Polymeric micelle delivery system for combinatorial therapy after traumatic brain injury

摘要

Statement of Purpose: Traumatic brain injury (TBI) continues to represent one of the leading causes of disability and death following injury. The progression of TBI neurotoxic cytokines can be inhibited by increasing levels of cyclic adenosine monophosphate (cAMP)Pl. Rolipram (Rm), a phosphodiesterase inhibitor, prevents the degradation of cAMP and is able to inhibit production of potentially neurotoxic cytokines and also lead to increased axonal regeneration. An additional treatment for TBI is the administration of exogenous nerve growth factor (NGF), which has shown neuroprotective function and can reduce edema following primary injury. We propose a novel polymeric carrier system combining the delivery of Rolipram and plasmid DNA encoding NGF to neural cells present at the injury site. This carrier will be formed from amphiphilic copolymers composed of poly(lactide-co-glycolide)-g-polyethylenimine (PgP), which has previously demonstrated its ability for efficient nucleic acid delivery. Methods: PgP was synthesized from branched PEI (25kDa) and PLGA (4kDA) and characterized by 1H- NMR as previously described. The transfection efficiency of PgP was evaluated using pBLAST44-hNGF (NGFB (InvivoGen), 2ug/mL) in C8-B4 (ATCC® CRL-2540) in the presence of 10% serum. Transfection was performed by complexing pDNA with PgP at N/P ratios 25/1 and 30/1 that showed the highest transfection without cytotoxicity in previous studies. At each time point, culture medium was collected and replaced with fresh medium. The NGF concentration was determined by ELISA assay and cytotoxicity using MTT assay. Rm was loaded in PgP micelles by solvent evaporation method and loading efficiency was measured by high performance liquid chromatography (HPLC). To evaluate the effect of Rm-loaded PgP (Rm-PgP) on neuron survival, neurite length, and inflammatory cytokine expression, we used hypoxia condition as an in vitro TBI model. Primary cerebellar neurons (CBNs) from post-natal day 3 rat pups were incubated in a hypoxia chamber (gas mixture of 95% Nitrogen and 5% CO2) for 48 hours. To assess the effect of hypoxia on neuron survival and cAMP level, neurite projection length was measured using ImageJ after fluorescent staining of neuron-specific beta ⅡI tubulin, and cAMP level was evaluated via ELISA assay (R&D Systems). CBN cells in normoxic conditions were used as a control. Also, induction of hypoxia was verified by Western blotting for Hypoxia Inducible Factor-1 α (HIF-1 α). Results: NGF expression was increased significantly over control for both PgP/pNGF and PEI/pNGF transfection. PgP/pNGF polyplexes at N/P ratios 25/1 and 30/1 was significantly higher than that of PEI at N/P 5/1, and persisted for 5 days. As seen in Figure 2, following incubation in hypoxia, there was a significant loss in both neurite length and cAMP level following hypoxia. Conclusions: We demonstrated that PgP polymeric micelle is a promising carrier for pNGF to C8B4 microglia cells in 10% serum condition with persistent elevation of NGF expression over a course of 120 hours. We successfully generated hypoxia condition for TBI model and demonstrated that both neurite length and the cAMP level was reduced in primary rat CBN cells cultured in hypoxia as compared to normoxia condition. Currently, we are testing the effect of Rolipram loaded PgP (Rm-PgP) on neurite length and cAMP level compared to free rolipram dissolved in DMSO in primary CBN cultured in hypoxia condition.