Luminescent Alendronic Acid-Conjugated Micellar Nanostructures for Potential Application in the Bone-Targeted Delivery of Cholecalciferol

Abstract

Vitamin D, an essential micronutrient crucial for skeletal integrity and various non-skeletal physiological functions, exhibits limited bioavailability and stability in vivo. This study is focused on the development of polyethylene glycol (PEG)-grafted phospholipid micellar nanostructures co-encapsulating vitamin D3 and conjugated with alendronic acid, aimed at active bone targeting. Furthermore, these nanostructures are rendered optically traceable in the UV-visible region of the electromagnetic spectrum via the simultaneous encapsulation of vitamin D3 with carbon dots, a newly emerging class of fluorescents, biocompatible nanoparticles characterized by their resistance to photobleaching and environmental friendliness, which hold promise for future in vitro bioimaging studies. A systematic investigation is conducted to optimize experimental parameters for the preparation of micellar nanostructures with an average hydrodynamic diameter below 200 nm, ensuring colloidal stability in physiological media while preserving the optical luminescent properties of the encapsulated carbon dots. Comprehensive chemical-physical characterization of these micellar nanostructures is performed employing optical and morphological techniques. Furthermore, their binding affinity for the principal inorganic constituent of bone tissue is assessed through a binding assay with hydroxyapatite nanoparticles, indicating significant potential for active bone-targeting. These formulated nanostructures hold promise for novel therapeutic interventions to address skeletal-related complications in cancer affected patients in the future.

Keywords: active bone targeting; alendronate; cholecalciferol; hydroxyapatite nanoparticles; luminescent carbon dots; micellar nanostructures.

Figure 1

(A) PL spectra of the ‘as synthesized’ C-dots dispersed in organic solvent and recorded at increasing excitation wavelengths (λ) of 300 nm (black line), 320 nm (red line), 340 nm (blue line), 360 nm (magenta line), 375 nm (olive line), 380 nm (brown line), 400 nm (violet line), 420 nm (lime green line), 440 nm (light blue line), 460 nm (orange line), 480 nm (dark blue line), 500 nm (pink line), 520 nm (heavenly blue line), and 540 nm (purple line). Inset: UV-Vis absorption spectrum. (B) TEM micrograph of oil-soluble C-dots cast from chloroform.

Figure 2

(A) DLS analysis and ζ-potential values for micellar nanostructures prepared at different starting C-dot/PEG–phospholipid weight ratio percentages. (B) Picture of the vial containing micellar nanostructures encapsulating C-Dots, acquired under 400 nm light exposure along with a representative scheme. (C) PL emission spectra recorded at excitation wavelengths ranging from 300 to 540 nm (1:3 dilution of sample C in PBS) and (D) representative TEM micrograph and corresponding close-up view in the inset cast from the water from sample C.

Figure 3

(A) Encapsulation efficiency (EE%) and loading (DL%) percentages of the VitD3 for samples of micellar nanostructures co-encapsulating VitD3 and C-Dots, prepared at different starting VitD3/phospholipid weight ratio percentages. (B) Size distribution by intensity of the micellar nanostructures co-encapsulating VitD3 and C-Dots obtained by using a VitD3/phospholipid weight ratio percentage of 3.3% and dispersed in PBS (10 mM, pH 7.4). (C) representative TEM micrograph along with (D) corresponding close-up and schematic sketch. (E) PL emission spectra recorded at excitation wavelengths ranging from 300 to 440 nm. Inset: UV-Vis absorbance spectrum. (F) In vitro release profile of the VitD3 recorded for the VitD3/C-dot micellar nanostructures (VitD3/phospholipid weight ratio percentage of 3.3%, C-dot/phospholipid weight ratio percentage of 21%).

Scheme 1

Reaction scheme: (a) phthalic anhydride, triethylamine (Et₃N_(cat.₎), toluene, reflux, 5 h; (b) thionyl chloride, reflux, 2 h; (c) tris(trimethylsilyl)phosphite, tetrahydrofuran (THF), 0 °C to r. t., 15 min–15 h; (d) methanol (MeOH), r. t., 1 h; (e) 12 N HCl, reflux, 5.5–20 h.

Figure 4

(A) FT-IR spectra of free VitD3, luminescent micellar structures and loaded with VitD3, synthesized ALE and ALE conjugated to the surface of the luminescent micellar formulation loaded with VitD3. (B) Representative size distribution based on intensity. (C) Determination of the ALE concentrations in the micellar formulations based on complexation with Fe(III) ions in acidic conditions.

Figure 5

Representative FE-SEM micrograph (A), EDX element spectrum (A1), element content table (A2), and representative TEM micrograph of the HA nanoparticles (B). PL spectra, before and after incubation with HA NPs, of the non-conjugated (C) and ALE-conjugated (D) VitD3/C-dot micellar nanostructures. Table reporting the binding percentage for the non-conjugated and ALE-conjugated samples obtained by estimating the integrated area under the PL spectrum, before and after the incubation of each sample with the HA NPs. The data are presented as means ± S.D (n = 3) (E).