Sustained Release of Minor-Groove-Binding Antibiotic Netropsin from Calcium-Coated Groove-Rich DNA Particles

Abstract

Control of the release properties of drugs has been considered a key factor in the development of drug delivery systems (DDSs). However, drug delivery has limitations including cytotoxicity, low loading efficiency, and burst release. To overcome these challenges, nano or micro-particles have been suggested as carrier systems to deliver chemical drugs. Herein, nano-sized DNA particles (DNAp) were manufactured to deliver netropsin, which is known to bind to DNA minor grooves. The rationally designed particles with exposed rich minor grooves were prepared by DNAp synthesis via rolling circle amplification (RCA). DNAp could load large quantities of netropsin in its minor grooves. An analytical method was also developed for the quantification of netropsin binding to DNAp by UV-visible spectrometry. Moreover, controlled release of netropsin was achieved by forming a layer of Ca²⁺ on the DNAp (CaDNAp). As a proof of concept, the sustained release of netropsin by CaDNAp highlights the potential of the DNAp-based delivery approach.

Keywords: DNA particle (DNAp); calcium; minor groove binder; netropsin; rolling circle amplification (RCA).

Figure 1

Schematic illustration of CaDNAp@Ne synthesis. (A) Scheme for DNA particle (DNAp) synthesis via rolling circle amplification (RCA). (B) Scheme of netropsin loading and netropsin-loaded DNAp (DNAp@Ne) synthesis. Because netropsin is a well-known minor groove binder of double-strand DNA, DNAp was used as a supporting matrix for netropsin. (C) Scheme for synthesizing DNAp@Ne from netropsin loading to DNAp and CaDNAp@Ne from Ca²⁺ coating of DNAp@Ne.

Figure 2

Stoichiometric analysis of netropsin. (A) UV–vis spectra of 0 to 100 μM netropsin in aqueous solution. Netropsin showed major peaks at 245 and 294 nm, and 294 nm was selected as it did not overlap with the major peak of DNAp (260 nm). (B) Linear regression plot for the netropsin concentration dependence of the absorbance at 294 nm.

Figure 3

Stoichiometric analysis of netropsin loading on DNAp. (A) UV–vis spectra of DNAp and DNAp@Ne. The netropsin treatment time for synthesizing DNAp@Ne was 6 h. DNAp and DNAp@Ne were adjusted to final concentrations of 100 ng μL⁻¹ (DNAp: red, DNAp@Ne: blue). (B) Stoichiometric analysis of netropsin in DNAp@Ne. The increase in UV–vis absorbance of DNAp@Ne as a function of the absorbance of DNAp (black line) was similar to that of the free netropsin (netropsin 40 μM: purple, 30 μM: blue, 20 μM: red). (C) Time-dependent netropsin loading profile on DNAp@Ne where 100 μM of netropsin was added to a solution of 100 ng μL⁻¹ of DNAp and measured at various time points.

Figure 4

Synthesis and characterization of CaDNAp via Ca²⁺ coating of DNAp. (A) Schematic illustration for synthesis of CaDNAp (top) and SEM (middle) and TEM images (bottom) of DNAp, low-CaDNAp, and high-CaDNAp (scale bar: 1 μm). (B) Analysis of Ca²⁺ content quantification coated on CaDNAp. (C) TEM-EDS mapping images of DNAp (top row), low-CaDNAp (middle row), and high-CaDNAp (bottom row). Each element is indicated above the image. (D) The atomic ratios of magnesium and calcium to phosphorus for DNAp, low-CaDNAp, and high-CaDNAp. Atomic percentage data were obtained from TEM-EDS.

Figure 5

Netropsin release efficiency of DNAp@Ne and CaDNAp@Ne. A cumulative graph of the three samples produced under the same conditions is shown (n = 3). For each time point, the sample supernatant was measured using a UV–vis spectrometer (DNAp: black, DNAp@Ne: blue, low-CaDNAp@Ne: light green, high-CaDNAp@Ne: green).