Despite considerable progress in comprehending the mechanisms and treatment of osteosarcoma cancer, it remains a significant public health concern, posing a severe risk to human lives, especially children. The escalating mortality rate of osteosarcoma is primarily attributed to the inefficacy of treatment methods during the cancer’s advanced stages. Current treatments, including chemotherapy and radiation therapy, not only impact cancer cells but also normal, healthy tissues, leading to severe side effects, such as Myeloid Suppression, Neurotoxicity, Anaphylaxis, Anemia, Intoxication, Cardiac toxicity, Mucositis, pain, Halotherapy, and Anorexia [21]. Furthermore, conventional osteosarcoma treatments fail to specifically target the molecular pathways and genes implicated in bone tumor development. Recent advancements have demonstrated the effectiveness of treatments that directly regulate gene expression in various diseases like Alzheimer’s at clinical stages. Thus, it appears that treatments with minimal side effects that can modulate molecular pathways could be the most advantageous for osteosarcoma [22]. In this context, the nanoscience in combination with medicine has emerged in the realm of cancer therapy. It is a novel and interesting scientific field focusing on designing targeted delivery systems for drugs and the transportation of small and large molecules that are effective in diagnosing and improving cancer therapy process.

Flavonoids, being natural phenolic compounds, are regarded as potent agents for cancer prevention. Despite this, the application of these biodegradable plant-derived anti-cancer compounds has been somewhat overlooked. Numerous herbs and spices contain flavonoids in their chemical makeup. These compounds exhibit pharmacological and biochemical properties, such as antioxidant and anti-inflammatory activities, which are believed to contribute to their anti-cancer and anti-mutagenic effects. Given that the progression of osteosarcoma (OS) cancer is strongly associated with inflammation and oxidative stress, a compound possessing anti-inflammatory and antioxidant properties could potentially serve as an anti-cancer agent [23]. The primary objective of cancer prevention using natural or man-made materials is to decelerate, inhibit, or reverse the process of cancer development. According to findings, certain plant compounds like curcumin inherently possess the ability to modulate these pathways, thereby treating or delaying the carcinogenic process in various species. For instance, a study by Taebpour et al. in 2022 demonstrated that curcumin could significantly impede the growth of MG-6 bone cancer cells [3]. Curcumin’s tumor-inhibiting effect is attributed to its influence on substances, such as MMPs, NF-κß, AP-1, TNF-α, Lox, COX-2, chemokines, and growth factor, like HER2 and EGFR, as well as matrix metalloproteases, which can inhibit the activity of N-Terminal of tyrosine kinase [24]. However, its direct effects on oncogenes and tumor suppressors have received less scrutiny.

The ideal composition of PLGA nanoparticles is influenced by a multitude of factors. The desired nanoparticles should meet criteria in terms of drug encapsulation efficiency, regulated release profiles, as well as the dimensions and zeta potential of the nanoparticles, and their stability. Each of these aspects is interconnected with numerous variables. The solvent used in synthesized NPs is a crucial factor that significantly affects the loading capacity of nanoparticles. In this study, it was observed that by switching the solvent from chloroform to dichloromethane, both the encapsulation and release rates in the system increased. This is due to the fact that chloroform, being a highly volatile organic solvent, evaporates rapidly during the stirring stage of the system synthesizing. The proportion of PVA used in synthesizing PLGA also plays a vital role in its encapsulation efficiency and release rate. The percentage of PVA has a direct relationship with the loading capacity of the system and an inverse relationship with the release rate. By increasing the amount of polyvinyl alcohol from 2 to 5%, the system’s loading capacity increased. This is because this polymer hinders drug release by forming a tighter and more intricate coil around the system, naturally increasing its encapsulation efficiency and reversely, reducing drug release. Sonication time also has a significant impact on these parameters. Sonication duration is highly influential on the loading capacity of the system as an increase in sonication duration results in polymer loss and formation of polymer sediment, which reduces the loading capacity of the system [25, 26]. The encapsulation efficiency of synthesized PLGA in this study was 91.5 ± 1.16% and its highest amount of drug release in 48 h was 71 ± 1% at 42 ̊C and pH 4.5 which shows how effective the synthesized NPs were.

In our research, we examined the influence of the drug-to-polymer ratio on the curcumin loading rate in PLGA nanoparticles. We synthesized nanoparticles using drug-to-polymer ratios of 1/10 and 10/25. Our findings revealed a direct correlation between the drug-to-polymer ratio and the curcumin loading in nanoparticles, with loading rates increasing from 63% to 91.5%. Polyvinyl alcohol (PVA) emulsifiers significantly affect the size of synthesized polymer, loading capacity, and fabricated polymer NPs’ stability. The PVA forms a protective layer around the nanoparticles, enhancing their cohesion and slowing solvent evaporation. However, overuse of emulsifiers can lead to particle agglomeration. The concentration and molecular weight of PVA are also crucial determinants of nanoparticle size. Excessive use can result in particle agglomeration [27].

Another effective factor in NPs as drug delivery carriers is their size. PVA forms aggregate layers around nanoparticles, increasing their hydrodynamic diameter. As a result, nanoparticles containing higher molecular weight PVA may be larger in size. Sonication time during nanoparticle synthesis is another factor that influences nanoparticle size. Ultrasonic waves are used to produce small, homogeneously distributed nanoparticles with high confinement efficiency. The strength and duration of sonication affect nanoparticle size, with longer sonication and higher strengths resulting in smaller particles. However, excessive sonication strength can damage nanoparticles through shear effects [28]. The fabricated polymer NPs in this scientific project have an average size of 321.2 ± 12.5 nm. The PDI serves as a quantifier for the heterogeneity in the size of NPs within a specific sample. An elevated PDI value signifies a broad spectrum of NPs size, potentially leading to aggregation. This could subsequently result in diminished stability of the nanoparticle suspension and a lack of uniformity. In the case of PLGA nanoparticles, it is important to have a low PDI value to ensure that the nanoparticles are uniform in size and have a stable suspension. Indeed, the size and the stability of NPs as drug delivery carriers are crucial factors in drug delivery systems. These characteristic can influence the NPs’ ability to target desirable sites and control drug release effectively. This underlies the importance of maintaining a low PDI, as a higher PDI number can lead to aggregation and reduce stability, potentially impact the efficacy of the drug delivery [29]. The PDI of NPs in this project was 0.107 which is practically considered an appropriate number for a drug delivery carrier.

During the optimization process, we observed alterations in the zeta potential. In theory, a high absolute value (> 30mV) surface electrical charge indicates increased colloidal stability because of enhance electrostatic repulsion. However, from a biological perspective, iotas with extremely enormous negative or positive electrical charge are willing to detection and removal from the living organism system. For nanomedical systems, a range of −5 to −45 mV of surface charge is typically deemed ideal, as it aligns with the zeta potential of most cells. The significance of a negative zeta potential is underscored in capacity of prevent non-specific attachment to blood cells and debris in circulating system, thereby reducing the chances of opsonization and immune system scavenging [30, 31]. In 2008, Ozkan and colleagues conducted an experiment on breast cancer cells (MCF-7), which have a zeta potential of −20.3 mV. They treated these cells with NPs with −13.5 mV surface charge. Interestingly, they observed that the zeta potential of the cells became increasingly negative after 30 min, 4 h, and a day decreasing to −24.5, −25.4, and −26.3 mV, respectively. Electron Microscopy images confirmed the attachment of NPs to the cells and showed their entry into the mentioned cancerous cells via endocytosis. Despite the theoretical electrostatic repulsion that should prevent the endocytosis of negatively charged vesicles into cells with negative surface charge, they found that the certain protein and membrane ligands could facilitate this process [32]. Consequently, NPs that possess a negative charge similar to the zeta potential of the target cells could effectively improve cellular uptake process. In current project, the synthesized polymer containing curcumin exhibit an average zeta potential of −38.9 ± 2.6 mV, which falls within the desired range. During optimization, despite the reduction in nanoparticle size leading to an almost twofold increase in their negative charge’s numerical value, this augment did not exhibit a consistent template at various stages.

Responding to various stimuli has turned into a tremendous indispensable characteristic of drug delivery carriers. One of the main advantages of using nanocarriers, such as PLGA, is their ability to enhance drug efficacy in targeted tissues. NPs possess the ability to alter the rate of encapsulated drugs release process, adapting in accordance with changes in environmental conditions such as temperature and pH levels, optimizing drug usage and minimizing potential side effects. Elevated temperatures influence the permeability on NPs’ membranes, thereby accelerating they release speed. The acidity of the environment can also strength the membrane permeability by establishing a proton gradient in the nanocarrier membrane. This occurs through the protonation of specific amino groups in chemical constituents of the encapsulated active component, which in turn boost the release rate of drug from NPs. Cancer cells typically exhibit higher temperatures and lower pH levels compared to normal human cells. This characteristic allows thermo-pH-sensitive NPs to distinguish between normal and cancerous cells. In essence, the development of NPs that are sensitive to both pH and temperature presents a promising approach to address the challenge of differentiating between normal and cancerous cells. This is prevalent issue in chemotherapy and radiotherapy and such advancements could potentially mitigate the side effects associated with these treatments [20, 33]. In current research, the highest amount of drug release was in warmer conditions (42 ̊C) and more acidic environment (4.5) at 71 ± 1%, while the lowest percentage of drug release was in physiological circumstances (pH 7.4, 37C) at 38.54 ± 1.12%, demonstrating how smart and effectively the synthesized nanosystem responded to various stimuli.

Encapsulation of active substances in PLGA polymer nanoparticles has been shown to significantly enhance their effectiveness and bioavailability. For instance, a study found that loading Berberin in PLGA polymer heighten its toxicity impacts on MCF-7 cancer cells in comparison to the active component in free form. Other studies demonstrated similar improvements in the anti-cancer effects of curcumin and quercetin when encapsulated in PLGA nanoparticles [34]. Encapsulation can protect against light, increase stable release and solubility and overcome issues, such as low hydrophobicity and poor permeability, leading to increased effectiveness of the encapsulated substances [35]. These findings suggest that encapsulation can have a positive impact on the effectiveness of materials with low bioavailability. In our investigation after the encapsulation of curcumin into PLGA, its anti-tumor effects on SAOS-2 significantly increased. The IC50 of encapsulated curcumin was at 7.32 μg/ml almost half of its free form with 13.96 μg/ml.

As previously stated, genes are crucial elements in cancer progress. This research compared the impact of loaded and free of an active herbal component namely curcumin on the relative expression of P53 and Mcl-1 genes, with the goal of developing new approaches to cancer treatment. The outcomes of this study showed that PLGA-Cur significantly increased the expression of P53 compared to the control group and the free form of curcumin. Additionally, this nanoparticle reduced MCL-1 expression compared to the control group and also free form of bioactive component. The higher the concentration of curcumin and nanosystem was, the more genetic effects were imposed. Curcumin, a bioactive compound found in turmeric, has been demonstrated to modulate the function of the p53 tumor suppressor gene. Research has demonstrated that curcumin has a significant impact on colon cancer cells, specifically, curcumin interferes with the formation of P53 protein, which is crucial for its Serine phosphorylation, DNA binding, and activation of gene responsive to p53. This interference disrupts the ability of P53 to halt the cell cycle effectively. Additionally, curcumin hinders the activity of Thioredoxin reductase and prevents the proper folding of wild-type P53 protein into necessary conformation for its phosphorylation, DNA attachment, and activation gene responsible for suppressing tumor growth [36].

It is important to note that curcumin has also been shown to exhibit anti-cancer impacts by alleviating the regulation of anti-apoptotic gene products, caspase activation, and upregulating tumor suppressor gene, such as P53. The effects of curcumin on cancer cells can be different based on the kind and the progress level of cancer as well as other various factors. Therefore, further studies are necessary to completely elucidate the intricate interactions between curcumin and p53. In Caki cells, a type of human renal carcinoma cell, curcumin has been shown to enhance apoptosis induced by NVP-BEZ235, a dual PI3K/Akt and mTOR inhibitor. This effect is achieved through the down-regulation of Bcl-2 expression, which is dependent on p53, and the inhibition of Mcl-1 protein stability [37]. Another study found that curcumin could induce apoptosis in cells overexpressing Mcl-1 or Bcl-2 when treated with curcumin in the presence of PP242, a mTORC1/2 inhibitor [38]. However, the mechanisms by which curcumin affects Mcl-1 expression are intricate and probably affected various molecular pathways. Further studies are needed to help to know these interactions.