimodipine particle-loaded gels yielded particle size-dependent, biphasic release profiles, which reflected rapid release of the solubilized drug followed by the slow, dissolution-limited release of solid nimodipine. This suggests that injectable hydrogel matrices can act as polymeric excipients that accelerate the delivery of poorly soluble drugs and yield highly tunable release rates.

Poly(ethylene glycol) modification enhances penetration of fibroblast growth factor 2 to injured spinal cord tissue from an intrathecal delivery system

There is no effective treatment for spinal cord injury and clinical drug delivery techniques are limited by the blood-spinal cord barrier. Our lab has developed an injectable drug delivery system consisting of a biopolymer blend of hyaluronan and methylcellulose (HAMC) that can sustain drug release for up to 24h in the intrathecal space. Fibroblast growth factor 2 (FGF2) has great potential for treatment of spinal cord injury due to its angiogenic and trophic effects, but previous studies showed no penetration into spinal cord tissue when delivered locally. Conjugation to poly(ethylene glycol) (PEG) is known to improve penetration of proteins into tissue by reducing clearance and providing immunogenic shielding. We investigated conjugation of PEG to FGF2 and compared its distribution relative to unmodified FGF2 in injured spinal cord tissue when delivered intrathecally from HAMC. Importantly, PEG conjugation nearly doubled the concentration of FGF2 in the injured spinal cord when delivered locally and, contrary to previous reports, we show that some FGF2 penetrated into the injured spinal cord using a more sensitive detection technique. Our results suggest that PEGylation of FGF2 enhanced tissue penetration by reducing its rate of elimination.

The effects of intrathecal injection of a hyaluronan-based hydrogel on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis

Traumatic spinal cord injury (SCI) comprises a heterogeneous condition caused by a complex array of mechanical forces that damage the spinal cord - making each case somewhat unique. In addition to parenchymal injury, a subset of patients experience severe inflammation in the subarachnoid space or arachnoiditis, which can lead to the development of fluid-filled cavities/syringes, a condition called post-traumatic syringomyelia (PTS). Currently, there are no therapeutic means to address this devastating complication in patients and furthermore once PTS is diagnosed, treatment is often prone to failure. We hypothesized that reducing subarachnoid inflammation using a novel bioengineered strategy would improve outcome in a rodent model of PTS. A hydrogel of hyaluronan and methyl cellulose (HAMC) was injected into the subarachnoid space 24 h post PTS injury in rats. Intrathecal injection of HAMC reduced the extent of fibrosis and inflammation in the subarachnoid space. Furthermore, HAMC promoted improved neurobehavioural recovery, enhanced axonal conduction and reduced the extent of the lesion as assessed by MRI and histomorphometric assessment. These findings were additionally associated with a reduction in the post-traumatic parenchymal fibrous scar formation as evidenced by reduced CSPG deposition and reduced IL-1α cytokine levels. Our data suggest that HAMC is capable of modulating inflammation and scarring events, leading to improved functional recovery following severe SCI associated with arachnoiditis.

Localized and Sustained Delivery of Fibroblast Growth Factor-2 from a Nanoparticle-Hydrogel Composite for Treatment of Spinal Cord Injury

After traumatic spinal cord injury, grossly injured blood vessels leak blood and fluid into the parenchyma, leading to a large cystic cavity. Fibroblast growth factor-2 (FGF2) can reduce immediate vasoconstriction of vessels in the tissue surrounding the primary injury and promote angiogenesis. A localized delivery system would both achieve restricted delivery of FGF2 to the spinal cord and limit possible systemic effects such as mitogenesis. To enhance the endogenous angiogenic response after spinal cord injury, FGF2 was encapsulated in poly(lactide-co-glycolide) (PLGA) nanoparticles which were embedded in a biopolymer blend of hyaluronan and methylcellulose (HAMC) and then injected into the intrathecal space. Treatment began immediately after a 26 g clip compression spinal cord injury in rats and consisted of intrathecal delivery of FGF2 from the HAMC/PLGA/FGF2 composite. Control animals received intrathecal HAMC loaded with blank nanoparticles, intrathecal HAMC alone or intrathecal artificial cerebrospinal fluid alone. Sustained and localized delivery of FGF2 from composite HAMC/PLGA/FGF2 achieved higher blood vessel density in the dorsal horns 28 days post-injury, due to either greater angiogenesis near the epicenter of the injury or vasoprotection acutely after spinal cord injury. Importantly, delivery of FGF2 from composite HAMC/PLGA/FGF2 did not produce proliferative lesions that had been previously reported for FGF2 delivered locally using a minipump/catheter. These results suggest that localized and sustained delivery with composite HAMC/PLGA/FGF2 is an excellent system to deliver biomolecules directly to the spinal cord, thereby circumventing the blood spinal cord barrier and avoiding systemic side effects.

Intrathecal drug delivery strategy is safe and efficacious for localized delivery to the spinal cord

Neuroprotective and neuroregenerative strategies for spinal cord injury repair are limited in part by poor delivery techniques. A novel drug delivery system is being developed in our laboratory that can provide localized release of therapeutically relevant molecules from an injectable hydrogel. Design criteria were established for the hydrogel to be--injectable, fast-gelling, biocompatible, biodegradable and able to release biologically active therapeutics when injected into the intrathecal space that surrounds the spinal cord. This novel way of localized drug delivery to the spinal cord was tested first with a collagen gel and then with a new hydrogel blend of hyaluronan and methylcellulose (HAMC). The underlying principle that this novel methodology is both safe and able to provide localized delivery was proven with a fast gelling collagen solution. Using a recombinant human epidermal growth factor, rhEGF, dispersed in collagen, we demonstrated localized release to the injured spinal cord. We extended this technology to other fast-gelling systems and found that HAMC was injectable due to the shear thinning property of hyaluronan (HA), biocompatible and had some therapeutic benefit when injected into the intrathecal space using a compression injury model in rats.

Spinal Cord Blood Flow and Blood Vessel Permeability Measured by Dynamic Computed Tomography Imaging in Rats after Localized Delivery of Fibroblast Growth Factor

Following spinal cord injury, profound vascular changes lead to ischemia and hypoxia of spinal cord tissue. Since fibroblast growth factor 2 (FGF2) has angiogenic effects, its delivery to the injured spinal cord may attenuate the tissue damage associated with ischemia. To limit systemic mitogenic effects, FGF2 was delivered to the spinal cord via a gel of hyaluronan and methylcellulose (HAMC) injected into the intrathecal space, and compared to controls receiving HAMC alone and artificial cerebrospinal fluid (aCSF) alone. Dynamic perfusion computed tomography (CT) was employed for the first time in small animals to serially measure blood flow and permeability in the injured and uninjured spinal cord. Spinal cord blood flow (SCBF) and permeability-surface area (PS) measurements were obtained near the injury epicenter, and at two regions rostral to the epicenter in animals that received a 26-g clip compression injury. As predicted, SCBF measurements decreased and PS increased after injury. FGF2 delivered via HAMC after injury restored SCBF towards pre-injury values in all regions, and increased blood flow rates at 7 days post-injury compared to pre-injury measurements. PS was stabilized at regions rostral to the epicenter of injury when FGF2 was delivered with HAMC, with significantly lower values than aCSF controls at 7 days in the region farthest from the epicenter. Laminin staining for blood vessels showed a qualitative increase in vessel density after 7 days when FGF2 was locally delivered. Additionally, permeability stains showed that FGF2 moderately decreased permeability at 7 days post-injury. These data demonstrate that localized delivery of FGF2 improves spinal cord hemodynamics following injury, and that perfusion CT is an important technique to serially measure these parameters in small animal models of spinal cord injury.

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.