Propolis-Loaded Liposomes (ProLip): A Nanoformulated Immunomodulator Targeting Breast Cancer via Macrophage Activation

Propolis-Loaded Liposomes (ProLip): A Nanoformulated Immunomodulator Targeting Breast Cancer via Macrophage Activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Propolis-Loaded Liposomes (ProLip): A Nanoformulated Immunomodulator Targeting Breast Cancer via Macrophage Activation Hamidah Mohd Zain, Musthahimah Muhamad, Nik Nur Syazni Nik Mohamed Kamal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6817917/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Propolis, a natural remedy derived from bee by-products, is known for its immunomodulatory and anticancer properties. However, its clinical application is hindered by poor solubility and bioavailability. This study formulated a propolis-loaded liposome (ProLip) using the thin-film hydration technique (soy phospholipid-to-cholesterol ratio 6:1) to enhance its therapeutic effect. Encapsulation reduced the particle size of propolis from 402.77 ± 7.53 nm to 249.67 ± 5.79 nm and enhanced physicochemical properties, including a low polydispersity index (0.098 ± 0.02), highly negative zeta potential (-50.80 ± 0.10 mV), and improved solubility (water contact angle of 50.247°). FTIR analysis confirmed intermolecular interactions between phenolic groups in propolis and phospholipid carbonyl groups, while electron microscopy and surface morphology analysis revealed uniform structure and phagosomal localization in macrophages. Functionally, ProLip enhanced the secretion of anti-inflammatory cytokines IL-10 (49.429 ± 0.38 pg/mL) and IL-6 (40.488 ± 0.10 pg/mL), while suppressing pro-inflammatory mediators TNF-α and IL-1β by more than 80% compared to the LPS-treated group, highlighting ProLip as a potential immunoregulatory agent. Electron microscopy confirmed phagosomal localization of ProLip and reduced macrophage morphological damage compared to unencapsulated propolis, validating targeted delivery and protection capacity. Additionally, conditioned media from ProLip-treated macrophages significantly induced apoptosis (>50%) and inhibited migration and invasion in MCF-7 breast cancer cells, supporting immune-mediated anticancer effects. These findings highlight ProLip’s potential as a nanocarrier to enhance the bioavailability, cellular targeting, and therapeutic efficacy of stingless bee propolis in cancer immunotherapy. Biological sciences/Biotechnology Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Drug discovery propolis liposome targeted delivery immunomodulation anti-cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Immunotherapy has revolutionized cancer treatment by offering innovative solutions to the challenges posed by conventional regimens such as systemic toxicity, limited efficacy, and resistance development 1 , 2 . Among these strategies, immunomodulation-based therapies harness the host immune system to selectively target and eliminate cancer cells 3 . Macrophages, as central regulators of immune responses and the tumor microenvironment, are particularly promising targets for these therapies 4 , 5 . However, the limitations of existing approaches underscore the need for novel therapeutic agents that are both effective and specific. Propolis, a natural resinous product produced by bees, has garnered attention for its potent anti-inflammatory, antioxidant, and anticancer properties 6 – 8 . Propolis derived from Heterotrigona itama (H. itama) stingless bees is particularly rich in phenolic and flavonoid compounds, which confer its pharmacological activity 9 , 10 . Despite its promise, the clinical application of propolis remains limited due to poor water solubility, low bioavailability, and variability in chemical composition 11 , 12 . These challenges have hindered its consistent therapeutic outcomes and broader adoption in oncology and immunotherapy. Moreover, mechanistic insights into its interaction with immune cells, particularly macrophages, remain underexplored, creating a critical gap in its translation to clinical applications. Nanotechnology offers a transformative approach to address these limitations. Liposomal encapsulation, leveraging biocompatible phospholipid bilayers, can enhance the solubility, stability, and bioavailability of hydrophobic compounds such as propolis 13 , 14 . This study introduces ProLip, a novel liposomal formulation of H. itama propolis, engineered to overcome the solubility barrier and improve its interaction with immune cells. ProLip not only protects the bioactive components of propolis but also facilitates efficient macrophage uptake, enabling targeted delivery and controlled release. This dual-targeted system amplifies propolis's immunomodulatory and anticancer effects, addressing the limitations of conventional therapies and current propolis-based studies. The novelty of this work lies in its exploration of ProLip’s dual therapeutic potential: modulating immune responses and inhibiting cancer progression. Unlike previous studies that focus exclusively on either immunomodulation or anticancer activity, this research investigates the synergistic interplay between these mechanisms. By combining natural bioactive with advanced nanotechnology, this study addresses critical challenges in drug delivery, immune modulation, and cancer therapy. ProLip exemplifies a novel therapeutic strategy capable of overcoming the limitations of traditional propolis formulations, offering enhanced solubility, stability, and targeted delivery. The findings not only highlight the clinical relevance of liposomal propolis but also establish its potential as a multifunctional nanotherapeutic for inflammation-driven diseases and cancer. This research provides a foundation for future studies to translate natural product-based therapies into clinical applications, bridging the gap between laboratory research and real-world healthcare needs. Results Physicochemical analysis and performance assessment of ProLip The encapsulation of Pro into Lip demonstrated significant changes in the physicochemical characteristics of the ProLip formulation. Based on Fig. 1 (a), a significant reduction in the average particle size of Pro was observed after encapsulation into the liposomal carrier, which was from 402.77 ± 7.53 nm to 249.67 ± 5.79 nm. Besides, all samples exhibited monodisperse distribution of particles with PDI values < 0.12 (Fig. 1 (b)). Surface charge determination, through zeta potential measurement showed an overall negative value with Lip and ProLip possessed more negative zeta potential at -51.37 ± 1.11 mV and − 50.80 ± 0.10 mV respectively compared to Pro alone (-1.49 ± 0.11 mV) as observed in Fig. 1 (c). The degree of wettability of ProLip revealed a lower contact angle (50.247° ± 2.76) after encapsulation compared to before encapsulation (Pro: 81.629° ± 3.16), indicating improved solubility. The loading capacity (LC) and encapsulation efficiency (EE) of ProLip demonstrated high loading (72.98 ± 0.72%) and encapsulation (83.52 ± 0.66%) of Pro into Lip. Surface morphology of ProLip via SEM The surface morphological evaluation of Pro, Lip and ProLip was visualized by SEM analysis (Fig. 2 ). Based on the results, Pro exhibited an irregular circular morphology with a rough surface and heterogeneous particle sizes (Fig. 2 (a)). In contrast, the propolis-free liposome (Lip) displayed a nearly circular smooth surface with well-defined, uniform particles (Fig. 2 (b)). The ProLip formulation maintained a comparable uniformity in particle size but exhibited a slightly textured surface, differentiating it from the smoother morphology of Lip (Fig. 2 (c)). Chemical interaction of ProLip via FTIR spectra The interaction between Pro, Lip and ProLip were observed through the distinct changes in the peaks and intensities, as depicted in Fig. 3 . According to the results, identical bands were identified in both Pro and ProLip at 3308 cm − 1 , corresponding to O-H stretching. However, there was a reduction in the intensity of ProLip, characterized by a broader absorption band. Additionally, C-H stretching vibrations at 2924 cm − 1 and 2848 cm − 1 were present in all samples, with a more pronounced peak similarity between Pro and ProLip. Further spectral shifts were observed in Lip and ProLip, particularly with the presence of carbonyl (C = O) stretching at 1737 cm − 1 , carboxyl (C = C) stretching at 1638 cm − 1 , and aromatic C-H bending at 1446 cm − 1 . In addition, a distinct O-H bending was observed at 1400 cm − 1 in Lip and 1365 cm − 1 in ProLip. The characteristic peaks of Pro at 1065 cm − 1 (C-O), 981 cm − 1 (C = C), and 795 cm − 1 (C = C) were also detected in ProLip. Immunomodulatory activities in THP-1 macrophages The effects of ProLip and associated controls including Pro, and Doxorubicin were evaluated on selected biomarkers involved in immune response by ELISA assay (Fig. 4 ). LPS was added in the subsequent assays as it acts as a potent inflammatory stimulus 15 . In this assay, LPS served as an inflammatory stimulus to assess the response of PMA-differentiated THP-1 macrophages to ProLip and the associated controls. When exposed to LPS, PMA-differentiated THP-1 macrophages, similar to primary macrophages, activated signalling pathways that led to the production of pro-inflammatory cytokines, such as TNF-α and IL-1β 16 , 17 . Supporting this, the study herewith demonstrated that LPS enhanced the expression of TNF-α (154.54 pg/mL, ***p < 0.001) and IL-1β (231.37 pg/mL, ***p < 0.001) in PMA-differentiated THP-1 macrophages, as illustrated in Fig. 4 (a) and 4(b) respectively. Figure 4 (a) illustrated the expression of TNF-α cytokine in PMA-differentiated THP-1 macrophages following treatment with ProLip and the respective controls. Following 24 hr incubation with ProLip + LPS, TNF-α levels were reduced by 20.48 pg/mL relative to the LPS-treated macrophages (154.54 pg/mL, ***p < 0.001). Similar findings were observed in THP-1 macrophages treated with ProLip without LPS stimulation, where TNF-α cytokine expression was measured at 30.26 pg/mL. This suggests that ProLip may exert an anti-inflammatory effect by suppressing the production of this key pro-inflammatory cytokine. Meanwhile, Pro and Doxorubicin treatments resulted in TNF-α cytokine expression levels 46.13 pg/mL and 75.12 pg/mL respectively. Figure 4 (b) illustrated the expression of IL-1β cytokine in THP-1 macrophages following treatment with ProLip treatment and the respective controls. IL-1β is a pro-inflammatory cytokine, and its elevated levels have been associated with various inflammatory disease, including rheumatoid arthritis and development of cancer 18 . Treatment with ProLip + LPS led to a significant reduction in IL-1β production to 14.94 pg/mL in THP-1 macrophages compared to the LPS-treated macrophages (231.37 pg/mL, ***p < 0.001). Similar findings were observed in THP-1 macrophages treated with ProLip without LPS stimulation where IL-1β cytokine expression was measured at 30.46 pg/mL. This suggests that ProLip may exert an anti-inflammatory effect by suppressing the production of this key pro-inflammatory cytokine. Meanwhile, Pro and Doxorubicin treatments resulted in IL-1β cytokine expression levels of 61.89 pg/mL and 98.03 pg/mL respectively. Figure 4 (c) illustrated the expression of IL-10 cytokine in THP-1 macrophages following treatment with ProLip treatment and the respective controls. IL-10 is an anti-inflammatory cytokine produced by a variety of immune cells, including macrophages, T cells, B cells, and dendritic cells 19 . Treatment with ProLip resulted in an increase in IL-10 production (13.46 pg/mL, **p < 0.01) in THP-1 macrophages compared to LPS-treated macrophages (8.67 pg/mL). Comparable results were obtained in THP-1 macrophages treated with ProLip under LPS stimulation, where IL-10 levels were measured at 49.43 pg/mL (***p < 0.001). Based on these findings, it is hypothesized that ProLip may promote the anti-inflammatory function of IL-10 of THP-1 macrophages, by reducing excessive inflammation in an already activated pro-inflammatory environment, as evidenced by the LPS-induced response. Meanwhile, Pro and Doxorubicin treatment resulted in IL-10 expression levels of 9.98 pg/mL and 4.16 pg/mL, respectively. Figure 4 (d) illustrated the expression of IL-6 cytokine in THP-1 macrophages following treatment with ProLip treatment and the respective controls. IL-6 is a pleiotropic cytokine, exhibiting both pro-inflammatory and anti-inflammatory activities depending on the specific cellular context, including the microenvironment, the presence of other cytokines, and the target cell type 20 , 21 . Treatment with ProLip resulted in an increase in IL-6 production (24.17 pg/mL, ***p < 0.001) in THP-1 macrophages compared to LPS-treated macrophages (10.80 pg/mL). Comparable results were obtained in THP-1 macrophages treated with ProLip under LPS stimulation, where IL-6 levels were measured at 40.49 pg/mL (***p < 0.001). Based on these findings, it is hypothesized that ProLip may promote the anti-inflammatory function of IL-6 of THP-1 macrophages, by reducing excessive inflammation in an already activated pro-inflammatory environment, as evidenced by the LPS-induced response. Meanwhile, Pro and Doxorubicin treatment resulted in IL-6 expression levels of 12.58 pg/mL and 7.13 pg/mL, respectively. Morphological Analysis of ProLip Uptake Figure 5 TEM of phagocytic uptake and intracellular trafficking of treatments in THP-1 macrophages following 24 hr exposure. (A) Schematic illustration of the key stages of phagocytosis. Macrophage formed an actin-mediated membrane ruffling to sense particles and initiate engulfment. Progressive pseudopod extension led to the engulfment of particles forming a phagosome. Figure 5 (A) is adapted from Moreno-Mendieta et al., (2022) 22 . (B-H) Each treatment condition is represented by two images: a low-magnification overview (left) and a high-magnification image (right) showing subcellular details. Untreated control cells showed no morphological abnormalities (B(i-ii)). Pro and ProLip were deposited within the membrane-bound structures and identified as electron dense particles. Magnified electron micrograph showed phagolysosome containing Pro (C(ii)), ProLip (D(ii)), Pro + LPS (E(ii)), ProLip + LPS (F(ii)), and Doxorubicin (H(ii)). Activated macrophages showed extensive pseudopods and phagocytic activity (F(i-ii) and G(i-ii)). Magnification: B(i): x2000, B(ii): x4000, C(i): x2000, C(ii): x6300, D(i): x1600, D(ii): x6300, E(i): x2500, E(ii): x6300, F(i): x1600, F(ii): x8000, G(i): x2500, G(ii): x6300, H(i): x2500, H(ii): x6300. The intracellular localization and morphological changes induced by Pro and ProLip in THP-1 macrophages, both in the presence and absence of LPS was conducted by TEM analysis. As shown in Fig. 5 , untreated (UT) THP-1 macrophages exhibited a healthy and viable cellular structure, characterized by intact organelles, a large nucleus with well-defined chromatin, abundant mitochondria, and an intact plasma membrane (Fig. 4.21 B(i-ii)) 23 , 24 . Macrophages, being phagocytic cells, engulf foreign materials such as Pro, encapsulating them within a membrane-bound vesicle known as a phagosome. As depicted in Fig. 5 C(i-ii), Pro was found inside phagosomes. The phagosome usually fuses with lysosomes to form phagolysosome, where the engulfed material undergoes enzymatic degradation and digestion 25 . This process may explain why some Pro was also observed in the macrophage cytoplasm. In addition to this process, it may also indicate phagolysosomal membrane disruption 26 , leading to the release of Pro into the cytoplasm. The presence of larger vesicles, likely phagosomes, indicated that phagocytosis was the primary uptake mechanism of ProLip by THP-1 macrophages (Fig. 5 D(i-ii)). This observation was further supported by pseudopod-like structures surrounding the large vesicles, suggesting active phagocytosis. These structures likely represented the macrophage membrane extending and enclosing the ProLip during the engulfment process. No free ProLip was detected in the cytoplasm; it was only found within phagocytic vacuoles. LPS is a potent immunostimulant that activates THP-1 macrophages, resulting in enhanced phagocytic activity and increased lysosomal enzyme production 27 . Lysosomes have the ability to fuse with the plasma membrane, resulting in the extracellular release of their contents 28 . This can occur as part of a cellular response to inflammation, potentially exacerbated by LPS stimulation. The combined challenge of Pro and LPS may exert an overwhelming impact, potentially leading to macrophage cell death (cytotoxicity) and the subsequent release of lysosomal and cytoplasmic contents, as depicted in Fig. 5 E(i-ii). Interestingly, ProLip was exclusively located within membrane-bound vesicles, phagosomes, even in the presence of LPS stimulation (Fig. 5 F(i-ii)). Similar to ProLip alone, no free ProLip + LPS was detected in the macrophage cytoplasm. These findings suggest efficient containment and processing. Liposomes are commonly utilized as drug and nanoparticle carrier due to their biocompatibility and efficient uptake by macrophages through phagocytosis 29 . Liposomal encapsulation likely enhanced ProLip uptake compared to free Pro. As reported by Marrocco & Ortiz, (2022), LPS stimulation can enhance macrophage activity, which may contribute to the observed synergistic effect of liposomal delivery under LPS exposure in facilitating the efficient engulfment of ProLip within phagosomes 30 . Figure 5 G(i-ii) depicts THP-1 macrophages under LPS exposure. It was observed that activated macrophages underwent morphological changes, becoming larger and flatter. The cell membrane formed ruffles and extensions, known as filopodia 31 , 32 . These dynamic membrane protrusions play a crucial role in capturing and engulfing pathogens and other targets 33 . Figure 4.21 H(i-ii) presents a TEM micrograph of a THP-1 macrophage after exposure to Doxorubicin. As observed, different stages of phagosomes and lysosomes were present, and some Doxorubicin appeared to have escaped the phagocytic pathway, dispersing throughout the macrophage cytoplasm and accumulating on the outer layer of the phagosome/lysosome membrane. Adjuvant activity and effect on Breast Cancer Cells Building on the observed effects of Pro and ProLip, conditioned media from Pro, ProLip, and other treated macrophages (including Pro + LPS, ProLip + LPS, LPS, and Doxorubicin) were investigated on MCF-7 breast cancer cells (Fig. 6 ). It was hypothesized that ProLip modulated macrophage activity, which in turn impacted the behavior of neighbouring breast cancer cells. This co-culture assay aimed to characterize the interactions between ProLip-treated macrophages and breast cancer cells, focusing on migration and invasion activities, as well as mechanisms of cell death. Based on the results, treatment with Pro, ProLip, Pro + LPS, and ProLip + LPS macrophage conditioned media, exhibited a pronounced suppression of MCF-7 cells migration after a 24-hr incubation, with minimal wound closure observed as compared to the baseline scratch area at 0 hr (Fig. 6 (c,d)). In contrast, the untreated control group (UT) and Doxorubicin showed nearly complete wound closure by 24 hr. LPS -conditioned media group, in particular, showed a gradual migration after 24 hr, in comparison to the control. Meanwhile, the Transwell invasion assay demonstrated that, treatment with Pro and ProLip significantly inhibited MCF-7 invasion compared to the untreated control, 16.77% (***p < 0.001) and 11.38% (***p < 0.001), respectively (Fig. 6 (e,f)). The inhibitory effect was further enhanced in the presence of LPS. Specifically, Pro + LPS reduced invasion by 10.21% compared to the UT control, while ProLip + LPS exhibited the most pronounced reduction, decreasing invasion by 7.98%. While the effects of LPS alone and Doxorubicin were less pronounced compared to ProLip and the combination treatments, the results demonstrated a 63.31% (***p < 0.001) and 73.58% (**p < 0.01) reduction in invasion, respectively, compared to the untreated cells. As for cell death mechanism, MCF-7 cells treated with Pro-macrophage conditioned media showed a significant increase in which 38.56% ± 2.58 (***p < 0.001) of cells undergoing apoptotic cell death compared to the untreated control cells observed (Fig. 6 (a,b)). Interestingly, apoptosis was significantly higher in MCF-7 cells following incubation with ProLip-macrophage conditioned media, reaching 55.83% (***p < 0.001). Under LPS stimulation, treatment with Pro- and ProLip macrophage conditioned media resulted in a significant increase in apoptotic cell death in MCF-7 cells, reaching 47.41% (***p < 0.001) and 69.36% (***p < 0.001), respectively. In contrast, conditioned media from LPS alone or the chemotherapeutic agent doxorubicin induced lower levels of apoptosis in MCF-7 cells, with 13.07% ± 4.02 and 28.56% ± 2.21% of apoptotic cells, respectively. Discussion This study presents a comprehensive integration of nanotechnology with natural immunotherapeutic by employing liposomal encapsulation of propolis (ProLip), aiming to overcome the physicochemical limitations of crude propolis (Pro) and enhance its biological efficacy in modulating immune cell behavior. Propolis, a resinous natural product from bees, is widely recognized for its potent anti-inflammatory and anticancer properties; however, its clinical translation has been limited by poor aqueous solubility, instability, and reduced bioavailability 34 , 35 . These challenges significantly affect its cellular uptake and therapeutic consistency. Our findings demonstrate that the physicochemical modifications achieved through liposomal encapsulation significantly improved the bioavailability, stability, and cellular interaction of propolis. Crude propolis, when directly exposed to cells, exhibited uncontrolled cytotoxicity and induced cellular stress, likely due to its non-specific interaction with cellular membranes and tendency to form intracellular aggregates, as previously reported 36 – 38 . By encapsulating propolis in liposomes, these challenges were effectively mitigated. The ProLip formulation displayed enhanced hydrophilicity and uniform dispersion of propolis within the liposomal bilayer, transforming the aggregated resinous particles of crude Pro into a stable nanosystem. This reconfiguration significantly improved delivery characteristics, as evidenced by the mean particle size of 249.67 ± 5.79 nm, an optimal size range for cellular uptake via endocytosis and enhanced accumulation at sites of inflammation or tumorigenesis 39 , 40 . Nanosized particles within this range are known to exhibit prolonged systemic circulation and enhanced permeability and retention (EPR) effects, facilitating passive targeting of inflammatory tissues and tumors 41 , 42 . Furthermore, macrophages are highly phagocytic and naturally internalize nanoparticles within this size spectrum, enabling ProLip to be preferentially taken up through actin-mediated phagocytosis and macropinocytosis 43 . The ProLip formulation also exhibited a highly negative zeta potential (− 50.80 ± 0.10 mV), attributed to the presence of deprotonated phenolic groups and negatively charged phospholipids. This surface charge enhanced colloidal stability through electrostatic repulsion and prevented nanoparticle aggregation, contributing to a low polydispersity index (PDI) and uniform particle distribution 44 , 45 . In addition to its role in stability, negative zeta potential has been shown to improve macrophage uptake efficiency by 5.3-fold compared to neutral liposomes 46 . The hydrophilicity of ProLip, validated by contact angle measurement, further supported its suitability for biological applications. Increased wettability allowed for better interaction with aqueous biological environments, improving cell surface adherence and internalization efficiency 47 , 48 . Enhanced aqueous compatibility is a critical feature for any nanoformulation targeting immune cells, particularly macrophages, which thrive in dynamic fluidic environments and regulate immune responses through both direct contact and soluble mediators 49 . FTIR and SEM analyses confirmed successful molecular integration and morphological reconfiguration of propolis within the liposomal matrix. FTIR spectra revealed spectral shifts and band broadening, particularly in the carbonyl and hydroxyl regions, suggesting the formation of hydrogen bonding and van der Waals interactions between propolis phenolics and phospholipid components 50 . These molecular interactions indicate strong compatibility between the payload (Pro) and carrier (Lip), crucial for maintaining structural integrity during circulation and ensuring controlled drug release. Meanwhile, SEM analysis revealed distinct morphological differences between ProLip and the unloaded liposome (Lip). The surface of ProLip exhibited increased roughness and slight irregularities, likely attributable to the deposition of complex organic constituents present in propolis onto the liposomal surface 50 . These morphological alterations suggest effective integration of Pro within the hydrophobic domains of the lipid bilayer. Biologically, ProLip outperformed crude Pro in terms of immunomodulatory potency and cellular compatibility. Propolis is known for its immunomodulatory properties, with its polyphenolic constituents exerting dual effects by suppressing excessive inflammation while promoting immune homeostasis 51 , 52 . In this study, ELISA results revealed significant upregulation of anti-inflammatory cytokines (IL-10 and IL-6) in ProLip-treated macrophages compared to the crude Pro. This suggests that encapsulation facilitated a macrophage phenotype that promoted immune resolution while preventing prolonged inflammation 53 . Notably, the co-treatment of macrophages with ProLip and LPS elicited a synergistic increase in IL-10 and IL-6 expression, indicating the formulation's capacity to modulate macrophage activation even under pro-inflammatory stimulation 54 , 55 . ProLip also demonstrated an enhanced suppression on pro-inflammatory cytokines (IL-1β and TNF-α) compared to Pro, implying that the encapsulated formulation enhanced the bioavailability of active polyphenols and improved their regulatory impact on inflammatory signalling cascades 56 . From a therapeutic standpoint, the ability of ProLip to suppress inflammatory cytokines without completely abolishing immune responsiveness represented a significant advantage. This is due to, excessive pro-inflammatory cytokine expression contributes to chronic inflammation and immune exhaustion, while insufficient inflammation can impair pathogen clearance and tumor control 57 , 58 . The ability of ProLip to strike a functional balance between immunostimulation and immune suppression highlights its potential as a versatile immunotherapeutic agent 59 . TEM imaging provided further insight into the intracellular behavior of Pro and ProLip. While Pro formed irregular and undefined aggregates in the cytoplasm, causing structural disruption to cellular organelles, ProLip was internalized more uniformly and trafficked toward lysosomal and phagosomal compartments. ProLip preserved membrane integrity and mitochondrial structure, suggesting that liposomal encapsulation shielded macrophages from potential damage while retaining propolis's biological activity 36 . This behavior indicates that, the encapsulation of Pro within liposomes likely facilitated its uptake through endocytic pathways, particularly clathrin- and caveolin-mediated endocytosis, mechanisms commonly observed in liposomal drug delivery systems 60 , 61 . By preventing premature degradation and enabling sustained intracellular release, ProLip’s encapsulation enhances bioavailability and mitigates cytotoxic stress 29 . The preferential localization within lysosomal compartments suggested that macrophages actively processed and degraded ProLip, allowing for sustained intracellular release of bioactive compounds. In comparison, doxorubicin, a commonly used chemotherapeutic, demonstrated widespread intracellular dispersion and nuclear condensation, confirming its passive diffusion and DNA intercalation-based mechanism of cytotoxicity 62 , 63 . These findings emphasize the difference in cellular handling between synthetic drugs and biologically encapsulated therapeutics like ProLip. While doxorubicin causes significant genotoxic stress, ProLip achieved effective intracellular delivery without inducing similar levels of cellular damage. To further assess the therapeutic potential of ProLip, this study utilized the human monocytic cell line THP-1 as a platform to investigate the interactions between MCF-7 breast cancer cells and THP-1-derived macrophages treated with ProLip. By recreating the inflammatory condition established by stimulation with LPS, the present study aimed to determine whether ProLip enhances or mitigates the anti-tumorigenic activities of M1-like macrophages within a model of the tumor microenvironment. The present study found that the conditioned media significantly inhibited MCF-7 cell migration. Consistently, results from the transwell invasion assay demonstrated that THP-1 macrophages treated with ProLip more effectively suppressed the invasive behavior of MCF-7 cells compared to unencapsulated propolis. Interestingly, this growth inhibitory effect correlated with an increased apoptotic population in MCF-7 cells following exposure to conditioned media derived from ProLip-treated THP-1 macrophages. Macrophages, particularly the classically activated M1 subtype, have been documented to play a key role in inducing apoptosis in cancer cells through the release of pro-inflammatory cytokines such as TNF-α and IFN-γ. These cytokines activate intracellular signalling pathways that ultimately trigger programmed cell death in cancer cells 64 . Moreover, M1 macrophages are also known to possess both phagocytic and antigen-presenting capabilities, allowing them to produce pro-inflammatory cytokines and exert cytotoxic effects. Through these functions, they can directly eliminate target cells via the generation of ROS, reactive nitrogen species (RNS), IL-1β and TNF-α. Alternatively, M1 macrophages can also promote indirect cytotoxicity by activating other immune cells, such as NK cells and T cells 65 . In the present study, exposure to ProLip significantly increased the levels of TNF-α and IL-1β in THP-1 macrophages compared to untreated cells. This elevated production of pro-inflammatory cytokines strongly indicates that ProLip promotes a pro-inflammatory, M1-like macrophage phenotype. These findings also suggest that ProLip may enhance the cytotoxic capacity of M1 macrophages against MCF-7 breast cancer cells, either directly or indirectly, through the activation of other immune cells. Such multifaceted signalling pathways underscore TNF-α’s role as a potent anticancer cytokine 66 . The dual capability of ProLip to reduce inflammation in resting macrophages while enhancing pro-inflammatory, tumor-suppressive responses in inflamed or LPS-stimulated environments underscores its adaptive immunomodulatory profile. Conclusion This study established liposomal encapsulation as a viable strategy to enhance the therapeutic potential of Heterotrigona itama propolis by addressing its physicochemical limitations and improving its biological performance. The development of Propolis Liposome (ProLip) significantly improved the aqueous solubility, stability, and cellular bioavailability of crude propolis, enabling efficient macrophage-targeted delivery while minimizing nonspecific cytotoxicity. Physicochemical characterization confirmed successful integration of propolis into the liposomal bilayer, resulting in a nanosized, stable formulation with enhanced hydrophilicity and favourable surface charge for immune cell interaction. This ProLip also exhibited a dual modulatory effect on cytokine secretion in THP-1 macrophages, promoting both anti-inflammatory and pro-inflammatory responses. While the anti-inflammatory cytokines contribute to immune regulation, the elevated pro-inflammatory mediators may enhance the immune system’s cytotoxic activity, thereby facilitating breast cancer cell death – as evidenced by increased apoptosis and reduced migration and invasion in functional assays. Collectively, these findings demonstrates that liposomal encapsulation not only optimizes the pharmacological profile of propolis but also enhances its functional impact on immune modulation and cancer cell suppression. This work provides a promising foundation for the development of propolis-based nanotherapeutics in immunomodulatory and cancer-targeted clinical applications. Nevertheless, further in vivo validation and pharmacological evaluations are warranted to confirm ProLip’s therapeutic efficacy, safety and ADME (absorption, distribution, metabolism and excretion) profiles. Material and Methods Materials Propolis from Heterotrigona itama (H. itama) stingless bee species were purchased from a local beekeeping company, Bayu Gagah Sdn Bhd (Kulim Hightech, Kedah, Malaysia). Soy phospholipids (≥ 99%), phosphate-buffered saline (PBS) tablets, lipopolysaccharides (LPS), and phorbol 12-myristate 13-acetate (PMA) (≥ 99%) were acquired from Sigma-Aldrich (St. Louis, USA). Cholesterol (≥ 99%) was sourced from Nacalai Tesque (Kyoto, Japan). Methanol (≥ 99.9%) and chloroform (≥ 99.9%) were purchased from Merck Millipore (Germany). THP-1 and MCF-7 cells were purchased from American Type Culture Collection (ATCC®) (Porton Down, Salisbury, UK). Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle Medium (DMEM), Penicillin-Streptomycin, and Fetal bovine serum (FBS), and trypsin were procured from Capricorn Scientific (Ebsdorfergrund, Germany). Enzyme-linked Immunosorbent assay (ELISA) and apoptosis kits were acquired from ElabScience Biotechnology Co., Ltd (China). Transwell polycarbonate membrane cell culture inserts were purchased from Corning (Massachusetts, USA). Preparation of Propolis Liposome (ProLip) Propolis encapsulation was carried out by the thin-film hydration technique 50 . A mixture of 3 mg/mL ethanolic extract of propolis (EEP) with (6: 1) ratio of phospholipid, and cholesterol was solubilised in 10 mL methanol: chloroform (1:1 v/v), followed by evaporation at 45ºC using a rotary evaporator (Buchi, USA). A lipid thin film was formed and subsequently hydrated using 10 mL of phosphate-buffered saline (PBS) (10 mL), while continuously stirring at 150 rpm for 30 min. The resulting suspension of propolis liposome (ProLip) was homogenized using a QSonica ultrasonic homogenizer (Q700, USA) for 30 min at 60% amplitude on ice to prevent temperature elevation. ProLip was harvested at 9000 rpm by centrifugation for 1 hr at 4 ºC, washed several times, recentrifuged, and finally resuspended in PBS. The liposomal suspension was lyophilized for 72 hr to obtain dried ProLip extract. To evaluate the loading capacity (LC) and encapsulation efficiency (EE) of ProLip, assessment was calculated by the following formula: EE (%) = (Amount of encapsulated propolis / Initial amount of ProLip) × 100 LC (%) = (Amount of encapsulated propolis / Weight of lyophilized ProLip) × 100 Briefly, after liposomal formulation, ProLip was appropriately collected by centrifugation at 4000 rpm for 20 min at 4°C using a refrigerated centrifuge, enabling the separation of encapsulated propolis within the liposomes (pellet) from free, unencapsulated propolis present in the supernatant. Then, the supernatant and pellet were separately lyophilized and redissolved in distilled water to measure the optical density (OD) at 420 nm. The total flavonoid contents (TFC) in both supernatant and pellet was quantified by referencing the calibration curve constructed using standard solutions. To determine the weight of the liposomal formulations, the pellet obtained after centrifugation was lyophilized for 72 hr, and the dried samples were weighed using a precision analytical balance. All measurements were performed in triplicate to ensure reliability and reproducibility of the data. Determination of particle size, Polydispersity index (PDI), and zeta potential of ProLip The particle size, polydispersity index (PDI), and zeta potential of the liposomal formulations were measured using a Zetasizer (Ver. 7.11, Malvern Instruments, Worcestershire, UK). For these measurements, a small amount of the liposomal suspension was redispersed in deionized water and sonicated briefly to ensure uniform dispersion. The particle size and PDI were determined using dynamic light scattering (DLS), while the zeta potential was measured through electrophoretic light scattering (ELS). Measurement of dynamic contact angle Dynamic contact angle measurements were performed using the DataPhysics DCAT21 (DataPhysics Instruments GmbH, Germany) to assess the wettability of propolis after its encapsulation into a liposomal nanocarrier. Powder contact angle measurements were conducted for propolis (Pro), while solid contact angle measurements were carried out for the liposome (Lip) and ProLip formulations. For the powder measurement, 400 mg of Pro was weighed and compressed into a sample glass tube. Calibration was performed using hexane as the reference liquid. For the solid samples, Lip and ProLip were spread on a plastic strip with dimensions of 0.6 cm in height and 0.5 cm in width. All samples were tested with distilled water, using both advancing and receding sequences. The contact angle values were automatically calculated by the software. Scanning electron microscopy (SEM) The surface characteristic of ProLip were observed using scanning electron microscopy (SEM) (Quanta FEG 650, Fei, USA). The freeze-dried ProLip samples were mounted onto adhesive-taped stubs and sputter-coated with platinum film using an automated coater (JFC 11600) to prevent charging up by the electron beam. The SEM images were captured at various magnifications to observe the surface structure, shape, and morphological characteristics of the liposomal particles. Attenuated total reflection Fourier-Transform Infrared (ATR-FTIR) spectroscopy Attenuated total reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy was employed for chemical composition analysis of Pro, Lip, and ProLip (Bruker, Germany). The sample was prepared in spectroscopic grade potassium bromide (KBr) disks and the spectra were recorded in the range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹. Preparation of PMA-differentiated THP-1 Macrophages Model The leukemic monocyte cell line, THP-1 was retrieved from ATCC (Porton Down, Salisbury, UK). The cells were cultured in RPMI 1640 medium (Capricorn, Germany) enriched with 1% (v/v) penicillin/streptomycin (Capricorn, Germany), and 10% (v/v) FBS (Capricorn Scientific, Germany) at seeding density of 3 × 10 5 cells/mL. The cells were kept at 37°C in a humidified atmosphere with 5% CO 2 . For differentiation, the cells were treated with PMA (Sigma-Aldrich, USA). Briefly, 3 x 10 6 cells/mL of cells were seeded in T-25 plate and induced for 48 hr by PMA (60 ng/mL). After differentiation, cells were rested for another 24 hr before being subjected for further assays. Measurement of Cytokine Production To investigate the ability of ProLip on the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, and IL-1ß in human macrophage model, cells were co-stimulated with lipopolysaccharide (LPS) (Sigma-Aldrich, USA) at 500 ng/mL. The level of secreted TNF-α, IL-6, IL-10, and IL-1ß were measured using commercial ELISA kit (ElabScience Biotechnology Co., Ltd, China). Absorbance was recorded at 450 nm using a microplate reader, with a wavelength correction at 550 nm. Cytokine concentrations were determined by comparing the absorbance values to a standard curve. Transmission electron microscopy (TEM) analysis of THP-1 Macrophages Macrophage morphology was observed by transmission electron microscopy (TEM). The cell pellets were fixed in McDowell-Trump fixative overnight and post-fixed with 1% osmium tetroxide for 1 hr at room temperature. After washing thoroughly, the cells were dehydrated using a series of graded ethanol solutions (50%, 70%, and 95%) for 15 min each, followed by 30 min in 100% ethanol. Cells were further dehydrated using 100% acetone for 10 min, and then infiltrated overnight with a 1:1 mixture of acetone and Spurr’s resin. Subsequently, the cells were further infiltrated with a fresh change of pure Spurr’s resin for 3 days and embedded in pure Spurr’s resin at 60°C overnight. Specimen blocks were ultrathin-sectioned using a PowerTome XL ultramicrotome (RMC Boeckeler Instruments, Inc., Tucson, Arizona, USA) with an Ultra 45 Diatome diamond knife (Diatome, Biel, Switzerland), producing sections of 70–90 nm. The sections were collected on copper grids and stained with uranyl acetate and lead citrate, then imaged using energy-filtered transmission electron microscopy (EFTEM) on a Libra 120 (Carl Zeiss Meditec AG, Jena, Germany). In vitro Adjuvant Activity of macrophages with breast cancer cells Three separate assays were conducted including apoptosis assay, scratch wound assay, and invasion assay. First, MCF-7 cells were cultured in DMEM high glucose supplemented with 1% (v/v) penicillin/streptomycin (Capricorn, Germany), and 10% (v/v) FBS (Capricorn Scientific, Germany) at 37°C in a 5% CO 2 atmosphere. MCF-7 cells (1 × 10 6 /mL) were seeded into a T-25 culture flask and allowed to adhere for 24 hr. Then, cells were treated for another 24 hr with macrophage conditioned media treated with Pro, ProLip, Pro + LPS, ProLip + LPS, LPS, and doxorubicin. Apoptosis was assessed by flow cytometry using Annexin V and propidium iodide (PI) co-staining. For scratch wound assay, MCF-7 cells were scratched and then incubated for 24 hr to observe the change in the wounded area. Image of the cells were captured using an inverted microscope, and the healed area was quantified using Image J software (version 1.54g, National Institute of Health, USA). As for the Transwell invasion assay, THP-1 cells were seeded into the lower compartment of a 6-well plate, followed by differentiation. After the differentiation was completed, MCF-7 cells (5 × 10 5 cells per well) were seeded onto the upper inserts. The assay was carried out for 24 hr. After the incubation period, the invasive cells that passed through the membrane to the lower compartment were fixed in 100% methanol (Sigma-Aldrich, St. Louis, USA) for 20 min. After re-washing with PBS and air drying, chambers were photographed under an inverted microscope at 20 × magnification for five random fields per insert. The percentage of invaded cells were calculated by Image J software (version 1.54g, National Institute of Health, USA). Statistical Analysis Data were expressed as means ± standard deviation (SD) and analysed using one-way analysis of variance (ANOVA) to compare the different among the groups, followed by the Tukey’s test as the post hoc test. A p-value of < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS software package (version 20.0, SPSS, Chicago, IL, USA), and graphs were created using Microsoft Excel. Declarations Competing Interests The author(s) declare no competing interests. Author Contribution H.M.Z. performed the experimental work, prepared, and analysed the data, and participated in the preparation of the manuscript. M.M. provided technical assistance with cell culture techniques. N.N.S.N.M.K. conceived and designed the study, funding acquisition, interpreted the data, and participated in the final editing of the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGEMENT This project was funded by the Ministry of Higher Education (MOHE), Malaysia, under the Fundamental Research Grant Scheme (FRGS) (FRGS; Reference code: FRGS/1/2021/STG01/USM/02/11), Account no. 203.CIPPT.6711976. HMZ received the Graduation Research Assistance allowance under FRGS scheme for her postgraduate study. Data Availability The image supporting Fig. 5 (A) is adapted from Moreno-Mendieta et al. (2022) and publicly available in https://link.springer.com/article/10.1007/s11095-022-03301-2 [22].All data generated and analysed during this study are included in the manuscript. References Khan, M., Maker, A. V. & Jain, S. The Evolution of Cancer Immunotherapy. Vaccines 9, (2021). Garg, P. et al. Next-Generation Immunotherapy: Advancing Clinical Applications in Cancer Treatment. J Clin. Med 13 , (2024). Gupta, S. L., Basu, S., Soni, V. & Jaiswal, R. K. Immunotherapy: an alternative promising therapeutic approach against cancers. Mol. Biol. Rep. 49 , 9903–9913 (2022). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6817917","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473509215,"identity":"699a0bb7-de1b-4a9e-b35a-a156c23b9edc","order_by":0,"name":"Hamidah Mohd Zain","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Hamidah","middleName":"Mohd","lastName":"Zain","suffix":""},{"id":473509216,"identity":"0ba1691c-b3b1-4a3f-ab6b-c6c1a4f0a590","order_by":1,"name":"Musthahimah Muhamad","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Musthahimah","middleName":"","lastName":"Muhamad","suffix":""},{"id":473509217,"identity":"b01ea58c-d426-4855-9638-5fa7aaadb3d5","order_by":2,"name":"Nik Nur Syazni Nik Mohamed Kamal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYDACZjYIzcbeQLIWngNEWwPVwiCRQKQGc3a25M88NXV5fJJvjD/dYNiWSNB9ls1sx6R5jrEVs0nnmEnnMNwmrMXgMHsbMw8bT2IbUAszsVqaP/P8k0hskzxj/JlILWwHpHnbDBLbJHgMiHUYW5rk3L6ExDaetDLpHIPbxoS1nD9m/OHNt7rE+e2HN3/OqbgtS1ALCDDxIExgcCRKC+MPJI49MTpGwSgYBaNgZAEAk+04rEODtWMAAAAASUVORK5CYII=","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Nik","middleName":"Nur Syazni Nik Mohamed","lastName":"Kamal","suffix":""}],"badges":[],"createdAt":"2025-06-04 08:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6817917/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6817917/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-19867-x","type":"published","date":"2025-10-15T15:58:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85079801,"identity":"80aa7a48-bd82-4a9a-804d-77be69530398","added_by":"auto","created_at":"2025-06-20 17:25:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71478,"visible":true,"origin":"","legend":"\u003cp\u003eThe bar charts of average particle size value (a), polydispersity index (b), zeta potential (c), contact angle (d) and, loading capacity and encapsulation efficiency of ProLip (e). Data were presented as mean ± SD, with statistical significance indicated (**p \u0026lt; 0.01, ***p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/0030d1f5fd32dc6851ec2c61.png"},{"id":85080053,"identity":"02c6d4d6-71d0-47dc-a3a4-e2c366caf145","added_by":"auto","created_at":"2025-06-20 17:33:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":654368,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological visualization of (a) Pro, (b) Lip, and (c) ProLip under SEM at a magnification of 20,000x. Scale bar: 5 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/8011fe65396378ce63c742da.png"},{"id":85080824,"identity":"82107715-bda4-4cc5-a1a1-02d96c080bfa","added_by":"auto","created_at":"2025-06-20 17:41:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99237,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristic functional group variations of the FTIR spectra in (a) Pro, (b) Lip, and (c) ProLip.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/08a853d0a95bef28ab8f5148.png"},{"id":85079803,"identity":"044b185a-b9f8-41b2-b697-462a921f7183","added_by":"auto","created_at":"2025-06-20 17:25:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":194351,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine production in THP-1 macrophages treated with Pro, ProLip, Pro + LPS, ProLip + LPS, LPS, and Doxorubicin. (a) TNF-α, (b) IL-1β, (c) IL-10 and (d) IL-6 levels were quantified. Data was presented as mean ± SD (n= 3). Statistical analysis was analysed using one-way ANOVA followed by Tukey’s post hoc test (*p \u0026lt; 0.05; ns = not significant, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/8c6abb195f92e5bb4bd4a9d9.png"},{"id":85080823,"identity":"5aaa7910-4578-41cb-99f1-bf4190d54b98","added_by":"auto","created_at":"2025-06-20 17:41:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":799592,"visible":true,"origin":"","legend":"\u003cp\u003eTEM of phagocytic uptake and intracellular trafficking of treatments in THP-1 macrophages following 24 hr exposure. (A) Schematic illustration of the key stages of phagocytosis. Macrophage formed an actin-mediated membrane ruffling to sense particles and initiate engulfment. Progressive pseudopod extension led to the engulfment of particles forming a phagosome. Figure 5 (A) is adapted from Moreno-Mendieta et al., (2022)\u003csup\u003e22\u003c/sup\u003e. (B-H) Each treatment condition is represented by two images: a low-magnification overview (left) and a high-magnification image (right) showing subcellular details. Untreated control cells showed no morphological abnormalities (B(i-ii)). Pro and ProLip were deposited within the membrane-bound structures and identified as electron dense particles. Magnified electron micrograph showed phagolysosome containing Pro (C(ii)), ProLip (D(ii)), Pro + LPS (E(ii)), ProLip + LPS (F(ii)), and Doxorubicin (H(ii)). Activated macrophages showed extensive pseudopods and phagocytic activity (F(i-ii) and G(i-ii)).\u0026nbsp; Magnification: B(i): x2000, B(ii): x4000, C(i): x2000, C(ii): x6300, D(i):\u0026nbsp; x1600, D(ii): x6300, E(i): x2500, E(ii): x6300, F(i): x1600, F(ii): x8000, G(i):\u0026nbsp; x2500, G(ii): x6300, H(i): x2500, H(ii): x6300.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/414fc959664927904da74313.png"},{"id":85080828,"identity":"89580771-6e66-4901-ba05-1d1f594e51cf","added_by":"auto","created_at":"2025-06-20 17:41:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":642901,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Assessment of ProLip’s immunomodulatory and anticancer activities in breast cancer cells using different formulations of macrophages conditioned media. (a) Flow cytometric analysis of apoptosis in MCF-7 cells. The quadrants represent viable, early apoptotic, late apoptotic, and necrotic cell populations. (b) Quantification of apoptotic cells. ProLip enhanced apoptotic activity compared to other groups. (c) Wound healing assay of MCF-7 cells. Representative images at 0 and 24 hr show reduced wound closure in ProLip-treated cells. (d) Quantification of wound healing (mm\u003csup\u003e2\u003c/sup\u003e) after 24 hr. ProLip showed significant inhibition of MCF-7 cell migration compared to controls and other treatments. (e) Transwell invasion assay of MCF-7 cells. Images show reduced invasive cell numbers in ProLip-treated groups. (f) Quantification of invasive cells. UT: untreated; Pro: propolis; ProLip: propolis liposome; LPS: lipopolysaccharide. Data were presented as mean ± SD (n = 3). Statistical significance was indicated as ***p \u0026lt; 0.001 compared to the untreated control (UT).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/dd966061bfe8758be9047a2e.png"},{"id":93956191,"identity":"c034ce8a-ab2f-4181-94ad-07dd587f7beb","added_by":"auto","created_at":"2025-10-20 16:11:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3388458,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6817917/v1/e3cbca32-352f-4813-bbd7-8452184d0d72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Propolis-Loaded Liposomes (ProLip): A Nanoformulated Immunomodulator Targeting Breast Cancer via Macrophage Activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmunotherapy has revolutionized cancer treatment by offering innovative solutions to the challenges posed by conventional regimens such as systemic toxicity, limited efficacy, and resistance development \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among these strategies, immunomodulation-based therapies harness the host immune system to selectively target and eliminate cancer cells \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Macrophages, as central regulators of immune responses and the tumor microenvironment, are particularly promising targets for these therapies \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, the limitations of existing approaches underscore the need for novel therapeutic agents that are both effective and specific.\u003c/p\u003e \u003cp\u003ePropolis, a natural resinous product produced by bees, has garnered attention for its potent anti-inflammatory, antioxidant, and anticancer properties \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Propolis derived from \u003cem\u003eHeterotrigona itama (H. itama)\u003c/em\u003e stingless bees is particularly rich in phenolic and flavonoid compounds, which confer its pharmacological activity \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Despite its promise, the clinical application of propolis remains limited due to poor water solubility, low bioavailability, and variability in chemical composition \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These challenges have hindered its consistent therapeutic outcomes and broader adoption in oncology and immunotherapy. Moreover, mechanistic insights into its interaction with immune cells, particularly macrophages, remain underexplored, creating a critical gap in its translation to clinical applications.\u003c/p\u003e \u003cp\u003eNanotechnology offers a transformative approach to address these limitations. Liposomal encapsulation, leveraging biocompatible phospholipid bilayers, can enhance the solubility, stability, and bioavailability of hydrophobic compounds such as propolis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This study introduces ProLip, a novel liposomal formulation of \u003cem\u003eH. itama\u003c/em\u003e propolis, engineered to overcome the solubility barrier and improve its interaction with immune cells. ProLip not only protects the bioactive components of propolis but also facilitates efficient macrophage uptake, enabling targeted delivery and controlled release. This dual-targeted system amplifies propolis's immunomodulatory and anticancer effects, addressing the limitations of conventional therapies and current propolis-based studies.\u003c/p\u003e \u003cp\u003eThe novelty of this work lies in its exploration of ProLip\u0026rsquo;s dual therapeutic potential: modulating immune responses and inhibiting cancer progression. Unlike previous studies that focus exclusively on either immunomodulation or anticancer activity, this research investigates the synergistic interplay between these mechanisms. By combining natural bioactive with advanced nanotechnology, this study addresses critical challenges in drug delivery, immune modulation, and cancer therapy. ProLip exemplifies a novel therapeutic strategy capable of overcoming the limitations of traditional propolis formulations, offering enhanced solubility, stability, and targeted delivery. The findings not only highlight the clinical relevance of liposomal propolis but also establish its potential as a multifunctional nanotherapeutic for inflammation-driven diseases and cancer. This research provides a foundation for future studies to translate natural product-based therapies into clinical applications, bridging the gap between laboratory research and real-world healthcare needs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical analysis and performance assessment of ProLip\u003c/h2\u003e \u003cp\u003eThe encapsulation of Pro into Lip demonstrated significant changes in the physicochemical characteristics of the ProLip formulation. Based on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), a significant reduction in the average particle size of Pro was observed after encapsulation into the liposomal carrier, which was from 402.77\u0026thinsp;\u0026plusmn;\u0026thinsp;7.53 nm to 249.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.79 nm. Besides, all samples exhibited monodisperse distribution of particles with PDI values\u0026thinsp;\u0026lt;\u0026thinsp;0.12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). Surface charge determination, through zeta potential measurement showed an overall negative value with Lip and ProLip possessed more negative zeta potential at -51.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 mV and \u0026minus;\u0026thinsp;50.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mV respectively compared to Pro alone (-1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 mV) as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c). The degree of wettability of ProLip revealed a lower contact angle (50.247\u0026deg; \u0026plusmn; 2.76) after encapsulation compared to before encapsulation (Pro: 81.629\u0026deg; \u0026plusmn; 3.16), indicating improved solubility. The loading capacity (LC) and encapsulation efficiency (EE) of ProLip demonstrated high loading (72.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72%) and encapsulation (83.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66%) of Pro into Lip.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurface morphology of ProLip via SEM\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eThe surface morphological evaluation of Pro, Lip and ProLip was visualized by SEM analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on the results, Pro exhibited an irregular circular morphology with a rough surface and heterogeneous particle sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a)). In contrast, the propolis-free liposome (Lip) displayed a nearly circular smooth surface with well-defined, uniform particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b)). The ProLip formulation maintained a comparable uniformity in particle size but exhibited a slightly textured surface, differentiating it from the smoother morphology of Lip (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c)).\u003c/p\u003e\n\u003ch3\u003eChemical interaction of ProLip via FTIR spectra\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eThe interaction between Pro, Lip and ProLip were observed through the distinct changes in the peaks and intensities, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. According to the results, identical bands were identified in both Pro and ProLip at 3308 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to O-H stretching. However, there was a reduction in the intensity of ProLip, characterized by a broader absorption band. Additionally, C-H stretching vibrations at 2924 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2848 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were present in all samples, with a more pronounced peak similarity between Pro and ProLip. Further spectral shifts were observed in Lip and ProLip, particularly with the presence of carbonyl (C\u0026thinsp;=\u0026thinsp;O) stretching at 1737 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, carboxyl (C\u0026thinsp;=\u0026thinsp;C) stretching at 1638 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and aromatic C-H bending at 1446 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In addition, a distinct O-H bending was observed at 1400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Lip and 1365 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in ProLip. The characteristic peaks of Pro at 1065 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C-O), 981 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;C), and 795 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;C) were also detected in ProLip.\u003c/p\u003e\n\u003ch3\u003eImmunomodulatory activities in THP-1 macrophages\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects of ProLip and associated controls including Pro, and Doxorubicin were evaluated on selected biomarkers involved in immune response by ELISA assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). LPS was added in the subsequent assays as it acts as a potent inflammatory stimulus \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In this assay, LPS served as an inflammatory stimulus to assess the response of PMA-differentiated THP-1 macrophages to ProLip and the associated controls. When exposed to LPS, PMA-differentiated THP-1 macrophages, similar to primary macrophages, activated signalling pathways that led to the production of pro-inflammatory cytokines, such as TNF-α and IL-1β \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Supporting this, the study herewith demonstrated that LPS enhanced the expression of TNF-α (154.54 pg/mL, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and IL-1β (231.37 pg/mL, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in PMA-differentiated THP-1 macrophages, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a) and 4(b) respectively.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a) illustrated the expression of TNF-α cytokine in PMA-differentiated THP-1 macrophages following treatment with ProLip and the respective controls. Following 24 hr incubation with ProLip\u0026thinsp;+\u0026thinsp;LPS, TNF-α levels were reduced by 20.48 pg/mL relative to the LPS-treated macrophages (154.54 pg/mL, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar findings were observed in THP-1 macrophages treated with ProLip without LPS stimulation, where TNF-α cytokine expression was measured at 30.26 pg/mL. This suggests that ProLip may exert an anti-inflammatory effect by suppressing the production of this key pro-inflammatory cytokine. Meanwhile, Pro and Doxorubicin treatments resulted in TNF-α cytokine expression levels 46.13 pg/mL and 75.12 pg/mL respectively.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b) illustrated the expression of IL-1β cytokine in THP-1 macrophages following treatment with ProLip treatment and the respective controls. IL-1β is a pro-inflammatory cytokine, and its elevated levels have been associated with various inflammatory disease, including rheumatoid arthritis and development of cancer \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Treatment with ProLip\u0026thinsp;+\u0026thinsp;LPS led to a significant reduction in IL-1β production to 14.94 pg/mL in THP-1 macrophages compared to the LPS-treated macrophages (231.37 pg/mL, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar findings were observed in THP-1 macrophages treated with ProLip without LPS stimulation where IL-1β cytokine expression was measured at 30.46 pg/mL. This suggests that ProLip may exert an anti-inflammatory effect by suppressing the production of this key pro-inflammatory cytokine. Meanwhile, Pro and Doxorubicin treatments resulted in IL-1β cytokine expression levels of 61.89 pg/mL and 98.03 pg/mL respectively.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c) illustrated the expression of IL-10 cytokine in THP-1 macrophages following treatment with ProLip treatment and the respective controls. IL-10 is an anti-inflammatory cytokine produced by a variety of immune cells, including macrophages, T cells, B cells, and dendritic cells \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Treatment with ProLip resulted in an increase in IL-10 production (13.46 pg/mL, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in THP-1 macrophages compared to LPS-treated macrophages (8.67 pg/mL). Comparable results were obtained in THP-1 macrophages treated with ProLip under LPS stimulation, where IL-10 levels were measured at 49.43 pg/mL (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Based on these findings, it is hypothesized that ProLip may promote the anti-inflammatory function of IL-10 of THP-1 macrophages, by reducing excessive inflammation in an already activated pro-inflammatory environment, as evidenced by the LPS-induced response. Meanwhile, Pro and Doxorubicin treatment resulted in IL-10 expression levels of 9.98 pg/mL and 4.16 pg/mL, respectively.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d) illustrated the expression of IL-6 cytokine in THP-1 macrophages following treatment with ProLip treatment and the respective controls. IL-6 is a pleiotropic cytokine, exhibiting both pro-inflammatory and anti-inflammatory activities depending on the specific cellular context, including the microenvironment, the presence of other cytokines, and the target cell type \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Treatment with ProLip resulted in an increase in IL-6 production (24.17 pg/mL, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in THP-1 macrophages compared to LPS-treated macrophages (10.80 pg/mL). Comparable results were obtained in THP-1 macrophages treated with ProLip under LPS stimulation, where IL-6 levels were measured at 40.49 pg/mL (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Based on these findings, it is hypothesized that ProLip may promote the anti-inflammatory function of IL-6 of THP-1 macrophages, by reducing excessive inflammation in an already activated pro-inflammatory environment, as evidenced by the LPS-induced response. Meanwhile, Pro and Doxorubicin treatment resulted in IL-6 expression levels of 12.58 pg/mL and 7.13 pg/mL, respectively.\u003c/p\u003e\n\u003ch3\u003eMorphological Analysis of ProLip Uptake\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e TEM of phagocytic uptake and intracellular trafficking of treatments in THP-1 macrophages following 24 hr exposure. (A) Schematic illustration of the key stages of phagocytosis. Macrophage formed an actin-mediated membrane ruffling to sense particles and initiate engulfment. Progressive pseudopod extension led to the engulfment of particles forming a phagosome. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (A) is adapted from Moreno-Mendieta et al., (2022)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. (B-H) Each treatment condition is represented by two images: a low-magnification overview (left) and a high-magnification image (right) showing subcellular details. Untreated control cells showed no morphological abnormalities (B(i-ii)). Pro and ProLip were deposited within the membrane-bound structures and identified as electron dense particles. Magnified electron micrograph showed phagolysosome containing Pro (C(ii)), ProLip (D(ii)), Pro\u0026thinsp;+\u0026thinsp;LPS (E(ii)), ProLip\u0026thinsp;+\u0026thinsp;LPS (F(ii)), and Doxorubicin (H(ii)). Activated macrophages showed extensive pseudopods and phagocytic activity (F(i-ii) and G(i-ii)). Magnification: B(i): x2000, B(ii): x4000, C(i): x2000, C(ii): x6300, D(i): x1600, D(ii): x6300, E(i): x2500, E(ii): x6300, F(i): x1600, F(ii): x8000, G(i): x2500, G(ii): x6300, H(i): x2500, H(ii): x6300.\u003c/p\u003e \u003cp\u003eThe intracellular localization and morphological changes induced by Pro and ProLip in THP-1 macrophages, both in the presence and absence of LPS was conducted by TEM analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, untreated (UT) THP-1 macrophages exhibited a healthy and viable cellular structure, characterized by intact organelles, a large nucleus with well-defined chromatin, abundant mitochondria, and an intact plasma membrane (Fig.\u0026nbsp;4.21 B(i-ii)) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Macrophages, being phagocytic cells, engulf foreign materials such as Pro, encapsulating them within a membrane-bound vesicle known as a phagosome. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC(i-ii), Pro was found inside phagosomes. The phagosome usually fuses with lysosomes to form phagolysosome, where the engulfed material undergoes enzymatic degradation and digestion \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This process may explain why some Pro was also observed in the macrophage cytoplasm. In addition to this process, it may also indicate phagolysosomal membrane disruption \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, leading to the release of Pro into the cytoplasm.\u003c/p\u003e \u003cp\u003eThe presence of larger vesicles, likely phagosomes, indicated that phagocytosis was the primary uptake mechanism of ProLip by THP-1 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(i-ii)). This observation was further supported by pseudopod-like structures surrounding the large vesicles, suggesting active phagocytosis. These structures likely represented the macrophage membrane extending and enclosing the ProLip during the engulfment process. No free ProLip was detected in the cytoplasm; it was only found within phagocytic vacuoles.\u003c/p\u003e \u003cp\u003eLPS is a potent immunostimulant that activates THP-1 macrophages, resulting in enhanced phagocytic activity and increased lysosomal enzyme production \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Lysosomes have the ability to fuse with the plasma membrane, resulting in the extracellular release of their contents \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This can occur as part of a cellular response to inflammation, potentially exacerbated by LPS stimulation. The combined challenge of Pro and LPS may exert an overwhelming impact, potentially leading to macrophage cell death (cytotoxicity) and the subsequent release of lysosomal and cytoplasmic contents, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE(i-ii).\u003c/p\u003e \u003cp\u003eInterestingly, ProLip was exclusively located within membrane-bound vesicles, phagosomes, even in the presence of LPS stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF(i-ii)). Similar to ProLip alone, no free ProLip\u0026thinsp;+\u0026thinsp;LPS was detected in the macrophage cytoplasm. These findings suggest efficient containment and processing. Liposomes are commonly utilized as drug and nanoparticle carrier due to their biocompatibility and efficient uptake by macrophages through phagocytosis \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Liposomal encapsulation likely enhanced ProLip uptake compared to free Pro. As reported by Marrocco \u0026amp; Ortiz, (2022), LPS stimulation can enhance macrophage activity, which may contribute to the observed synergistic effect of liposomal delivery under LPS exposure in facilitating the efficient engulfment of ProLip within phagosomes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG(i-ii) depicts THP-1 macrophages under LPS exposure. It was observed that activated macrophages underwent morphological changes, becoming larger and flatter. The cell membrane formed ruffles and extensions, known as filopodia \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These dynamic membrane protrusions play a crucial role in capturing and engulfing pathogens and other targets \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;4.21 H(i-ii) presents a TEM micrograph of a THP-1 macrophage after exposure to Doxorubicin. As observed, different stages of phagosomes and lysosomes were present, and some Doxorubicin appeared to have escaped the phagocytic pathway, dispersing throughout the macrophage cytoplasm and accumulating on the outer layer of the phagosome/lysosome membrane.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdjuvant activity and effect on Breast Cancer Cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBuilding on the observed effects of Pro and ProLip, conditioned media from Pro, ProLip, and other treated macrophages (including Pro\u0026thinsp;+\u0026thinsp;LPS, ProLip\u0026thinsp;+\u0026thinsp;LPS, LPS, and Doxorubicin) were investigated on MCF-7 breast cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It was hypothesized that ProLip modulated macrophage activity, which in turn impacted the behavior of neighbouring breast cancer cells. This co-culture assay aimed to characterize the interactions between ProLip-treated macrophages and breast cancer cells, focusing on migration and invasion activities, as well as mechanisms of cell death.\u003c/p\u003e \u003cp\u003eBased on the results, treatment with Pro, ProLip, Pro\u0026thinsp;+\u0026thinsp;LPS, and ProLip\u0026thinsp;+\u0026thinsp;LPS macrophage conditioned media, exhibited a pronounced suppression of MCF-7 cells migration after a 24-hr incubation, with minimal wound closure observed as compared to the baseline scratch area at 0 hr (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (c,d)). In contrast, the untreated control group (UT) and Doxorubicin showed nearly complete wound closure by 24 hr. LPS -conditioned media group, in particular, showed a gradual migration after 24 hr, in comparison to the control. Meanwhile, the Transwell invasion assay demonstrated that, treatment with Pro and ProLip significantly inhibited MCF-7 invasion compared to the untreated control, 16.77% (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 11.38% (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (e,f)). The inhibitory effect was further enhanced in the presence of LPS. Specifically, Pro\u0026thinsp;+\u0026thinsp;LPS reduced invasion by 10.21% compared to the UT control, while ProLip\u0026thinsp;+\u0026thinsp;LPS exhibited the most pronounced reduction, decreasing invasion by 7.98%. While the effects of LPS alone and Doxorubicin were less pronounced compared to ProLip and the combination treatments, the results demonstrated a 63.31% (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 73.58% (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) reduction in invasion, respectively, compared to the untreated cells.\u003c/p\u003e \u003cp\u003eAs for cell death mechanism, MCF-7 cells treated with Pro-macrophage conditioned media showed a significant increase in which 38.56% \u0026plusmn; 2.58 (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of cells undergoing apoptotic cell death compared to the untreated control cells observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a,b)). Interestingly, apoptosis was significantly higher in MCF-7 cells following incubation with ProLip-macrophage conditioned media, reaching 55.83% (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Under LPS stimulation, treatment with Pro- and ProLip macrophage conditioned media resulted in a significant increase in apoptotic cell death in MCF-7 cells, reaching 47.41% (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 69.36% (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively. In contrast, conditioned media from LPS alone or the chemotherapeutic agent doxorubicin induced lower levels of apoptosis in MCF-7 cells, with 13.07% \u0026plusmn; 4.02 and 28.56% \u0026plusmn; 2.21% of apoptotic cells, respectively.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study presents a comprehensive integration of nanotechnology with natural immunotherapeutic by employing liposomal encapsulation of propolis (ProLip), aiming to overcome the physicochemical limitations of crude propolis (Pro) and enhance its biological efficacy in modulating immune cell behavior. Propolis, a resinous natural product from bees, is widely recognized for its potent anti-inflammatory and anticancer properties; however, its clinical translation has been limited by poor aqueous solubility, instability, and reduced bioavailability \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These challenges significantly affect its cellular uptake and therapeutic consistency. Our findings demonstrate that the physicochemical modifications achieved through liposomal encapsulation significantly improved the bioavailability, stability, and cellular interaction of propolis. Crude propolis, when directly exposed to cells, exhibited uncontrolled cytotoxicity and induced cellular stress, likely due to its non-specific interaction with cellular membranes and tendency to form intracellular aggregates, as previously reported \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. By encapsulating propolis in liposomes, these challenges were effectively mitigated.\u003c/p\u003e \u003cp\u003eThe ProLip formulation displayed enhanced hydrophilicity and uniform dispersion of propolis within the liposomal bilayer, transforming the aggregated resinous particles of crude Pro into a stable nanosystem. This reconfiguration significantly improved delivery characteristics, as evidenced by the mean particle size of 249.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.79 nm, an optimal size range for cellular uptake via endocytosis and enhanced accumulation at sites of inflammation or tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Nanosized particles within this range are known to exhibit prolonged systemic circulation and enhanced permeability and retention (EPR) effects, facilitating passive targeting of inflammatory tissues and tumors \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Furthermore, macrophages are highly phagocytic and naturally internalize nanoparticles within this size spectrum, enabling ProLip to be preferentially taken up through actin-mediated phagocytosis and macropinocytosis \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe ProLip formulation also exhibited a highly negative zeta potential (\u0026minus;\u0026thinsp;50.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mV), attributed to the presence of deprotonated phenolic groups and negatively charged phospholipids. This surface charge enhanced colloidal stability through electrostatic repulsion and prevented nanoparticle aggregation, contributing to a low polydispersity index (PDI) and uniform particle distribution \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In addition to its role in stability, negative zeta potential has been shown to improve macrophage uptake efficiency by 5.3-fold compared to neutral liposomes \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The hydrophilicity of ProLip, validated by contact angle measurement, further supported its suitability for biological applications. Increased wettability allowed for better interaction with aqueous biological environments, improving cell surface adherence and internalization efficiency \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Enhanced aqueous compatibility is a critical feature for any nanoformulation targeting immune cells, particularly macrophages, which thrive in dynamic fluidic environments and regulate immune responses through both direct contact and soluble mediators \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFTIR and SEM analyses confirmed successful molecular integration and morphological reconfiguration of propolis within the liposomal matrix. FTIR spectra revealed spectral shifts and band broadening, particularly in the carbonyl and hydroxyl regions, suggesting the formation of hydrogen bonding and van der Waals interactions between propolis phenolics and phospholipid components \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. These molecular interactions indicate strong compatibility between the payload (Pro) and carrier (Lip), crucial for maintaining structural integrity during circulation and ensuring controlled drug release. Meanwhile, SEM analysis revealed distinct morphological differences between ProLip and the unloaded liposome (Lip). The surface of ProLip exhibited increased roughness and slight irregularities, likely attributable to the deposition of complex organic constituents present in propolis onto the liposomal surface \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. These morphological alterations suggest effective integration of Pro within the hydrophobic domains of the lipid bilayer.\u003c/p\u003e \u003cp\u003eBiologically, ProLip outperformed crude Pro in terms of immunomodulatory potency and cellular compatibility. Propolis is known for its immunomodulatory properties, with its polyphenolic constituents exerting dual effects by suppressing excessive inflammation while promoting immune homeostasis \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In this study, ELISA results revealed significant upregulation of anti-inflammatory cytokines (IL-10 and IL-6) in ProLip-treated macrophages compared to the crude Pro. This suggests that encapsulation facilitated a macrophage phenotype that promoted immune resolution while preventing prolonged inflammation \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Notably, the co-treatment of macrophages with ProLip and LPS elicited a synergistic increase in IL-10 and IL-6 expression, indicating the formulation's capacity to modulate macrophage activation even under pro-inflammatory stimulation \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. ProLip also demonstrated an enhanced suppression on pro-inflammatory cytokines (IL-1β and TNF-α) compared to Pro, implying that the encapsulated formulation enhanced the bioavailability of active polyphenols and improved their regulatory impact on inflammatory signalling cascades \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. From a therapeutic standpoint, the ability of ProLip to suppress inflammatory cytokines without completely abolishing immune responsiveness represented a significant advantage. This is due to, excessive pro-inflammatory cytokine expression contributes to chronic inflammation and immune exhaustion, while insufficient inflammation can impair pathogen clearance and tumor control \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The ability of ProLip to strike a functional balance between immunostimulation and immune suppression highlights its potential as a versatile immunotherapeutic agent \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTEM imaging provided further insight into the intracellular behavior of Pro and ProLip. While Pro formed irregular and undefined aggregates in the cytoplasm, causing structural disruption to cellular organelles, ProLip was internalized more uniformly and trafficked toward lysosomal and phagosomal compartments. ProLip preserved membrane integrity and mitochondrial structure, suggesting that liposomal encapsulation shielded macrophages from potential damage while retaining propolis's biological activity \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This behavior indicates that, the encapsulation of Pro within liposomes likely facilitated its uptake through endocytic pathways, particularly clathrin- and caveolin-mediated endocytosis, mechanisms commonly observed in liposomal drug delivery systems \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. By preventing premature degradation and enabling sustained intracellular release, ProLip\u0026rsquo;s encapsulation enhances bioavailability and mitigates cytotoxic stress \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The preferential localization within lysosomal compartments suggested that macrophages actively processed and degraded ProLip, allowing for sustained intracellular release of bioactive compounds. In comparison, doxorubicin, a commonly used chemotherapeutic, demonstrated widespread intracellular dispersion and nuclear condensation, confirming its passive diffusion and DNA intercalation-based mechanism of cytotoxicity \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. These findings emphasize the difference in cellular handling between synthetic drugs and biologically encapsulated therapeutics like ProLip. While doxorubicin causes significant genotoxic stress, ProLip achieved effective intracellular delivery without inducing similar levels of cellular damage.\u003c/p\u003e \u003cp\u003eTo further assess the therapeutic potential of ProLip, this study utilized the human monocytic cell line THP-1 as a platform to investigate the interactions between MCF-7 breast cancer cells and THP-1-derived macrophages treated with ProLip. By recreating the inflammatory condition established by stimulation with LPS, the present study aimed to determine whether ProLip enhances or mitigates the anti-tumorigenic activities of M1-like macrophages within a model of the tumor microenvironment. The present study found that the conditioned media significantly inhibited MCF-7 cell migration. Consistently, results from the transwell invasion assay demonstrated that THP-1 macrophages treated with ProLip more effectively suppressed the invasive behavior of MCF-7 cells compared to unencapsulated propolis. Interestingly, this growth inhibitory effect correlated with an increased apoptotic population in MCF-7 cells following exposure to conditioned media derived from ProLip-treated THP-1 macrophages.\u003c/p\u003e \u003cp\u003eMacrophages, particularly the classically activated M1 subtype, have been documented to play a key role in inducing apoptosis in cancer cells through the release of pro-inflammatory cytokines such as TNF-α and IFN-γ. These cytokines activate intracellular signalling pathways that ultimately trigger programmed cell death in cancer cells \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Moreover, M1 macrophages are also known to possess both phagocytic and antigen-presenting capabilities, allowing them to produce pro-inflammatory cytokines and exert cytotoxic effects. Through these functions, they can directly eliminate target cells via the generation of ROS, reactive nitrogen species (RNS), IL-1β and TNF-α. Alternatively, M1 macrophages can also promote indirect cytotoxicity by activating other immune cells, such as NK cells and T cells \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. In the present study, exposure to ProLip significantly increased the levels of TNF-α and IL-1β in THP-1 macrophages compared to untreated cells. This elevated production of pro-inflammatory cytokines strongly indicates that ProLip promotes a pro-inflammatory, M1-like macrophage phenotype. These findings also suggest that ProLip may enhance the cytotoxic capacity of M1 macrophages against MCF-7 breast cancer cells, either directly or indirectly, through the activation of other immune cells. Such multifaceted signalling pathways underscore TNF-α\u0026rsquo;s role as a potent anticancer cytokine \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The dual capability of ProLip to reduce inflammation in resting macrophages while enhancing pro-inflammatory, tumor-suppressive responses in inflamed or LPS-stimulated environments underscores its adaptive immunomodulatory profile.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study established liposomal encapsulation as a viable strategy to enhance the therapeutic potential of \u003cem\u003eHeterotrigona itama\u003c/em\u003e propolis by addressing its physicochemical limitations and improving its biological performance. The development of Propolis Liposome (ProLip) significantly improved the aqueous solubility, stability, and cellular bioavailability of crude propolis, enabling efficient macrophage-targeted delivery while minimizing nonspecific cytotoxicity. Physicochemical characterization confirmed successful integration of propolis into the liposomal bilayer, resulting in a nanosized, stable formulation with enhanced hydrophilicity and favourable surface charge for immune cell interaction. This ProLip also exhibited a dual modulatory effect on cytokine secretion in THP-1 macrophages, promoting both anti-inflammatory and pro-inflammatory responses. While the anti-inflammatory cytokines contribute to immune regulation, the elevated pro-inflammatory mediators may enhance the immune system\u0026rsquo;s cytotoxic activity, thereby facilitating breast cancer cell death \u0026ndash; as evidenced by increased apoptosis and reduced migration and invasion in functional assays. Collectively, these findings demonstrates that liposomal encapsulation not only optimizes the pharmacological profile of propolis but also enhances its functional impact on immune modulation and cancer cell suppression. This work provides a promising foundation for the development of propolis-based nanotherapeutics in immunomodulatory and cancer-targeted clinical applications. Nevertheless, further in vivo validation and pharmacological evaluations are warranted to confirm ProLip\u0026rsquo;s therapeutic efficacy, safety and ADME (absorption, distribution, metabolism and excretion) profiles.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003ePropolis from \u003cem\u003eHeterotrigona itama (H. itama)\u003c/em\u003e stingless bee species were purchased from a local beekeeping company, Bayu Gagah Sdn Bhd (Kulim Hightech, Kedah, Malaysia). Soy phospholipids (\u0026ge;\u0026thinsp;99%), phosphate-buffered saline (PBS) tablets, lipopolysaccharides (LPS), and phorbol 12-myristate 13-acetate (PMA) (\u0026ge;\u0026thinsp;99%) were acquired from Sigma-Aldrich (St. Louis, USA). Cholesterol (\u0026ge;\u0026thinsp;99%) was sourced from Nacalai Tesque (Kyoto, Japan). Methanol (\u0026ge;\u0026thinsp;99.9%) and chloroform (\u0026ge;\u0026thinsp;99.9%) were purchased from Merck Millipore (Germany). THP-1 and MCF-7 cells were purchased from American Type Culture Collection (ATCC\u0026reg;) (Porton Down, Salisbury, UK). Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle Medium (DMEM), Penicillin-Streptomycin, and Fetal bovine serum (FBS), and trypsin were procured from Capricorn Scientific (Ebsdorfergrund, Germany). Enzyme-linked Immunosorbent assay (ELISA) and apoptosis kits were acquired from ElabScience Biotechnology Co., Ltd (China). Transwell polycarbonate membrane cell culture inserts were purchased from Corning (Massachusetts, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Propolis Liposome (ProLip)\u003c/h2\u003e \u003cp\u003ePropolis encapsulation was carried out by the thin-film hydration technique \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. A mixture of 3 mg/mL ethanolic extract of propolis (EEP) with (6: 1) ratio of phospholipid, and cholesterol was solubilised in 10 mL methanol: chloroform (1:1 v/v), followed by evaporation at 45\u0026ordm;C using a rotary evaporator (Buchi, USA). A lipid thin film was formed and subsequently hydrated using 10 mL of phosphate-buffered saline (PBS) (10 mL), while continuously stirring at 150 rpm for 30 min. The resulting suspension of propolis liposome (ProLip) was homogenized using a QSonica ultrasonic homogenizer (Q700, USA) for 30 min at 60% amplitude on ice to prevent temperature elevation. ProLip was harvested at 9000 rpm by centrifugation for 1 hr at 4 \u0026ordm;C, washed several times, recentrifuged, and finally resuspended in PBS. The liposomal suspension was lyophilized for 72 hr to obtain dried ProLip extract.\u003c/p\u003e \u003cp\u003eTo evaluate the loading capacity (LC) and encapsulation efficiency (EE) of ProLip, assessment was calculated by the following formula:\u003c/p\u003e \u003cp\u003eEE (%) = (Amount of encapsulated propolis / Initial amount of ProLip) \u0026times; 100\u003c/p\u003e \u003cp\u003eLC (%) = (Amount of encapsulated propolis / Weight of lyophilized ProLip) \u0026times; 100\u003c/p\u003e \u003cp\u003eBriefly, after liposomal formulation, ProLip was appropriately collected by centrifugation at 4000 rpm for 20 min at 4\u0026deg;C using a refrigerated centrifuge, enabling the separation of encapsulated propolis within the liposomes (pellet) from free, unencapsulated propolis present in the supernatant. Then, the supernatant and pellet were separately lyophilized and redissolved in distilled water to measure the optical density (OD) at 420 nm. The total flavonoid contents (TFC) in both supernatant and pellet was quantified by referencing the calibration curve constructed using standard solutions. To determine the weight of the liposomal formulations, the pellet obtained after centrifugation was lyophilized for 72 hr, and the dried samples were weighed using a precision analytical balance. All measurements were performed in triplicate to ensure reliability and reproducibility of the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of particle size, Polydispersity index (PDI), and zeta potential of ProLip\u003c/h2\u003e \u003cp\u003eThe particle size, polydispersity index (PDI), and zeta potential of the liposomal formulations were measured using a Zetasizer (Ver. 7.11, Malvern Instruments, Worcestershire, UK). For these measurements, a small amount of the liposomal suspension was redispersed in deionized water and sonicated briefly to ensure uniform dispersion. The particle size and PDI were determined using dynamic light scattering (DLS), while the zeta potential was measured through electrophoretic light scattering (ELS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of dynamic contact angle\u003c/h2\u003e \u003cp\u003eDynamic contact angle measurements were performed using the DataPhysics DCAT21 (DataPhysics Instruments GmbH, Germany) to assess the wettability of propolis after its encapsulation into a liposomal nanocarrier. Powder contact angle measurements were conducted for propolis (Pro), while solid contact angle measurements were carried out for the liposome (Lip) and ProLip formulations. For the powder measurement, 400 mg of Pro was weighed and compressed into a sample glass tube. Calibration was performed using hexane as the reference liquid. For the solid samples, Lip and ProLip were spread on a plastic strip with dimensions of 0.6 cm in height and 0.5 cm in width. All samples were tested with distilled water, using both advancing and receding sequences. The contact angle values were automatically calculated by the software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThe surface characteristic of ProLip were observed using scanning electron microscopy (SEM) (Quanta FEG 650, Fei, USA). The freeze-dried ProLip samples were mounted onto adhesive-taped stubs and sputter-coated with platinum film using an automated coater (JFC 11600) to prevent charging up by the electron beam. The SEM images were captured at various magnifications to observe the surface structure, shape, and morphological characteristics of the liposomal particles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAttenuated total reflection Fourier-Transform Infrared (ATR-FTIR) spectroscopy\u003c/h2\u003e \u003cp\u003eAttenuated total reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy was employed for chemical composition analysis of Pro, Lip, and ProLip (Bruker, Germany). The sample was prepared in spectroscopic grade potassium bromide (KBr) disks and the spectra were recorded in the range of 4000\u0026ndash;400 cm⁻\u0026sup1; at a resolution of 4 cm⁻\u0026sup1;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of PMA-differentiated THP-1 Macrophages Model\u003c/h2\u003e \u003cp\u003eThe leukemic monocyte cell line, THP-1 was retrieved from ATCC (Porton Down, Salisbury, UK). The cells were cultured in RPMI 1640 medium (Capricorn, Germany) enriched with 1% (v/v) penicillin/streptomycin (Capricorn, Germany), and 10% (v/v) FBS (Capricorn Scientific, Germany) at seeding density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL. The cells were kept at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. For differentiation, the cells were treated with PMA (Sigma-Aldrich, USA). Briefly, 3 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL of cells were seeded in T-25 plate and induced for 48 hr by PMA (60 ng/mL). After differentiation, cells were rested for another 24 hr before being subjected for further assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Cytokine Production\u003c/h2\u003e \u003cp\u003eTo investigate the ability of ProLip on the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, and IL-1\u0026szlig; in human macrophage model, cells were co-stimulated with lipopolysaccharide (LPS) (Sigma-Aldrich, USA) at 500 ng/mL. The level of secreted TNF-α, IL-6, IL-10, and IL-1\u0026szlig; were measured using commercial ELISA kit (ElabScience Biotechnology Co., Ltd, China). Absorbance was recorded at 450 nm using a microplate reader, with a wavelength correction at 550 nm. Cytokine concentrations were determined by comparing the absorbance values to a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM) analysis of THP-1 Macrophages\u003c/h2\u003e \u003cp\u003eMacrophage morphology was observed by transmission electron microscopy (TEM). The cell pellets were fixed in McDowell-Trump fixative overnight and post-fixed with 1% osmium tetroxide for 1 hr at room temperature. After washing thoroughly, the cells were dehydrated using a series of graded ethanol solutions (50%, 70%, and 95%) for 15 min each, followed by 30 min in 100% ethanol. Cells were further dehydrated using 100% acetone for 10 min, and then infiltrated overnight with a 1:1 mixture of acetone and Spurr\u0026rsquo;s resin. Subsequently, the cells were further infiltrated with a fresh change of pure Spurr\u0026rsquo;s resin for 3 days and embedded in pure Spurr\u0026rsquo;s resin at 60\u0026deg;C overnight. Specimen blocks were ultrathin-sectioned using a PowerTome XL ultramicrotome (RMC Boeckeler Instruments, Inc., Tucson, Arizona, USA) with an Ultra 45 Diatome diamond knife (Diatome, Biel, Switzerland), producing sections of 70\u0026ndash;90 nm. The sections were collected on copper grids and stained with uranyl acetate and lead citrate, then imaged using energy-filtered transmission electron microscopy (EFTEM) on a Libra 120 (Carl Zeiss Meditec AG, Jena, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eAdjuvant Activity of macrophages with breast cancer cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThree separate assays were conducted including apoptosis assay, scratch wound assay, and invasion assay. First, MCF-7 cells were cultured in DMEM high glucose supplemented with 1% (v/v) penicillin/streptomycin (Capricorn, Germany), and 10% (v/v) FBS (Capricorn Scientific, Germany) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. MCF-7 cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/mL) were seeded into a T-25 culture flask and allowed to adhere for 24 hr. Then, cells were treated for another 24 hr with macrophage conditioned media treated with Pro, ProLip, Pro\u0026thinsp;+\u0026thinsp;LPS, ProLip\u0026thinsp;+\u0026thinsp;LPS, LPS, and doxorubicin. Apoptosis was assessed by flow cytometry using Annexin V and propidium iodide (PI) co-staining.\u003c/p\u003e \u003cp\u003eFor scratch wound assay, MCF-7 cells were scratched and then incubated for 24 hr to observe the change in the wounded area. Image of the cells were captured using an inverted microscope, and the healed area was quantified using Image J software (version 1.54g, National Institute of Health, USA).\u003c/p\u003e \u003cp\u003eAs for the Transwell invasion assay, THP-1 cells were seeded into the lower compartment of a 6-well plate, followed by differentiation. After the differentiation was completed, MCF-7 cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) were seeded onto the upper inserts. The assay was carried out for 24 hr. After the incubation period, the invasive cells that passed through the membrane to the lower compartment were fixed in 100% methanol (Sigma-Aldrich, St. Louis, USA) for 20 min. After re-washing with PBS and air drying, chambers were photographed under an inverted microscope at 20 \u0026times; magnification for five random fields per insert. The percentage of invaded cells were calculated by Image J software (version 1.54g, National Institute of Health, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analysed using one-way analysis of variance (ANOVA) to compare the different among the groups, followed by the Tukey\u0026rsquo;s test as the post hoc test. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analysis was performed using the SPSS software package (version 20.0, SPSS, Chicago, IL, USA), and graphs were created using Microsoft Excel.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.M.Z. performed the experimental work, prepared, and analysed the data, and participated in the preparation of the manuscript. M.M. provided technical assistance with cell culture techniques. N.N.S.N.M.K. conceived and designed the study, funding acquisition, interpreted the data, and participated in the final editing of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThis project was funded by the Ministry of Higher Education (MOHE), Malaysia, under the Fundamental Research Grant Scheme (FRGS) (FRGS; Reference code: FRGS/1/2021/STG01/USM/02/11), Account no. 203.CIPPT.6711976. HMZ received the Graduation Research Assistance allowance under FRGS scheme for her postgraduate study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe image supporting Fig. 5 (A) is adapted from Moreno-Mendieta et al. (2022) and publicly available in https://link.springer.com/article/10.1007/s11095-022-03301-2 [22].All data generated and analysed during this study are included in the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhan, M., Maker, A. V. \u0026amp; Jain, S. 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Prolif.\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e, e12441 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"propolis, liposome, targeted delivery, immunomodulation, anti-cancer","lastPublishedDoi":"10.21203/rs.3.rs-6817917/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6817917/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePropolis, a natural remedy derived from bee by-products, is known for its immunomodulatory and anticancer properties. However, its clinical application is hindered by poor solubility and bioavailability. This study formulated a propolis-loaded liposome (ProLip) using the thin-film hydration technique (soy phospholipid-to-cholesterol ratio 6:1) to enhance its therapeutic effect. Encapsulation reduced the particle size of propolis from 402.77 ± 7.53 nm to 249.67 ± 5.79 nm and enhanced physicochemical properties, including a low polydispersity index (0.098 ± 0.02), highly negative zeta potential (-50.80 ± 0.10 mV), and improved solubility (water contact angle of 50.247°). FTIR analysis confirmed intermolecular interactions between phenolic groups in propolis and phospholipid carbonyl groups, while electron microscopy and surface morphology analysis revealed uniform structure and phagosomal localization in macrophages. Functionally, ProLip enhanced the secretion of anti-inflammatory cytokines IL-10 (49.429 ± 0.38 pg/mL) and IL-6 (40.488 ± 0.10 pg/mL), while suppressing pro-inflammatory mediators TNF-α and IL-1β by more than 80% compared to the LPS-treated group, highlighting ProLip as a potential immunoregulatory agent. Electron microscopy confirmed phagosomal localization of ProLip and reduced macrophage morphological damage compared to unencapsulated propolis, validating targeted delivery and protection capacity. Additionally, conditioned media from ProLip-treated macrophages significantly induced apoptosis (\u0026gt;50%) and inhibited migration and invasion in MCF-7 breast cancer cells, supporting immune-mediated anticancer effects. These findings highlight ProLip’s potential as a nanocarrier to enhance the bioavailability, cellular targeting, and therapeutic efficacy of stingless bee propolis in cancer immunotherapy.\u003c/p\u003e","manuscriptTitle":"Propolis-Loaded Liposomes (ProLip): A Nanoformulated Immunomodulator Targeting Breast Cancer via Macrophage Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 17:25:24","doi":"10.21203/rs.3.rs-6817917/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-07T08:01:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-12T11:32:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T05:16:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T07:43:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16831837201273798473005603532785998362","date":"2025-06-21T22:43:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248998379405379788748230096597755968160","date":"2025-06-20T21:47:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137927927234422935978536524011480855801","date":"2025-06-18T22:46:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212218794118738439714047842993915034434","date":"2025-06-18T21:39:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T20:32:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-18T18:17:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-18T09:15:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-08T00:42:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-08T00:38:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56879b8d-3597-456e-9480-0ffc4f4f838f","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50366923,"name":"Biological sciences/Biotechnology"},{"id":50366924,"name":"Biological sciences/Cancer"},{"id":50366925,"name":"Biological sciences/Cell biology"},{"id":50366926,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2025-10-20T16:07:44+00:00","versionOfRecord":{"articleIdentity":"rs-6817917","link":"https://doi.org/10.1038/s41598-025-19867-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-15 15:58:21","publishedOnDateReadable":"October 15th, 2025"},"versionCreatedAt":"2025-06-20 17:25:24","video":"","vorDoi":"10.1038/s41598-025-19867-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-19867-x","workflowStages":[]},"version":"v1","identity":"rs-6817917","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6817917","identity":"rs-6817917","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}