Development of dihydrochalcone-functionalized gold nanoparticles for augmented antineoplastic activity

Abstract

Background: Phloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodium-glucose linked transport 2 (SGLT2) inhibitor. While the aglycone metabolite of phloridzin, phloretin, displays a reduced capacity of SGLT2 inhibition, this nutraceutical displays enhanced antineoplastic activity in comparison to phloridzin.

Purpose: The objective of this study was to develop gold nanoparticle (AuNP) mediated delivery of phloridzin and phloretin and explore their anticancer mechanism through conjugation of the dihydrochalcones and the AuNP cores.

Methods: Phloridzin and phloretin conjugated AuNPs (Phl-AuNP and Pht-AuNP) were synthesized in single-step, rapid, biofriendly processes. The synthesized AuNPs morphology was characterized via transmission electron microscopy and ultraviolet-visible spectroscopy. The presence of phloridzin or phloretin was confirmed using scanning electron microscopy-energy dispersive x-ray spectroscopy. The percentage of organic component (phloridzin/phloretin) onto AuNPs surface was characterized using thermogravimetric analysis. Assessment of the antineoplastic potency of the dihydrochalcones conjugated AuNPs against cancerous cell lines (HeLa) was accomplished through monitoring via flow cytometry.

Results: The functionalized AuNPs were synthesized via a single-step method that relied only upon the redox potential of the conjugate itself and required no toxic chemicals. The synthesized Phl-AuNPs were found to be in the size range of 15±5 nm, whereas the Pht-AuNP were found to be 8±3 nm, placing both conjugated AuNPs well within the size range necessary for successful pharmaceutical applications. These assays demonstrate a significant increase in the cancerous cell toxicities as a result of the conjugation of the drugs to AuNPs, as indicated by the 17.45-fold increase in the efficacy of Pht-AuNPs over pure phloretin, and the 4.49-fold increase in efficacy of Phl-AuNP over pure phloridzin.

Conclusion: We report a simple, biofriendly process using the reducing and capping potential of the dihydrochalcones, phloridzin and phloretin, to synthesize stable AuNPs that have promising futures as potential antineoplastic agents.

Keywords: cancer; gold nanoparticles; phloretin; phloridzin.

Figure 1

Chemical structures of the two dihydrochalcones. Note: (A) Phloretin molecular weight: 274.26 g mol⁻¹; (B) phloridzin molecular weight: 342.30 g mol⁻¹.

Figure 2

Illustration of the morphological and size characterization of synthesized Pht-AuNPs and Phl-AuNPs through TEM, UV-Vis, and DLS analysis. Notes: (A) TEM image showing formation of well-dispersed spherical Pht-AuNPs with a core size range of 8±3 nm (scale bar =50 nm). (B) TEM image showing formation of well-dispersed spherical Phl-AuNPs with a core size range of 15±5 nm (scale bar =100 nm). (C) UV-Vis spectrogram of Pht-AuNPs showing a SPR event occurring at 536 nm correlating to spheroid-shaped NPs. (D) UV-Vis spectrogram of Phl-AuNPs showing a SPR event occurring at 542 nm correlating to spheroid-shaped NPs. (E) Pht-AuNP DLS analysis shows the average hydrodynamic diameter as 8±2 nm. (F) Phl-AuNP DLS analysis depicts the average hydrodynamic diameter as 12±2 nm. Abbreviations: Pht-AuNPs, phloretin-conjugated gold nanoparticles; Phl-AuNPs, phloridzin-conjugated gold nanoparticles; TEM, transmission electron microscopy; UV-Vis, ultraviolet-visible spectroscopy; DLS, dynamic light scattering; SPR, surface plasmon resonance.

Figure 3

Characterization of Pht-AuNPs and Phl-AuNPs. Notes: (A) EDS spectra of Pht-AuNPs showing the presence of an elemental peak for carbon (C) and gold (Au) at 0.2 keV and 2.1 keV respectively. Figure in the inset shows SEM image of spin coated sample of Pht-AuNPs on silicon chip obtained at an accelerating voltage of 20 keV with a magnification of 5 kX. (B) EDS spectra of Pht-AuNPs showing the presence of an elemental peak for C and Au at 0.2 keV and 2.1 keV respectively. Figure in the inset shows SEM image of spin coated sample of Pht-AuNPs on silicon chip obtained at an accelerating voltage of 20 keV with a magnification of 5 kX. (C) A comparison of TGA showing loss of organic material for Pht-AuNP (—) and phloretin (- - -) respectively. The samples were heated from room temperature to 650°C at a rate of 10°C min⁻¹ under nitrogen flow followed by heating to 850°C under air. (D) A comparison of TGA showing loss of organic material for Phl-AuNP (—) and phloridzin (- - -) respectively. The samples were heated from room temperature to 650°C at a rate of 10°C min⁻¹ under nitrogen flow followed by heating to 850°C under air. Abbreviations: EDS, energy dispersive spectroscopy; TGA, thermo-gravimetric analysis; Pht-AuNPs, phloretin-conjugated gold nanoparticles; Phl-AuNPs, phloridzin-conjugated gold nanoparticles; SEM, scanning electron microscopy; Pht, phloretin.

Figure 4

Surface zeta potential analysis. Notes: (A) Surface zeta potential analysis of Pht-AuNPs determined to be −31.7 mV, and (B) of Phl-AuNPs determined to be −38.2 mV. The magnitude of the zeta potential greater than 30 mV is indicative of a moderately stable AuNP suspension. Abbreviations: Pht-AuNPs, phloretin-conjugated gold nanoparticles; Phl-AuNPs, phloridzin-conjugated gold nanoparticles; AuNP, gold nanoparticle.

Figure 5

HeLa live/dead cell fluorescence staining analysis via flow-cytometry. Notes: (A) Comparison of the pure drug forms of phloretin and phloridzin to non-inoculated cells. (B) Comparison of the AuNP functionalized forms of the drugs to non-inoculated cells. (C) Comparison of the functionalized AuNP to pure AuNP to determine the synergistic activity of the Pht-AuNPs and Phl-AuNPs. Abbreviations: AuNP, gold nanoparticle; Pht-AuNP, phloretin-conjugated gold nanoparticles; Phl-AuNPs, phloridzin-conjugated gold nanoparticles.