Reveiw of Synergistic Cancer Therapy: Combining Betulinic Acid and Gold Nanoparticles for Improved Efficacy

INTRODUCTION
Cancer therapy encompasses a variety of treatments aimed at eliminating malignant cells and preventing recurrence. These treatments include surgery, radiation therapy, chemotherapy, immunotherapy, hormone therapy, and targeted therapy. The choice of treatment depends on the type, stage, and location of the cancer, as well as the patient’s overall health and preferences [1]. Despite significant advances, current cancer treatments face several challenges. These include the development of drug resistance, severe side effects, limited efficacy in certain cancer types, and the high cost of treatment. Additionally, the heterogeneity of tumors and the complexity of cancer biology make it difficult to achieve a cure for all patients [2].
Betulinic acid (BA) is a pentacyclic triterpenoid derived from the bark of birch trees, known for its anticancer properties. It has shown promise in inducing apoptosis in cancer cells and inhibiting tumor growth [3]. Gold nanoparticles (GNPs) are tiny particles of gold that can be used as carriers for drug delivery due to their unique physical and chemical properties [4]. When functionalized with BA, GNPs can enhance the bioavailability and therapeutic efficacy of BA, making it a potential candidate for cancer therapy [5]. 
The aim of this article is to explore the synergistic effects of betulinic acid-functionalized gold nanoparticles in cancer therapy. The novelty lies in the innovative approach of combining BA with GNPs to improve drug delivery and therapeutic outcomes. This study investigates the mechanisms by which BA-GNPs induce cancer cell death and assesses their potential as a more effective and targeted cancer treatment.

PROPERTIES OF BETULINIC ACID
Chemical Structure and Mechanism of Action
Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with the molecular formula C30H48O3. Its structure consists of five fused rings, including a hydroxyl group at the C-3 position and a carboxyl group at the C-28 position. This complex molecular structure supports its biological activity [6]. 
Betulinic acid induces apoptosis primarily through the mitochondrial (intrinsic) pathway. It disrupts the mitochondrial membrane potential, leading to the release of cytochrome c into the cytosol. This event triggers the activation of caspase-9 and subsequently caspase-3, which execute apoptosis [7]. BA can also interact with death receptors on the cell surface, such as Fas (CD95) and TRAIL receptors, leading to the activation of caspase-8 and the extrinsic apoptotic pathway. Betulinic acid increases the intracellular levels of ROS, which contribute to oxidative stress and damage to cellular components, ultimately leading to cell death [8]. The elevated ROS levels can also activate signaling pathways that promote apoptosis. BA inhibits topoisomerase I and II, enzymes crucial for DNA replication and transcription. By stabilizing the cleavage complexes formed by these enzymes on DNA, BA prevents the relegation of DNA strands, leading to DNA damage and inhibition of cell proliferation [9]. BA inhibits the nuclear factor-kappa B (NF-κB) pathway, which is involved in cell survival, proliferation, and inflammation. By inhibiting this pathway, BA reduces the expression of anti-apoptotic genes and promotes apoptosis [10]. BA modulates the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which is critical for cytokine signaling and cancer cell growth. Inhibition of this pathway contributes to the suppression of tumor growth. BA downregulates the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, which is involved in cell survival and metabolism [11]. This downregulation promotes apoptotic cell death. BA affects the mitogen-activated protein kinase (MAPK) pathway, which regulates cell proliferation, differentiation, and survival. Modulation of this pathway contributes to the antiproliferative effects of BA [12].

Anticancer Properties
Betulinic acid demonstrates significant anticancer activity through several mechanisms like, selectively induces apoptosis in cancer cells while sparing normal cells. This selectivity is partly due to the higher oxidative stress levels in cancer cells, making them more susceptible to BA-induced ROS generation and mitochondrial disruption [3]. BA inhibits angiogenesis, the process of new blood vessel formation, which is essential for tumor growth and metastasis. It achieves this by downregulating pro-angiogenic factors such as vascular endothelial growth factor (VEGF) [13]. BA reduces the metastatic potential of cancer cells by inhibiting the expression of matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix and facilitate cancer cell invasion [14]. Also enhances the immune system’s ability to recognize and destroy cancer cells. It increases the activity of natural killer (NK) cells and cytotoxic T lymphocytes, which play crucial roles in immune surveillance and cancer cell elimination [15].
Emili Pisha et al [16], studied of bioassay–guided fractionation, betulinic acid, a pentacyclic triterpene, turned into identified as a cancer–precise cytotoxic agent. In observe–up research performed with athymic mice carrying human melanomas, tumour increase was completely inhibited without toxicity. As judged by way of a selection of cellular responses, antitumour interest turned into mediated by way of the induction of apoptosis. Betulinic acid is inexpensive and available in considerable supply from common natural sources, considerably the bark of white birch trees.
Valentina Zuco et al [17], studied the betulinic acid interest become evaluated, in contrast with doxorubicin, on specific human neoplastic and non-neoplastic cell traces and on proliferating everyday lymphocytes. Boom inhibition become evident in all the neoplastic cellular lines independently on p53 popularity and histotype. Antiproliferative activity of betulinic acid became related to a cytotoxic impact on p53 wild-type and on one p53 mutant mobile strains and to a cytostatic impact on one p53 mutant cancer clone. On the same concentrations, ordinary cells have been unaffected indicating a selective impact of this agent. A cytotoxic pastime of doxorubicin turned into obvious on all of the examined systems. In vivo experiments, achieved on such a cell lines, confirmed the antineoplastic activity of this drug. Those facts help in addition preclinical research of betulinic acid now not restricted to cancer and neuroectodermal tumors independently of p53 status.

PROPERTIES OF GOLD NANOPARTICLES
Synthesis and Characterization
Gold nanoparticles (AuNPs) can be synthesized through different approaches, each with its specific mechanisms and characteristics [18]. Citrate Reduction Method involves the reduction of chloroauric acid (HAuCl₄) in an aqueous medium using sodium citrate as the reducing agent. The citrate ions, apart from reducing HAuCl₄ to elemental gold, simultaneously act as stabilizing agents, preventing the aggregation of nanoparticles [19]. This dual role of citrate ions contributes to the formation of spherical gold nanoparticles, which are often preferred due to their simplicity of synthesis and uniformity in size [20].  
Seed-Mediated Growth Method, a two-step process is employed, beginning with the creation of gold nanoparticle seeds. These seeds, which serve as nucleation points, are formed by reducing HAuCl₄ with a strong reducing agent [21]. Following this, the growth phase involves enlarging the seed particles by adding a solution containing HAuCl₄ and a weaker reducing agent, such as ascorbic acid. The shape and size of the nanoparticles during this growth phase are further influenced by the use of shape-directing agents, such as cetyltrimethylammonium bromide (CTAB). This method provides precise control over the morphology of the nanoparticles, allowing for diverse shapes, including rods, cubes, and stars [22].  
Biological Synthesis method uses biological entities, such as microorganisms, plant extracts, or biomolecules, to reduce HAuCl₄ into gold nanoparticles. Unlike chemical methods, biological synthesis eliminates the need for toxic chemicals and provides a more environmentally friendly alternative [20]. Furthermore, biomolecules naturally act as reducing and stabilizing agents, simplifying the synthesis process. However, the complexity of biological systems can sometimes lead to variations in nanoparticle size and shape [23].  
The characterization of AuNPs involves the use of advanced analytical techniques to determine their properties, such as size, shape, and stability [24]. UV-Vis-NIR spectroscopy is widely used due to its ability to monitor the surface plasmon resonance (SPR) peak, which provides insights into the size and aggregation state of the nanoparticles [25]. Dynamic light scattering (DLS) is another common technique, offering precise measurements of particle size distribution and zeta potential, which indicates the stability of colloidal suspensions [26].  

Biomedical Applications
Gold nanoparticles (AuNPs) exhibit several biomedical applications due to their unique optical, chemical, and biological properties. Notably, their potential in cancer diagnosis and treatment [27]. 
Bioimaging, Gold nanoparticles enhance imaging capabilities due to their strong surface plasmon resonance (SPR) effect, which amplifies contrast in imaging techniques such as optical coherence tomography (OCT), dark-field microscopy, and photoacoustic imaging [28]. These properties allow precise visualization of tumors, aiding in early cancer detection. Functionalized AuNPs can be targeted to cancer cells, improving the specificity and accuracy of imaging [29].  
Biosensing, the sensitivity of gold nanoparticles to environmental changes enables their use in detecting cancer biomarkers. AuNP-based biosensors can identify specific proteins, DNA, or RNA associated with cancer, providing a rapid and non-invasive diagnostic tool [30]. The aggregation or functionalization of AuNPs results in measurable optical or electrical signals, enabling accurate cancer diagnosis [31].  
Therapeutic Agents of Gold nanoparticles exhibit potent anticancer properties through their application in targeted drug delivery, photothermal therapy (PTT), and photodynamic therapy (PDT) [32].  
Targeted Drug Delivery makes Functionalized AuNPs can selectively deliver anticancer drugs to tumor cells by binding to overexpressed receptors on cancer cells. This targeted approach minimizes off-target effects and enhances therapeutic efficacy [33]. Photothermal Therapy (PTT) of Gold nanoparticles absorb near-infrared (NIR) light and convert it into localized heat, effectively destroying cancer cells without harming surrounding healthy tissues. Their biocompatibility and efficient heat generation make them ideal for minimally invasive cancer treatments [32].  
Photodynamic Therapy (PDT), AuNPs are used to deliver photosensitizing agents to tumor sites. Upon light activation, these agents generate reactive oxygen species (ROS), inducing apoptosis or necrosis in cancer cells [34]. Studies have shown that AuNPs can directly induce cancer cell death by interacting with cellular components such as mitochondria, disrupting energy production and initiating apoptosis. Functionalized AuNPs can also interfere with angiogenesis, the process by which tumors develop blood vessels to sustain growth [35]. While primarily researched for their anticancer applications, AuNPs also exhibit antimicrobial properties that help prevent infections during cancer treatments, such as chemotherapy or surgery [36].  

Advantages in Drug Delivery Systems
Gold nanoparticles offer distinct advantages in drug delivery systems due to their unique physicochemical properties and functional versatility like Biocompatibility, Targeted Delivery, Controlled Release, and Enhanced Stability which explained each part sepratly [20].
Functionalization of AuNPs with biomolecules, such as antibodies, peptides, or small ligands, enables them to selectively bind to specific receptors on target cells. This targeted approach not only enhances the efficacy of drug delivery but also reduces off-target effects, making them particularly beneficial for treating diseases like cancer and inflammatory disorders [33]. The surface chemistry of gold nanoparticles allows for the attachment of drug molecules through covalent or non-covalent interactions. These interactions can be designed to release the drug in response to specific stimuli, such as pH changes, temperature variations, or enzymatic activity, ensuring a sustained therapeutic effect over time [37].  Gold nanoparticles are generally regarded as biocompatible and non-toxic, which is crucial for their safe use in medical applications. However, the biocompatibility of AuNPs is largely influenced by their size, shape, and surface modifications, which must be carefully optimized for each application [38]. The chemical stability of gold nanoparticles, particularly their resistance to oxidation and degradation, makes them suitable for long-term storage and use in drug formulations. This stability also contributes to their robustness in physiological conditions, where they maintain their functional integrity [39].  

COMBINING BETULINIC ACID AND GOLD NANOPARTICLES
Synergistic Effects 
The combination of betulinic acid and gold nanoparticles offers a unique therapeutic approach by integrating the anticancer properties of betulinic acid with the delivery and functional versatility of gold nanoparticles. This system aims to improve the therapeutic efficacy of betulinic acid, enhance its bioavailability, and leverage the additional benefits of gold nanoparticles in cancer therapy [40]. The rationale for combining betulinic acid with gold nanoparticles is rooted in the complementary nature of their properties and mechanisms. Betulinic acid, a naturally occurring pentacyclic triterpenoid, exhibits potent anticancer activity through its ability to induce apoptosis in cancer cells via mitochondrial pathways [41]. However, its poor solubility, low bioavailability, and rapid degradation in biological environments limit its therapeutic potential. Gold nanoparticles, on the other hand, provide a stable and versatile platform for drug delivery due to their biocompatibility, tunable size, and ability to be functionalized with biomolecules [42]. When conjugated with gold nanoparticles, betulinic acid benefits from enhanced solubility, improved stability, and targeted delivery to tumor sites. Additionally, gold nanoparticles possess intrinsic anticancer properties and can be employed in advanced therapeutic modalities such as photothermal and photodynamic therapy [43]. 
The combination of betulinic acid and gold nanoparticles generates synergistic effects that enhance their anticancer efficacy. Gold nanoparticles act as carriers, ensuring efficient delivery of betulinic acid to tumor cells while protecting it from premature degradation. This targeted delivery increases the local concentration of betulinic acid in the tumor microenvironment, amplifying its therapeutic effects [44]. Additionally, the cytotoxic effects of gold nanoparticles complement those of betulinic acid, resulting in enhanced cancer cell death. Gold nanoparticles induce oxidative stress and disrupt cellular homeostasis, while betulinic acid triggers apoptosis through mitochondrial dysfunction. Together, these mechanisms create a multi-pronged attack on cancer cells, overcoming limitations associated with monotherapy and reducing the likelihood of drug resistance [45]. Moreover, the combination allows for stimuli-responsive drug release, where gold nanoparticles can release betulinic acid in response to environmental triggers such as pH changes in the tumor microenvironment or external stimuli like light. This controlled release further enhances therapeutic precision and minimizes systemic toxicity. The combined approach also reduces the required dosage of each agent, decreasing potential side effects and improving overall safety [46].  

Mechanisms of Action
The anticancer activity of the betulinic acid and gold nanoparticle combination is achieved through multiple interconnected mechanisms such as, Enhanced Delivery and Cellular Uptake that Gold nanoparticles improve the solubility and stability of betulinic acid, facilitating its efficient delivery to cancer cells. Functionalized gold nanoparticles are designed to target specific tumor markers, enabling selective accumulation in cancerous tissues while sparing healthy cells. Their small size and surface properties enhance cellular uptake through endocytosis, ensuring effective intracellular delivery of betulinic acid [40]. Induction of Apoptosis which Betulinic acid induces programmed cell death by targeting the mitochondria, disrupting the mitochondrial membrane potential, and activating caspases. This mitochondrial dysfunction leads to the release of pro-apoptotic factors such as cytochrome c, which initiates the apoptotic cascade. Gold nanoparticles amplify apoptosis by generating reactive oxygen species (ROS) and interfering with cellular redox balance [47].  Oxidative Stress that Gold nanoparticles generate ROS, which damage cellular components such as DNA, proteins, and lipids. This oxidative stress creates a hostile environment for cancer cells, sensitizing them to the apoptotic effects of betulinic acid. The combination enhances ROS-mediated damage, increasing the cytotoxicity of the treatment [48]. Photothermal and photodynamic effects when used in photothermal therapy, gold nanoparticles absorb near-infrared light and convert it into localized heat, causing thermal ablation of cancer cells [34]. This effect synergizes with the apoptosis induced by betulinic acid, leading to more effective tumor eradication. In photodynamic therapy, gold nanoparticles can deliver photosensitizers that produce ROS upon light activation, further amplifying cancer cell death [49]. Anti-Angiogenic Activity of Betulinic acid inhibits angiogenesis, preventing the formation of new blood vessels that supply nutrients to tumors. This effect is enhanced by gold nanoparticles, which improve the delivery of betulinic acid to angiogenic sites and may disrupt endothelial cell function [50]. Overcoming Drug Resistance through combination therapy addresses drug resistance by employing multiple mechanisms that target cancer cells at different levels. The ability of gold nanoparticles to deliver drugs directly to resistant tumor cells and enhance the cytotoxic effects of betulinic acid makes this system highly effective against multidrug-resistant cancers [41].  

MECHANISTIC INSIGHTS
Cellular Uptake and Distribution
The cellular uptake and intracellular distribution of the betulinic acid–gold nanoparticle system play a critical role in its therapeutic efficacy. Gold nanoparticles are small enough to be internalized by cells via endocytosis, where they exploit active transport mechanisms, such as clathrin-mediated or caveolae-mediated pathways [51]. Functionalization of the nanoparticles with targeting ligands or biomolecules, such as antibodies or peptides, enhances their specificity toward cancer cells, enabling selective accumulation within tumors while sparing healthy tissues [52].  
Upon cellular uptake, the nanoparticles are transported to endosomes and lysosomes, where the acidic environment can trigger the release of betulinic acid. This release is facilitated by the surface chemistry of gold nanoparticles, which allows for controlled drug release in response to the tumor’s microenvironmental conditions, such as low pH or high levels of enzymes [41]. Once released, betulinic acid diffuses through the cytoplasm, ensuring its availability for mitochondrial targeting. The efficient internalization and intracellular distribution of the system ensure a high local concentration of betulinic acid within cancer cells, enhancing its therapeutic effects [53].  

Mitochondrial Targeting
Mitochondria serve as critical regulators of energy production and apoptosis, making them a prime target for anticancer therapies. Betulinic acid exhibits a strong affinity for mitochondria, where it disrupts the mitochondrial membrane potential. This disruption compromises mitochondrial integrity, leading to the release of pro-apoptotic factors such as cytochrome c into the cytosol [54].  
Gold nanoparticles enhance mitochondrial targeting by facilitating the delivery of betulinic acid directly to the mitochondria. Functionalization with mitochondrial-targeting moieties, such as triphenylphosphonium (TPP), can further increase the system’s specificity for these organelles [55]. The combination of betulinic acid and gold nanoparticles amplifies mitochondrial damage by generating reactive oxygen species (ROS). ROS disrupt mitochondrial DNA, proteins, and lipids, exacerbating mitochondrial dysfunction and initiating the intrinsic apoptotic pathway [56, 57].  
The precise targeting of mitochondria not only enhances the cytotoxicity of the system but also reduces the risk of off-target effects, ensuring that healthy cells remain largely unaffected [58].   

Apoptosis Induction
The induction of apoptosis is the primary mechanism by which the betulinic acid–gold nanoparticle system exerts its anticancer effects. Betulinic acid primarily activates the intrinsic apoptotic pathway by targeting mitochondria [40]. The loss of mitochondrial membrane potential leads to the release of apoptogenic factors, including cytochrome c, which activates caspase-9. This, in turn, triggers the caspase cascade, culminating in the activation of caspase-3, the executioner caspase responsible for DNA fragmentation, cell shrinkage, and other hallmarks of apoptosis [59].  Gold nanoparticles amplify apoptosis through their ability to generate oxidative stress. The ROS produced by gold nanoparticles further damage cellular components, increasing the susceptibility of cancer cells to apoptosis [60]. Additionally, gold nanoparticles may interfere with cellular signaling pathways involved in survival and proliferation, such as the PI3K/Akt pathway, further sensitizing cancer cells to apoptosis [61]. The dual mechanism of action—mitochondrial disruption by betulinic acid and ROS generation by gold nanoparticles—creates a robust apoptotic response that is highly effective against cancer cells. This combination minimizes the likelihood of resistance, as it simultaneously targets multiple cellular pathways critical for cancer cell survival [52]. The mechanistic synergy of cellular uptake, mitochondrial targeting, and apoptosis induction in the betulinic acid–gold nanoparticle system ensures precise, efficient, and selective cancer cell elimination [40, 62].

CLINICAL TRIALS
Olakunle Oladimeji et al [41], investigated the mitochondrial-targeted delivery of betulinic acid (BA) using TPP+-functionalized, epigallocatechin gallate (EGCG)-capped gold nanoparticles (AuNPs) and compared the efficacy of polyethylene glycol (PEG) and poly-L-lysine-graft-polyethylene glycol (PLL-g-PEG) copolymers in enhancing delivery. The system was tested on Caco-2, HeLa, and MCF-7 cancer cell lines, showing significantly improved cancer cell growth inhibition compared to free BA. The targeted nanocomplexes achieved lower IC50 values (3.12–13.2 µM) versus free BA (9.74–36.31 µM), indicating enhanced therapeutic efficacy. The mechanisms of action included mitochondrial depolarization, activation of caspases 3/7, and cell cycle arrest at the G0/G1 phase. This system demonstrates the potential of mitochondrial targeting as a strategy for delivering BA in cancer therapy, offering improved efficacy and precision.
Marius Mioc et al [63], studied Betulin-conjugated gold nanoparticles have been proposed as a potential therapeutic strategy for melanoma treatment. These nanoparticles were synthesized using a citrate reduction method with chloroauric acid as the gold precursor, resulting in stable metallic cores with sizes of 14–15 nm. Dynamic light scattering (DLS) measurements showed larger diameters (50–70 nm) due to factors such as surface modifications (e.g., PEG-SH coating) and water dispersion effects. In vitro studies demonstrated that the betulin-conjugated gold nanoparticles exhibited dose- and time-dependent cytotoxic and apoptotic effects on all tested cell lines. Conversely, unmodified (naked) gold nanoparticles or PEG-coated nanoparticles had minimal impact on cell viability. This highlights the therapeutic potential of betulin-conjugated gold nanoparticles in melanoma therapy.
Roxana Ghiulai et al [40], conducted a biological in vitro evaluation of betulinic acid (BA)-functionalized gold nanoparticles (GNP) was conducted. BA-GNP were synthesized by grafting BA onto pre-synthesized citrate-capped GNP using cysteamine as a linker. The nanoformulation was tested on HaCaT human keratinocytes and RPMI-7951 human melanoma cells, demonstrating selective cytotoxicity and stronger antiproliferative effects compared to free BA. Further analysis showed a pro-apoptotic effect, evident from morphological changes in melanoma cells, and supported by Western blot data indicating a decrease in anti-apoptotic Bcl-2 expression and an increase in pro-apoptotic Bax levels. Additionally, GNPs significantly inhibited mitochondrial respiration, confirming their targeted activity toward mitochondria.
Yanping Liu et al [64], studied, Gold nanoshell-coated betulinic acid liposomes (AuNS-BA-Lips), mediated by glutathione, were developed and characterized. The AuNS-BA-Lips exhibited a favorable size distribution (149.4 ± 2.4 nm) and demonstrated excellent photothermal conversion properties, enabling synergistic chemo-photothermal therapy. The absorption wavelength of the AuNS-BA-Lips was significantly red-shifted into the near-infrared (NIR) region, allowing strong absorption of NIR laser and efficient conversion into localized heat. This facilitated controlled drug release and antitumor thermotherapy. Additionally, NIR irradiation significantly enhanced cellular uptake of the nanocarriers, leading to increased intracellular drug accumulation. In tumor-bearing mice, NIR-irradiated AuNS-BA-Lips exhibited highly effective antitumor effects, achieving an inhibition rate of 83.02%. This study highlights the potential of AuNS-BA-Lips for synergistic chemotherapy and thermotherapy, providing a promising approach for multifunctional antitumor drug development.

CONCLUSION AND FUTURE PERSPECTIVES 
This review underscores the potential of combining betulinic acid (BA) and gold nanoparticles (AuNPs) in cancer therapy. The conjugation of these agents enhances the efficacy of treatment by leveraging the distinct properties of each component. Betulinic acid’s ability to induce apoptosis and target mitochondria, combined with the exceptional drug delivery capabilities of gold nanoparticles, results in a synergistic therapeutic effect. The preliminary in vitro and in vivo studies reveal promising outcomes, demonstrating the potential for this combination therapy to overcome limitations of current cancer treatments. This novel approach offers a promising strategy for improving cancer treatment outcomes and warrants further investigation.
The integration of nanotechnology and natural compounds in cancer therapy represents a significant advancement in the field. Future research should focus on optimizing the synthesis and functionalization of BA-AuNP complexes to maximize their therapeutic potential. Clinical trials are needed to validate the efficacy and safety of this combination in human subjects. Additionally, exploring the use of BA-AuNPs in conjunction with other therapeutic agents could further enhance treatment outcomes. Advancements in nanotechnology and a deeper understanding of cancer biology will likely lead to the development of more effective and targeted cancer therapies. This study highlights the importance of continued research and innovation in the quest to improve cancer treatment and patient outcomes.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.