Size- and charge-dependent non-specific uptake of PEGylated nanoparticles by macrophages

Abstract

The assessment of macrophage response to nanoparticles is a central component in the evaluation of new nanoparticle designs for future in vivo application. This work investigates which feature, nanoparticle size or charge, is more predictive of non-specific uptake of nanoparticles by macrophages. This was investigated by synthesizing a library of polymer-coated iron oxide micelles, spanning a range of 30–100 nm in diameter and −23 mV to +9 mV, and measuring internalization into macrophages in vitro. Nanoparticle size and charge both contributed towards non-specific uptake, but within the ranges investigated, size appears to be a more dominant predictor of uptake. Based on these results, a protease-responsive nanoparticle was synthesized, displaying a matrix metalloproteinase-9 (MMP-9)-cleavable polymeric corona. These nanoparticles are able to respond to MMP-9 activity through the shedding of 10–20 nm of hydrodynamic diameter. This MMP-9-triggered decrease in nanoparticle size also led to up to a six-fold decrease in nanoparticle internalization by macrophages and is observable by T₂-weighted magnetic resonance imaging. These findings guide the design of imaging or therapeutic nanoparticles for in vivo targeting of macrophage activity in pathologic states.

Keywords:

• macrophage targeting
• poly(ethylene glycol) (PEG)
• poly(propylene sulfide) (PPS)
• iron oxides
• opsonization

Acknowledgments

This work was supported by a Vanderbilt University Intramural Discovery Grant (4-48-999-9132), the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-08-1-0502), and a Whitaker International Scholarship to SNT. CML acknowledges support through a fellowship from the Vanderbilt University Undergraduate Summer Research Program (VUSRP). Dynamic light scattering and TEM were conducted through the use of the core facilities of the Vanderbilt Institute of Nanoscale Sciences and Engineering (VINSE), using facilities renovated under NSF ARI-R2 DMR-0963361. We thank Dr Daniel Colvin of the Vanderbilt University Institute of Imaging Science (VUIIS) for his assistance with MRI imaging and analysis. We also acknowledge the laboratories of Professors Hak-Joon Sung and Craig L Duvall (Vanderbilt Biomedical Engineering), whose equipment were instrumental to the execution of this work.

Disclosure

The authors have no conflicts of interest to disclose.

Supplementary figures

Online supplementary materials include the following Figures: (S1) control over iron oxide nanoparticle size, (S2) Lowry protein assay standard curves, (S3) nanoparticle binding experiments, (S4) nanoparticle cytotoxicity assay, and (S5) control internalization experiments involving MMP-9, MMP-9 inhibitor, and protease-insensitive nanoparticles.

Figure S1. Feed ratio of oleic acid surfactant to iron pentacarbonyl precursors and resulting USPIO diameters. A 6 mmol quantity of Fe(CO)₅ was introduced into reactors containing 40 mL octyl ether and varying amounts of oleic acid at 100°C. USPIO cores were allowed to grow and then oxidize as described in materials and methods, and then imaged by HRTEM. Core diameters were measured via ImageJ software.

Abbreviations: HRTEM, high resolution transmission electron microscope; USPIO, ultrasmall superparamagnetic iron oxides.

Figure S2. Lowry protein assay standard curves. BSA was dissolved in PBS and treated with either 0.1 N NaOH or 6 N HCl prior to performance of the Lowry protein assay. While the assay is typically run under alkaline conditions (blue), strong acidic conditions do not significantly affect the sensitivity or reliability of this assay.

Abbreviations: BSA, bovine serum albumin; PBS, phosphate buffered saline.

Figure S3. Twenty-four hour uptake of nanoparticles by THP-1 macrophages. Cells were treated with 40 nm PEG-PPS-USPIOs for 24 hours, and then measured for iron content via the phenanthroline assay. Iron content was normalized to cell number indirectly via a protein assay. To confirm that the phenanthroline assay measures internalized nanoparticles and not just nanoparticles that have bound to macrophage receptors, some cells were incubated with nanoparticles at 4°C. Results showed about ten-fold lower iron content in these samples relative to samples treated at 37°C, indicating that the protocol successfully lyses cells and enables measurements of internalized iron.

Note: Error bars indicate standard deviation of three independent experiments (*P < 0.01).

Abbreviations: PEG, poly(ethylene glycol); PPS, poly(propylene sulfide); USPIO, ultrasmall superparamagnetic iron oxides; THP, human acute monocytic leukemia cell line.

Figure S4. Cell viability measurements on nanoparticle-treated THP-1 cells, normalized to untreated cells (media + PBS). Cells were treated with increasing doses of 100 nm PEG-PPS-USPIOs for 24 hours, prior to removal of unbound nanoparticles and assessment of cell viability via quantification of calcein-AM/ethidium homodimer staining. Dosage on the x-axis represents actual iron concentration within the samples. No statistically significant differences in viability were observed between any of the treatment groups (n = 3).

Abbreviations: PBS, phosphate buffered saline; PEG, poly(ethylene glycol); PPS, poly(propylene sulfide); USPIO, ultrasmall superparamagnetic iron oxides; THP, human acute monocytic leukemia cell line.

Figure S5. Co-administration of 40 nm PEG-PPS-USPIOs (do not contain MMP- 9-cleavable peptide) with MMP-9 does not significantly affect internalization of nanoparticles. THP-1 cells were treated with media only (untreated), nanoparticles only, or nanoparticles co-administered with 200 ng/mL MMP-9 and/or 300 ng/mL MMP-9 inhibitor. Because these nanoparticles do not contain MMP-9-cleavable elements, their diameter is unaffected by treatment (data not shown). MMP-9 treatment does not change the properties of the THP-1 cell membrane in a way that affects their interactions with nanoparticles. Error bars indicate standard deviation for three independent experiments.

Abbreviations: MMP-9, matrix metalloproteinase-9; PEG, poly(ethylene glycol); PPS, poly(propylene sulfide); USPIO, ultrasmall superparamagnetic iron oxides; THP, human acute monocytic leukemia cell line.