(352d) DNA-Mediated, on-Membrane Sequential Assemblyof Conjugated Polymer Nanoparticles for Sensitive Detectionof Cell Surface Markers

Cell surface markers are proteins that are expressed on cell membrane. They have been reported to be associated with diseases such as cancer. Also, they can be labeled with fluorescent probes. Therefore, they have been targets for diagnosis and therapy. Flow cytometry is a gold standard to detect these markers. Although more than several thousand markers need to be expressed on a cell to detect, some crucial markers do not meet this detection limit. Therefore, improvement of detection sensitivity is necessary. To amplify fluorescent signals, in previous studies, antibody was modified with multiple fluorescent dyes. However, excessive modification made antibodies lose their binding ability. On the other hand, use of large fluorescent particles (Ca. >60 nm) with high bright fluorescence tends to inhibit the access to neighboring makers due to steric hindrance. Therefore, alternative strategy for fluorescent amplification for antibodies is needed. Conjugated polymer nanoparticles (Pdots) are one of the new fluorescent probes ^[1]. Pdots have been investigated in the biomedical field because of their highly bright fluorescence, excellent biocompatibility, photostability, and size-tunability. Recently, DNA-mediated assembly of nanoparticles has attracted attention. Modification of single-stranded DNA on nanoparticle surface and subsequent hybridization with linker DNA that has complementary sequences enables flexible arrangement of nanoparticles in superstructure. Since the properties of photo-functional nanoparticles such as gold nanoparticles and quantum dots change with steric configuration, such as the inter-particle distance, their assembly can be expected to expand their functions ^[2]. It is expected that employment of this technology for Pdots could enable highly bright labeling. However, to the best of our knowledge, there is no report on not only the assembly of Pdots using DNA, but also fluorescent nanoparticle assembly on cell membrane. In this study, we aim to achieve highly sensitive surface marker detection via sequential on-membrane assembly of Pdots mediated by DNA.

Experimental

1. Synthesis of Pdots

Pdots was synthesized by nanoprecipitation method previously reported by our group ^[3]. Poly [2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) and poly (styrene-co-maleic anhydride) (PSMA) were used as fluorescent conjugated polymer and dispersion stabilizer, respectably. The mixture of MEH-PPV (0.04 mg/mL) and PSMA (0.01 mg/mL) dissolved in tetrahydrofuran (THF) was added to 100 mL of pure water under sonication and vigorous stirring to form a homogenous Pdots solution. After purification by ultracentrifugation, the particle size distribution and morphology were evaluated by DLS and TEM observation.

2. DNA modified Pdots (DNA-Pdots)
The Pdot surface were modified with DNA by carbodiimide reaction. Amine-functionalized single stranded DNAs, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinyl imide (NHS) were added to the Pdots dispersion for the reaction. After centrifugation to remove free DNA, surface evaluation was conducted by FT-IR and gel electrophoresis.

3. Study of Pdot assembly on cells
CD19 expressed on Nalm-6, a cell line derived from human B-cell acute lymphoblastic leukemia cells, was used as a target for demonstrating Pdot assembly on cells membrane. First, Nalm-6 cells were treated with 4 % paraformaldehyde solution. Then, DNA conjugated anti-CD19 antibody were added to cell suspension in PBS containing 1 % BSA. After the purification, linker-DNA and DNA-Pdots that have complemental sequences were added one by one, followed by the hybridization at room temperature to label CD19 with Pdots. Afterwards, linker-DNA and DNA-Pdots were further added sequentially to achieve on-membrane Pdot assembly.

Results and Discussion

1. Synthesis of Pdots
TEM image revealed that the obtained nanoparticles have an average diameter of 28 nm. Absorption and fluorescent spectrum of the obtained MEH-PPV Pdots were also evaluated by UV-vis spectroscopy and fluorometer. Pdots exhibit broad absorption band ranging from 400 nm to 550 nm, which is convenient for fluorescent microscopy and laser excitation. The maximum fluorescent emission peak was 590 nm, which is longer than autofluorescence of cells. These results illustrated the proper optical property of Pdots for surface marker detection.

2. DNA modified Pdots (DNA-Pdots)
FT-IR spectra of free DNA and Pdots before and after DNA modification were measured. Spectrum of DNA-Pdots had peaks at 1090 cm^-1 and 1200 cm^-1 derived from the phosphate group of DNA, and the peak at 3400 cm^-1 derived from the amide bond. Gel electrophoresis was also conducted in agarose gel for Pdots before and after DNA conjugation. After the conjugation of DNA, position of the band shifted to downstream region, which is caused by the increased negative surface charge. These results suggested successful DNA modification on the surface of Pdots.

3. Study of Pdot assembly on cells
To investigate if it is possible to assemble Pdots on actual cell surface markers, CD 19 expressed on Nalm-6 cells was used as a target. Confocal laser microscope was used for obtaining fluorescence image of Pdot assembly on cells at each step. Fluorescent intensity was weak at the initial step, whereas it was enhanced with the number of assemblies. Therefore, Pdots assembly on Nalm-6 cells were suggested. Fluorescent evaluation was further conducted by flow cytometry. The fluorescent signal in flow cytometry was increased with increasing number of assembly steps, demonstrating the successfully fluorescent amplification by sequential Pdot assembly on cell surface markers. By optimizing the reaction conditions for the assembly, up to 22-fold fluorescent intensity increase was achieved.

To further investigate the advantage of sequential assembly, we compared the results with one-step labeling with larger Pdots. Because the average number of Pdots (28 nm) after 2 step assembly on a marker was estimated to be 22, 95 nm Pdots, whose volume is almost equivalent to that of total assembled 28 nm Pdots, was chosen for the comparison. As a result, fluorescent intensity obtained by one-step labeling using 95 nm Pdots was much lower than that by sequential 28 nm Pdot assembly, which would be due to the steric hindrance between particles. These results further highlighted the usefulness of the proposed sequential assembly strategy of Pdots mediated by DNA.

Summary

Pdots and antibodies were modified with single stranded DNAs. After sequential assembly of Pdots on cell membranes via complementary DNA linkers, the fluorescent signal in flow cytometry was successfully amplified, up to 22-fold compared with the first step, giving a much stronger signal than conventional fluorescent dye-modified antibody. The obtained results are expected to contribute for early disease diagnostics.

References

[1] C. Wu, J. McNeill, ACS Nano, 2, 11,2415－2423, (2008).

[2] S. Ohta, D. Glancy, W.C.W. Chan, Science, 351, 841－845, (2016).

[3] N. Nakamura, N. Tanaka, S. Ohta, RSC Adv., 12, 11606－11611, (2022).