Lipid-Coated Polymeric Nanoparticles for the Photodynamic Therapy of Head and Neck Squamous Cell Carcinomas

Abstract

Next to alcohol and tobacco abuse, infection with human papillomaviruses (HPVs) is a major risk factor for developing head and neck squamous cell carcinomas (HNSCCs), leading to 350,000 casualties worldwide each year. Limited therapy options and drug resistance raise the urge for alternative methods such as photodynamic therapy (PDT), a minimally invasive procedure used to treat HNSCC and other cancers. We prepared lipid-coated polymeric nanoparticles encapsulating curcumin as the photosensitizer (CUR-LCNPs). The prepared CUR-LCNPs were in the nanometer range (153.37 ± 1.58 nm) and showed an encapsulation efficiency of 92.69 ± 0.03%. Proper lipid coating was visualized using atomic force microscopy (AFM). The CUR-LCNPs were tested in three HPV^pos and three HPV^neg HNSCC lines regarding their uptake capabilities and in vitro cell killing capacity, revealing a variable but highly significant tumor cell inhibiting effect in all tested HNSCC cell lines. No significant differences were detected between the HPV^pos and HPV^neg HNSCC groups (mean IC₅₀: (9.34 ± 4.73 µmol/L vs. 6.88 ± 1.03 µmol/L), suggesting CUR-LCNPs/PDT to be a promising therapeutic option for HNSCC patients independent of their HPV status.

Keywords: HNSCC; LED; PDT; PLGA; cancer; curcumin; human papillomavirus; liposomes.

Figure 1

Characterization of nanoparticles by height, amplitude, and phase using atomic force microscopy. (A) and (B): LIPOs, (C) and (D): CUR-NPs with an overall smooth and even surface. Scale bars in row (A) + (C) represent 200 nm. Scale bars in row (B) + (D) represent 100 nm.

Figure 2

Characterization of nanoparticles by height, amplitude, and phase using atomic force microscopy. (A) and (B): CUR-LCNPs. The white arrowhead in the amplitude image in B indicates a lipid layer on the nanoparticle’s surface. Scale bars in row (A) represent 200 nm. Scale bars in row (B) represent 100 nm.

Figure 3

Confocal laser scanning microscope images of six different HNSCC cell lines treated for 4 h with 10 µmol/L curcumin-loaded lipid-coated polymeric nanoparticles (CUR-LCNPs). The merged images show the taken-up curcumin (green fluorescence) and DAPI-stained cell nuclei (blue fluorescence).

Figure 4

Drug uptake by flow cytometry. Treated (blue) cells were exposed to 10 µmol/L curcumin-loaded lipid-coated polymeric nanoparticles (CUR-LCNPs) for 4 h and compared to non-treated (red) cells. Drug uptake is expressed as mean percentage ± SD, calculated using the FlowJo Overton algorithm.

Figure 5

Evaluation of in vitro cell viability via MTT assay in UM-SCC-3, UM-SCC-27, and UT-SCC-26A cells (HPV^neg subgroup) and UM-SCC-47, UM-SCC-104, and UPCI:SCC-154 cells (HPV^pos subgroup) treated with curcumin-loaded lipid-coated polymeric nanoparticles (CUR-LCNPs) or free curcumin (Free CUR) and irradiated (IR) with λ = 457 nm for 6.5 min, resulting in a radiant exposure of 8.6 J/cm², or without irradiation (DARK). The half maximal inhibitory concentration (IC₅₀) was calculated for CUR-LCNP and free CUR (curcumin dissolved in 1% DMSO).

Figure 6

Biocompatibility of curcumin-loaded lipid-coated polymeric nanoparticles (CUR-LCNPs) and curcumin-loaded polymeric nanoparticles (CUR-NPs) with curcumin concentrations ranging from 0 to 50 µmol/L was tested in L929 cells. Cells were kept in the dark throughout the experiment.

Figure 7

Production of reactive oxygen species inside UM-SCC-3 cells treated with PBS (control), curcumin-loaded lipid-coated polymeric nanoparticles (CUR-LCNPs), free curcumin (free CUR) (curcumin dissolved in 1% DMSO), lipid-coated polymeric nanoparticles (LCNPs), unloaded nanoparticles (NPs), and liposomes (LIPOs) and irradiated (IR) with λ = 457 nm for 6.5 min, resulting in a radiant exposure of 8.6 J/cm², or without irradiation (DARK). Statistical significance is calculated with the relative fluorescence of control IR vs. the respective fluorescence of samples IR. Statistical differences are denoted as “*” p < 0.05, “***” p < 0.001, and “****” p < 0.0001.

Figure 8

Mitochondrial transmembrane potential of UM-SCC-3 cells treated with FCCP (positive control), curcumin-loaded lipid-coated polymeric nanoparticles (CUR-LCNPs), free curcumin (free CUR) (curcumin dissolved in 1% DMSO), lipid-coated polymeric nanoparticles (LCNPs), unloaded nanoparticles (NPs), and liposomes (LIPOs) and irradiated (IR) with λ = 457 nm for 6.5 min, resulting in a radiant exposure of 8.6 J/cm², or without irradiation (DARK). Results are normalized and compared to PBS as the negative control (100%). Statistical differences are denoted as “**” p < 0.01.