Cancer is a disease of significant social importance whose treatment requires multidisciplinary expertise. Its distinguishing characteristics include uncontrolled cell division, loss of differentiation, invasiveness, metastatic potential, and increased synthesis of macromolecules from nucleosides and amino acids. The diverse causes encompass genetic mutations, exposure to carcinogens (chemicals, radiation, viruses), immune suppression, and chronic inflammation [1], [2], [3].

The primary methods for treating oncological diseases include surgery, radiation therapy, and chemotherapy [4], [5]. Chemotherapy is widely used because of its high efficacy in treating tumors of various locations, nosologies, and stages [6]. The primary limitations of chemotherapy are the high systemic toxicity of cytostatics and the development of resistance, which drives the active search for alternative therapeutic approaches [7].

In clinical practice, anthracycline antibiotics such as doxorubicin, daunorubicin, idarubicin, epirubicin, mitomycin, etc. are widely used as first-line chemotherapy agents [8]. Doxorubicin is an effective broad-spectrum antitumor antibiotic. Doxorubicin is used both individually and in combination with other drugs to treat various types of cancer [9]. Anthracycline antibiotics, such as doxorubicin, exhibit cytotoxic activity through various mechanisms directed against tumor cells, including disruption of DNA replication [10], [11], [12], and oxidative stress caused by reactive oxygen species (ROS) [13], [14].

Doxorubicin is an effective anticancer drug, but its use is limited by serious side effects, especially dose-dependent and irreversible heart damage [15], [16], [17], [18]. This heart damage, caused by low tumor selectivity, can show up as cardiomyopathy and arrhythmias and is affected by treatment plan and patient age. Suggested mechanisms include mitochondrial dysfunction, ROS production, and topoisomerase II-related damage in heart cells. Since there are no established ways to prevent this effect, doxorubicin is not advised for elderly patients or those with heart problems, and severe cases may need a heart transplant [[19], [20], [21]].

Various strategies have been used to reduce the toxicity of anthracycline antibiotics such as doxorubicin, including the use of cardioprotectors (e.g., dexrazoxane) to protect the heart muscle [[22], [23]], combination therapy with antioxidants (N-acetylcysteine, resveratrol) to combat oxidative stress [24], and nanostructured delivery systems (liposomes, e.g., Doxil, polymeric and metal nanoparticles) for more selective drug accumulation in tumors [[25], [26], [27]]. Prodrug forms activated in the tumor microenvironment [28] or by light exposure [[29], [30], [31]] are promising, as are innovative approaches such as photodynamic therapy (PDT) using phthalocyanine photosensitizers (PS) [[32], [33], [34]] or targeted delivery using antibodies and peptides [35]. Despite significant advances, the cardiotoxicity of doxorubicin remains a serious problem requiring further research.

PDT is a minimally invasive treatment for solid tumors, which relies on the selective accumulation of PS in tumor tissue. Subsequent laser irradiation induces the generation of ROS, which exert a cytotoxic effect by damaging the cell at the molecular level [36]. The main advantages of PDT include its relative selectivity due to the local activation of the PS only within the irradiation zone [[37], [38]], minimal invasiveness [[39], [40]], and the possibility of combination with other treatment methods [41]. Since monotherapy is usually ineffective in oncology, the use of PDT in a single regimen has a number of limitations associated with the small depth of light penetration into tissue, photosensitivity of the skin leading to photodermatitis, and low treatment efficacy in hypoxic tumor tissue [42]. Currently, tetrapyrrole macrocyclic compounds of the chlorin series are used as the PS in clinical practice due to a number of advantages, including intense absorption in the near infrared region of the spectrum, which ensures deeper penetration of light into tissue and high biocompatibility [[43], [44], [45], [46]].

Previously, our research team obtained and studied a conjugate of bacteriochlorin with doxorubicin, which demonstrated a significant increase in antitumor activity both in vitro and in vivo compared to doxorubicin or bacteriochlorin alone [47]. Building upon these promising results and aiming to further enhance the efficacy and selectivity of combined photodynamic and chemotherapy, this study focuses on the development of a novel conjugate platform based on natural chlorins. The central concept is to achieve efficient and controlled release of doxorubicin directly within the tumor microenvironment. For this purpose, we synthesized a series of conjugates where a natural chlorin derivative, acting as a photoactive and tumor-targeting component, is covalently linked to doxorubicin, a first-line cytotoxic agent, via specially designed labile linker molecules. These linkers were designed to be sensitive to specific biochemical stimuli: reduction induced by elevated intracellular glutathione (GSH) levels and oxidation by ROS generated in tumor cells or during PDT.

The incorporation of such stimuli-responsive linkers is hypothesized to ensure the spatiotemporally controlled release of the anthracycline antibiotic, thereby minimizing its systemic exposure and reducing dose-limiting side effects, particularly cardiotoxicity.

To design and synthesize a novel series of conjugates combining derivatives of natural chlorins with doxorubicin using three types of linkers: i) a stable linker based on succinic acid, ii) a reductively cleavable disulfide-based linker, and iii) an oxidatively cleavable thioketal-based linker.

To comprehensively characterize the synthesized conjugates using modern analytical techniques, including NMR spectroscopy and high-resolution mass spectrometry.

To investigate the photophysical properties of the conjugates, specifically determining the quantum yield of singlet oxygen generation, a key parameter for PDT efficacy.

To experimentally verify the controlled cleavage of the labile (disulfide and thioketal) linkers under conditions simulating the reductive or oxidative (ROS upon irradiation) tumor microenvironment.

To evaluate the in vitro activity of the conjugates, including their dark cytotoxicity and photoinduced cytotoxicity against human breast adenocarcinoma MCF-7 cells, and also to analyze their combined photodynamic/chemotherapeutic effect.

To study the intracellular distribution and internalization dynamics of the conjugates using fluorescence microscopy, with a focus on tracking the release and nuclear accumulation of doxorubicin following linker cleavage.