Polymer-Conjugated Carbon Nanotubes for Biomolecule Loading

Abstract

Nanomaterials have emerged as an invaluable tool for the delivery of biomolecules such as DNA and RNA, with various applications in genetic engineering and post-transcriptional genetic manipulation. Alongside this development, there has been an increasing use of polymer-based techniques, such as polyethylenimine (PEI), to electrostatically load polynucleotide cargoes onto nanomaterial carriers. However, there remains a need to assess nanomaterial properties, conjugation conditions, and biocompatibility of these nanomaterial-polymer constructs, particularly for use in plant systems. In this work, we develop mechanisms to optimize DNA loading on single-walled carbon nanotubes (SWNTs) with a library of polymer-SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. Counterintuitively, we demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and we describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the stress response of plants to polymer-based nanomaterials and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

Keywords: DNA loading; carbon nanotubes; nanomaterials; plant; polymer; toxicity.

Figure 1.

Synthesis and characterization of polymer-SWNTs: (a) scheme of polymer-SWNT synthesis using EDC-NHS chemistry and subsequent DNA loading; (b) ζ potential measurements of initial COOH-SWNT constructs, after conjugation with BPEI-25k via EDC-NHS chemistry and after addition of DNA; (c) scheme of polymer-SWNT synthesis using triazine chemistry and subsequent DNA loading; (d) ζ potential measurements of triazine-functionalized SWNTs, after conjugation with BPEI-25k via nucleophilic substitution and after addition of DNA.

Figure 2.

Quantification of amorphous carbon and SWNT carboxylation: (a) TGA measurements of COOH-SWNTs either purchased commercially or carboxylated in-house via reflux in nitric acid and washed with 1.0 M NaOH; (b) bar plot displaying carboxyl group peak area, normalized in relation to sp² C peak area in C 1s XPS spectra.

Figure 3.

Quantification of free polymer removal from polymer-SWNT complexes. (a) Schematic of polymer-SWNT washing via spin filtration. (b) Schematic of polymer-SWNT washing via vacuum frit filtration. (c) Filtrate from the first polymer-SWNT wash step loaded with DNA and run on an agarose gel for all polymers. (d) Filtrate from the sixth polymer-SWNT wash step loaded with DNA and run on an agarose gel for all polymers. From left to right: (1) free plasmid, (2) LPEI-5000, (3) LPEI-800, (4) BPEI-800, (5) low-phi-BPEI, (6) branched polylysine, (7) med-phi-BPEI, (8) BPEI-750k, (9) BPEI-25k. (e) Measurements taken after each wash step for BPEI-25k polymer-SWNTs show a steady increase in the plasmid migration distance, corresponding to a decrease in free polymer, after each wash step.

Figure 4.

Long-term stability of polymer-SWNT nanoparticles and protein adsorption to nanoparticles for different polymer attachments: (a) ζ potential measurements of BPEI-25k polymer-SWNT immediately after synthesis and after 30 days; (b) N 1s XPS spectra of a fresh and aged BPEI-25k polymer-SWNT sample, normalized to the respective C 1s peak; (c) ζ potential of a BPEI-25k polymer-SWNT immediately after synthesis and after storage at −80 °C for 30 days; (d) quantification of the area under the curve overlap in ζ potential spectra peaks between days 1 and 30 for each polymer-SWNT construct; (e) ζ potential measurements of BPEI-25k-Trz-SWNT immediately after synthesis and after 30 days; (f) concentration of adsorbed FAM-FBG on 5 μg mL⁻¹ polymer-SWNT. Initial concentrations of FAM-FBG added to solution were 20, 40, and 60 μg mL⁻¹ respectively. Error bars represent standard deviation of the mean (N = 3).

Figure 5.

DNA loading capacity of polymer-SWNTs: (a) ζ potential measurements, in water, of polymer-SWNT constructs before and after addition of DNA; (b) DLS measurements, in water, of polymer-SWNT constructs before and after addition of DNA, where error bars represent standard deviation of the mean (N = 3); (c) agarose gel of polymer-SWNTs (10 μg mL⁻¹) loaded with DNA (10 μg mL⁻¹).

Figure 6.

Plant toxicity induced by polymer-SWNT nanoparticles measured by stress gene response in Nicotiana benthamiana: (a) graphic illustration of leaf infiltration with polymer-SWNTs; (b) plant leaves immediately after infiltration; (c) qPCR analysis quantifying mRNA fold-change for PR1A gene 2 days after infiltration; (d) qPCR analysis quantifying mRNA fold change for NbAGP41 gene 2 days after infiltration. Error bars represent standard deviation of the mean (N = 4).