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SC BioCRAFT

Project II

JEOUNG SOO LEE, PhD Assistant Professor of Bioengineering Clemson University Targeted Nano-therapeutics for Neural Regeneration

Target Investigator

JEOUNG SOO LEE, PhD
Assistant Professor of Bioengineering
Clemson University

Email: ljspia@clemson.edu   
Phone: 864-656-3213

PROGRESS REPORT

Neuron-specific Polymeric Micelle Delivery System for Neural Regeneration
Physical trauma or ischemia results in significant damage to the central nervous system (CNS). The regenerative capacity of the injured adult CNS is extremely limited, due to both extrinsic microenvironmental factors and intrinsic, age-related changes in plasticity. With few existing therapies available, CNS injury commonly leads to permanent loss of cognitive, motor, and/or sensory function. Strategies for improving axonal regeneration include antagonism of growth-inhibitory molecules and their receptors; delivery of growth-promoting stimuli through cell transplantation and neurotrophic factor delivery; and manipulation of cyclic nucleotide levels.  While all these approaches have achieved varying degrees of improvement in plasticity, regeneration, and function; it is clear that no single therapeutic will achieve adequate functional recovery. Recently, combinatorial strategies incorporating two or more of the above therapeutic modalities have achieved synergistic increases in axonal growth and functional recovery. In order to realize the full potential of combinatorial therapy, there is a need for the development of platform technologies capable of simultaneously delivering multiple bioactive molecules targeting different barriers to axonal regeneration. The objective of this proposal is to gather critical preliminary data to provide improved evidence of feasibility and enable the development of a competitive R01 submission applying this drug delivery technology for the treatment of traumatic brain injury (TBI).

The specific aims are as follows:

Aim 1: To synthesize and characterize the physical/chemical properties of rolipram-loaded PGP-Ab/siRNA complex nanoparticles as a drug and nucleic acid delivery carrier;

Aim 2: To evaluate the specificity, transfection efficiency and cytotoxicity of PGP-Ab/siRNA complex nanoparticle using biotin conjugated siRNA in rat DRG neurons in vitro; and

Aim 3: To evaluate the distribution of PGP-Ab/siRNAs complex nanotherapeutics in a rat direct cortical impact model of TBI.

The outcome of this research is expected to be the development of multi-functional nanotherapeutics capable of delivering multiple therapeutic agents (neuron-targeting antibodies, siRNA, and hydrophobic drug) within a single formulation and therapeutic intervention (localized injection). This contribution will be significant because it offers a simplified platform for testing and implementing combinatorial therapies with the potential for relatively rapid clinical translation, as well as design flexibility allowing for optimization and adaptation to address regenerative barriers that may be discovered in the future. Therapies capable of transiently increasing plasticity and promoting axonal growth can be applied to diverse types of neural trauma including TBI, spinal cord injury, and stroke.  Finally, the basic capability to efficiently deliver siRNA to neurons may be beneficial for inhibiting the expression of genes implicated in other CNS disorders such as Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis.

SUMMARY & HIGHLIGHTS:

Neuron-specific Polymeric Micelle Delivery System for Neural Regeneration:
We have shown that PGP is a promising nucleic acid carrier for both pDNA and siRNA using pGFP and siGLO ® transfection indicator capable of transfecting neuroglioma (C6) cells and primary chick forebrain neuron cell (E8 CFN) in non-serum/5% serum condition with low cytotoxicity. The degree of antibody functionalization of PGP-Ab may significantly impact the stability and complex formation of micelles with siRNA and/or cellular uptake. We conjugated 5 different amount of ant We will evaluate effect of antibody conjugation on micelle stability, particle size and surfacbody to PGP evaluate the stability and physico-chemical properties of PGP-Ab/pGFP complexes. The particle size and zeta-potential of PGP-Ab were evaluated and antibody conjugation did not change particle size and zeta-potential of PGP micelles. Currently, we are in the process of optimizing the amount of antibody conjugation to the PGP and evaluating transfection and cytotoxicity of PGP-Ab/siRNA complexes in C6 and CFN cells (Aim 1). We will also evaluate rolipram loading efficiency in PGP-Ab/siRNA complexes. We will conjugate NgR antibody to PGP to evaluate the specificity, transfection efficiency and cytotoxicity of PGP-Ab/siRNA complex in rat DRG neurons and rat DRG/astrocyte co-cultures in vitro. Drs. Hoffman and Visconti will provide the NgR antibody (Aim2). Currently, we are studying the biodistribution of the fluorescently labeled PGP/siRNA polyplexes after systemic injection by tail vein. We will evaluate the distribution of fluorescently labeled PGP-Ab/siRNAs complex nanotherapeutics before/after TBI by systemic injection /local injection in a rat direct cortical impact model. Dr. Kindy (Project consultant) will help us to set up and conduct these experiments (Aim3).

Target-specific Polymeric Micelle Delivery System for Breast Cancer Therapy:

Metastatic breast cancer is the leading cause of death among women and women with Her2-positive breast cancer are at greater risk for disease progression and death. Treatment strategies have been developed to block Her2 in combination with chemotherapy. However, the usage of anticancer drugs has been limited by their side effects in normal organs and drug resistance acquired by cancer cells. The objective of this proposal is to develop two multi-functional polymeric nanoparticles as simultaneous delivery carriers for combinatorial therapy of anticancer drug and siRNA for the efficient treatment of various types of drug resistant breast cancers.  We evaluated the transfection efficiency of PGP/pGFP in MCF-7 (breast cancer cell) cells and found that transfection efficiency increased with increasing N/P ratio in non-serum/5% serum condition in vitro, while maintaining low cytotoxicity. Currently, we are evaluating the silencing efficiency of PGP/mGFP siRNA in mGFP-transfected MCF cells in non-serum/5% serum condition in vitro.

Colon-specific bi-functional amebicidal therapeutic (Unrelated to COBRE):
Funded by NIH/NIAID R03 (1R03 A1076869)

Entamoeba histolytica is the causative agent of hemorrhagic enteric colitis and liver abscess and has been classified as a category B priority pathogen. Therefore, there is a continued need for the development of innovative therapeutic strategies for E. histolytica in order to counter large scale outbreaks. The objective of this project is to develop a novel bi-functional polymeric amebicidal therapeutic, Galactose-Dextran-Metronidazole (Gal-Dextran-MM) prodrug as a colon-specific therapeutic to efficiently treat E. histolytica infection   through two mechanisms, 1) delivery of the amebicidal agent, metronidazole, to kill the amoebae, and 2) delivery of galactose which may inhibit adhesion of the parasite to host cells. We synthesized novel bi-functional colon-specific polymeric prodrug, Gal-Dex-MM and demonstrated that Gal-Dex-MM is chemically and enzymatically stable in vitro. We also demonstrated, for the first time, that Mz released from Gal-Dex-MM showed amebicidal activity against E. histolytica and the released Gal inhibited ameba adhesion to CHO cells in vitro. We also demonstrated that Gal-Dex-MM was stable during transit through the stomach and the small intestine and that Mz was released from Gal-Dex-MM after reaching the lower intestine in rats in vivo. Currently, we are in the process of evaluating the efficacy and cytotoxicity of Gal-Dex-MM in a mouse amebic ulcer model.

GRANTS / PAPERS (PUBLISHED/SUBMITTED):

Papers:

E. Cho, J. S. Lee, and K. Webb “Formulation and characterization of poloxamine-based hydrogels as tissue sealants”, Acta Biomaterialia 8:2223-2232 (2012).

J. Zhang, K Webb, and J.S. Lee “Synthesis and characterization of target-specific polymeric micelles

as gene carriers.” manuscript in preparation to submit to Biomaterials

J. Zhang, A. Sen, E. Cho, J. S. Lee, and K. Webb. “Poloxamine/fibrin hybrid hydrogels for controlled release of nonviral vectors”, submitted to Journal of Tissue Engineering and Regenerative Medicine.

J. Zhang, J. S. Lee, and K. Webb. “Effect of Tetronic T904 of polyplex transfection efficiency and gene expression.” Submitted Journal of Gene Medicine.

H. Lee, A. Sen, S. Bae, J. S. Lee, and K. Webb “The effect of network composition on cellular remodeling in PEG-diacrylate/hyaluronic acid semi-interpenetrating networks.” For March submission to Acta Biomaterialia.

Grants/Proposals:

  • R01 application “Bioactive hydrogels for nonviral gene delivery” (Co-PI) submitted 2/13.
  • R01 application (revised) “Hybrid hydrogels for tissue engineering” (Co-PI) to be submitted 3/13.
  • R01 application (new) “Neuron-specific combinatorial nanothereapeutics for neural regeneration to be submitted 6/13.

FUTURE DIRECTIONS:

Once we gather critical preliminary data to provide improved evidence of feasibility of PGP-Ab as a target-specific drug and nucleic acid delivery carrier, we can apply this delivery system for many other applications to alleviate pathological conditions and promote functional tissue regeneration by changing 1) targeting moiety on the PGP micelle surface, 2) therapeutic nucleic acids in the hydrophilic shell, and 3) any hydrophobic drug in the micelle core.

In terms of funding opportunities, I will submit two new R01 grants and one new R21 grant. One R01 applications related to Neuron-specific Polymeric Micelle Delivery System for Neural Regeneration will be submitted to NIH/NINID (6/2013).  I will submit R01 proposal to NIAID/NIH based on data obtained my funding (NIH R03) from NIH/NIAID (10/2013). I will also submit R21 proposal, “Target-specific Polymeric Micelle Delivery System for breast cancer therapy” to NIH/NCI (10/2013).

SPECIFIC AIMS FOR RENEWAL

The goal of this research is to develop novel neuron-specific nanotherapeutics for combinatorial therapy of drug and small interfering RNAs (siRNAs) for the efficient treatment of traumatic brain injury. Traumatic or ischemic damage to the adult central nervous system (CNS) commonly leads to permanent loss of cognitive, sensory, and/or motor function. The limited ability of the injured adult CNS to achieve functional repair results from the presence of growth inhibitory molecules in the extracellular microenvironment and intrinsic, age-dependent changes in neuronal biochemistry. Major changes in both of these characteristics are closely associated with the loss of plasticity that occurs after completion of critical periods of CNS development. While these mechanisms stabilize and preserve developmental patterns, they also limit regenerative capacity. Two major classes of growth inhibitors are myelin-associated proteins (Nogo A, myelin associated glycoprotein, and oligodendrocyte myelin glycoprotein) that bind to the axonal Nogo-66 receptor (NgR1) and chondroitin sulfate proteoglycans (CSPGs) expressed by reactive cell types during glial scarring. Recently, both families of inhibitors have been found to act through common intracellular signal transduction pathways involving Rho A/Rho Kinase, Protein Kinase C (PKC), and epidermal growth factor receptor (EGFR).

Strategies for improving neural plasticity and axonal regeneration include antagonism of growth-inhibitory molecules and their receptors; delivery of growth-promoting stimuli through cell transplantation and neurotrophic factor delivery; and manipulation of cyclic nucleotide levels. While all these approaches have achieved varying degrees of improvement in plasticity, regeneration, and function; it is clear that no single therapeutic will achieve adequate functional recovery. Recently, combinatorial strategies incorporating two or more of the above therapeutic modalities have achieved synergistic increases in growth and recovery. In order to realize the full potential of combinatorial therapy, there is a need for the development of technologies capable of simultaneously delivering multiple bioactive molecules targeting different barriers to axonal regeneration.

We designed a novel neuron-specific nanotherapeutics for combinatorial delivery of drug and siRNAs to target molecules common to intracellular signaling pathways of both myelin and CSPGs. First, anti-NgR1 antibody (Ab) conjugated to the nanoparticle will specifically deliver the nanotherapeutics to neurons and interfere with the function of existing NgR1 receptors by antagonizing the binding of myelin associated inhibitors. Second, to target common intracellular signal transduction pathways for both myelin and CSPGs, siRNAs for RhoA, PKC, and EGFR will be used. Third, to increase intrinsic neuronal growth capacity by preventing injury-induced reductions in cAMP levels, rolipram, a phosphodiesterase inhibitor will be employed.

In preliminary studies, we have described the synthesis and characterization of poly (lactide-co-glycolide)-graft-polyethylenimine (PGP) and PGP-Ab and shown its ability to deliver siRNA to primary CNS neurons with high transfection efficiency and minimal cytotoxicity. Our central hypothesis is that combinatorial therapy of drug and siRNAs that increase neural plasticity will improve functional recovery following traumatic brain injury.

The specific aims are as follows:

Aim 1: To synthesize and evaluate novel neuron-specific nanoparticles (PGP-Ab) as a drug and siRNA delivery carrier. These studies will characterize the physical/chemical properties of PGP-Ab/siRNA complexes, evaluate rolipram loading efficiency and release kinetics, and evaluate transfection efficiency and cytotoxicity in rat DRG neurons in vitro, and evaluate targeting specificity in rat DRG neuron/astrocyte co-cultures;

Aim 2: To evaluate the ability of PGP-Ab/siRNA complex nanotherapeutics to knockdown gene expression of signaling intermediaries implicated in growth inhibition, elevate cAMP, and stimulate neurite outgrowth on inhibitory substrates. These studies will evaluate the effect of rolipram-loaded PGP-Ab /siRNA nanotherapeutics on RhoA, PKC, and EGR mRNA and protein expression levels and cAMP levels. Compartmented culture chambers will be used to test neuronal capacity for retrograde transport of the nanoparticles and their ability to overcome CNS myelin and/or CSPG inhibition of neurite outgrowth; and

Aim 3: To evaluate axonal regeneration and functional recovery in response to delivery of rolipram-loaded PGP-Ab/siRNAs complex nanotherapeutics in a rat direct cortical impact model of TBI. The biodistribution, histopathological analysis, and motor and cognitive functional outcomes will be evaluated in rats injected systemically or locally with rolipram-loaded PGP-Ab / siRNA nanotherapeutics after TBI compared to saline-injected control rats.

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