Redox-Switchable Multicolor Fluorescent Polymers for Theragnosis of Osteoarthritis

By Nidhi DhullReviewed by Laura ThomsonNov 29 2024

A recent article published in Nature Communications explored an emission mechanism in bioactive polycysteine (PCys), an aliphatic polymer exhibiting high quantum yield, polymerization- and aggregation-induced emission, and multicolor emission characteristics. The polymer's anti-inflammatory and antioxidative activities are promising for in vivo theragnosis of osteoarthritis (OA).

Background

Many biological macromolecules like green fluorescent protein (GFP) display fluorescent properties.

Jellyfish-derived GFP is a popular biomarker and imaging tool for investigating protein localization and gene expression.

Inspired by such fluorophores, synthetic luminescent polymers have been fabricated for several applications, including biosensing, imaging, optoelectronics, molecular detection, and anti-counterfeiting. However, most of these polymers have limitations of poor water solubility, potential toxicity, and complicated synthesis.

Nonconjugated and nonaromatic fluorescent materials without common chromophores offer superior processability, molecular flexibility, biocompatibility, and biodegradability. However, the basic mechanism of their unique luminescence remains mostly unknown.  

Nonclassical aliphatic fluorescent polymers have been barely used for disease diagnosis and treatment in vivo.

This study examined the intrinsic fluorescence and color-switching characteristics and in vivo OA theragnosis potential of PCys derived from natural amino acids.

Methods

S-carbobenzoxy-L-cysteine (L-Cys-Cbz) powder was synthesized from L-Cysteine and benzyl chloroformate. It was used with tetrahxydrofuran and triphosgene to synthesize S-carbobenzoxy-L-cysteine-N-carboxyanhydride (L-Cys-Cbz NCA) via the Fuchs-Farthing method.

To obtain MPEG-PCys-Cbz, ring-opening polymerization of L-Cys-Cbz NCA, initiated by methoxy polyethylene glycol amine (MPEG-NH2), was performed.

This was treated with HBr in CF₃COOH to obtain MPEG-PCys (PCysx).

The x in PCysx polymers represented the number of cysteine residues per chain. Finally, PCys_x assemblies were prepared using a dialysis method.

The PCys' fluorescence quantum yield (QY) was measured in H₂O relative to quinine sulfate in H₂SO₄. Their fluorescence spectra were recorded in dimethylformamide (DMF)-ether mixtures. Meanwhile, time-dependent density functional theory (TD-DFT) was employed to elucidate the corresponding fluorescence mechanism.

4T1 cancer cells and chondrocytes were used for cellular assays, including cell viability, multicolor cell imaging, intracellular reactive oxygen species (ROS) detection and scavenging activity, intracellular glutathione (GSH) detection, and cell apoptosis assay.

Male Sprague Dawley rats were selected for the OA model and in vivo therapy using the PCys. OA was induced in the rats via a single intra-articular injection of monosodium iodoacetate (MIA) in a knee joint. After three weeks of MIA injection, the rats were treated with PCys weekly (a total of five times). Apart from continuous analysis via in vivo imaging and micro-computed tomography (micro-CT) arthrography, histological studies were performed to assess the biological safety of the treatment.

Results and Discussion

PCys exhibited strong visible fluorescence in solid, solution, and water dispersion forms under ultraviolet irradiation. The fluorescence intensity for PCys in water was considerably higher with a red-shifted optimal excitation wavelength than in DMF. This was attributed to the aggregation of PCys segments via self-assembly of copolymer in water.

Dynamic laser scattering and transmission electron microscopy measurements revealed that PCys_x self-assembled into spherical nanoparticles with a unimodal distribution. These nanoparticles exhibited diverse fluorescent colors under varying excitation wavelengths, indicating that PCys could emit multicolor fluorescence. The emission peaks shifted to longer wavelengths with increasing excitation wavelength, which is favorable for biomedical applications.

The TD-DFT simulations revealed that polymerization reduced the distance between discrete sulfhydryl groups of Cys and PCys, enhancing electron delocalization and electron cloud overlap and thus lowering the energy gap. This facilitated electron transition and luminescence generation.

Considering biomedical applications, PCys did not show any inhibitory effect against the 4T1 cells, even at a high concentration. Alternatively, PCys exhibited distinct intracellular fluorescence and outlined the cell contours under different excitation wavelengths, demonstrating their great potential for multicolor cell imaging. Moreover, PCys could successfully detect the intracellular redox microenvironment comprising H₂O₂ or GSH in 4T1 cells.

In the OA model analysis and in vivo therapy on rats, PCys exhibited enhanced anti-inflammatory, anti-oxidative, and anti-apoptotic activities. Simultaneously, they provided a linear fluorescence response to the redox microenvironment. Furthermore, micro-CT imaging confirmed that PCys could effectively prevent OA progression and cartilage degradation. Therefore, PCys effectively treated OA and enabled real-time monitoring of ROS levels in the joint cavity.

Conclusion

The researchers proposed a non-classical luminescent aliphatic polymer employing aggregated thiols as chromophores. The developed polymer exhibited molecular weight-dependent high QY, exceptional multicolor luminescence, and multicolor imaging properties.

The polymer's fluorescence could be reversibly switched on and off under reductive and oxidative conditions, respectively, facilitating quantitative detection of ROS and GSH in cells and in vivo. Additionally, the bioactive polymer exhibited superior and more stable antioxidant properties than first-line clinical antioxidants, enabling long-term treatment and diagnosis of OA.

The fluorescent polymer had a simple structure, distinct luminescence mechanism, good biocompatibility, and numerous active sites for functionalization. It can help advance bioactive imaging materials with a potential for clinical translation in biosensing, drug delivery, and disease theragnosis applications.

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