Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

Figures & data

Figure 1 Preparation and functionalization of nanodiamonds (NDs) with lysine in the presence of a three-carbon-length linker: (A) synthesis of compound 1 [N′-(N^α,N^ε-bis-Boc-lysyl),N″-(Fmoc)-diaminopropane], reagents, and solvents – Fmoc-NH(CH₂)₃NH₂ · HCl, HATU [N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate], diisopropylethylamine, and dimethylformamide (DMF); (B) Fmoc deprotection, reagents, and solvents – 50% (v/v) piperidine-DMF; (C) reoxidation of pristine carboxylated NDs, reagents – concentrated sulfuric acid and concentrated nitric acid, and preparation of acid chloride–functionalized NDs, reagents – thionyl chloride; (D) synthesis of Boc-protected lysine-functionalized NDs, solvent – DMF; (E) deprotection of Boc on lysine-functionalized NDs, reagents, and solvents – hydrochloric acid and dichloromethane.

Figure 2 Baseline-corrected static scan Raman spectra of the pristine carboxylated nanodiamonds (pNDs), reoxidized nanodiamonds (rNDs), lysine-functionalized nanodiamonds (fNDs), and compound 1 [N′-(N^α,N^ε-bis-Boc-lysyl),N″-(Fmoc)-diaminopropane] using a 514.5 nm excitation source. A total of 128 accumulations were collected for pNDs and rNDs, while the spectrum of fNDs was obtained from a single acquisition.

Figure 3 Baseline-corrected absorbance and attenuated total reflectance: (A) infrared spectra of the pristine carboxylated nanodiamonds (pNDs), reoxidized nanodiamonds (rNDs), lysine-functionalized nanodiamonds (fNDs), and compound 1 [N′-(N^α,N^ε-bis-Boc-lysyl),N″-(Fmoc)-diaminopropane]; (B) second derivatives of all spectra.

Notes: A total of 512 individual interferograms were collected and averaged for each sample. The featureless region between wave numbers of 2700 and 1900 cm⁻¹ is not shown.

Table 1 Zeta potential measurements of nanodiamonds (NDs)

Sample	Zeta potential (mV) in reverse osmosis-purified water
pNDs	−21.1 ± 0.2
rNDs	−23.7 ± 0.5
fNDs	48.9 ± 0.1

Note: Each value represents the mean plus or minus standard deviation of three measurements (n ≥ 10).

Abbreviations: pNDs, pristine carboxylated nanodiamonds; rNDs, reoxidized nanodiamonds; fNDs, lysine-functionalized nanodiamonds.

Table 2 Surface loading of nanodiamonds (NDs), as calculated from their respective thermograms

Sample	Surface loading (mmol/g)
pNDs	0.35
rNDs	1.00
fNDs	1.70

Note: Weight loss below 115°C is excluded.

Abbreviations: pNDs, pristine carboxylated nanodiamonds; rNDs, reoxidized nanodiamonds; fNDs, lysine-functionalized nanodiamonds.

Figure 4 Thermograms of pristine carboxylated nanodiamonds (pNDs), reoxidized nanodiamonds (rNDs), and lysine-functionalized nanodiamonds (fNDs).

Figure 5 Dispersion of 2 mg/mL nanodiamonds (NDs) in water after 20 minutes of conventional bath sonication and 3-day incubation.

Abbreviations: pNDs, pristine carboxylated nanodiamonds; rNDs, re-oxidized nanodiamonds; fNDs, lysine-functionalized nanodiamonds.

Figure 6 Size distributions of pristine carboxylated nanodiamonds (pNDs), reoxidized nanodiamonds (rNDs), and lysine-functionalized nanodiamonds (fNDs) in water, as measured by dynamic light scattering.

Note: Each curve is derived from three measurements (n ≥ 10).

Figure 7 (A) Intermittent contact mode atomic force microscopy topographic images of nanodiamond samples and (B) line-scan profiles of the particles indicated in each image. From left to right: (i) pristine carboxylated, (ii) reoxidized, and (iii) lysine-functionalized nanodiamonds.

Note: The bar (solid white line) represents 500 nm.

Figure 8 Results of agarose gel electrophoresis of (A) lysine-functionalized nanodiamond (fND)–plasmid DNA and (B) fND–small interfering RNA complexes (“diamoplexes”) with increasing weight ratios of fNDs to nucleic acid: 1:1 (lane 2), 5:1 (lane 3), 10:1 (lane 4), 15:1 (lane 5), 20:1 (lane 6), 25:1 (lane 7), 30:1 (lane 8), 35:1 (lane 9), 40:1 (lane 10), 45:1 (lane 11), and 50:1 (lane 12). Lane 1 consists of standard nucleic acid only, and lane 13 is empty.

Figure 9 Size and zeta potential measurements using various weight ratios of (A) lysine-functionalized nanodiamonds and plasmid DNA (fNDs:pDNA) and (B) fNDs and small interfering RNA (fNDs:siRNA).

Notes: Each value represents mean plus or minus standard deviation of four measurements (n ≥ 10).

Table 3 Ratios of lysine linker residues on the nanodiamonds (NDs) per base pair of genetic material, as calculated from surface loading measurements

Weight ratio of fNDs:genetic material	Calculated lysine linker per pDNA base pair ratio	Calculated lysine linker per siRNA base pair ratio
1:1	1.1	1.1
5:1	NR	5.4
6:1	6.6	NR
10:1	11	11
20:1	22	22
30:1	33	32
40:1	44	43
50:1	55	54

Abbreviations: fNDs, lysine-functionalized nanodiamonds; pDNA, plasmid DNA; siRNA, small interfering RNA; NR, not reported.

Figure 10 Schematic representation of the different binding behavior of plasmid DNA (pDNA) and small interfering RNA (siRNA) to positively charged lysine-functionalized nanodiamonds (fNDs).

Figure S1 Proton nuclear magnetic resonance spectra of compound 1 [N′-(N^α,N^ε-bis-Boc-lysyl),N″-(Fmoc)-diaminopropane] at 500 MHz.

Figure S2 Mass spectrum of compound 1 [N′-(N^α,N^ε-bis-Boc-lysyl),N″-(Fmoc)-diaminopropane].

Abbreviations: max, maximum; m/z, mass-to-charge ratio.