Cancer still remains one of the leading causes of morbidity worldwide despite great efforts and advancements in cancer prevention and treatment over the years. Conventional chemotherapeutic agents have numerous disadvantages, such as poor solubility, high dosage needs, severe side effects, low therapeutic indices, development of multi-drug resistance, and non-specific targeting of cancer cells. Targeted, functional drug delivery systems based on nanocarriers sensitive to intrinsic and extrinsic stimuli are widely studied due to the increasing cancer prevalence and emerging issues with conventional chemotherapy. Using nanotechnological methods in drug delivery systems provides advantages for the encapsulation of therapeutic agents that cause improvements in drug circulation time, the increased uptake of nanoparticles (NPs) into tumor cells, avoidance of the reticuloendothelial system (RES), and minimizing toxicity [1].

Delivering a high dose of an anticancer drug directly to the tumor, increasing drug uptake by malignant cells, and minimizing drug uptake by non-malignant cells are at the focus of engineering strategies for targeted cancer therapy [[2], [3], [4]]. Such delivery systems typically require the chemical conjugation of drugs or drug carriers to the targeting moiety [5]. Nanocarriers’ surface is open to chemical modifications with specific recognition ligands, functional groups, and polymers of different sizes and charges to increase their specificity for target sites [6]. Proteins, sugars, or lipids found in diseased organs or on the surface of cells may be target molecules. Representative ligands include small molecules such as antibodies, proteins, peptides, nucleic acids, sugars, and vitamins (Epidermal growth factor receptor-EGFR, folic acid (FA), transferrin receptors) [7].

NPs of biological origin can be produced from biopolymers, proteins, polysaccharides (chitosan, carboxymethyl cellulose, etc.), and lipids. Protein-based NPs as drug delivery systems have attracted attention as a research subject of cancer studies in recent years [8]. Protein-based NPs, such as silk, keratin, collagen, zein, and albumin, have advantages such as biodegradability, non-toxicity, high bioavailability, and relatively low costs. Many protein NPs are easily processable and suitable for modifications to achieve the desired specifications, including size, morphology, and weight [9]. Furthermore, Arg-Gly-Asp (RGD) sequences in proteins can regulate cell adhesion and support cell recognition sites [10].

Protein-based bovine serum albumin (BSA) will be used, and active substance-loaded albumin nanoparticles (BSANPs) will be prepared within the scope of the article. BSA is a transport protein found in large amounts in the blood plasma of humans and other mammals. It regulates the blood's osmotic pressure and ensures the transport of various substances in the blood. BSA is a very important surface modifying agent for carrier systems, which prolongs the circulation time of carrier systems. On the other hand, research on albumin accumulation in these regions has been done in parallel with the increased nutritional and oxygen needs in tumor tissues [9,11,12].

BSA is a compound with active and passive targeting properties. Studies have demonstrated that tumors and inflamed tissues metabolize significant amounts of BSA in the body as a source of nitrogen and energy. Other studies conducted on tumor-bearing animals have provided evidence that BSA accumulates at tumor sites because of the altered physiology and metabolism of tumor sites. Hence, using BSA in a polymeric-based drug delivery system has come to the forefront to promote retention in cancerous cells compared to healthy cells of the body [11]. BSA-drug conjugates, BSA-metal conjugates, BSA-polymer conjugates, BSA micelles, core-shell NPs, and BSA-based prodrugs can be given as examples of BSA-based nanocarriers [10].

Folic acid (FA) can be used as a targeting ligand for the targeted release of drugs. FA plays an essential role in DNA synthesis and replication, cell division and growth, particularly in carbon transfer reactions in rapidly dividing cells. It is known that folate receptors are overexpressed in different cancer cells, including breast cancer. Furthermore, whereas only reduced folate is transported in healthy cells, cancer cells can internalize folate conjugates via receptor-mediated endocytosis. Through this mechanism, FA-coated and drug-loaded NPs can overcome drug resistance caused by P-glycoprotein efflux pumps. The natural properties of FA and FA receptors on cancer cells render them effective agents for drug targeting, mitigating the severe side effects of free drugs and overcoming drug resistance. FA-modified drug-loaded NPs are known to exhibit high cytotoxicity and cellular uptake efficiency [13,14].

In a study published in 2019, Baneshi et al. prepared BSANPs loaded with iron oxide and gold NPs and functionalized with AS1411 aptamer and researched the targeted release of doxorubicin [15]. Ano et al. prepared parvifloron D-loaded BSANPs by the desolvation method for use in treating pancreatic and breast cancer [16]. Rokon et al. synthesized BSANPs by the coacervation/nanoprecipitation method and investigated oxytocin release for treating autism spectrum disorder [17]. Bolin et al. prepared resveratrol-loaded, folate-conjugated BSANPs for targeting liver tumors [18]. Kushwah et al. modified BSA by the chemical conjugation method with anacardic acid (AA) and gemcitabine (GEM), provided the development of docetaxel-loaded AA-GEM-BSANPs, and investigated the effectiveness of this formulation on breast cancer [19]. Yang et al. prepared folic acid-conjugated, doxorubicin-loaded, magnetic iron oxide bovine serum albumin nanospheres (FA-DOX-BSA MNPs) according to the desolvation-cross-linking method, and researched their effectiveness in nasopharyngeal carcinoma [20]. The studies summarized above have shown that BSANPs are generally used for the active targeting of chemotherapeutic drugs.

It has been reported that curcumin (CUR), a flavonoid polyphenol with a yellow phenolic pigment [21], exerts numerous pharmacological activities such as anti-inflammatory, antioxidant, anticancer, and antitumor properties. It is also stated that CUR can prevent carcinogenesis, sensitize cancer cells to chemotherapy, and protect normal cells from the damage caused by chemotherapy [21]. CUR is hydrophobic and usually soluble in oil, ethanol, acetone, and dimethyl sulfoxide (DMSO) [22]. The antitumor activity of CUR on breast, lung, gastrointestinal, head and neck, prostate cancer, melanoma, and brain tumors and its ability to target multiple cancer cell lines have been reported [23].

CUR primarily fights against cancer by promoting apoptotic pathways in cancer cells and inhibiting pre-cancerous processes such as inflammation, angiogenesis and metastasis. CUR targets several pathways involved in cancer treatment. These include those involved in p53, Ras, protein kinase B, the Wnt-beta-catenin signalling pathway, phosphatidylinositol 3-kinase and mTOR (mammalian target of rapamycin protein complex). Breast cancer tissues show overexpression of cyclin-dependent kinases (CDKs) and underexpression of the tumour suppressor protein p53. At the same time, some proteins regulating cell cycle are downregulated, including p21, p27 and p57 CDK inhibitors [24]. It is thought that it may be possible to cure breast cancer by targeting these particular molecules. CUR works by inhibiting the growth of breast cancer cells in the following ways i) inducing cell cycle arrest and p53-dependent apoptosis; ii) modulating the expression of signalling proteins, including Ras, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and Wnt/β-catenin; iii) downregulating transcription factors; and iv) inhibiting tumour growth and angiogenesis [25].

The mechanism studies of the interaction between CUR and BSA indicate the static quenching occurred between BSA and CUR. Hydrogen bonds and van der Waals forces play a significant role in the binding reaction between BSA and CUR, as indicated by the negative values of ΔH and ΔS. The value of ΔG < 0 indicates the spontaneity of the interaction between the molecules [26,27].

Proteins and polysaccharides are especially preferred for use in nanotechnological drug delivery systems intended for use in healthcare because they are biodegradable, biocompatible, and non-toxic natural substances. Although there are some studies in the literature on the targeted forms of protein-based nano-drug delivery systems for use in cancer treatment, they are more limited compared to other biopolymeric materials. There is a need for more studies for the methods used to denature proteins, in addition to the difficulties in reproducibility and size control while preparing protein nanoparticles [12]. In the present study, a drug delivery system is designed in which the carrier material, the flavonoid active ingredient with anticancer and chemopreventive properties and the targeting ligand are soft materials of biological origin. Thus, the harmful impacts of the active ingredient and the carrier system on healthy cells are attempted to be completely eliminated. In the published studies on albumin nanoparticle synthesis, very different particle size distributions and average particle sizes of BSANPs were reported. The albumin protein allows the encapsulation of compounds with different charges due to the negatively and positively charged functional groups in its structure. This study investigates CUR release from CUR-loaded BSANPs (CUR-BSANPs) at the acidic tumor microenvironment pH of 5.6 and physiological blood pH of 7.4. The surface of CUR-BSANPs was conjugated with FA to provide the direct transport of the active substance to the target cells. The fit of CUR release kinetics to zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell models was researched. The CUR release mechanism from CUR-BSANPs is suggested by calculating the values of kinetic constants. BSANPs have also started to be preferred in dual drug release studies because of their anticipated advantages. Moreover, it is thought that the results acquired from this study will contribute to the literature by providing preliminary knowledge on the issues related to dual active substance release studies and will form the basis of studies carried out in the further development of the system.