There has been a growing interest in the field of women’s health recently owing to the increasing health threats driven by the major changes in the lifestyle, nutrition, and the excessive environmental hazards [1]. Breast cancer is one of the most common threats affecting the lives of millions of women and their families across the globe. In 2022, the American Cancer Society revealed that despite the fact that the mortality rate of breast cancer has been significantly reduced thanks to the advanced therapies, the incidence rate has been increased in contrary [2]. The increased number of cases exerts a burden on the national and private healthcare budgets, especially post COVID-19 pandemic and its severe impact on the global economy. Moreover, the conventional chemotherapy has multiple unfavorable adverse effects and holds the risk of development of chemoresistance [3]. Furthermore, the increased number of breast cancer survivors are still in need of years-long prophylactic treatment with hormonal therapies or immunotherapies to prevent recurrence, which carry numerous side effects that adversely affect their quality of live [4], [5].

Almost all mammalian cells require L-lactic acid to be transported across their plasma membranes. On one hand, tissues like heart, neurons and red muscle fibers import lactic acid for oxidation, for gluconeogenesis (kidney and liver), or for lipid generation (adipose tissue). On the other hand, tissues like white muscle use glycolysis for the ATP production and for this reason, the large lactic acid accumulation should be exported [6], [7]. Interestingly, previous studies also showed that cancer cells are programmed to conduct glycolysis for rapid energy generation due to the fast growth [8], [9], and the generated lactate will be exported outside of the cells, which is conducted due to the high acidity of the environment [10]. For lactic acid rapid transportation across the plasma membrane, the cells utilize monocarboxylate transporters (MCT) which can export or import lactate in combination with protons. There are different MCTs (MCT1–16) from which only MCTs 1–4 are well exploited and have been confirmed as lactic acid transporters [11]. The well-characterized member MCT4 is expressed at high levels in tissues that rely on glycolysis and in white skeletal muscle. Moreover, previous studies showed increased expression of MCT4 in cells in response to hypoxia, which coincides with high glycolysis [7], [12]. Previous studies showed that MCT4 was overexpressed in prostate and cervical cancer as well as in renal carcinoma and other cancer cells [13], [14], [15], [16]. Interestingly, the most aggressive tumors mainly express MCT4 which is increased by over-expression of hypoxia-condition [17], [18]. Furthermore, solid tumors could show heterogeneity in lactate metabolism; the hypoxic center produces lactate and exports it via MCT4 to be taken up for oxidation by peripheral cells, which overexpresses more MCT1 [19], [20]. Taken all together, lactate transport has become a critical regulator of cancer cell growth, and metastasis, and inhibition of this process was a potential target for chemotherapy [21], [22], [23]. Nevertheless, most chemotherapeutics are limited by their off-target toxicities as well as the possibility of emergence of chemoresistance [24]. Therefore, innovative therapeutic approaches have become a must in the battle against breast cancer.

There has been a growing evidence that the immune signaling in the tumor microenvironment plays pivotal roles in the etiology, growth, and treatment of tumors. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that exerts critical roles in the host defense immunity against stress. Recent studies reported on an aberrant signaling of IL-6 in breast cancer, which is associated with the enrichment of cancer stem cells and the activation of the oncogenic pathway, Signal transducer and activator of transcription 3 (STAT-3) [25], [26]. Tumor necrosis factor-α (TNF-α) is another pro-inflammatory cytokine that is enriched in the breast cancer microenvironment and contributes to all stages of the tumor proliferation, survival, and metastasis through the promotion of the epithelial-to-mesenchymal transition (EMT) [27]. Therefore, the concept of “Cytokine remodeling” has come to the forefront as a potential strategy for the treatment of breast cancer [28].

Recently, there has been a growing interest in gene therapy as a promising therapeutic alternative in order to tackle the drawbacks of conventional therapies [29]. Viral-based delivery vectors have originally dominated the research on gene therapy from 1990 s until 2010. However, the paradigm has been shifted later in favor of non-viral delivery vectors owing to their higher safety, economic cost, and easier manufacture [30]. Lipid nanoparticles (LNPs) have proven to be the gold standard in this area, especially after the FDA approval of several LNPs encapsulating nucleic acid therapeutics including Onpattro®, Comirnaty®, and Moderna® COVID-19 vaccine [31], [32], [33]. The fundamental technology in these delivery systems was the ionizable cationic lipids, which possess a protonable amino groups with pKa˂7.4. Thus, they demonstrate a neutral surface charge in the physiological pH with minimum non-specific interactions and side effects. Following their cellular uptake, these lipids respond to the acidic environment in the endosome and regain their cationic nature for an improved endosomal escape function and delivery of the nucleic acid cargo into the cytosol [34], [35]. 1,2-dioleoyl-3-dimethylammonium propane (DODAP) is one of the earliest models of ionizable cationic lipids, with a pKa that can be tailored in response to the ionic strength of the solution [36], [37]. DODAP has shown success in the delivery of a wide diversity of nucleic acid cargos, including plasmid DNA (pDNA), small interfering RNA (siRNA), and messenger RNA (mRNA) [38], [39], [40]. Despite the recent advancements in the technology of LNPs, their clinical translation is still challenged by the classic targeting methods (e.g. targeting ligands and antibodies) that increase the complexity of the delivery vectors and compromise their scale-up [34].

In the present study, we designed and optimized ligand-free DODAP-based LNPs for the delivery of siRNA to the breast cancer cells so as to knockdown the expression of MCT4 gene. The pH-sensitive nature of the designed LNPs enables hitting two birds with a single stone. First, the relative acidity of the tumor microenvironment in comparison with the healthy tissues is exploited in achieving a tumor-selective gene delivery in an economic and scalable approach, in contrary to the classic ligand-based targeting. Second, the neutral surface charge of the developed LNPs in the normal physiological conditions increases their biotolerability and minimizes the non-specific interactions with off-target cells. The selectivity of the proposed delivery concept was evaluated in breast cancer cells versus normal cells, in the presence of serum to mimic the in vivo conditions and allow the adsorption of serum proteins to the surface of LNPs; generating a targeting protein corona that can also contribute to the selective delivery of LNPs to the target cells. Then, the anticancer activities of the LNPs were assessed against an aggressive breast cancer cell line, 4T1. Furthermore, we report on a novel impact of MCT4 knockdown on the remodeling of the cytokine profile in the breast cancer.