Functional protein delivery into neurons using polymeric nanoparticles

Abstract

An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.

FIGURE 1.

Uptake of FITC-labeled PBCA NPs by primary neurons is temperature-dependent. A, fluorescence and phase-contrast micrographs of primary rat hippocampal neurons incubated with 250 μg/ml FITC-NPs (green) at 37°C versus 15 °C. Cells were fixed and counterstained with DAPI (blue) and the neuron-specific marker NeuN (red). Only neurons (purple nuclei), are co-labeled with FITC, while glia (blue nuclei) are unlabeled. Scale bar = 10 μm. B, fluorescence intensity was measured for each group of cells over time. FITC incorporation was calculated as a percentage of the total nanoparticle dose in medium alone, and plotted as mean ± S.D. ^**, p < 0.0001; ^*, p < 0.01, by one-way analysis of variance with post-hoc Tukey test.

FIGURE 2.

LDL receptor blockade inhibits uptake of PBCA NPs. A, fluorescence and phase-contrast microscopy images of primary hippocampal neurons treated with FITC-NPs, following pretreatment with anti-LDL receptor antibody (middle), anti-EGF receptor antibody (bottom), or without antibody pretreatment (top). Cells were counterstained with anti-NeuN (red). Scale bar = 10 μm. B, time course of incorporation of ³H-nanoparticles in the presence or absence of blocking antibodies against LDL receptor and EGF receptor is plotted as mean ± S.D. At the 45-min time point and beyond, LDL receptor blockade resulted in significantly reduced uptake relative to EGF receptor blockade or no antibody treatment (p < 0.05, by one-way analysis of variance with post-hoc Tukey test).

FIGURE 3.

Intraneuronal delivery of functional protein. A, fluorescence and phase-contrast microscopy images of live primary neurons treated with β-gal loaded nanoparticles (top row) or β-gal only with Polysorbate 80 (bottom row) and stained for enzymatic activity using the substrate C₁₂FDG; viability of assessed with Live/Dead™ Red, which is excluded from live cells. Fixed cells were stained with X-gal and visualized via light microscopy. B, decay of β-gal activity in neurons after 2 h of treatment with β-gal-loaded NPs. Data are plotted as mean ± S.D. B (inset), Western blot analysis of lysates from cells treated with β-gal-loaded NPs (lane 1), β-gal alone with surfactant (lane 2), and β-gal-loaded nanoparticles after pre-treatment with a blocking antibody against the LDL receptor (lane 3). Only cells treated with β-gal-loaded NPs in the absence of anti-LDL receptor contained detectable levels of β-gal enzyme. β-Actin was also probed for as a loading control. Scale bar = 10 μm.

FIGURE 4.

Delivery of rhoG into PC12 cells produces neurite outgrowth and differentiation. A, phase-contrast microscopy images of untreated PC12 cells; B, PC12 cells treated with empty nanoparticles; C, with Myc-rhoG protein plus Polysorbate 80, with NGF (D), treated with nanoparticles loaded with Myc-rhoG protein (E), and transfected with rhoG DNA (F). G (top), quantification of neurite outgrowth in cultures treated as above. Values are reported as mean ± S.D. (^*, p < 0.01, by one-way analysis of variance with post-hoc Tukey test). G (bottom), Western blot analyses of PC12 cell lysates treated as above; lysates were incubated with GTPγS to activate rho, subjected to pull-down with GST-rhotekin-RBD, and probed with anti-rho (first row). Whole lysates were also probed with an anti-Myc antibody to detect exogenous Myc-rhoG (third row) with β-actin as a loading control (second row). All samples were assayed at 5 days. Scale bar = 50 μm.

FIGURE 5.

Delivery of anti-synuclein antibody H3C into primary neurons. A, fluorescence microscopy images of primary neurons treated with nanoparticles loaded with a monoclonal antibody H3C against α-synuclein or (B) with H3C and Polysorbate 80 alone and stained with a Alexa Fluor 594-conjugated secondary and co-stained with DAPI. C, Western blot analysis of whole-cell lysates (W) and protein-G pulldown assays (G) from neurons treated with H3C-NPs (lanes 3 and 4), empty nanoparticles (lane 5 and 6), or H3C-NPs after preincubation with anti-LDL receptor antibody (lanes 7 and 8). Lane 1 = H3C antibody (50 ng), lane 2 = recombinant human α-synuclein (50 ng). Blot was probed with a goat antibody (SC7012) against N-terminal α-synuclein. D, immunocytochemistry of neurons treated with H3C-NPs then fixed and stained with SC7012, a goat antibody against the N terminus of α-synuclein. The primary antibodies were detected using Alexa Fluor 488-conjugated anti-mouse and Cy3-conjugated anti-goat, respectively. NP-delivered H3C colocalizes with SC7012. Scale bar = 50 μm.