Research Papers

Cellular Uptake and Intracellular Cargo Release From Dextran Based Nanogel Drug Carriers

[+] Author and Article Information
M. Carme Coll Ferrer

Department of Anesthesiology and Critical Care
and Department of Materials Science and Engineering,
University of Pennsylvania,
Philadelphia, PA 19104

Peter Sobolewski

Department of Anesthesiology and Critical Care,
University of Pennsylvania,
Philadelphia, PA 19104

Russell J. Composto

Department of Materials Science and Engineering,
University of Pennsylvania,
Philadelphia, PA 19104

David M. Eckmann

Department of Anesthesiology and Critical Care,
University of Pennsylvania,
PA 19104
e-mail: eckmanndm@uphs.upenn.edu

1These authors contributed equally to this work.

2Corresponding author.

Manuscript received September 11, 2012; final manuscript received December 10, 2012; published online July 11, 2013. Assoc. Editor: Malisa Sarntinoranont.

J. Nanotechnol. Eng. Med 4(1), 011002 (Jul 11, 2013) (8 pages) Paper No: NANO-12-1120; doi: 10.1115/1.4023246 History: Received September 11, 2012; Revised December 10, 2012

Nanogels (NG) hold great promise as a drug delivery platform. In this work, we examine the potential of lysozyme-dextran nanogels (LDNG) as drug carriers in vitro using two cell lines: a model target tissue, human umbilical cord vein endothelial cells (HUVEC) and a model of the mononuclear phagocyte system (phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells). The LDNG (∼100 nm) were prepared with rhodamine-label dextran (LRDNG) via Maillard reaction followed by heat-gelation reaction and were loaded with a fluorescent probe, 5-hexadecanoylaminofluorescein (HAF), as a mock drug. Epifluorescence microscopy confirmed rapid uptake of LRDNG by HUVEC. Although LysoTracker Green staining indicated a lysosomal fate for LRDNG, the mock drug cargo (HAF) diffused extensively inside the cell within 15 min. Flow cytometry and confocal microscopy indicated slow uptake of LRDNG in PMA-stimulated THP-1 cells, with only 41% of cells containing LRDNG after 24 h exposure. Finally, 24 h exposure to LRDNG did not affect the viability of either cell type at the dose studied (20 μg/ml). At a higher dose (200 μg/ml), LRDNG resulted in a marked loss of viability of HUVEC and THP-1, measuring 30% and 38%, respectively. Collectively, our results demonstrate the great potential of LRDNG as a drug delivery platform, combining simple production, rapid uptake and cargo release in target cells with “stealth” properties and low cytotoxicity.

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Fig. 1

Characterization of LRDNG. (a) Size distribution as measured by DLS following synthesis (line) and after 6 month storage at 4  °C (open circles). (b) TEM micrographs showing particles after drying on holy carbon grid. The scale bars correspond to 0.5 μm and 50 nm (inset). (c) Histogram of particle size distribution as measured from TEM micrographs. The hydrodynamic diameter (DH) of the LRDNG is 107 ± 26 nm whereas the average particle size in the dried state (d) is 57 ± 13 nm.

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Fig. 2

Viability of cells after 24 h exposure to a range of LRDNG concentrations. (a) Viability of HUVEC, determined using alamarBlue® indicator. (b) Viability of PMA-stimulated, GFP-actin THP-1 cells, determined using Calcein Violet. * indicates significant difference from control, p < 0.05.

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Fig. 3

Summary of loading amount, defined as the weight ratio of loaded HAF to LRDNG ([HAF]/[LRDNG]) and loading efficiency, defined as the weight ratio of loaded HAF to initial HAF ([HAF]/[HAF]0) as a function of the loading concentration

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Fig. 4

HUVEC uptake of HAF-LRDNG by epifluorescence microscopy. (a) Representative epifluorescence photomicrograph of cells after 1 h incubation with LRDNG (red) loaded with mock drug, HAF, (green), namely HAF-LRDNG. The scale bar corresponds to 10 μm. (b) Uptake of HAF-LRDNG and (c) intracellular (closed circles) and extracellular (open circles) release of mock drug HAF as a function of incubation time.

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Fig. 5

HUVEC uptake of HAF-LRDNG and intracellular HAF release after 15 min by epifluorescence microscopy. Representative epifluorescence photomicrograph of cells after 15 min incubation with LRDNG (red) loaded with mock drug, HAF, (green), namely HAF-LRDNG. The scale bar corresponds to 20 μm.

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Fig. 6

Localization of LRDNG (red) inside HUVEC after 1 h. RDNG are pseudocolored red, lysozymes are pseudocolored green, and colocalization, as determined by the Costes image analysis, is highlighted in white. (a) Single, middle confocal slice of a representative cell. (b) Zoomed in view of the region highlighted in panel A. (c) XZ rendering for the plane indicated by the yellow line in panel B.

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Fig. 7

Uptake of LRDNG by PMA-stimulated, GFP-actin THP-1 cells by confocal microscopy following 24 h incubation. (a) Representative epifluorescence photomicrograph of middle slice of confocal z stacks (b) 3D image of a single cell. (c) Sum of fluorescence intensities of green (GFP-actin) and red (LRDNG) channels for all z heights (0.2 μm steps) of the 3D image, indicating LRDNG inside the cell body and not on the surface.

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Fig. 8

Flow cytometry analysis of LRDNG uptake by PMA-stimulated, GFP-actin THP-1 cells. Histograms of (a) control cells and cells incubated with LRDNG for (b) 1 h, (c) 3 h, (d) 6 h, and (e) 24 h. (f) Summary of percentage of cells containing LRDNG as a function of incubation time.



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