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 polyethyleneimine (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 describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the toxicity of polymer-based nanomaterials in plants and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

Carbon nanotube biocompatibility in plants is determined by their surface chemistry

“…Our results demonstrate that toxicity generated by PEI in PEI-SWNTs can become a limiting factor when high concentrations of PEI-SWNTs are required to ensure efficient biomolecule delivery. To find more biocompatible SWNT surface chemistries we infiltrated Arabidopsis leaves with several PEI polymer variants covalently bound to SWNTs with similar zeta potential and DNA binding capabilities to PEI -SWNTs 51 . We infiltrated 50mg/L of low molecular weight linear PEI (800 Da; L-PEI 800), hydrophobically modified branched PEI (25-30 kDa; H-PEI) and high molecular weight branched PEI (750 kDa; PEI 750k).…”

“…Plasmid DNA adsorbed PEI-SWNTs were prepared as described in 29 . Other polymer-SWNTs were prepared as described in 51 .…”

Importance of Standardizing Analytical Characterization Methodology for Improved Reliability of the Nanomedicine Literature

“…However, many studies, particularly those claiming cytotoxicity, lack analytical validation of nanoparticle purity and/or surface chemistry [ 21 ]. Meanwhile, nanomaterial purity can vary greatly from the specifications provided by the manufacturer, such as commercially-procured carboxylated nanotubes (COOH-SWNT) that were found to contain over 80% more amorphous carbon contamination than specified by its supplier, Sigma-Aldrich [ 22 ]. Therefore, it is possible that reports of carbon nanotube toxicity may originate from the toxicity of residual nanotube synthesis by-products such as amorphous carbon and residual metal precursor catalysts, or from intended or unintended modifications to nanotube surface chemistry [ 23 ].…”

“…The characterization of nanomaterials in scientific papers, however, can vary widely from study to study since these materials are mostly used in preclinical animal models. One common omission from many academic studies is analytical assessments of nanomaterials when used as-procured from commercial sources, despite large possible variabilities in the quality and purity of commercially procured nanomaterials from supplier-to-supplier, from batch-to-batch, and even from the vendor-provided spec sheet [ 22 ]. Efforts to increase the rigor and reproducibility of nanomaterials science research have been initiated both from the scientific community and from national regulatory bodies as described below.…”

Targeted Carbon Nanostructures for Chemical and Gene Delivery to Plant Chloroplasts

“…9,11,19,24,36 High aspect ratio nanomaterials functionalized with highly positively charged polymers have been reported to enable the delivery of genetic elements into plants nuclear and chloroplast genomes. 11,19,37,38 The delivery of a DNA plasmid encoding a green fluorescent protein (GFP) to the plant nuclear genome was mediated by single-walled carbon nanotubes (SWCNTs) that were covalently modified with a cationic polymer (polyethylenimine, PEI). 24,37 The Figure 1.…”