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. 2012 Dec 2:10:44.
doi: 10.1186/1477-3155-10-44.

Fabrication and characterization of DNA-loaded zein nanospheres

Mary C Regier  1 , Jessica D Taylor, Tyler Borcyk, Yiqi Yang, Angela K Pannier
Affiliations

Fabrication and characterization of DNA-loaded zein nanospheres

Mary C Regier et al. J Nanobiotechnology. .

Abstract

Background: Particulates incorporating DNA are promising vehicles for gene delivery, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Zein, a hydrophobic protein from corn, is biocompatible and has properties that make it a promising candidate material for particulate delivery, including its ability to form nanospheres through coacervation and its insolubility under physiological conditions, making it capable of sustained release of encapsulated compounds. Due to the promise of this natural biomaterial for drug delivery, the objective of this study was to formulate zein nanospheres encapsulating DNA as the therapeutic compound, and to characterize size, charge, sustained release, cell cytotoxicity and cellular internalization of these particles.

Results: Zein nanospheres encapsulating DNA were fabricated using a coacervation technique, without the use of harsh solvents or temperatures, resulting in the preservation of DNA integrity and particles with diameters that ranged from 157.8 ± 3.9 nm to 396.8 ± 16.1 nm, depending on zein to DNA ratio. DNA encapsulation efficiencies were maximized to 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. The spheres protected encapsulated DNA from DNase I degradation and exhibited sustained plasmid release for at least 7 days, with minimal burst during the initial phase of release. Zein/DNA nanospheres demonstrated robust biocompatibility, cellular association, and internalization.

Conclusions: This study represents the first report on the formation of zein particles encapsulating plasmid DNA, using simple fabrication techniques resulting in preservation of plasmid integrity and tunable sizes. DNA encapsulation efficiencies were maximized to acceptable levels at higher zein to DNA ratios, while loading was comparable to that of other hydrophilic compounds encapsulated in zein and that of DNA incorporated into PLGA nano- and microspheres. The hydrophobic nature of zein resulted in spheres capable of sustained release of plasmid DNA. Zein particles may be an excellent potential tool for the delivery of DNA with the ability to be fine-tuned for specific applications including oral gene delivery, intramuscular delivery, and in the fabrication of tissue engineering scaffolds.

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Figures

Figure 1
Figure 1
SEM of zein-DNA nanospheres formed at 20:1 (A, 90 k magnification, scale bar 500 nm), 40:1 (B, 90 k, scale bar 500 nm), 80:1 (C, 60 k, scale bar 500 nm), 160:1(D, 60 k, scale bar 500 nm), 250:1 (E, 30 k, scale bar 1 μm) zein:DNA ratios; average diameter for each ratio measured from SEM images (F), reported as mean ± standard error of the mean.
Figure 2
Figure 2
Size, PdI, and zeta potential for zein/DNA nanospheres formed at various zein to DNA ratios measured directly after resuspension (0 hours) and three hours after resuspension (3 hours) in wate (A, C, E) or PBS (B, D, F). Data points labeled with the same letter are not significantly different, while those labeled with asterisks vary significantly (p < 0.05) between the zero hour and 3 hour measurements. All data are reported as mean ± standard error of the mean, with n = 6.
Figure 3
Figure 3
Percent encapsulation and loading of nanospheres prepared at various zein to DNA ratios. Data labeled with the same letter are not significantly different. Data are reported as mean ± standard error of the mean, with n = 6.
Figure 4
Figure 4
Agarose gel electrophoresis images of extracted samples for spheres made at various zein to DNA ratios (A): lane 1, ladder; lane 2, stock DNA; lane 3, 20:1 spheres; lane 4, 20:1 supernatant; lane 5, 40:1 spheres; lane 6, 40:1 supernatant; lane 7, 80:1 spheres; lane 8, 80:1 supernatant; lane 9, 160:1 spheres; lane 10, 160:1 supernatant; lane 11, 250:1 spheres; lane 12, 250:1 supernatant; lane 13, stock DNA; lane 14 ladder. Agarose gel image of DNA extracted from spheres (B) and supernatants (C) at various time points in the PBS release study: lane 1, ladder; lane 2, stock DNA; lane 3, 0 hr; lane 4, 1 hr; lane 5, 3 hr; lane 6, 6 hr; lane 7, 9 hr; lane 8, 12 hr; lane 9, 24 hr; lane 10, ladder; lane 11, 48 hr; lane 12, 72 hr; lane 13, 96 hr; lane 14, 120 hr; lane 15, 144 hr; lane 16, 168 hr; lane 17, stock DNA; lane 18, ladder. Agarose gel electrophoresis images of pDNA in DNase I degradation assay : lane 1, ladder; lane 2, stock DNA; lane 3, Naked DNA + DNase I; lane 4, 80:1 spheres + DNase I; lane 5, blank spheres (zein with no DNA); lane 6, 80:1 spheres ; lane 7, stock DNA; lane 8, ladder.
Figure 5
Figure 5
Release of DNA from 80:1 zein spheres incubated in PBS at 37°C over 7 days. Data reported as mean ± standard error of the mean, with n = 5.
Figure 6
Figure 6
Cytotoxicity of zein-DNA nanospheres quantified by WST-1 assay for (A) HEK 293T and (B) Caco-2 cells as a function of time and DNA dose. Control condition represents cells without the addition of particles. Data points labeled with the same letter are not significantly different (p < 0.05). Data reported as mean ± standard error of the mean, with n = 3.
Figure 7
Figure 7
Confocal images of HEK 293T cells with associated fluorescent 80:1 spheres (A, 600X magnification, 0.1 μg DNA/well) and 250:1 spheres (C, 600X, 0.1 μg DNA/well), and Caco-2 cells with associated 80:1 spheres (B, 600X, 0.1 μg DNA/well) and 250:1 spheres (D, 600X, 0.1 μg DNA/well). It should be noted that not all of the particles fluoresced in the images as some were out of the focal plane of the microscope. Scale bars represent 20 μm.
Figure 8
Figure 8
Analysis of sphere internalization within Caco-2 cells, with DiO membrane staining, using merged fluorescence confocal images of entire z-stack with orthogonal views, XZ and YZ (A) and a digital magnification of the area outlined by a white square in A (B). Punctate green staining within cells are indicative of endosomes. Filled arrows indicate internalized, autofluorescent nanospheres. Scale bars in A and B represent 50 and 20 μm, respectively.
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