Surgery is the frontline treatment for a large number of cancers. The objective of these excisional surgeries is the complete removal of the primary tumor with sufficient safety margins. Removal of the entire tumor is essential to improve the chances of a full recovery. To help surgeons achieve this objective, near-infrared fluorescence-guided surgical techniques are of great interest. The concomitant use of fluorescence and indocyanine green (ICG) has proved effective in the identification and characterization of tumors. Moreover, ICG is authorized by the Food and Drug Administration and the European Medicines Agency and is therefore the subject of a large number of studies. ICG is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, it also has some disadvantages, such as limited photostability, a moderate fluorescence quantum yield, a high plasma protein binding rate, and undesired aggregation in aqueous solution. In addition, ICG does not specifically target tumor cells. One way to exploit the capabilities of ICG while offsetting these drawbacks is to develop high-performance near-infrared nanocomplexes formulated with ICG (with high selectivity for tumors, high tumor-to-background ratios, and minimal toxicity). In this review article, we focus on recent developments in ICG complexation strategies to improve near-infrared fluorescence-guided tumor surgery. We describe targeted and nontargeted ICG nanoparticle models and ICG complexation with targeting agents.

Conventional 3D multicellular tumor spheroids of head and neck squamous cell carcinoma (HNSCC) consisting exclusively of cancer cells have some limitations. They are compact cell aggregates that do not interact with their extracellular milieu, thus suffering from both insufficient extracellular matrix (ECM) deposition and absence of different types of stromal cells. In order to better mimic in vivo HNSCC tumor microenvironment, we have constructed a 3D stroma-rich in vitro model of HNSCC, using cancer-associated MeWo skin fibroblasts and FaDu pharynx squamous cell carcinoma. The expression of stromal components in heterospheroids was confirmed by immunochemical staining. The generated co-culture FaDu/MeWo spheroids were applied to study penetration, distribution and antitumor efficacy of photoactive drugs such as Temoporfin and Chlorin e6 used in the photodynamic therapy flow cytometry and fluorescence microscopy techniques. We also investigated the distribution of photodiagnostic agent Indocyanine Green. We demonstrated that the presence of stroma influences the behavior of photoactive drugs in different ways: (i) No effect on Indocyanine Green distribution; (ii) lower accumulation of Chlorin e6; (iii) better penetration and PDT efficiency of Temoporfin. Overall, the developed stroma-rich spheroids enlarge the arsenal of in vitro pre-clinical models for high-throughput screening of anti-cancer drugs.

There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic photodynamic therapy. Therefore, we aimed to develop covalent conjugates between the photosensitizer chlorin e6 (Ce6) and PAMAM G4.5 dendrimers. Singlet oxygen generation (SOG) efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. Compared to free Ce6, PAMAM anchored Ce6 displays a much higher photodynamic effect, which is ascribable to better internalization in a tumor cell model. Intracellular fate and internalization pathway of our different compounds were investigated using specific inhibition conditions and confocal fluorescence microscopy. Free Ce6 was shown to enter the cells by a simple diffusion mechanism, while G4.5-Ce6-PEG internalization was dependent on the caveolae pathway, whereas G4.5-Ce6 was subjected to the clathrin-mediated endocytosis pathway. Subcellular localization of PAMAM anchored Ce6, PEGylated or not, was very similar suggesting that the nanoparticles behave similarly in the cells. As a conclusion, we have demonstrated that PEGylated G4.5 PAMAM-Ce6 dendrimers may offer effective biocompatible nanoparticles for improved photodynamic treatment in a preclinical tumor model.

The main goal of this study was to use hybrid delivery system for effective transportation of temoporfin (meta-tetrakis(3-hydroxyphenyl)chlorin, mTHPC) to target tissue. We suggested to couple two independent delivery systems (liposomes and inclusion complexes) to achieve drug-in-cyclodextrin-in-liposome (DCL) nanoconstructs. We further optimized the composition of DCLs, aiming to alter in a more favorable way a distribution of temoporfin in tumor tissue. We have prepared DCLs with different compositions varying the concentration of mTHPC and the type of β-cyclodextrin (β-CD) derivatives (Hydroxypropyl-, Methyl- and Trimethyl-β-CD). DCLs were prepared by thin-hydration technique and mTHPC/β-CD complexes were added at hydration step. The size was about 135 nm with the surface charge of (−38 mV). We have demonstrated that DCLs are stable and almost all mTHPC is bound to β-CDs in the inner aqueous liposome core. Among all tested DCLs, trimethyl-β-CD-based DCL demonstrated a homogenous accumulation of mTHPC across tumor spheroid volume, thus supposing optimal mTHPC distribution.