The water solubility of drugs is important to consider during their development and application because they should be completely solubilized without aggregates for injection into the human body. Unfortunately, many hydrophobic drugs are poorly soluble in water [1]. Many approaches have been tested by researchers to overcome this situation, and nanoparticle (NP) encapsulation has been a promising solution [2]. Many NP types have been developed using materials including phospholipids, polymers, proteins, nucleotides, metals, silicas, and carbon materials [3]. Many of them demonstrated complete solubilization of hydrophobic drugs in buffer solution for injection into the body. Their nanoscale size is suitable for long blood circulation, and high accumulation in angiogenic tissues like tumors by the enhanced permeability and retention (EPR) effect made them promising drug carriers [4]. In addition, versatile modification of NP surface with biological ligands or addition of stimuli-responsive properties further increased their ability to target disease sites [5,6]. Doxil, Abraxane, and recently emerged COVID-19 vaccines using lipid NPs are representative examples of the clinical application of NP carriers [7]. However, considering the numerous trials in laboratory-scale research, the number of clinically approved NPs is small. This may originate from the difficulties in their FDA approval, and safety issues are the biggest hurdle [8].

In this point of view, developing NPs using biocompatible materials would be advantageous [9]. This could be a reason why clinically approved NPs are comprised of phospholipids and albumins. Recently, some researchers have focused on casein, a protein present in breast milk. Various molecules are transferred from mother to baby through breast milk, many of which are hydrophobic. Such molecules can be stably solubilized in the body in the form of a casein micelle (CM) with hydrophobic pockets [10]. CM is composed of αS1, αS2, β, κ-casein and amorphous calcium phosphate. Hydrophobic α, β-caseins make inner pockets for drug loading and hydrophilic κ-casein surrounded them with hairy structure [10]. It suggests that CM is a nature-designed carrier for hydrophobic molecules, and it is expected to be biocompatible. Therefore, several studies applying CMs to drug delivery have been reported [11]. Various hydrophobic drugs including paclitaxel, SN38, curcumin, celecoxib, alfuzosin have been loaded into the inner pocket of CM for drug delivery [[12], [13], [14], [15], [16]].

Photodynamic therapy (PDT) is a laser-based therapeutic modality that is used in the treatment of various cancers in the head and neck, skin, prostate, breast, and lung [17]. Photosensitizer (PS) is a special chemical dye that generates cytotoxic singlet oxygen along with fluorescence upon laser irradiation during PDT. However, the poor water solubility of many PSs is a critical hurdle in PDT, and their efficient delivery to target tissue and cells is also important. For this purpose, various NPs have been developed and used, which were summarized in other reviews [18,19]. However, CM has been rarely used in PDT until now [20].

Therefore, in this study, we developed CM containing pheophorbide a (Pba), a representative PS with an emission wavelength in the near-infrared region, which is useful for in vivo applications (Scheme 1) [21]. The stability of the Pba-loaded CM (CM-Pba) and its photodynamic effect were evaluated in vitro. Its biocompatibility and tumor cell-killing effect were observed in SCC7 (murine squamous cell carcinoma) cells. In vivo live imaging was performed to analyze its biodistribution after injection into SCC7 tumor-bearing mice, and therapeutic results after laser irradiation and in vivo PDT were obtained with the same mouse model. To the best of our knowledge, this is the first study to demonstrate the potential of CM as a carrier for PS delivery and PDT.