Acrylic resin-coated rhodamine-6G/isorhamnetin/nido-carborane fluorescent complexes: Photophysical properties, drug release, and tumor cell imaging studies

1. Introduction

Sea buckthorn and Ginkgo biloba are two plants that contain the flavonoid isorhamnetin (Gong et al., 2020, Liga et al., 2023, Lei et al., 2024), which has a lot of medicinal potential. It has a lot of biological and pharmacological effects, including immune-modulating, anti-tumor, antiviral, anti-myocardial hypoxia, cholesterol-lowering, and antiallergic effects (Yang et al., 2023, Liu et al., 2023, Biswas et al., 2024, Zhao et al., 2018, Yan et al., 2024).

Boron neutron capture therapy (BNCT) (Dymova et al., 2020) is a radiation therapy for biological targets (Xu et al., 2024, Mushtaq et al., 2023). Its function involves precise, targeted radiation at the cellular level, which enables single-cell selective tumoricidal therapy as a state-of-the-art, minimally invasive cancer treatment. Using B¹⁰ isotopes to produce high-energy particles (α and Li nuclei) (Wang et al., 2025) upon neutron irradiation, BNCT is a binary radiotherapy that targets and kills tumor cells (Shi et al., 2023, Chiang et al., 2021). Because carborane has a high boron content and can target tumors, it has been a focus of global cancer research. Carborane (C₂H₁₂B₁₀), a novel tumor-targeting medication, is known for its effectiveness and higher survival rates, when treating refractory tumors (Wang et al., 2024, Calabrese et al., 2012, Gozzi et al., 2021). Its dodecahedral structure comes in three different forms: o-carborane, m-carborane, and p-carborane. These forms resemble small versions of the C₆₀ and benzene rings (Sha et al., 2022). Although it is naturally hydrophobic due to its large steric hindrance backbone, chemical modifications can increase its solubility and biocompatibility (Shao et al., 2021, Chai et al., 2024). However, the limited water solubility of isorhamnetin and carborane limits their potential medical uses. Rhodamine-6G was added to fluorescent probes to bypass these solubility restrictions, allowing for both therapeutic and diagnostic uses. Real-time drug visualization was made possible by the creation of fluorescent medications through the incorporation of rhodamine (Chai et al., 2024, Ouyang et al., 2023). These substances make accu possible through fluorescence imaging (Chi et al., 2023, Deng et al., 2019) (Fig. 1).

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Fig. 1.

Isorhamnetin, rhodamine 6G, and nido-carborane structures.

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Water solubility can be improved by a variety of formulations, such as emulsions, solutions, chemical changes, and nanotechnology-based methods, with a focus on nanoparticles Wang et al. (2021). Kaniowski et al., 2020. Acrylic resin is widely recognized for its remarkable resistance to heat, water, and corrosion as a drug coating material. The hydrophilic shell that the acrylic resin coating forms around the hydrophobic core improves solubility while shielding the medication from enzymatic breakdown. Instead of being absorbed or broken down by the liver, it can withstand digestive enzymes and swell in bodily fluids to form channels when used as a drug encapsulation layer (Jiao et al. Lu et al. (2021) in 202). Two physical interactions involved in the coating of inclusion complexes during the dynamic process of materials entering and leaving the cavity are hydrogen bonding and van der Waals (Zhang et al. in 2024). The solubility problems of the rhodamine 6G/isorhamnetin/o-carborane complex are addressed by structural modifications. Biological activity can be maintained by repurposing potassium carborane salts and incorporating carbonyl groups into the fluorophore (Chai et al. in 2024). The biocompatibility and selectivity of the nanocrystalline forms are evaluated using tumor cell imaging following the application of an acrylic resin coating (Hu et al., 2023, Hu et al., 2024).

The objective of this study is to utilize previously documented advancements in polymorphism strategies of Eudragit®-encapsulated ionic polymer IR775@nido-carborane and encapsulated polyindole@nido-carborane fluorescent polymer nanocapsules to address multiple problems, including low inhibition rates and low biocompatibility. By using acrylic resin coating and nanotechnology, the anti-cancer compounds isorhamnetin and o-carborane will become more stable and soluble, increasing their efficacy. A novel approach to treating cancer and the use of different hydrophobic drug formulations could result from the successful development of this technique.

2. Materials and Methods

All solvents and reagents were procured from commercial sources and utilized as received, without additional purification. The specific reagents employed in this study include isorhamnetin (98%, RG), acrylic resin (RG), carborane (98%, RG), rhodamine 6g (98%, RG), and HeLa cells (product code: FH0314, Specification: 1×10⁶cells/T25 culture bottle), PC-3 cells (product code: FH0195, Specification: 1×10⁶cells/T25 culture bottle) and LO2 cells (product code: FH0109, Specification: 1×10⁶cells/T25 culture bottle) were procured from Shanghai Fuheng Biotechnology Co., LTD. These materials were acquired from suppliers like Titan Technologies. The bioconsumables used in this research encompass DMEM/High Glucose Medium (Product Code: AF29498406), RPMI 1640 Medium (Product Code: 70080192), Trypsin for Cell Digestion (Product Code: 70080900), and PBS (Product Code: PBSGNM20012), sourced from vendors including Hyclone, Biosharp, Bioshop, and Genomcellbio. Prior to usage, all glassware was air-dried and subsequently stored in a drying cabinet.

2.1 Synthesis

2.1.1 Synthesis of RIC-1 complexes

O-carborane (1.0 g, 0.7 mmol) and potassium hydroxide (0.4 g, 7.14 mmol) were dissolved in 5 mL of EtOH and stirred at 80°C for 2 h. Isorhamnetin (0.22 g, 0.7 mmol) and potassium hydroxide (0.14 g, 2.5 mmol) were dissolved in 3 mL of EtOH and stirred at room temperature for 1 h. Rhodamine 6g (0.28 g, 1.2 mmol) was then added and stirred for 1 h. The EtOH was concentrated to yield a 2.0 g complex. Subsequently, 50 mg of the isorhamnetin-o-carborane-rhodamine 6g complex (RIC) was reacted with 100 mg each of L100-55, EPO, RS, and RL in 3 mL of EtOH for 3 h. The solids were collected, dried under vacuum, and 30 mg of a dark reddish-brown solid (RIC-1) was obtained (Scheme 1).

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Scheme 1.

Preparation routes of four fluorescent complexes.

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FC-2, FC-3, and FC-4 were obtained using the same method. The reaction yields for RIC-1 to RIC-4 ranged from 85-92% after purification by ethanol recrystallization.

2.1.2 UV-Vis spectroscopy, fluorescence spectroscopy, and fluorescence stability tests

The UV-Vis spectrum of the fluorescent complex was recorded using a UV-2550 spectrophotometer equipped with a 1 cm quartz cuvette. For further experiments, a concentration gradient was created by dissolving 10 mg of the complex in tetrahydrofuran (THF), dichloromethane (MC), ethyl acetate (EA), MeOH, EtOH, and dimethyl sulfoxide (DMSO). The 400–700 nm range spectra of these solutions were acquired at different concentrations. The fluorescence characteristics were measured using a 10 mm cuvette with an excitation wavelength of 450 nm and an emission range of 400–800 nm. Three separate measurements of the UV-Vis spectra were made, with standard deviations less than 1%.

2.1.3 Transmission electron microscopy (TEM)

TEM analysis was carried out using a Zeiss Ultra Plus microscope and an Oxford Instruments X-Max 60 mm² SDD X-ray microanalysis system at an accelerating voltage of 15 keV. The sample, made by depositing the precipitate from the EtOH suspension onto a silicon wafer connected to a conductive adhesive, was subjected to image analysis at two point magnifications of 0 μm and 200 nm. A copper grid was ultrasonically treated for 10 to 30 min following the application of a thin supporting film. In a separate process, THF and powder were mixed in a beaker for 3-5 min. The copper grid was then covered with two to three drops, which were left to dry for over 15 min. The dehydrated sample was examined under an electron microscope.

2.1.4 Zeta potential and atomic force microscopy (AFM) testing

A Zetasizer Nano ZS90 device (Malvern Instruments, France) was used to collect zeta potential, or ZP. Deionized water solutions with the same mass concentration of each of the six compounds were measured three times. To conduct the AFM test, mix the RIC-1 sample with atomic-grade water in a 1:100 mass ratio. After 30 min of ultrasonication, the supernatant was poured onto freshly separated mica sheets and naturally dried. Dump mode scanning was used after the mica piece was placed on the AFM stage.

2.1.5 Cell imaging

In 96-well plates, HeLa and PC3 cells that reached the logarithmic phase of growth were used for trypsinization and round coverslip seeding operations. These cells were then cultured at 37°C with 5% CO₂ for 1 day to promote adherence. A stock solution containing 10 mg/mL of the polymer was prepared in DMSO and subsequently diluted to achieve the desired concentration. Each well was replaced with a new medium containing 10 μg/mL of different sample polymers and incubated for 36 h. The cells were then rinsed twice with PBS, and the medium was discarded. The cells were fixed for 25 min using a 5% paraformaldehyde solution. After removing the fixative and giving the cells two PBS washes, they were stained with DAPI for 20 min in a dark environment. The cells further underwent staining, an antifluorescence quencher treatment, and two PBS washes. To capture fluorescence images of cells, fluorescence microscopes were utilized.

Image J software was used to process the captured images and analyze the data. To quantify the correlation in the imaging results, the Pearson correlation coefficient, a measure of the strength of a linear relationship between two variables, was computed.

3. Result and Discussion

Lipophilic drugs, such as those taken orally and those used for targeted treatment, frequently have low bioavailability. This study aims to improve the aqueous solubility and targeting of fat-soluble drugs, including isorhamnetin and nido-carborane. By encasing these drugs in innovative carrier materials, the study looks to bypass the limitations imposed by their lipophilicity. The simplicity and affordability of the synthetic method set it apart from other nanopreparation techniques. This approach produces the desired outcomes while also providing new insights for drug development and medical applications.

Fluorescent complexes were synthesized in this study using the continuous method, as illustrated in Scheme 1. Treating isorhamnetin with a strong base to address solubility issues is one of several crucial steps involved. This step results in the formation of small zwitterionic molecules. Then, these molecules were mixed with rhodamine-6G/nido-carboran to ensure complete ionic binding. These complexes were then reacted with various types of acrylic resins to produce the desired coating. Comparing the color properties of these four fluorescent complexes under two distinct lighting conditions, daylight and UV light with a wavelength of 356 nm, was another aspect of the study. When the composite is exposed to natural sunlight, Fig. 2 shows its clear and vibrant orange-red coloring.

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Fig. 2.

Comparison of four fluorescent complexes under sunlight and 356 nm irradiation.

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The findings show that the four fluorescent complexes’ maximum absorption peaks are all roughly located at 555 nm, as shown in Figs. 3 and 4 and Tables 1 and 2. In particular, RIC-1, RIC-2, RIC-3, and RIC-4 have maximum absorption wavelengths of 555–557 nm, 554–560 nm, and 555–559 nm, respectively. The solvent that a compound is placed in has a significant impact on its absorbance. RIC-1 was the most efficient UV absorber; at the same concentration, its absorbance was noticeably higher than that of RIC-3 and RIC-4. According to the theory that the dipole moment of polar solvent molecules enhances the polarity of the compound molecules, changing the molecular energy levels, the absorption bands of RIC-1 and RIC-2 both redshifted as the solvent’s polarity increased. The four fluorescent complexes’ maximum absorption wavelengths, which varied by only 558 ± 2 nm in buffers with varying pH values, indicate that the compounds’ maximum absorption wavelengths at the same concentration are barely affected by pH changes. Furthermore, the four compounds’ UV absorption spectra all rise in tandem with an increase in compound concentration in the same solvent or pH level.

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Fig. 3.

Ultraviolet absorption of RIC-1, RIC-2, RIC-3, and RIC-4 in different solvents

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Fig. 4.

UV absorption of four fluorescent complexes in buffers with different pH (pH = 4.5, 5.0, 6.5, 7.4, 8.0)

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By scanning at an excitation wavelength of 550 nm, the emission spectra of RIC-1, RIC-2, RIC-3, and RIC-4 were acquired (Figs. 5 and 6 and Tables 3 and 4). In a variety of solvents, the emission wavelengths of RIC-1 and RIC-2 are concentrated in the 556–557 nm range, RIC-3’s in the 558–563 nm range, and RIC-4’s in the 553–562 nm range. The compounds RIC-1, RIC-2, RIC-3, and RIC-4 exhibited the highest fluorescence intensity in THF and the lowest in DMSO when dissolved in different solvents at the same concentration. This could be impacted by the solvent’s polarity, which can decrease the compounds’ stability and cause non-radiative transitions, decreasing the fluorescence intensity. At the same concentration, there was no discernible impact of pH on the fluorescence intensity of the complex. In buffers with varying pH levels, the four fluorescent polymers’ emission wavelengths are concentrated between 553 and 563 nm. Additionally, the fluorescence intensity rises as the polymer concentration does when the complexes are in the same solvent and pH buffer solution.

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Fig. 5.

Fluorescence emission spectrum of RIC-1, RIC-2, RIC-3, and RIC-4 in different solvents

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Fig. 6.

Fluorescence emission spectrum of RIC-1, RIC-2, RIC-3, and RIC-4 in buffers with different pH

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All four complexes had fairly good fluorescence stability, according to fluorescence stability analysis (Fig. 7). This was demonstrated by the fluorescence intensity lines, which were nearly horizontal during the 5- and 10-min testing periods. However, it was found that the fluorescence intensities of both compounds decreased with increasing pH, with RIC-1 showing the strongest tendency. Based on the stability of their fluorescence under test conditions, these complexes seemed to be well-suited for both in vitro and in vivo imaging applications, despite their sensitivity to pH. They are appealing choices for a range of imaging investigations due to their responsiveness to pH variations and their consistent performance over short periods.

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Fig. 7.

Fluorescence stability (5 min and 10 min) of two compounds in three different pH PBS buffers

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To examine the microscopic morphology of the compounds and confirm the encapsulation of the isorhamnetin/nido-carborane/rhodamine 6G complex, the inner structure of the RIC-1 fluorescent complex was examined using TEM. The TEM images showed that RIC-1 has a distinct grid-like structure. It was discovered that the RIC-1 complex, which mostly takes the form of an amorphous, flocculent form, was uniformly distributed throughout the acrylic resin system and maintained a stable grid-like structure. Furthermore, these complexes displayed a porous morphology, as seen in Fig. 8, suggesting an expansion that could increase their surface area. The complex’s ability to load drugs and interact with tumor cells is improved by the grid-like morphology, which increases the complex’s surface area while maintaining the same volume. This particular structural feature is anticipated to enhance the RIC-1 complex’s functional capabilities, particularly in scenarios where surface area and external interactions are crucial. The grid-like and porous characteristics of RIC-1 may be advantageous for a variety of applications where efficient substance exchange and reactivity are crucial, including targeted drug delivery and imaging.

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Fig. 8.

TEM diagram of fluorescent complex RIC-1

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Zeta potentials of the RIC-1 were obtained to gain a preliminary understanding of its stability in aqueous solution. Aqueous solution of compound RIC-1 coated with acrylic resin L100-55 demonstrates a relatively stable surface charge of -33 mV, as illustrated in Fig. 9(a). The zeta potential of −33 mV indicates strong electrostatic repulsion between particles, preventing aggregation and ensuring colloidal stability in aqueous solutions. The effects of RIC-1 on the growth of HeLa, PC3, and LO₂ cells were evaluated using the CCK-8 assay. In general, RIC-1 compounds exhibit significantly lower toxicity to human LO₂ cells while having a strong inhibitory effect on PC3 and HeLa cell proliferation. The proliferation rates of PC3 and HeLa cells treated with this substance were 49.56% and 55.62%, respectively, as indicated in Fig. 9(b) and Table 5, whereas the proliferation rate of LO₂ cells remained at 65.36%. This indicates that this material has some selectivity for tumor cells and strong biological activity. At 24 μg/mL, RIC-1 demonstrated similar inhibition rates (49–62%) to cisplatin (55–68%), indicating its potential as a chemotherapeutic substitute.

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Fig. 9.

(a) The zeta potential value of RIC-1 in aqueous solution was measured 3 times. (b) Proliferation rate of RIC-1-treated HeLa, PC-3, and LO2 cells. In the figure, blue represents HeLa cells and green represents PC-3 cells

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Fig. 10 displays the AFM image of RIC-1. The reticular distribution in the TEM spectrum is indirectly confirmed by the white dots in the image, which also shows that the RIC-1 particles are uniformly distributed and have a smooth surface.

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Fig. 10.

AFM imaging of RIC-1 in aqueous solution. (a) and (b) are AFM images of RIC-1 at a size of 5x5 μm².

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The gastrointestinal microenvironment was simulated using pH values of 4.5, 5.5, and 6.5 to observe the effect of RIC-1 on drug release. Fig. 11 shows that at pH 4.5 and pH 5.5, the release amounts are 28% and 41%, respectively. Under pH = 6.5, the maximum release effect was 81%.

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Fig. 11.

(a) The standard curve of the RIC-1 and cumulative release. (b) RIC-1 in buffers with different pH (pH =4.5, 5.5, 6.5).

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A drug’s effectiveness depends critically on its ability to cross the biological barrier and enter cells, particularly if it can enter the cell nucleus and kill cancer cells. Fig. 12 illustrates how we stained HeLa cells using laser confocal microscopy in conjunction with different fluorescence microscopy techniques to see how the cells absorbed the complex. Brightfield, DAPI, green channel, red channel, and a combination of these modes are among the imaging modes used. The RIC-1 complex’s selectivity and biocompatibility clearly show how HeLa cells interact with it. Blue dots in the microscope image represent the RIC complex entering and interacting with cancer cells. More significantly, clear orange bright spots inside the cancer cells can be seen in the combined and overlapped images. This is explained by the fact that carboranes are naturally selective for cancer cells, which enhances complex targeting. The targeting and biocompatibility of isorhamnetin and nido-carborane molecules are greatly enhanced by the addition of rhodamine 6G, allowing them to enter cellular domains in zwitterionic forms. Through cell imaging, the structural properties of this fluorescent complex were methodically examined to assess their biocompatibility, selectivity, and affinity in cancer cells. Both HeLa cell staining and laser confocal microscopy show this.

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Fig. 12.

Fluorescence imaging of HELA cells in different channels by RIC-1 complex, with a DAPI of 340–390 nm at 325 nm excitation, green channel of 450–480 nm at 405 nm excitation, and red channel of 550–590 nm at 535 nm excitation

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In both the red and green channels, the polymers produced intense and observable fluorescence. The combined imaging showed how the fluorescent polymers looked inside the cells, indicating that both polymers were able to enter the cells. Image J software was used to perform the co-localization analysis of the polymers with the cell nucleus, as shown in Fig. 13. The range of the Pearson correlation coefficient is [-1, 1]. The degree of co-localization between the polymer and the cell nucleus in the corresponding channel increases with the value’s proximity to 1, indicating a more consistent spatial distribution. The degree of co-localization decreases as the value approaches 0. This polymer’s high biocompatibility and ability to partially enter the cell nucleus are demonstrated by the fact that all the coefficients above are greater than 0.

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Fig. 13.

Scatter plot of red channels and green channels co-located with blue nuclei cells

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4. Conclusion

In conclusion, by streamlining the synthesis process, four fluorescent polymers were effectively created. Nido-carborane markers coated with acrylic resin were employed in this study to increase the bioavailability of medications containing boron. Rhodanine 6G/nido-carborane resolves the water solubility issue of carborane, making in vivo monitoring more tolerant by emitting fluorescence. TEM and AFM imaging reveal spherical smooth structures that facilitate efficient drug delivery. RIC-1 can penetrate two types of tumor cells in CCK8. This development offers an efficient method for imaging tumor cells and has great potential in drug fluorescence imaging. RIC-1 demonstrated excellent stability and release properties among the four fluorescent complexes. These results offer a solid theoretical foundation for the application of carborane fluorescence complexes in the future. The delivery carriers for oral or intravenous administration of isorhamnetin, as well as the suitability of this carrier design as a novel delivery system for other hydrophobic drugs, will be further investigated in the future.