Unsymmetrical, monocarboxyalkyl meso-arylporphyrins in the photokilling of breast cancer cells using permethyl-β-cyclodextrin as sequestrant and cell uptake modulator

Highlights

• New unsymmetrically substituted, water insoluble, non-ionic tetraphenylporphyrins bearing a single carboxyalkyl side-arm are synthesized

• Permethylated β-cyclodextrin (pMβCD) forms highly stable (K > 10¹² M^-2) isomeric 2:1 assemblies with new complexation modes

• The complexes show remarkable photostability and remain intact in the presence of cell culture proteins

• The complexes show enhanced endoplasmic reticulum preference in MCF-7 cells and ~ 90% photokilling efficiency

• The pMβCD/porphyrin complexes are interesting prospective components of advanced drug delivery systems

Abstract

In the search for photosensitizers with chemical handles to facilitate their integration into complex drug delivery nanosystems, new, unsymmetrically substituted, water insoluble meso-tetraphenylporphyrin and meso-tetra(m-hydroxyphenyl)porphyrin derivatives bearing one carboxyalkyl side chain were synthesized. Permethyl-β-cyclodextrin (pMβCD) was their ideal monomerizing host and highly efficient shuttle to transfer them into water. New assembly modes of the extremely stable (K_binding > 10¹² M⁻²) 2:1 complexes were identified. The complexes are photostable and do not disassemble in FBS-containing cell culture media for 24 h. Incubation of breast cancer MCF-7 cells with the complexes results in intense intracellular fluorescence, strongly enhanced in the endoplasmic reticulum (ER), high photokilling efficiency (~90%) and low dark toxicity. pMβCD stands out as a very capable molecular isolator of mono-carboxyalkyl-arylporphyrins that increases uptake and modulates their localization in the cells. The most efficient porphyrins are envisaged as suitable photosensitizers that can be linked to biocompatible drug carriers for photo- and chemo-therapy applications.

Introduction

Photoactive molecules and nanomaterials based on porphyrinoid compounds and cyclodextrins have been synthesized as components in biomimetic systems or, more commonly, used in phototherapy modalities in vitro and in vivo, aiming to provide photomedicine, bioimaging, and drug delivery applications (Mavridis & Yannakopoulou, 2020). Porphyrinoids are particularly suitable and effective photosensitizers (PSs) for photodynamic therapy (PDT), a clinically approved treatment of tumors (Agostinis et al., 2011; Dolmans et al., 2003) and other diseases such as psoriasis (Choi et al., 2015), microbial infections (Wainwright et al., 2017) and macular degeneration (Newman, 2016). Porphyrins possess desirable photophysical properties for such applications because upon illumination at a suitable wavelength (λ) in the region 400–800 nm, they produce toxic singlet oxygen or other reactive oxygen species (ROS) (Dolmans et al., 2003; Martinez De Pinillos Bayona et al., 2017). Moreover, porphyrins have high preferable accumulation in tumor tissues (Allison & Moghissi, 2013; Dolmans et al., 2003) and upon excitation they emit red light, thus they render fluorescence microscopy studies and bioimaging naturally feasible. Pre-clinical and clinical studies have confirmed that PDT is also capable of stimulating the immune system, a property that can increase the beneficial effects of PDT and provide many advantages for its use in clinical practice (Reginato et al., 2014). Despite their great potential, the majority of porphyrin PSs, being strongly amphiphilic or hydrophobic, are scarcely soluble in aqueous media and even the ionic, water soluble derivatives have a high propensity to aggregate. As a result, their systemic administration is problematic and their phototoxic efficiency is severely compromised (Dabrowski et al., 2016; Martinez De Pinillos Bayona et al., 2017). Therefore, suitable biocompatible, water-soluble molecular carriers that can render porphyrins sufficiently water-soluble, minimize aggregation and enable optimal delivery without altering their PS activity (Dabrowski et al., 2016) are much desired.

Cyclodextrins (CDs) are macrocyclic carbohydrate hosts that form inclusion complexes by enclosing guest molecules in their hollow structure. The CDs improve the aqueous solubility, physicochemical stability and bioavailability of the enclosed guests, thus controlling their release (Saokham et al., 2018; Tian et al., 2020). Alkylation of the peripheral hydroxyl groups interrupts the intra-glucosidic H-bonding network that keeps the CDs rigid and endows them with increased aqueous solubility as well as ability to adopt an optimal macrocyclic conformation in order to engulf most effectively suitable guest molecules (“induced fit”). It has been established that the inclusion complexation of certain ionic, water soluble meso-arylporphyrins inside heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (pMβCD) is a particularly strong and specific event, characterized by unusually high binding constants (K > 10⁷ M⁻¹) in aqueous media (Carofiglio et al., 1996; Kano et al., 2002; Kano et al., 2005). The binding motif typically involves two pMβCD macrocycles enclosing one meso-arylporphyrin molecule in a trans fashion (2:1 stoichiometry). Such CD/porphyrin systems reported in the literature refer to symmetrically substituted, mainly anionic or cationic, water soluble meso-arylporphyrins designed for the detailed study of photophysical and structural properties of the complexes (Carofiglio et al., 1996; Horiguchi et al., 2018; Kano, 2004; Kano et al., 2002; Kano et al., 2005; Tsuchiya et al., 2012), as PDT models in vitro (Kitagishi et al., 2015; Ikeda et al., 2017; Yumoto et al., 2020) or as integral components of imaginative biomimetic assemblies (Kitagishi et al., 2016; Minegishi et al., 2018; Vonesch et al., 2019). Only few reports can be found on unsymmetrical arylporphyrin complexes (Kitagishi et al., 2013) and, to the best of our knowledge, none on insoluble meso-tetraphenylporphyrin (TPP) derivatives.

It is recognized that amphiphilicity constitutes one of the significant features for high PDT activity of PSs. Amphiphilic PSs prefer to anchor into the lipid bilayers of the membranes of organelles. Membrane anchoring has been skillfully exploited by Berg's group to introduce PCI technology for photoinduced drug delivery (Sultan et al., 2016). Amphiphilicity and non-symmetrical substitution in the PSs' structure deeply affect the cellular uptake and the subcellular localization of porphyrins and in several systems improve the PDT activity considerably (Martinez De Pinillos Bayona et al., 2017).

In the search of porphyrin photosensitizers that can become parts of more advanced drug delivery systems, we thought that attachment of a single lipophilic arm of variable length with a terminal carboxyl group as chemical handle on neutral, water insoluble porphyrins such as TPP will result in an interesting class of compounds that could be used as components of 3rd generation PSs (Yakavets et al., 2019; Theodossiou et al., 2015). We hypothesized that despite lack of functional groups and water solubility, some of the designed unsymmetrical porphyrins could become water soluble with fully functioning photophysical properties via inclusion into the pMβCD host. It is worth noting that pMβCD is a host with a double solubility profile: it is highly soluble in water (310 mg/mL at 25 °C) (Saokham et al., 2018), but it is also freely soluble in organic solvents, including highly non-polar ones, such as octane (Griebenow et al., 1999). The aim is to highlight the pivotal role of pMβCD as molecular sequestrant and cell uptake modulator of minimally modified, insoluble arylporphyrins, steering them into cells independently of the presence of serum proteins.

This work presents the synthesis of new unsymmetrical, mildly amphiphilic porphyrins, derivatives of the water insoluble TPP and meso-tetra(m-hydroxyphenyl)porphyrin (mTHPP), as well as of their complexes with pMβCD, in aqueous media. The molecular and supramolecular systems were characterized by NMR, UV–Vis and fluorescence spectroscopy and DLS, and tested for photostability in solution and for intracellular distribution, phototoxicity and dark toxicity in MCF-7 breast cancer cells.