Enhancing drug delivery with supramolecular amphiphilic macrocycle nanoparticles: selective targeting of CDK4/6 inhibitor palbociclib to melanoma

Abstract

Drug delivery systems based on amphiphilic supramolecular macrocycles have garnered increased attention over the past two decades due to their ability to successfully formulate nanoparticles. Macrocyclic (MC) materials can self-assemble at lower concentrations without the need for surfactants and polymers, but surfactants are required to form and stabilize nanoparticles at higher concentrations. Using MCs to deliver both hydrophilic and hydrophobic guest molecules is advantageous. We developed two novel types of amphiphilic macrocycle nanoparticles (MC NPs) capable of delivering either Nile Red (NR) (a hydrophobic model) or Rhodamine B (RhB) (a hydrophilic model) fluorescent dyes. We extensively characterized the materials using various techniques to determine size, morphology, stability, hemolysis, fluorescence, loading efficiency (LE), and loading capacity (LC). We then loaded the CDK4/6 inhibitor Palbociclib (Palb) into both MC NPs using a solvent diffusion method. This yielded Palb-MC NPs in the size range of 65-90 nm. They exhibited high stability over time and in fetal bovine serum with negligible toxicity against erythrocytes. Cytotoxicity was minimal when tested against RAW macrophages, human fibroblast HDFn, and adipose stromal cells (ASCs) at higher concentrations of MC NPs. Cell viability studies were conducted with different concentrations of MC NPs, Palb-MC NPs, and free Palb against RAW macrophages, human U-87 GBM, and human M14 melanoma cell lines in vitro. Flow cytometry experiments revealed that blank MC NPs and Palb-MC NPs were selectively targeted to melanoma cells, resulting in cell death compared to the other two cell lines. Future work will focus on studying the biological effect of MC NPs including their binding affinity with molecules/receptors expressed on the M14 and other melanoma cell surfaces by molecular docking simulations. Subsequently, we will evaluate the MCs as a component of combination therapy in a murine melanoma model.

Figure 1.

(a) The chemical structure of MC compounds, (b) the formulation and purification processes of MC NPs by solvent diffusion method, and (c) the optimization of nanoformulations using different MC/surfactant weight ratios and organic solvents (THF and DMSO). Formulations were rated as (−) bad, (+) good, and (++) excellent based on particle size and PDI. Additionally, (d) digital photos of MC1 and MC2 materials (left) and MC1 and MC2 NPs aqueous dispersions (1–6) stabilized by PEGylated surfactant at the weight ratio of 36:64, respectively, are shown. Tubes 1, 3, and 4 represent MC1 NPs, NR dye-loaded MC1 NPs, and RhB dye-loaded MC1 NPs, respectively, while tubes 2, 5, and 6 represent MC2 NPs, NR dye-loaded MC2 NPs, and RhB dye-loaded MC2 NPs, respectively.

Figure 2.

The characterization of (a-d) MC1- and (e-h) MC2-based materials. TEM images of (a, e) pure MC1 and MC2 dried on a grid from THF, (b, f) unloaded MC1 and MC2-based NPs (both prepared in DMSO), and (d, h) NR-MC1 NPs and RhB-MC2 NPs (both prepared in DMSO) are presented at two different magnifications. Additionally, the intensity particle size distribution of empty MC1 and MC2 NPs (prepared in either THF or DMSO) is shown using DLS in (c, g).

Figure 3.

Fluorescence emission (λex 562 nm) spectra of MC NP loaded with (a) RhB and NR (b) dyes. All samples were prepared at MC (1 mg) : PEG (1.8 mg) : dye (1 mg) in a final volume of 1 mL of bi-distilled water. The excess of unloaded dyes was separated by ultracentrifugation. For the spectroscopic measurements. The spectra were recorded at the following final concentrations of the dyes (see Supplementary Table S1).

Figure 4.

(a,b) Fluorescence calibration curves of free NR and RhB, respectively (RhB Ex/Em 553/627 nm; NR Ex/Em 562/655 nm). (c) Dye loading capacities (LC) and loading efficiencies (LE) in MC NPs. (d) Dye release kinetics from MC NPs.

Figure 5.

The results of NTA of the blank and dye-loaded MC NPs. (a) Mean particle size of all nanoparticles immediately after preparation and after one month, indicating no variation in size. (b) Zeta potential, (c) particles’ concentration, and (d) stability of MC NPs in FBS over 48 h. The stock NPs solution was diluted 10-fold by FBS.

Figure 6.. Characterization of Palb-loaded MC NPs.

(a, d) Particle size distribution, (b, e) zeta potential (measured by NTA), and (c, f) TEM images of Palb-MC1 and Palb-MC2 NPs, respectively. (g) LC and LE percentages of Palb in MC1 and MC2 NPs. (h) Palb release profile from MC1 and MC2 NPs. (i) Hemolysis assay results for unloaded and Palb-MC NPs.

Figure 7.

Cell viability studies (measured by CCK-8 assay) using RAW264.7 macrophage cells treated with various concentrations of blank MC NPs, Palb-MC NPs, and free Palb drug. The cells were exposed to the test nanoparticles for 72 h, and the results of the 24 h exposure are presented in Supplementary Figure S3. The highlighted treatments in red rectangles were selected for flow cytometry analysis to quantify cell death caused by different treatments. Viability of cells treated with (a) blank MC1 NPs Palb-MC1 NPs. (b) blank MC2 NPs Palb-MC2 NPs, and (c) free Palb drug.

Figure 8.

Fluorescence microscopy images of M14-GFP melanoma, U-87 GBM-GFP, and RAW264.7 macrophage cells after 72 h of treatment, demonstrating the influence of the type and concentrations of MC NPs on cell death. The concentration of MC1 and MC2 in either drug-free or Palb-containing treatment solutions was either 0.02 mg/ml or 0.63 mg/ml as indicated. The Palb drug was used at a concentration of 0.78 μM as free or MC-formulated solution. The colored images show M14-GFP melanoma cells treated with different concentrations of MC NPs, as indicated in each image, while the black-and-white images depict RAW264.7 macrophages and U-87 GBM-GFP cells treated with the same concentrations. A scale bar of 200 μm is present in all panels.

Figure 9.

Representative flow cytometry profile and comparative cytotoxic effect after 72 h of treatment, demonstrating the selective killing of melanoma cells by MC1 and MC2 NPs. Panels (a) and (b) show the treatment of macrophages RAW264.7 cells with MC1 and MC2 NPs, while panels (c) and (d) show the treatment of U-87 GBM-GFP glioblastoma cells. Panels (e) and (f) depict the treatment of M14-GFP melanoma cells. The drug doses were the same as described in Figure 8.

Figure 10.

Cell viability of (a) HDFn), (b) Mel 29.1, (c) M14, and (d) U-87 MG cells treated with MC NPs for 72 hours of incubation. The experiment was done in triplicates.

Figure 11.

Confocal microscopy imaging of NR-MC1 and NR-MC2 NPs internalization in both M14 melanoma and HDFn fibroblast cell lines. The scale bar is 20 μm. Images in each row (left to right): CellMask (green) for staining cell membrane, NR-loaded MC NPs (red), overlapping (green-red), and bright field image (green-red).