Size Control and Fluorescence Labeling of Polydopamine Melanin-Mimetic Nanoparticles for Intracellular Imaging

Abstract

As synthetic analogs of the natural pigment melanin, polydopamine nanoparticles (NPs) are under active investigation as non-toxic anticancer photothermal agents and as free radical scavenging therapeutics. By analogy to the widely adopted polydopamine coatings, polydopamine NPs offer the potential for facile aqueous synthesis and incorporation of (bio)functional groups under mild temperature and pH conditions. However, clear procedures for the convenient and reproducible control of critical NP properties such as particle diameter, surface charge, and loading with functional molecules have yet to be established. In this work, we have synthesized polydopamine-based melanin-mimetic nanoparticles (MMNPs) with finely controlled diameters spanning ≈25 to 120 nm and report on the pH-dependence of zeta potential, methodologies for PEGylation, and the incorporation of fluorescent organic molecules. A comprehensive suite of complementary techniques, including dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), X-ray photoelectron spectroscopy (XPS), zeta-potential, ultraviolet-visible (UV-Vis) absorption and fluorescence spectroscopy, and confocal microscopy, was used to characterize the MMNPs and their properties. Our PEGylated MMNPs are highly stable in both phosphate-buffered saline (PBS) and in cell culture media and exhibit no cytotoxicity up to at least 100 μg mL^-1 concentrations. We also show that a post-functionalization methodology for fluorophore loading is especially suitable for producing MMNPs with stable fluorescence and significantly narrower emission profiles than previous reports, suggesting they will be useful for multimodal cell imaging. Our results pave the way towards biomedical imaging and possibly drug delivery applications, as well as fundamental studies of MMNP size and surface chemistry dependent cellular interactions.

Keywords: catechol; dopamine; melanin; nanoparticle.

Figure 1

Ultraviolet–visible (UV–Vis) absorbance and surface charge of polydopamine-based melanin-mimetic nanoparticles (MMNPs). (a) UV-Vis spectra of purified MMNPs and filtrate removed from crude product via 10 kDa centrifugal filtration. Arrows in (a) indicate the two peaks observed at 280 and 398 nm in the filtrate absorbance spectrum that are absent in the purified MMNP absorbance spectrum. A.U.: Arbitrary units. (b) Zeta potential of MMNPs at pH 2.5–9.0. The isoelectric point is approximately pH 4.0–4.1.

Figure 2

Dynamic light scattering (DLS) analysis of MMNPs. (a) Mean hydrodynamic diameters and (b) polydispersity indices (PDI) of multiple batches of MMNPs prepared at various dopamine·HCl (DA) concentrations and NaOH:DA ratios. n = 3−19 independently prepared batches of MMNPs were analyzed for each synthetic condition. Error bars represent standard deviations. Bars not sharing symbols in (a) differ significantly with p < 0.001.

Figure 3

Transmission electron microscopy (TEM) images of MMNPs and quantitative analysis of nanoparticle diameter grown at the conditions specified at the top of each column. (a–c) TEM images with uranyl acetate negative stain. (d–f) Cryo-TEM images were taken without staining. Nanoparticles are spherical but have rougher appearances as diameter decreases. (g–i) Distribution of MMNP diameters in cryo-TEM images.

Figure 4

MMNP@PEG vs. MMNP zeta potential, hydrodynamic diameter, morphology, and atomic composition. (a) Zeta potentials and (b) hydrodynamic diameters of MMNPs, MMNP@PEG, and control MMNPs treated with 10 mM NaOH base. Samples not sharing symbols are significantly different (p < 0.05). (c) TEM image of MMNP@PEG. (d) XPS survey scans of MMNP and MMNP@PEG with assignments for O 1s, N 1s, C 1s, and S 2s, and S 2p peaks. (e) C/O atomic ratios in MMNP vs. MMNP@PEG calculated from C 1s and O 1s signal ratios (* p < 0.01). at%: Atomic percent relative to total C, N, O, and S content. (f) Sulfur content in MMNP vs. MMNP@PEG calculated from S 2p signal intensity expressed as at% S (* p < 0.01). Error bars represent standard errors.

Figure 5

Approaches to synthesis of fluorescent MMNPs. (a) Structure of rhodamine 123 (RA123). (b) Structures of rhodamine B (RAB), including the fluorescent cationic acid, non-fluorescent neutral lactone, and fluorescent zwitterionic structures. (c) In situ approach and subsequent PEGylation: MMNP@RA123 and MMNP@RAB are first synthesized by DA polymerization in the presence of RA123 or RAB. These fluorescent NPs are subsequently PEGylated, forming MMNP@RA123@PEG and MMNP@RAB@PEG. (d) Post-functionalization approach: MMNP@PEG@RA123 and MMNP@PEG@RAB are formed by treatment of MMNP@PEG with RA123 or RAB in unbuffered ultrapure water or pH 8.5 buffer.

Figure 6

Fluorescence emission spectra and TEM images of in situ labeled MMNP@RA123 and MMNP@RAB. (a) Fluorescent emission spectra (λ_ex = 500 nm) of MMNP@RA123@PEG after seven day dialysis in 1× phosphate-buffered saline (PBS), rhodamine 123, and MMNP@PEG. (b) TEM image of MMNP@RAB. (c) Fluorescent emission spectra (λ_ex = 555 nm) of MMNP@RAB@PEG after seven day dialysis in 1× PBS, rhodamine B, and MMNP@PEG. (d) TEM image of MMNP@RA123.

Figure 7

Fluorescence, hydrodynamic diameter, and zeta potential of rhodamine post-functionalized MMNP@PEG. (a) Zeta potentials and (b) hydrodynamic diameters of rhodamine post-functionalized MMNP@PEG samples prepared in water or at pH 8.5 vs. unmodified MMNPs and MMNP@PEG. Groups not sharing symbols have significantly different values (p < 0.05). (c) Fluorescence emission spectra (λ_ex = 500 nm) of 25 µg mL⁻¹ samples of MMNP@PEG before and after modification with RA123 in water or at pH 8.5 followed by serial dialysis in ultrapure (UP) water for 72 h and 1× PBS for 72 h. Emission spectrum of RA123 was taken at 10 ng mL⁻¹. (d) Fluorescence emission spectra (λ_ex = 555 nm) of 25 µg mL⁻¹ samples of MMNP@PEG before and after modification with RAB in water or at pH 8.5 followed by serial dialysis in UP water for 72 h and 1× PBS for 72 h. Emission spectrum of RAB was taken at 10 ng mL⁻¹.

Figure 8

In vitro investigation of MMNP-cell interactions. (a) MMNP cytocompatibility with NIH/3T3 fibroblasts as measured by neutral red uptake viability assay. Error bars represent standard errors of triplicate experiments. (b) Representative confocal microscopy three-dimensional (3D) z-stack reconstruction image of Hoechst-stained NIH/3T3 fibroblasts treated with 20 µg mL⁻¹ MMNP@PEG@RA123. Hoechst stain (blue) and rhodamine fluorescence (red/pink) are shown here; Scale bar: 20 µm between gridlines.