Multimodal Analysis of PEI-Mediated Endocytosis of Nanoparticles in Neural Cells

Abstract

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.

Enabling dual cellular destinations of polymeric nanoparticles for treatment following partial injury to the central nervous system

“…MHA attachment to PGMA (PGMA-MHA) was confirmed using 1 H-NMR, by the presence of a singlet at δ 6.68 ppm corresponding to the olefin MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 protons on MHA (for further details see the NP synthesis and NP characterization sections in the Materials and Methods). PGMA-MHA was further functionalized with the fluorescent dye Rhodamine B (RhB), as reported previously, 49 and confirmed via 1 H-NMR. Dry functionalized PGMA-MHA with RhB was used to synthesize NP containing magnetite, with or without resveratrol, using spontaneous emulsification.…”

“…Dry functionalized PGMA-MHA with RhB was used to synthesize NP containing magnetite, with or without resveratrol, using spontaneous emulsification. 49 The size of the resultant NP was ~ 150 nm, as determined using DLS (specific SI size distributions listed in Table 1); the data describe single narrow peaks, indicating that the NP exist as distinct individual particles in solution. NP were purified and antibodies attached (Figure 1b), resulting in a marked increase in total NP diameter but no observable change in zeta potential (Table 1).…”

“…48 Polymer based nanoparticles (NPs) were prepared as described, 49 with the following modifications. PGMA (500 mg, M w 120,000 g/mol) was refluxed in MEK for 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 10 by suspending 200 μg of various NP preparations in 500 μL of albumin (6.0 mg/mL) and…”

“…NPs were synthesized from poly(glycidyl methacrylate) (PGMA): we have shown that our PGMA NP are non-toxic, stable under physiological conditions and readily functionalizable via the epoxide group. 40,49,56 We have functionalized the NP with antibodies to an extracellular epitope of AQP4, to facilitate targeting to astrocytes following injury to the CNS. The complex, folded protein structure of an antibody results in an interaction with the target antigen that is specific, high affinity and relatively resistant to enzymatic degradation.…”

RNA Interference Using c-Myc–Conjugated Nanoparticles Suppresses Breast and Colorectal Cancer Models

“…The PEI-PGMA nanoparticles were prepared following the procedure outlined by Evans and colleagues (16) with the magnetic cores synthesized as per the procedure outlined by Sun and colleagues (18). The nanoparticles were characterized using TEM (JOEL 2100) at 120 kV, dynamic light scattering for size and surface charge, and fluorescence spectrometry.…”