PEGylation of brain-derived neurotrophic factor for preserved biological activity and enhanced spinal cord distribution

Abstract

Brain-derived neurotrophic factor (BDNF) was covalently attached to polyethylene glycol (PEG) in order to enhance delivery to the spinal cord via the cerebrospinal fluid (intrathecal administration). By varying reaction conditions, mixtures of BDNF covalently attached to one (primary), two (secondary), three (tertiary), or more (higher order) PEG molecules were produced. The biological activity of each resulting conjugate mixture was assessed with the goal of identifying a relationship between the number of PEG molecules attached to BDNF and biological activity. A high degree of in vitro biological activity was maintained in mixtures enriched in primary and secondary conjugate products, while a substantial reduction in biological activity was observed in mixtures with tertiary and higher order conjugates. When a biologically active mixture of PEG-BDNF was administered intrathecally, it displayed a significantly improved half-life in the cerebrospinal fluid and an enhanced penetration into spinal cord tissue relative to native BDNF. Results from these studies suggest a PEGylation strategy that preserves the biological activity of the protein while also improving the half-life of the protein in vivo. Furthermore, PEGylation may be a promising approach for enhancing intrathecal delivery of therapeutic proteins with potential for treating disease and injury in the spinal cord.

Figure 1

Reaction diagrams of (a) conjugation with mPEG-ButyrALD and (b) conjugation with mPEG-SPA.

Figure 2

MALDI-TOF mass spectrometry of (a) PEG species used for conjugation, (b) BDNF and (c) PEG-BDNF conjugate mixture prepared with a 60-fold excess of PEG to BDNF and a 125-fold excess of reducing agent to BDNF.

Figure 3

SDS-PAGE analysis (10% Gels) of conjugate mixtures. (a) PEG-BDNF mixtures prepared with a 125-fold excess of reducing agent (RA) to BDNF with varied molar excesses of PEG to BDNF detected by coomassie staining. (b) PEG-BDNF mixtures prepared with a 60-fold excess of PEG to BDNF and either a 10-fold or a 60-fold molar excess of RA to BDNF detected by coomassie staining. (c) PEG-BDNF mixtures with a 60-fold molar excess of PEG to BDNF and a 600-fold molar excess of RA to BDNF detected by coomassie staining and immunoblotting.

Figure 4

PC12-trkB neurite extension results. (a) Application of BDNF at concentrations in the range of 0 to 0.5 ng/ml and (b) Application of 0.5 ng/ml of BDNF treated with PEG only or reducing agent (RA) only at the indicated molar excess values. * indicates p value < 0.05 relative to BDNF only condition (two-tailed t-test, error bars are standard error of the mean).

Figure 5

PC12-trkB neurite extension results after the application of BDNF and PEG-BDNF conjugate mixtures at a concentration of 0.5 mg/ml (total BDNF for all species). ‘--‘ represents the expected degree of neurite extension from residual free BDNF levels in each mixture (free BDNF level obtained from band density analysis of coomassie stained gels, and estimated degree of neurite extension obtained from the dose-response relationship in Fig. 4a). (a) PEG-BDNF mixtures enriched in primary conjugate species. (b) PEG-BDNF mixtures containing primary and secondary conjugate species. (c) PEG-BDNF mixtures containing tertiary and higher order conjugate species. (d) PEG-BDNF mixtures prepared with mPEG-SPA instead of mPEGButyrALD. m/n indicates m-fold excess PEG to BDNF and n-fold excess reducing agent to BDNF respectively. * indicates p value < 0.001 relative to control BDNF (two-tailed t-test, error bars are standard error of the mean).

Figure 6

ELISA detection of BDNF in lumbar CSF samples after intrathecal injections of BDNF or PEG-BDNF. * and ** indicate p values < 0.05 and <0.005, respectively, for time-matched PEG-BDNF compared to BDNF (unequal variance two-tailed t-test, error bars are standard error of the mean).

Figure 7

Confocal imaging along the periphery of BDNF immuno-stained lumbar spinal cord tissue sections. (a) Spinal cord cross-section 4 hours after intrathecally injected BDNF. (b) Spinal cord cross-section 4 hours after intrathecally injected PEG-BDNF. (c) ImageJ analysis of fluorescence intensity values relative to distance from the spinal cord cross-section periphery.