Enhanced Anticancer Activity in HR+/HER2+ Breast Cancer Cells Using Trastuzumab- Conjugated Liposomes co-loaded with siRNA and Tamoxifen

Enhanced Anticancer Activity in HR+/HER2+ Breast Cancer Cells Using Trastuzumab- Conjugated Liposomes co-loaded with siRNA and Tamoxifen | 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 Research Article Enhanced Anticancer Activity in HR+/HER2+ Breast Cancer Cells Using Trastuzumab- Conjugated Liposomes co-loaded with siRNA and Tamoxifen Gautam Kumar, Farmiza Begum, Prasada Chowdari Gurram, Bharath H. B., and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7025151/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Sep, 2025 Read the published version in BioNanoScience → Version 1 posted 10 You are reading this latest preprint version Abstract HR+/HER2 + breast cancer is characterized by the overexpression of hormone receptors (estrogen and progesterone) and the HER2 receptor, leading to aggressive tumor growth and poor prognosis. Conventional treatments often face challenges such as drug resistance and systemic toxicity. This study aims to develop and evaluate trastuzumab-conjugated siRNA and tamoxifen-loaded liposomes for targeted therapy in HR+/HER2 + breast cancer. Liposomes were prepared using the thin film hydration. Surface morphology was analyzed using scanning electron microscopy, while drug release profiles and serum stability of siRNA were assessed. In vitro cytotoxicity, cellular uptake, and Western blot analyses were conducted using HR+/HER2+ (BT474) breast cancer cells. Optimized trastuzumab-conjugated liposomes exhibited a particle size of 97.17 ± 0.2 nm, a zeta potential of 16.93 ± 0.25 mV, and high entrapment efficiencies for tamoxifen (79.02 ± 0.46%) and siRNA (79.85 ± 0.14%). Scanning electron microscopy confirmed spherical morphology. Drug release studies indicated controlled and sustained release, enhancing tamoxifen retention and stability. Serum stability tests demonstrated siRNA protection from degradation. In vitro cytotoxicity assays showed superior anticancer activity of TMX-FL compared to simple liposomes and pure tamoxifen. These findings suggest that TMX-FL liposomes offer a promising strategy for targeted cancer therapy, warranting further in vivo evaluations and clinical trials. Triple-positive breast cancer Tamoxifen-targeted delivery anti-ABCB1 siRNA Trastuzumab-conjugated liposomes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Background Breast cancer is the most prevalent cancer among women globally, with approximately 1 in 8 women receiving a diagnosis at some point in their lives. In 2022, there were 2.3 million new cases of breast cancer reported worldwide, resulting in 665,684 deaths attributed to the disease [1]. In the United States alone, 367,220 new cases of breast cancer were documented in 2024. Among these cases, HER2-positive breast cancer characterized by the overexpression of the human epidermal growth factor receptor 2 (HER2) and classified into subtypes such as HR+/HER2 + and HR-/HER2 + constitutes about 13% of all breast cancer cases [2], [3], [4]. This subtype is recognized for its aggressive nature and poorer prognosis compared to HER2-negative breast cancers [5]. The treatment regimen for triple-positive breast cancer typically involves a combination of therapies targeting hormone receptors and the HER2 protein. Hormone therapy, utilizing agents like tamoxifen and aromatase inhibitors, effectively blocks the actions of estrogen and progesterone, thereby slowing the proliferation of hormone receptor-positive cancer cells [6]. HER2-targeted therapies, including trastuzumab (Herceptin) and pertuzumab (Perjeta), specifically inhibit the HER2 protein, reducing the growth of HER2-positive cancer cells [7], [8]. Chemotherapy is frequently administered in conjunction with hormone and HER2-targeted therapies to eliminate rapidly dividing cancer cells [9], [10]. Surgical interventions such as lumpectomy or mastectomy are performed to excise tumors or affected breast tissue, while radiation therapy employs high-energy radiation to destroy any remaining cancer cells post-surgery [11], [12]. Despite these advancements, several challenges persist in managing HER2-positive breast cancer. Hormone therapies can lead to resistance over time, diminishing their effectiveness. Similarly, HER2-targeted therapies may encounter resistance mechanisms such as mutations in the HER2 gene or activation of alternative signaling pathways [13], [14], [15], [16]. Chemotherapy is often associated with significant side effects, including toxicity to healthy cells, which can limit its application [9]. Although surgical and radiation therapies effectively remove or destroy cancer cells, they do not address the underlying molecular mechanisms driving cancer progression and may result in recurrence [12], [17]. A promising strategy to overcome resistance involves the co-delivery of therapeutic agents using nanotechnology-based systems. Trastuzumab, a monoclonal antibody targeting HER2, has demonstrated efficacy in treating HER2-positive breast cancer; however, its effectiveness can be compromised by resistance mechanisms such as overexpression of the ATP-binding cassette subfamily B member 1 (ABCB1) protein [18], [19], [20], [21], [22]. This protein actively pumps therapeutic drugs out of cancer cells, reducing their intracellular concentrations and therapeutic efficacy [23], [24]. In this study, we explore the use of trastuzumab-decorated liposomes for the co-delivery of tamoxifen, a selective estrogen receptor modulator and anti-ABCB1 siRNA. Although tamoxifen is not a conventional chemotherapeutic agent, it plays a crucial role in inhibiting breast cancer cell proliferation by modulating estrogen receptor activity [25], [26]. The inclusion of anti-ABCB1 siRNA aims to downregulate ABCB1 expression, thereby enhancing tamoxifen's intracellular retention and effectiveness. Our approach seeks to reinforce anticancer potential in HER2-positive breast cancer cells by combining targeted delivery with gene silencing techniques. By leveraging trastuzumab's specificity alongside the therapeutic actions of tamoxifen and anti-ABCB1 siRNA, we aim to develop a novel treatment strategy that addresses drug resistance and improves patient outcomes. 2. Materials and Methods 2.1. Materials Soy-phosphatidylcholine (S-100) was kindly supplied by LIPOID GmbH, Germany. Didodecyldimethylammonium bromide (DDAB) was sourced from TCI Chemicals, Chennai, India. Cholesterol was acquired from Qualigens Fine Chemicals, Mumbai, India. DSPE-PEG(2000) Carboxylic Acid was obtained from Nanosoft Polymers, USA. Lyophilized Trastuzumab powder (Eleftha®) was sourced from the pharmacy at Kasturba Medical College and Hospital, Manipal, India. Tamoxifen citrate was generously provided as a gift by Neon Labs, Maharashtra, India. Anti-ABCB1-siRNA was purchased from CST, USA. The reagents EDC and NHS were purchased from Sigma-Aldrich, USA. High-performance liquid chromatography (HPLC) grade solvents were supplied by Merck India Private Limited, Mumbai, India. DMEM high-glucose media, fetal bovine serum (FBS), and antibiotic-antimycotic 100X solution were procured from Gibco, USA. Corning® Spin-X® UF concentrators (6 mL, 10 kDa molecular weight cut-off [MWCO]) were taken from Corning, Arizona, USA. Dialysis membranes (12 kDa MWCO) were obtained from HiMedia Laboratories, Maharashtra, India. BT-474 cell lines were generously provided by the Department of Pharmacology and Toxicology, NIPER Hyderabad, India. MCF10A cell lines were sourced from the NCCS Pune, Maharashtra, India. 2.2. Methods 2.2.1. Preparation of liposomes. The preparation of liposomal nanoformulations was conducted using a modified method involving thin film formation, followed by hydration using phosphate buffer saline (PBS) and probe sonication [27], [28]. In brief, S-100 (65.2 mg), DDAB (27.42 mg), cholesterol (7.34 mg), and HOOC-DSPE-PEG-2000 (5mg) were initially dissolved in 9 mL of chloroform. Concurrently, Tamoxifen (10 mg) was dissolved in 1 mL of methanol and subsequently incorporated into the lipid solution. The organic solvents, namely chloroform and methanol, were evaporated using a rotary evaporator set at 45°C, resulting in the formation of a thin lipid film. This film was then placed in a vacuum desiccator overnight to ensure complete removal of any residual organic solvents. Once the solvents were fully eliminated, the dried lipid films were hydrated with 10 mL of PBS pH 7.4, and heated to 60°C, to facilitate liposome formation. The resulting liposomes underwent sonication at 40% amp with a cycle of 10 seconds ON and 2 seconds OFF for a total duration ranging from 5 to 15 minutes, aimed at reducing their particle size. The liposomes were subsequently kept at a cool temperature. For purification, the liposomal suspension was transferred into Corning® Spin-X® UF tubes and centrifuged at 3000 rpm for one hour [29]. This step allowed for the separation of the supernatant containing unentrapped drugs from the liposomal formulation. The collected supernatant was then analyzed to determine the entrapment efficiency using an indirect method based on HPLC analysis (Eq. 1). \(\:\varvec{\%}\:\varvec{D}\varvec{r}\varvec{u}\varvec{g}\:\varvec{E}\varvec{n}\varvec{t}\varvec{r}\varvec{a}\varvec{p}\varvec{m}\varvec{e}\varvec{n}\varvec{t}=\frac{\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{d}\varvec{r}\varvec{u}\varvec{g}\:\varvec{a}\varvec{d}\varvec{d}\varvec{e}\varvec{d}\:\varvec{i}\varvec{n}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\varvec{s}\:-\:\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{d}\varvec{r}\varvec{u}\varvec{g}\:\varvec{i}\varvec{n}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\:\varvec{s}\varvec{u}\varvec{p}\varvec{e}\varvec{r}\varvec{n}\varvec{a}\varvec{t}\varvec{a}\varvec{n}\varvec{t}}{\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{d}\varvec{r}\varvec{u}\varvec{g}\:\varvec{a}\varvec{d}\varvec{d}\varvec{e}\varvec{d}\:\varvec{i}\varvec{n}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\varvec{s}}\varvec{X}100\) ………Eq. 1 2.2.2. siRNAs coupling to the liposomes. The prepared liposomes containing tamoxifen citrate were taken in Corning® Spin-X® UF tubes and centrifuged at 3000 rpm until the thick liposomes were left, and then fresh RNase-free water was added and centrifuged again to wash the liposomes twice. The thick suspension of washed liposomes was resuspended in 1 mL of RNase-free water and incubated with siRNA, approximately 3.5µg (20:1 N/P ratio) to 100mg of total lipid formulation, at room temperature for 2 hours for interacting the siRNAs with positively charged liposomes and bound to the surface by electrostatic interaction. After 2h, the liposomes were centrifuged, and the supernatant was used for the calculation of unentrapped siRNA (Eq. 2). The pellet was washed again with RNAs free water and resuspended in MES buffer pH 5.5 for surface modification with Trastuzumab monoclonal antibody [28], [30], [31]. $$\:\varvec{\%}\:\varvec{s}\varvec{i}\varvec{R}\varvec{N}\varvec{A}\:\varvec{E}\varvec{n}\varvec{t}\varvec{r}\varvec{a}\varvec{p}\varvec{m}\varvec{e}\varvec{n}\varvec{t}=$$ \(\:\frac{\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{s}\varvec{i}\varvec{R}\varvec{N}\varvec{A}\:\varvec{a}\varvec{d}\varvec{d}\varvec{e}\varvec{d}\:\varvec{t}\varvec{o}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\varvec{s}\:-\:\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{s}\varvec{i}\varvec{R}\varvec{N}\varvec{A}\:\varvec{i}\varvec{n}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\:\varvec{s}\varvec{u}\varvec{p}\varvec{e}\varvec{r}\varvec{n}\varvec{a}\varvec{t}\varvec{a}\varvec{n}\varvec{t}}{\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{s}\varvec{i}\varvec{R}\varvec{N}\varvec{A}\:\varvec{a}\varvec{d}\varvec{d}\varvec{e}\varvec{d}\:\varvec{t}\varvec{o}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\varvec{s}}\varvec{X}100\) …………………………Eq. 2 2.2.3. Trastuzumab functionalization of liposomes. Carboxyl-functionalized liposomes (1 mL) were activated through the addition of 200 µL of each EDC (4 mg/mL) and NHS (6 mg/mL). This activation mixture was incubated at room temperature with gentle shaking for 30 minutes to facilitate the formation of reactive intermediates [32], [33]. Following the activation step, the liposomes were subjected to centrifugation, and the resulting pellet was carefully washed twice with MES buffer (pH 5.5) to remove any unreacted reagents. The pellet was then resuspended in 1 mL of the same MES buffer to maintain optimal conditions for subsequent reactions. To the activated liposome suspension, 200 µL of trastuzumab solution (2.5 mg/mL) was added. The mixture was incubated at room temperature with shaking for a duration of 4 hours, allowing for effective conjugation of the antibody to the liposomal surface. After incubation, the trastuzumab-conjugated liposomes were centrifuged for 45 minutes at a cool temperature at 17,500 rpm to separate them from any unbound antibodies. The resulting pellet was washed twice with PBS at pH 7.4 and then resuspended in PBS for storage. The conjugated liposomes were kept at a temperature range of 2–8°C until further analysis. The efficiency of trastuzumab conjugation to the liposomes was quantified using Eq. 3. The conjugation chemistry utilized in this process, specifically the EDC-NHS method, is depicted in Fig. 1 , illustrating the mechanism by which the antibody is covalently linked to the liposomal surface [28], [34]. \(\:\varvec{\%}\:\varvec{T}\varvec{r}\varvec{a}\varvec{s}\varvec{t}\varvec{u}\varvec{z}\varvec{u}\varvec{m}\varvec{a}\varvec{b}\:\varvec{c}\varvec{o}\varvec{n}\varvec{j}\varvec{u}\varvec{g}\varvec{a}\varvec{t}\varvec{i}\varvec{o}\varvec{n}=\frac{\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{t}\varvec{r}\varvec{a}\varvec{s}\varvec{t}\varvec{u}\varvec{z}\varvec{u}\varvec{m}\varvec{b}\:\varvec{a}\varvec{d}\varvec{d}\varvec{e}\varvec{d}\:\varvec{t}\varvec{o}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\varvec{s}\:-\:\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{t}\varvec{r}\varvec{a}\varvec{s}\varvec{t}\varvec{u}\varvec{z}\varvec{u}\varvec{m}\varvec{b}\:\varvec{i}\varvec{n}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\:\varvec{s}\varvec{u}\varvec{p}\varvec{e}\varvec{r}\varvec{n}\varvec{a}\varvec{t}\varvec{a}\varvec{n}\varvec{t}}{\varvec{A}\varvec{m}\varvec{o}\varvec{u}\varvec{n}\varvec{t}\:\varvec{o}\varvec{f}\:\varvec{t}\varvec{r}\varvec{a}\varvec{s}\varvec{t}\varvec{u}\varvec{z}\varvec{u}\varvec{m}\varvec{b}\:\varvec{a}\varvec{d}\varvec{d}\varvec{e}\varvec{d}\:\varvec{t}\varvec{o}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{l}\varvec{i}\varvec{p}\varvec{o}\varvec{s}\varvec{o}\varvec{m}\varvec{e}\varvec{s}}\varvec{X}100\) ………………….Eq. 3 2.2.4. Characterization of liposomes. The developed liposomes were evaluated for zeta potential, particle size, and shape using a Malvern Zetasizer and Scanning Electron Microscopy. 2.2.5. Drug release study of liposomes. Tamoxifen release from liposomes was assessed using a dialysis method. A solution of tamoxifen (1 ml of 4 mg/mL) and a suspension of tamoxifen-loaded liposomes (1 mL, 4 mg/mL equivalent) were separately placed in dialysis bags (12 kDa MWCO). Each dialysis bag was then immersed in 40 mL of release medium consisting of PBS with 25% methanol (75:25 v/v). The release study was conducted at 100 rpm using a magnetic stirrer. At predetermined time points (0, 0.5, 1, 2, 4, 8, 12, 24, and 48 hours), 300 µL samples were withdrawn from the vials and replaced with an equal volume of fresh release medium [35], [36], [37], [38]. The concentration of tamoxifen in each sample was determined by HPLC using a standard calibration curve [28]. The release profile was generated using GraphPad Prism version 8.0.2. 2.2.6. Stability study of siRNAs in liposomes (TMX-FL). The stability of siRNA encapsulated in liposomes was assessed by incubating the liposomal formulations in fetal bovine serum (FBS) at a 1:1 ratio, maintained at 37°C. Samples were collected at predetermined time intervals of 2, 4, 8, and 12 hours to monitor the integrity of the siRNA. At each time point, the collected samples were treated with 10% sodium dodecyl sulfate (SDS) at a 1:10 ratio to facilitate the disruption of liposomal structures and inactivate serum components. The samples were then incubated in a water bath set to 55°C for 30 minutes, ensuring effective release of siRNA from the liposomes [39], [40], [41]. Following the incubation period, the samples were mixed with a loading dye and subjected to electrophoresis on a 1% agarose gel using TBE buffer (pH 8.3). The gel was run at a voltage of 90 V for 45 minutes to separate the siRNA based on size. After electrophoresis, the gel was visualized and imaged using a gel documentation system to assess the presence and integrity of siRNA. The results from this analysis provided insights into the stability of the siRNA within the liposomal formulation when exposed to biological conditions mimicking those found in vivo [28], [30], [42]. 2.2.7. Hemocompatibility study of liposomes. The hemocompatibility of the developed liposomes was assessed using a modified version of a previously established method [28], [43]. Blood samples were obtained from Sprague-Dawley (SD) rats in accordance with the Institutional Animal Ethics Committee approval (Ref No- IAEC/KMC/92/2021) through retro-orbital plexus puncture, collected into EDTA-containing microcentrifuge tubes. Erythrocytes were isolated by centrifuging the blood at 5000 rpm for 10 minutes. The resulting erythrocyte pellet was washed twice with an equal volume of PBS and subsequently resuspended in an equal volume of PBS to ensure uniformity for the hemolysis assay. For the hemolysis assay, 200 µL of the washed erythrocytes were allocated for each experimental condition. Negative control samples consisted of erythrocytes incubated with 800 µL of PBS, while positive control samples included erythrocytes treated with 800 µL of 2% Triton X-100, which is known to induce complete hemolysis. Test samples comprised erythrocytes incubated with 800 µL of tamoxifen-loaded liposomes at a concentration of 5 mg/mL in PBS. All sample conditions were prepared in triplicate within 2 mL tubes and incubated at 37°C for 2 hours with shaking to facilitate interaction [44], [45]. After the incubation period, the microcentrifuge tubes were left open to air for 10 minutes to allow for hemoglobin oxidation. Aliquots (20 µL) from each sample were then transferred to a 96-well microplate in triplicate, followed by the addition of 180 µL of PBS to each well. The plate was shaken for 5 minutes, and the optical density was measured at 540 nm using a microplate reader to quantify hemolysis. The percentage of hemolysis was calculated using Eq. 4. This systematic evaluation provides critical insights into the hemocompatibility profile of the liposomal formulations, essential for their potential therapeutic applications in vivo. The findings contribute to understanding how these liposomes interact with blood components, which is vital for ensuring their safety and efficacy in clinical settings [28], [43]. % Hamolysis = \(\:\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\left(\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\right)\:-\:\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\left(\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\left(\text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)\:-\:\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\left(\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}*100\) ……………..Eq. 4. 2.2.8. In vitro assessment of liposomes. 2.2.8.1. Cytotoxicity study. BT-474 and MCF10A cell lines were cultured in DMEM high glucose medium with supplements. After reaching 80% confluence, cells were harvested and seeded into 96-well plates for MTT assay cytotoxicity evaluation [28], [46], [47]. The cytotoxic effects of tamoxifen (TMX), tamoxifen-loaded stealth liposomes (TMX-SL), and trastuzumab-functionalized liposomes loaded with siRNA and tamoxifen (TMX-FL) were evaluated. Cells were seeded at a density of 1×10 4 cells per well. Tamoxifen citrate was initially dissolved in dimethyl sulfoxide (DMSO) and then diluted in DMEM complete medium, ensuring that the final DMSO concentration remained below 1%. The liposomal formulations were suspended in DMEM complete medium and serially diluted to achieve concentrations ranging from 50 µg/mL to 1.25 µg/mL. After seeding, the cells were incubated with the standard tamoxifen solution, liposomal formulations, or vehicle control for a period of 24 hours at 37°C in a humidified atmosphere with 5% CO₂. After incubation, the media was replaced with fresh medium, and 10 µL of MTT solution (5 mg/mL) was added to each well. The plates were incubated for 4 hours at 37°C. Then, the medium was removed, and 100 µL of DMSO was added to dissolve formazan crystals. Optical density (OD) was measured at 540 nm using an ELISA plate reader, and cell death percentage was calculated using Eq. 5 [28], [46]. \(\:\%\:Cell\:death\:=\frac{OD\:of\:control\:cells\:-\:OD\:of\:treated\:cells}{OD\:of\:control\:cells}*100\) …………………………………………. Eq. 5 Data analysis was performed using GraphPad Prism to determine the IC50 values and to compare the effects of standard tamoxifen with those of the liposomal formulations. 2.2.8.2. Cell uptake study. Sub-confluent BT-474 cells were cultured in 12-well plates and treated with either fluorescein isothiocyanate (FITC) or FITC-loaded liposomes at a concentration of 50 µg/mL. After a 4-hour incubation period, the cells were harvested using a trypsin-EDTA solution to detach them from the culture surface. Following detachment, the cells were washed twice with cold phosphate-buffered saline (PBS) to remove any residual trypsin and unbound FITC. The washed cells were then centrifuged at 1200 rpm for 3 minutes to pellet the cells, after which the supernatant was discarded. The cell pellets were resuspended in PBS to prepare them for flow cytometric analysis. The fluorescence intensity of the resuspended cells was subsequently analyzed using a BD Accuri flow cytometer. This analysis allowed for the quantification of FITC uptake by the BT-474 cells, providing insights into the efficiency of cellular internalization of both FITC and FITC-loaded liposomes [28], [48], [49]. 2.2.9. Biomarkers estimation. Western blot analysis was conducted to evaluate HER2, ABCB1, BCL2, and GAPDH expression in BT-474 cells. Cells were cultured in DMEM high glucose medium at 37°C with 5% CO₂ until 80% confluence, then treated with TMX (25 µg/mL), TMX-SL, or TMX-FL for 24 hours. Cells were lysed with cold RIPA buffer, and proteins were extracted and quantified using a BCA assay. Proteins were denatured, separated by SDS-PAGE at 70 V, and transferred to PVDF membranes at 90 V. Membranes were blocked with 3% BSA, incubated with primary antibodies overnight at a cool temperature, washed with 1X TBST, and incubated with secondary antibodies for 2 hours. Protein bands were visualized using chemiluminescence and analyzed with ImageJ and GraphPad Prism software [18], [28], [50]. 3. Results 3.1. Characterization of liposomes. The prepared liposomes were characterized in terms of particle size (PS) distribution, polydispersity index (PdI), zeta potential (ZP), and entrapment efficiency. The characteristics of the liposomes are summarized in Table 1 . Figures 2 A and 2 B show the PS, PdI, and ZP of the TMX-FL. The entrapment efficiency was determined to be 81.75 ± 0.48% for TMX-SL and 79.02 ± 0.46% for TMX-FL. For TMX-FL, the siRNA entrapment efficiency was 79.85 ± 0.14%, corresponding to approximately 13 ± 1 ng siRNA/mg liposomes. The trastuzumab conjugation efficiency to TMX-FL was 61.39 ± 2.18%, equivalent to approximately 4 µg trastuzumab/mg liposomes. Table 1 Particle size, PdI, and Zeta potential of optimized tamoxifen-loaded liposomes. Values are given as Mean ± SD, n = 3. Sample name PS (d.nm) PDI ZP (mV) %TMX Entrapped %siRNA Entrapped %Trastuzumab conjugated Simple Liposomes containing Tamoxifen (TMX-SL) 66.23 ± 0.07 0.29 ± 0.01 32.81 ± 0.38 81.75 ± 0.48 - - Trastuzumab-decorated Liposomes containing siRNA and Tamoxifen (TMX-FL) 97.17 ± 0.2 0.23 ± 0.002 16.93 ± 0.25 79.02 ± 0.46 79.85 ± 0.14 61.39 ± 2.18 3.2. Surface morphology of optimized liposomes. The surface morphology of the optimized liposomes (TMX-FL) was examined using scanning electron microscopy (SEM). The SEM image (Fig. 3 ) shows that the optimized liposomes exhibited a spherical morphology. 3.3. Drug release study of liposomes. The drug release percentages of the optimized liposomes at different time intervals are shown in Fig. 4 . The pure TMX group exhibited a burst release of 55.97 ± 6.19% in 0.5h, 83.38 ± 4.82% in 4h, and 99.75 ± 9.72% in 12h. The TMX-SL group showed a release of 3.28 ± 0.96% in 0.5h, 15.00 ± 1.98% in 4h, 24.97 ± 3.40% in 12h, and 58.18 ± 3.88% in 48h. The TMX-FL group showed a release of 2.50 ± 0.35% in 0.5h, 11.56 ± 2.06% in 4h, 20.41 ± 3.47% in 12h, and 53.43 ± 8.50% in 48h. This slow-release pattern demonstrates prolonged retention of tamoxifen within the liposomes, highlighting its potential for optimal delivery to HER2-positive cancer cells. 3.4. Stability study of siRNAs in liposomes (TMX-FL). The serum stability study demonstrated that the siRNA encapsulated within the liposomes remained stable for up to 12 hours. After 4 hours, approximately 58% of the encapsulated siRNA was still intact. This value decreased to 33% at 8 hours and 13% at 12 hours. Agarose gel electrophoresis (Figs. 5 and 6 ) shows the siRNA bands, relative band intensity, and the percentage of siRNA degradation over time. 3.5. In vitro assessment of liposomes. 3.5.1. Cytotoxicity study The optimized liposomes were evaluated for their anticancer efficacy against HER2-positive BT-474 breast cancer cells and HER2-negative MCF10A mammary epithelial cells to assess their selectivity and therapeutic potential. The percentage of cell death for each treatment is shown in Fig. 7 . Blank liposomes showed negligible cytotoxicity, with cell viability remaining high at both tested concentrations (cell death: 0.97 ± 0.26% at 30 µg/mL and 1.81 ± 0.20% at 50 µg/mL). In contrast, free tamoxifen (TMX) exhibited moderate cytotoxicity, inducing 23.65 ± 3.52% cell death at 30 µg/mL and 64.85 ± 1.39% at 50 µg/mL. The TMX-SL demonstrated enhanced anticancer activity compared to free TMX, with 43.77 ± 1.94% cell death at 30 µg/mL and 72.06 ± 0.93% at 50 µg/mL. Remarkably, TMX-FL displayed the highest cytotoxicity, achieving 58.97 ± 1.29% cell death at 30 µg/mL and 86.88 ± 0.50% at 50 µg/mL (p < 0.05 vs. TMX and TMX-SL), underscoring their superior therapeutic efficacy. In HER2-negative MCF10A cells, treatment with TMX-FL induced modest cytotoxicity, with 8.65 ± 1.55% cell death at 30 µg/mL and 19.70 ± 2.25% at 50 µg/mL (Fig. 8 ). In stark contrast, HER2-positive BT-474 cells exhibited significantly higher sensitivity to TMX-FL, showing 65.47 ± 1.34% cell death at 30 µg/mL and 90.74 ± 1.10% at 50 µg/mL. Statistical analysis (unpaired t-test with Welch’s correction) confirmed that TMX-FL has selective and enhanced cytotoxicity toward HER2-positive BT-474 cells compared to HER2-negative MCF10A cells (p < 0.05), highlighting its potential for targeted therapy. 3.5.2. Cell uptake study of liposomes. Cellular uptake studies in HER2-positive BT-474 cells revealed that trastuzumab-functionalized liposomes (FITC-FL) were internalized significantly more efficiently than non-targeted FITC-loaded stealth liposomes (FITC-SL) following a 4-hour incubation (Fig. 9 ). Statistical analysis (one-way ANOVA with Tukey’s post hoc test) confirmed highly significant differences in uptake between the groups (**p < 0.01, ***p < 0.001), underscoring the critical role of trastuzumab-mediated targeting in enhancing liposomal delivery to HER2-overexpressing cells. 3.5.3. Biomarkers estimation. After a 24-hour exposure to standard TMX, TMX-SL, or TMX-FL, cells were harvested for western blot analysis. The results demonstrated a marked downregulation of key proteins, including BCL2, ABCB1, and HER2, with the most pronounced reduction observed in TMX-FL-treated cells (Figs. 10 A and 10 B). Statistical analysis (two-way ANOVA with Bonferroni’s post hoc test) confirmed significant differences in protein expression across treatment groups (p < 0.05), highlighting the superior efficacy of TMX-FL in modulating these critical therapeutic targets. 4. Discussion Tamoxifen (TMX), a selective estrogen receptor modulator, has been a cornerstone in the treatment of hormone receptor-positive breast cancer. However, its clinical application is often limited by poor aqueous solubility, systemic toxicity, and the development of multidrug resistance, particularly in HER2-positive subtypes. To address these challenges, we developed a novel trastuzumab-conjugated liposomal formulation co-loaded with tamoxifen and anti-ABCB1 siRNA (TMX-FL) for targeted therapy against HER2-overexpressing breast cancer. The optimized TMX-FL liposomes exhibited a particle size of 97.17 ± 0.2 nm and a zeta potential of 16.93 ± 0.25 mV, which are within the optimal range for enhanced permeability and retention (EPR)-based tumor targeting. The entrapment efficiencies for tamoxifen and siRNA were 79.02 ± 0.46% and 79.85 ± 0.14%, respectively, with a trastuzumab conjugation efficiency of 61.39 ± 2.18%. These values are comparable to our previously reported study by Kumar et al. (2024) [28], who developed trastuzumab-conjugated liposomes co-loaded with paclitaxel and anti-ABCB1 siRNA, achieving similar conjugation efficiency but with a larger particle size (229 ± 4 nm), which may limit tumor penetration compared to our smaller-sized TMX-FL in the present study. Scanning electron microscopy confirmed the spherical morphology of TMX-FL, which is favorable for uniform biodistribution and cellular uptake. Drug release studies demonstrated a sustained release profile, with only 53.43 ± 8.50% of tamoxifen released over 48 hours, in contrast to the burst release observed with free tamoxifen (99.75 ± 9.72% in 12 hours). This controlled release is consistent with findings by Du et al. (2024) [51], who reported similar sustained release behavior in trastuzumab-functionalized liposomes loaded with pyrotinib, reinforcing the advantage of liposomal encapsulation in prolonging drug action. The serum stability study showed that siRNA encapsulated in TMX-FL remained protected for up to 12 hours, with 58% intact at 4 hours. This aligns with previous reports, such as those by Mainini and Eccles (2020) [52], which demonstrated that lipid-based nanocarriers can effectively shield siRNA from enzymatic degradation, thereby enhancing gene silencing efficiency. In vitro cytotoxicity assays revealed that TMX-FL exhibited significantly higher anticancer activity against HER2-positive BT-474 cells compared to free tamoxifen and non-targeted liposomes (TMX-SL). At 50 µg/mL, TMX-FL induced 86.88 ± 0.50% cell death, outperforming TMX-SL (72.06 ± 0.93%) and free TMX (64.85 ± 1.39%). These results are comparable to those reported by Kumar et al. (2024) [28], where their paclitaxel-based formulation also showed enhanced cytotoxicity in HER2-positive models. However, our study uniquely demonstrates the efficacy of tamoxifen in a similar dual-delivery system, offering a potentially safer and more cost-effective alternative. Importantly, TMX-FL showed selective cytotoxicity, with minimal toxicity in HER2-negative MCF10A cells, highlighting its targeted therapeutic potential. Cellular uptake studies confirmed that trastuzumab-functionalized liposomes were internalized significantly more efficiently than non-targeted liposomes, consistent with the findings of Du et al. (2024) [51] and Zafar et al. (2024) [53], who emphasized the role of HER2-targeting in enhancing intracellular delivery. Western blot analysis further validated the therapeutic efficacy of TMX-FL, showing significant downregulation of HER2, ABCB1, and BCL2 proteins. This multi-target modulation is critical for overcoming drug resistance and inducing apoptosis and is in line with the results of Gao et al. (2022) [20], who demonstrated that co-delivery of chemotherapeutics and siRNA can synergistically suppress oncogenic signaling pathways. In summary, our study demonstrates that TMX-FL liposomes offer a promising strategy for the targeted treatment of HER2-positive breast cancer by combining the benefits of controlled drug release, siRNA-mediated gene silencing, and trastuzumab-guided delivery. Compared to recent studies using paclitaxel or pyrotinib, our tamoxifen-based system achieves comparable efficacy with potentially improved safety and cost-effectiveness. 5. Conclusion and future perspectives To achieve targeted delivery of tamoxifen along with anti-ABCB1 siRNA, trastuzumab-decorated multifunctional liposomes (TMX-FL) were successfully developed and evaluated for their HER2-targeting efficiency and anticancer potential against HER2-positive breast cancer. The optimized liposomes met all critical formulation criteria, including appropriate particle size, surface charge, and high entrapment efficiencies for both tamoxifen and siRNA. Trastuzumab conjugation enabled selective targeting of HER2-overexpressing BT-474 cells, as confirmed by cellular uptake studies using flow cytometry. In vitro cytotoxicity assays demonstrated that TMX-FL exhibited significantly enhanced anticancer activity compared to free tamoxifen and non-targeted liposomes, owing to the synergistic effects of tamoxifen, anti-ABCB1 siRNA, and trastuzumab. Furthermore, western blot analysis revealed a marked downregulation of HER2, ABCB1, and BCL2 protein expression in TMX-FL-treated BT-474 cells, indicating effective modulation of key pathways involved in tumor progression and drug resistance. These findings suggest that the trastuzumab-decorated liposomes co-loaded with tamoxifen and siRNA represent a promising nanotherapeutic strategy for the treatment of HER2-positive breast cancer. Future in vivo studies and clinical translation efforts are warranted to further validate the therapeutic potential of this formulation. Declarations The authors declare no conflict of interest. Ethical Approval All animal experiments were conducted in compliance with the ARRIVE guidelines and the National Research Council’s Guide for the Care and Use of Laboratory Animals (Eighth Edition). The Institutional Animal Ethics Committee (IAEC) of the Kasturba Medical College, Manipal, India, reviewed and approved all procedures on October 23, 2021, under approval Reference No. IAEC/KMC/92/2021. Funding Declaration No funding was received for this work. Author Contribution G.K. wrote the main manuscript. F.B., P.C.G., and P.M. collected and acquired data. K.N., S.M., and C.M.R. guided and reviewed the manuscript. Acknowledgment The authors sincerely acknowledge the Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education (MAHE), Karnataka, India, for providing essential infrastructure, research facilities, and library resources. 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Supplementary Files 1.SupplementaryfileHER2Blots.jpg 2.SupplementaryfileABCB1Blots.jpg 3.SupplementaryfileBCL2bLOTS.jpg 4.SupplementaryfileGAPDHBlots.jpg 5.SupplementaryfilesiRNAstabilityAgaroseGel.tif Cite Share Download PDF Status: Published Journal Publication published 17 Sep, 2025 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 25 Jul, 2025 Reviews received at journal 23 Jul, 2025 Reviews received at journal 18 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 07 Jul, 2025 Editor assigned by journal 07 Jul, 2025 Submission checks completed at journal 04 Jul, 2025 First submitted to journal 02 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7025151","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481608002,"identity":"b087677f-0e62-4c3e-b0a6-db12d89daf94","order_by":0,"name":"Gautam Kumar","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Gautam","middleName":"","lastName":"Kumar","suffix":""},{"id":481608005,"identity":"d4d7a10e-0d46-4799-a8a5-ec0336a2a77c","order_by":1,"name":"Farmiza Begum","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Farmiza","middleName":"","lastName":"Begum","suffix":""},{"id":481608006,"identity":"f13d1452-c35c-41e7-b865-a07b22cefd2f","order_by":2,"name":"Prasada Chowdari Gurram","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Prasada","middleName":"Chowdari","lastName":"Gurram","suffix":""},{"id":481608008,"identity":"18192a8c-51ff-4555-b271-dc416f281591","order_by":3,"name":"Bharath H. B.","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Bharath","middleName":"H.","lastName":"B.","suffix":""},{"id":481608009,"identity":"06455dc4-766b-4b53-bfd1-230b161034ce","order_by":4,"name":"Prashansha Mullick","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Prashansha","middleName":"","lastName":"Mullick","suffix":""},{"id":481608010,"identity":"b7a506d4-06b4-4dd2-8d84-0fd82f0fa9e2","order_by":5,"name":"Krishnadas Nandakumar","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Krishnadas","middleName":"","lastName":"Nandakumar","suffix":""},{"id":481608011,"identity":"c15da0af-6825-4980-a724-9dd1b6143859","order_by":6,"name":"Srinivas Mutalik","email":"","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Srinivas","middleName":"","lastName":"Mutalik","suffix":""},{"id":481608012,"identity":"e35ec14d-72df-4249-999a-96f11093059b","order_by":7,"name":"Chamalamudi Mallikarjuna Rao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYFAC5sYDCQfADBApwcDADqR48GphbIBqYUuAaGEmRgsDWAuPAdRaAlp02w82HHhwhiGfv73n64afOyzy+JsZGB+8bcOtxexMItBhNxgsZ5w5u+1m7xmJYonDDMyGc/FpOQDS8oHBgOFG7rYbvG0SiQ2HGdikefFpOf8QokX+Rs6zm3+BWuYfZmD/jVfLDYjDDAxu5LDdBtmyAWgLM34tIFvOSBgYnjlmdlu2TaLY8DBjs+Scc/gclnzw4Y9jNgZyx5uf3XzbVpcHZBz88KYMtxYokICzEkAxRVA9CkggTfkoGAWjYBSMBAAAJjBdqI6kjQIAAAAASUVORK5CYII=","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":true,"prefix":"","firstName":"Chamalamudi","middleName":"Mallikarjuna","lastName":"Rao","suffix":""}],"badges":[],"createdAt":"2025-07-02 04:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7025151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7025151/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02149-1","type":"published","date":"2025-09-17T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86396490,"identity":"65b0fcd2-2e99-4d8d-b4a7-714784e59f31","added_by":"auto","created_at":"2025-07-10 07:59:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116451,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of EDC-NHS chemistry for trastuzumab (monoclonal antibody) conjugation on the surface of liposomes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/6e627f2aaae4a6d47824c5d9.png"},{"id":86395684,"identity":"0aecbb53-dde6-4527-b9be-4051c4a254ab","added_by":"auto","created_at":"2025-07-10 07:51:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eParticle size distribution of TMX-FL, and \u003cstrong\u003eB)\u003c/strong\u003e Zeta potential of TMX-FL.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/0090408492c7ffa8e3801f7a.png"},{"id":86395306,"identity":"a46bd6f6-ea34-4b62-beb8-5e48208b6d12","added_by":"auto","created_at":"2025-07-10 07:43:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1300739,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Microscopic images of Trastuzumab-decorated siRNA and Tamoxifen-loaded Liposomes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/100c3f45ec21a59098b4d00c.png"},{"id":86395314,"identity":"c7257a39-65ee-43f7-9ca7-174b2a649820","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23468,"visible":true,"origin":"","legend":"\u003cp\u003eTamoxifen release profile of optimized liposomes. \u003cem\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eTMX = Pure tamoxifen; TMX-FL = Trastuzumab-decorated liposomes containing siRNA and tamoxifen. Values are given as Mean ± SD, n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/022329b95e78210daedbfd5f.png"},{"id":86395688,"identity":"02d45d46-444d-4210-be71-e0a03f887f47","added_by":"auto","created_at":"2025-07-10 07:51:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":336879,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel for Serum stability of siRNA-loaded liposomes (TMX-FL).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/9000443a6ada36c9a8fe4428.png"},{"id":86395321,"identity":"7efba0a6-a880-42a4-b466-9ee799769211","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eRelative band intensity of siRNA;\u003cstrong\u003e B) \u003c/strong\u003e% siRNA degradation.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/8697e06cd07f7af16e78ae0a.png"},{"id":86395317,"identity":"8a40111f-89eb-4782-bb84-20db25a65d4d","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":46939,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytotoxicity of different concentrations of tamoxifen and TMX-liposomes in BT474 cell by MTT assay. \u003cem\u003eData are presented as mean ± standard deviation (SD) from three independent experiments (n=3). Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. The following notation indicates statistical significance: ns (non-significant), *p \u0026lt; 0.05, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/244554c9a22234ae53033fde.png"},{"id":86397036,"identity":"58c23810-0da8-4721-becb-4677f29cb1ec","added_by":"auto","created_at":"2025-07-10 08:07:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":38143,"visible":true,"origin":"","legend":"\u003cp\u003eThe comparison of cytotoxic potential of different concentrations of optimized tamoxifen-loaded liposomes in BT-474 and MCF10A cells after 24 h treatment by MTT assay. \u003cem\u003eStatistical analysis was performed using a t-test with the non-parametric Mann-Whitney test. A * indicates a p-value \u0026lt; 0.05, denoting statistically significant differences between the two cell lines.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/d5d701c4c2eb780d1fe52727.png"},{"id":86395312,"identity":"ec06dc7b-f7ed-46d4-9a74-b3fb8802ce5d","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":96094,"visible":true,"origin":"","legend":"\u003cp\u003eCellular uptake analysis of trastuzumab-decorated FITC-loaded liposomes in BT-474 cells \u003cem\u003ein vitro\u003c/em\u003e using flow cytometry. \u003cem\u003e(Values were taken as Mean ± SD, n=2).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/747088fa84f5e5e0b82664f7.png"},{"id":86395328,"identity":"4cb876b0-eb5e-4646-9ddc-0f7edbd39cb6","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":137327,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis of BT-474 cells samples after 24h of treatment. \u003cstrong\u003eA)\u003c/strong\u003e Blots showing expression of molecular markers and \u003cstrong\u003eB)\u003c/strong\u003eBar diagram showing significant difference in expression of molecular markers.\u003cem\u003e(Values were taken as Mean ± SD, n=3).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/ffd5b277d6b9d216ce155ec3.png"},{"id":91889759,"identity":"ecc53816-f74c-4682-8510-08bd9be11b56","added_by":"auto","created_at":"2025-09-22 15:59:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3098918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/8fb36f7b-3542-4211-a11f-149836f37923.pdf"},{"id":86395318,"identity":"6efd70c5-3b94-44a3-a0e4-a1f9a4d51f51","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":442989,"visible":true,"origin":"","legend":"","description":"","filename":"1.SupplementaryfileHER2Blots.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/1aa7ed98a135696f7c938976.jpg"},{"id":86395693,"identity":"91bcb445-6485-420c-b4c3-79b8eb6daed7","added_by":"auto","created_at":"2025-07-10 07:51:42","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":666272,"visible":true,"origin":"","legend":"","description":"","filename":"2.SupplementaryfileABCB1Blots.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/1eddb33df11972d331f1d21c.jpg"},{"id":86395682,"identity":"e93a3d34-adb3-4139-88c5-810ea078b0a8","added_by":"auto","created_at":"2025-07-10 07:51:40","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":77823,"visible":true,"origin":"","legend":"","description":"","filename":"3.SupplementaryfileBCL2bLOTS.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/8b4b7eaf0e0e2c81747e24bf.jpg"},{"id":86395341,"identity":"369deb20-2cd4-4344-a414-df52b13b3ec5","added_by":"auto","created_at":"2025-07-10 07:43:42","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":482845,"visible":true,"origin":"","legend":"","description":"","filename":"4.SupplementaryfileGAPDHBlots.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/86143e87aa1c2a8354201b36.jpg"},{"id":86395330,"identity":"599f114c-0c58-4f11-89cc-3f27a6e25c16","added_by":"auto","created_at":"2025-07-10 07:43:41","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12115824,"visible":true,"origin":"","legend":"","description":"","filename":"5.SupplementaryfilesiRNAstabilityAgaroseGel.tif","url":"https://assets-eu.researchsquare.com/files/rs-7025151/v1/ae70eec49b2cba97e2047fb3.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Anticancer Activity in HR+/HER2+ Breast Cancer Cells Using Trastuzumab- Conjugated Liposomes co-loaded with siRNA and Tamoxifen","fulltext":[{"header":"1. Background","content":"\u003cp\u003eBreast cancer is the most prevalent cancer among women globally, with approximately 1 in 8 women receiving a diagnosis at some point in their lives. In 2022, there were 2.3\u0026nbsp;million new cases of breast cancer reported worldwide, resulting in 665,684 deaths attributed to the disease [1]. In the United States alone, 367,220 new cases of breast cancer were documented in 2024. Among these cases, HER2-positive breast cancer characterized by the overexpression of the human epidermal growth factor receptor 2 (HER2) and classified into subtypes such as HR+/HER2\u0026thinsp;+\u0026thinsp;and HR-/HER2\u0026thinsp;+\u0026thinsp;constitutes about 13% of all breast cancer cases [2], [3], [4]. This subtype is recognized for its aggressive nature and poorer prognosis compared to HER2-negative breast cancers [5].\u003c/p\u003e\u003cp\u003eThe treatment regimen for triple-positive breast cancer typically involves a combination of therapies targeting hormone receptors and the HER2 protein. Hormone therapy, utilizing agents like tamoxifen and aromatase inhibitors, effectively blocks the actions of estrogen and progesterone, thereby slowing the proliferation of hormone receptor-positive cancer cells [6]. HER2-targeted therapies, including trastuzumab (Herceptin) and pertuzumab (Perjeta), specifically inhibit the HER2 protein, reducing the growth of HER2-positive cancer cells [7], [8]. Chemotherapy is frequently administered in conjunction with hormone and HER2-targeted therapies to eliminate rapidly dividing cancer cells [9], [10]. Surgical interventions such as lumpectomy or mastectomy are performed to excise tumors or affected breast tissue, while radiation therapy employs high-energy radiation to destroy any remaining cancer cells post-surgery [11], [12].\u003c/p\u003e\u003cp\u003eDespite these advancements, several challenges persist in managing HER2-positive breast cancer. Hormone therapies can lead to resistance over time, diminishing their effectiveness. Similarly, HER2-targeted therapies may encounter resistance mechanisms such as mutations in the HER2 gene or activation of alternative signaling pathways [13], [14], [15], [16]. Chemotherapy is often associated with significant side effects, including toxicity to healthy cells, which can limit its application [9]. Although surgical and radiation therapies effectively remove or destroy cancer cells, they do not address the underlying molecular mechanisms driving cancer progression and may result in recurrence [12], [17].\u003c/p\u003e\u003cp\u003eA promising strategy to overcome resistance involves the co-delivery of therapeutic agents using nanotechnology-based systems. Trastuzumab, a monoclonal antibody targeting HER2, has demonstrated efficacy in treating HER2-positive breast cancer; however, its effectiveness can be compromised by resistance mechanisms such as overexpression of the ATP-binding cassette subfamily B member 1 (ABCB1) protein [18], [19], [20], [21], [22]. This protein actively pumps therapeutic drugs out of cancer cells, reducing their intracellular concentrations and therapeutic efficacy [23], [24].\u003c/p\u003e\u003cp\u003eIn this study, we explore the use of trastuzumab-decorated liposomes for the co-delivery of tamoxifen, a selective estrogen receptor modulator and anti-ABCB1 siRNA. Although tamoxifen is not a conventional chemotherapeutic agent, it plays a crucial role in inhibiting breast cancer cell proliferation by modulating estrogen receptor activity [25], [26]. The inclusion of anti-ABCB1 siRNA aims to downregulate ABCB1 expression, thereby enhancing tamoxifen's intracellular retention and effectiveness. Our approach seeks to reinforce anticancer potential in HER2-positive breast cancer cells by combining targeted delivery with gene silencing techniques. By leveraging trastuzumab's specificity alongside the therapeutic actions of tamoxifen and anti-ABCB1 siRNA, we aim to develop a novel treatment strategy that addresses drug resistance and improves patient outcomes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003eSoy-phosphatidylcholine (S-100) was kindly supplied by LIPOID GmbH, Germany. Didodecyldimethylammonium bromide (DDAB) was sourced from TCI Chemicals, Chennai, India. Cholesterol was acquired from Qualigens Fine Chemicals, Mumbai, India. DSPE-PEG(2000) Carboxylic Acid was obtained from Nanosoft Polymers, USA. Lyophilized Trastuzumab powder (Eleftha\u0026reg;) was sourced from the pharmacy at Kasturba Medical College and Hospital, Manipal, India. Tamoxifen citrate was generously provided as a gift by Neon Labs, Maharashtra, India. Anti-ABCB1-siRNA was purchased from CST, USA. The reagents EDC and NHS were purchased from Sigma-Aldrich, USA. High-performance liquid chromatography (HPLC) grade solvents were supplied by Merck India Private Limited, Mumbai, India.\u003c/p\u003e\n \u003cp\u003eDMEM high-glucose media, fetal bovine serum (FBS), and antibiotic-antimycotic 100X solution were procured from Gibco, USA. Corning\u0026reg; Spin-X\u0026reg; UF concentrators (6 mL, 10 kDa molecular weight cut-off [MWCO]) were taken from Corning, Arizona, USA. Dialysis membranes (12 kDa MWCO) were obtained from HiMedia Laboratories, Maharashtra, India. BT-474 cell lines were generously provided by the Department of Pharmacology and Toxicology, NIPER Hyderabad, India. MCF10A cell lines were sourced from the NCCS Pune, Maharashtra, India.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Methods\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1. Preparation of liposomes.\u003c/h2\u003e\n \u003cp\u003eThe preparation of liposomal nanoformulations was conducted using a modified method involving thin film formation, followed by hydration using phosphate buffer saline (PBS) and probe sonication [27], [28]. In brief, S-100 (65.2 mg), DDAB (27.42 mg), cholesterol (7.34 mg), and HOOC-DSPE-PEG-2000 (5mg) were initially dissolved in 9 mL of chloroform. Concurrently, Tamoxifen (10 mg) was dissolved in 1 mL of methanol and subsequently incorporated into the lipid solution. The organic solvents, namely chloroform and methanol, were evaporated using a rotary evaporator set at 45\u0026deg;C, resulting in the formation of a thin lipid film. This film was then placed in a vacuum desiccator overnight to ensure complete removal of any residual organic solvents. Once the solvents were fully eliminated, the dried lipid films were hydrated with 10 mL of PBS pH 7.4, and heated to 60\u0026deg;C, to facilitate liposome formation. The resulting liposomes underwent sonication at 40% amp with a cycle of 10 seconds ON and 2 seconds OFF for a total duration ranging from 5 to 15 minutes, aimed at reducing their particle size. The liposomes were subsequently kept at a cool temperature. For purification, the liposomal suspension was transferred into Corning\u0026reg; Spin-X\u0026reg; UF tubes and centrifuged at 3000 rpm for one hour [29]. This step allowed for the separation of the supernatant containing unentrapped drugs from the liposomal formulation. The collected supernatant was then analyzed to determine the entrapment efficiency using an indirect method based on HPLC analysis (Eq.\u0026nbsp;1).\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{\\%}\\:\\varvec{D}\\varvec{r}\\varvec{u}\\varvec{g}\\:\\varvec{E}\\varvec{n}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{p}\\varvec{m}\\varvec{e}\\varvec{n}\\varvec{t}=\\frac{\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\:\\varvec{a}\\varvec{d}\\varvec{d}\\varvec{e}\\varvec{d}\\:\\varvec{i}\\varvec{n}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\varvec{s}\\:-\\:\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\:\\varvec{i}\\varvec{n}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\:\\varvec{s}\\varvec{u}\\varvec{p}\\varvec{e}\\varvec{r}\\varvec{n}\\varvec{a}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{t}}{\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\:\\varvec{a}\\varvec{d}\\varvec{d}\\varvec{e}\\varvec{d}\\:\\varvec{i}\\varvec{n}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\varvec{s}}\\varvec{X}100\\)\u003c/span\u003e\u003c/span\u003e\u0026hellip;\u0026hellip;\u0026hellip;Eq.\u0026nbsp;1\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2. siRNAs coupling to the liposomes.\u003c/h2\u003e\n \u003cp\u003eThe prepared liposomes containing tamoxifen citrate were taken in Corning\u0026reg; Spin-X\u0026reg; UF tubes and centrifuged at 3000 rpm until the thick liposomes were left, and then fresh RNase-free water was added and centrifuged again to wash the liposomes twice. The thick suspension of washed liposomes was resuspended in 1 mL of RNase-free water and incubated with siRNA, approximately 3.5\u0026micro;g (20:1 N/P ratio) to 100mg of total lipid formulation, at room temperature for 2 hours for interacting the siRNAs with positively charged liposomes and bound to the surface by electrostatic interaction. After 2h, the liposomes were centrifuged, and the supernatant was used for the calculation of unentrapped siRNA (Eq.\u0026nbsp;2). The pellet was washed again with RNAs free water and resuspended in MES buffer pH 5.5 for surface modification with Trastuzumab monoclonal antibody [28], [30], [31].\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\varvec{\\%}\\:\\varvec{s}\\varvec{i}\\varvec{R}\\varvec{N}\\varvec{A}\\:\\varvec{E}\\varvec{n}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{p}\\varvec{m}\\varvec{e}\\varvec{n}\\varvec{t}=$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{s}\\varvec{i}\\varvec{R}\\varvec{N}\\varvec{A}\\:\\varvec{a}\\varvec{d}\\varvec{d}\\varvec{e}\\varvec{d}\\:\\varvec{t}\\varvec{o}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\varvec{s}\\:-\\:\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{s}\\varvec{i}\\varvec{R}\\varvec{N}\\varvec{A}\\:\\varvec{i}\\varvec{n}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\:\\varvec{s}\\varvec{u}\\varvec{p}\\varvec{e}\\varvec{r}\\varvec{n}\\varvec{a}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{t}}{\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{s}\\varvec{i}\\varvec{R}\\varvec{N}\\varvec{A}\\:\\varvec{a}\\varvec{d}\\varvec{d}\\varvec{e}\\varvec{d}\\:\\varvec{t}\\varvec{o}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\varvec{s}}\\varvec{X}100\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;Eq.\u0026nbsp;2\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3. Trastuzumab functionalization of liposomes.\u003c/h2\u003e\n \u003cp\u003eCarboxyl-functionalized liposomes (1 mL) were activated through the addition of 200 \u0026micro;L of each EDC (4 mg/mL) and NHS (6 mg/mL). This activation mixture was incubated at room temperature with gentle shaking for 30 minutes to facilitate the formation of reactive intermediates [32], [33].\u003c/p\u003e\n \u003cp\u003eFollowing the activation step, the liposomes were subjected to centrifugation, and the resulting pellet was carefully washed twice with MES buffer (pH 5.5) to remove any unreacted reagents. The pellet was then resuspended in 1 mL of the same MES buffer to maintain optimal conditions for subsequent reactions. To the activated liposome suspension, 200 \u0026micro;L of trastuzumab solution (2.5 mg/mL) was added. The mixture was incubated at room temperature with shaking for a duration of 4 hours, allowing for effective conjugation of the antibody to the liposomal surface.\u003c/p\u003e\n \u003cp\u003eAfter incubation, the trastuzumab-conjugated liposomes were centrifuged for 45 minutes at a cool temperature at 17,500 rpm to separate them from any unbound antibodies. The resulting pellet was washed twice with PBS at pH 7.4 and then resuspended in PBS for storage. The conjugated liposomes were kept at a temperature range of 2\u0026ndash;8\u0026deg;C until further analysis. The efficiency of trastuzumab conjugation to the liposomes was quantified using Eq.\u0026nbsp;3. The conjugation chemistry utilized in this process, specifically the EDC-NHS method, is depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, illustrating the mechanism by which the antibody is covalently linked to the liposomal surface [28], [34].\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{\\%}\\:\\varvec{T}\\varvec{r}\\varvec{a}\\varvec{s}\\varvec{t}\\varvec{u}\\varvec{z}\\varvec{u}\\varvec{m}\\varvec{a}\\varvec{b}\\:\\varvec{c}\\varvec{o}\\varvec{n}\\varvec{j}\\varvec{u}\\varvec{g}\\varvec{a}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}=\\frac{\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{s}\\varvec{t}\\varvec{u}\\varvec{z}\\varvec{u}\\varvec{m}\\varvec{b}\\:\\varvec{a}\\varvec{d}\\varvec{d}\\varvec{e}\\varvec{d}\\:\\varvec{t}\\varvec{o}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\varvec{s}\\:-\\:\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{s}\\varvec{t}\\varvec{u}\\varvec{z}\\varvec{u}\\varvec{m}\\varvec{b}\\:\\varvec{i}\\varvec{n}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\:\\varvec{s}\\varvec{u}\\varvec{p}\\varvec{e}\\varvec{r}\\varvec{n}\\varvec{a}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{t}}{\\varvec{A}\\varvec{m}\\varvec{o}\\varvec{u}\\varvec{n}\\varvec{t}\\:\\varvec{o}\\varvec{f}\\:\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{s}\\varvec{t}\\varvec{u}\\varvec{z}\\varvec{u}\\varvec{m}\\varvec{b}\\:\\varvec{a}\\varvec{d}\\varvec{d}\\varvec{e}\\varvec{d}\\:\\varvec{t}\\varvec{o}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{l}\\varvec{i}\\varvec{p}\\varvec{o}\\varvec{s}\\varvec{o}\\varvec{m}\\varvec{e}\\varvec{s}}\\varvec{X}100\\)\u003c/span\u003e\u003c/span\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.Eq.\u0026nbsp;3\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.4. Characterization of liposomes.\u003c/h2\u003e\n \u003cp\u003eThe developed liposomes were evaluated for zeta potential, particle size, and shape using a Malvern Zetasizer and Scanning Electron Microscopy.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.5. Drug release study of liposomes.\u003c/h2\u003e\n \u003cp\u003eTamoxifen release from liposomes was assessed using a dialysis method. A solution of tamoxifen (1 ml of 4 mg/mL) and a suspension of tamoxifen-loaded liposomes (1 mL, 4 mg/mL equivalent) were separately placed in dialysis bags (12 kDa MWCO). Each dialysis bag was then immersed in 40 mL of release medium consisting of PBS with 25% methanol (75:25 v/v). The release study was conducted at 100 rpm using a magnetic stirrer. At predetermined time points (0, 0.5, 1, 2, 4, 8, 12, 24, and 48 hours), 300 \u0026micro;L samples were withdrawn from the vials and replaced with an equal volume of fresh release medium [35], [36], [37], [38]. The concentration of tamoxifen in each sample was determined by HPLC using a standard calibration curve [28]. The release profile was generated using GraphPad Prism version 8.0.2.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.6. Stability study of siRNAs in liposomes (TMX-FL).\u003c/h2\u003e\n \u003cp\u003eThe stability of siRNA encapsulated in liposomes was assessed by incubating the liposomal formulations in fetal bovine serum (FBS) at a 1:1 ratio, maintained at 37\u0026deg;C. Samples were collected at predetermined time intervals of 2, 4, 8, and 12 hours to monitor the integrity of the siRNA. At each time point, the collected samples were treated with 10% sodium dodecyl sulfate (SDS) at a 1:10 ratio to facilitate the disruption of liposomal structures and inactivate serum components. The samples were then incubated in a water bath set to 55\u0026deg;C for 30 minutes, ensuring effective release of siRNA from the liposomes [39], [40], [41]. Following the incubation period, the samples were mixed with a loading dye and subjected to electrophoresis on a 1% agarose gel using TBE buffer (pH 8.3). The gel was run at a voltage of 90 V for 45 minutes to separate the siRNA based on size. After electrophoresis, the gel was visualized and imaged using a gel documentation system to assess the presence and integrity of siRNA. The results from this analysis provided insights into the stability of the siRNA within the liposomal formulation when exposed to biological conditions mimicking those found in vivo [28], [30], [42].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.7. Hemocompatibility study of liposomes.\u003c/h2\u003e\n \u003cp\u003eThe hemocompatibility of the developed liposomes was assessed using a modified version of a previously established method [28], [43]. Blood samples were obtained from Sprague-Dawley (SD) rats in accordance with the Institutional Animal Ethics Committee approval (Ref No- IAEC/KMC/92/2021) through retro-orbital plexus puncture, collected into EDTA-containing microcentrifuge tubes. Erythrocytes were isolated by centrifuging the blood at 5000 rpm for 10 minutes.\u003c/p\u003e\n \u003cp\u003eThe resulting erythrocyte pellet was washed twice with an equal volume of PBS and subsequently resuspended in an equal volume of PBS to ensure uniformity for the hemolysis assay. For the hemolysis assay, 200 \u0026micro;L of the washed erythrocytes were allocated for each experimental condition. Negative control samples consisted of erythrocytes incubated with 800 \u0026micro;L of PBS, while positive control samples included erythrocytes treated with 800 \u0026micro;L of 2% Triton X-100, which is known to induce complete hemolysis. Test samples comprised erythrocytes incubated with 800 \u0026micro;L of tamoxifen-loaded liposomes at a concentration of 5 mg/mL in PBS. All sample conditions were prepared in triplicate within 2 mL tubes and incubated at 37\u0026deg;C for 2 hours with shaking to facilitate interaction [44], [45].\u003c/p\u003e\n \u003cp\u003eAfter the incubation period, the microcentrifuge tubes were left open to air for 10 minutes to allow for hemoglobin oxidation. Aliquots (20 \u0026micro;L) from each sample were then transferred to a 96-well microplate in triplicate, followed by the addition of 180 \u0026micro;L of PBS to each well. The plate was shaken for 5 minutes, and the optical density was measured at 540 nm using a microplate reader to quantify hemolysis. The percentage of hemolysis was calculated using Eq.\u0026nbsp;4. This systematic evaluation provides critical insights into the hemocompatibility profile of the liposomal formulations, essential for their potential therapeutic applications in vivo. The findings contribute to understanding how these liposomes interact with blood components, which is vital for ensuring their safety and efficacy in clinical settings [28], [43].\u003c/p\u003e\n \u003cp\u003e% Hamolysis = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\left(\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\right)\\:-\\:\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\left(\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\left(\\text{p}\\text{o}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)\\:-\\:\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\left(\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}*100\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;..Eq.\u0026nbsp;4.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.8. \u003cem\u003eIn vitro\u003c/em\u003e assessment of liposomes.\u003c/h2\u003e\n \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e\n \u003ch2\u003e2.2.8.1. Cytotoxicity study.\u003c/h2\u003e\n \u003cp\u003eBT-474 and MCF10A cell lines were cultured in DMEM high glucose medium with supplements. After reaching 80% confluence, cells were harvested and seeded into 96-well plates for MTT assay cytotoxicity evaluation [28], [46], [47]. The cytotoxic effects of tamoxifen (TMX), tamoxifen-loaded stealth liposomes (TMX-SL), and trastuzumab-functionalized liposomes loaded with siRNA and tamoxifen (TMX-FL) were evaluated. Cells were seeded at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well. Tamoxifen citrate was initially dissolved in dimethyl sulfoxide (DMSO) and then diluted in DMEM complete medium, ensuring that the final DMSO concentration remained below 1%. The liposomal formulations were suspended in DMEM complete medium and serially diluted to achieve concentrations ranging from 50 \u0026micro;g/mL to 1.25 \u0026micro;g/mL. After seeding, the cells were incubated with the standard tamoxifen solution, liposomal formulations, or vehicle control for a period of 24 hours at 37\u0026deg;C in a humidified atmosphere with 5% CO₂. After incubation, the media was replaced with fresh medium, and 10 \u0026micro;L of MTT solution (5 mg/mL) was added to each well. The plates were incubated for 4 hours at 37\u0026deg;C. Then, the medium was removed, and 100 \u0026micro;L of DMSO was added to dissolve formazan crystals. Optical density (OD) was measured at 540 nm using an ELISA plate reader, and cell death percentage was calculated using Eq. 5 [28], [46].\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\%\\:Cell\\:death\\:=\\frac{OD\\:of\\:control\\:cells\\:-\\:OD\\:of\\:treated\\:cells}{OD\\:of\\:control\\:cells}*100\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.\u003c/em\u003eEq.\u0026nbsp;5\u003c/p\u003e\n \u003cp\u003eData analysis was performed using GraphPad Prism to determine the IC50 values and to compare the effects of standard tamoxifen with those of the liposomal formulations.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e\n \u003ch2\u003e2.2.8.2. Cell uptake study.\u003c/h2\u003e\n \u003cp\u003eSub-confluent BT-474 cells were cultured in 12-well plates and treated with either fluorescein isothiocyanate (FITC) or FITC-loaded liposomes at a concentration of 50 \u0026micro;g/mL. After a 4-hour incubation period, the cells were harvested using a trypsin-EDTA solution to detach them from the culture surface. Following detachment, the cells were washed twice with cold phosphate-buffered saline (PBS) to remove any residual trypsin and unbound FITC. The washed cells were then centrifuged at 1200 rpm for 3 minutes to pellet the cells, after which the supernatant was discarded. The cell pellets were resuspended in PBS to prepare them for flow cytometric analysis. The fluorescence intensity of the resuspended cells was subsequently analyzed using a BD Accuri flow cytometer. This analysis allowed for the quantification of FITC uptake by the BT-474 cells, providing insights into the efficiency of cellular internalization of both FITC and FITC-loaded liposomes [28], [48], [49].\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.9. Biomarkers estimation.\u003c/h2\u003e\n \u003cp\u003eWestern blot analysis was conducted to evaluate HER2, ABCB1, BCL2, and GAPDH expression in BT-474 cells. Cells were cultured in DMEM high glucose medium at 37\u0026deg;C with 5% CO₂ until 80% confluence, then treated with TMX (25 \u0026micro;g/mL), TMX-SL, or TMX-FL for 24 hours. Cells were lysed with cold RIPA buffer, and proteins were extracted and quantified using a BCA assay. Proteins were denatured, separated by SDS-PAGE at 70 V, and transferred to PVDF membranes at 90 V. Membranes were blocked with 3% BSA, incubated with primary antibodies overnight at a cool temperature, washed with 1X TBST, and incubated with secondary antibodies for 2 hours. Protein bands were visualized using chemiluminescence and analyzed with ImageJ and GraphPad Prism software [18], [28], [50].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of liposomes.\u003c/h2\u003e\u003cp\u003eThe prepared liposomes were characterized in terms of particle size (PS) distribution, polydispersity index (PdI), zeta potential (ZP), and entrapment efficiency. The characteristics of the liposomes are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB show the PS, PdI, and ZP of the TMX-FL. The entrapment efficiency was determined to be 81.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48% for TMX-SL and 79.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46% for TMX-FL. For TMX-FL, the siRNA entrapment efficiency was 79.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14%, corresponding to approximately 13\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ng siRNA/mg liposomes. The trastuzumab conjugation efficiency to TMX-FL was 61.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18%, equivalent to approximately 4 \u0026micro;g trastuzumab/mg liposomes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eParticle size, PdI, and Zeta potential of optimized tamoxifen-loaded liposomes.\u003c/b\u003e \u003cem\u003eValues are given as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePS (d.nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZP (mV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%TMX Entrapped\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e%siRNA Entrapped\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e%Trastuzumab conjugated\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSimple Liposomes containing Tamoxifen (TMX-SL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e66.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e32.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e81.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrastuzumab-decorated Liposomes containing siRNA and Tamoxifen (TMX-FL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e97.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e16.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e79.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e79.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e61.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Surface morphology of optimized liposomes.\u003c/h2\u003e\u003cp\u003eThe surface morphology of the optimized liposomes (TMX-FL) was examined using scanning electron microscopy (SEM). The SEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) shows that the optimized liposomes exhibited a spherical morphology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Drug release study of liposomes.\u003c/h2\u003e\u003cp\u003eThe drug release percentages of the optimized liposomes at different time intervals are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The pure TMX group exhibited a burst release of 55.97\u0026thinsp;\u0026plusmn;\u0026thinsp;6.19% in 0.5h, 83.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.82% in 4h, and 99.75\u0026thinsp;\u0026plusmn;\u0026thinsp;9.72% in 12h. The TMX-SL group showed a release of 3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96% in 0.5h, 15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98% in 4h, 24.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40% in 12h, and 58.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88% in 48h. The TMX-FL group showed a release of 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35% in 0.5h, 11.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06% in 4h, 20.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47% in 12h, and 53.43\u0026thinsp;\u0026plusmn;\u0026thinsp;8.50% in 48h. This slow-release pattern demonstrates prolonged retention of tamoxifen within the liposomes, highlighting its potential for optimal delivery to HER2-positive cancer cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Stability study of siRNAs in liposomes (TMX-FL).\u003c/h2\u003e\u003cp\u003eThe serum stability study demonstrated that the siRNA encapsulated within the liposomes remained stable for up to 12 hours. After 4 hours, approximately 58% of the encapsulated siRNA was still intact. This value decreased to 33% at 8 hours and 13% at 12 hours. Agarose gel electrophoresis (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) shows the siRNA bands, relative band intensity, and the percentage of siRNA degradation over time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5. \u003cem\u003eIn vitro\u003c/em\u003e assessment of liposomes.\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.5.1. Cytotoxicity study\u003c/h2\u003e\u003cp\u003eThe optimized liposomes were evaluated for their anticancer efficacy against HER2-positive BT-474 breast cancer cells and HER2-negative MCF10A mammary epithelial cells to assess their selectivity and therapeutic potential. The percentage of cell death for each treatment is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eBlank liposomes showed negligible cytotoxicity, with cell viability remaining high at both tested concentrations (cell death: 0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26% at 30 \u0026micro;g/mL and 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% at 50 \u0026micro;g/mL). In contrast, free tamoxifen (TMX) exhibited moderate cytotoxicity, inducing 23.65\u0026thinsp;\u0026plusmn;\u0026thinsp;3.52% cell death at 30 \u0026micro;g/mL and 64.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39% at 50 \u0026micro;g/mL. The TMX-SL demonstrated enhanced anticancer activity compared to free TMX, with 43.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94% cell death at 30 \u0026micro;g/mL and 72.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93% at 50 \u0026micro;g/mL. Remarkably, TMX-FL displayed the highest cytotoxicity, achieving 58.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29% cell death at 30 \u0026micro;g/mL and 86.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50% at 50 \u0026micro;g/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. TMX and TMX-SL), underscoring their superior therapeutic efficacy.\u003c/p\u003e\u003cp\u003eIn HER2-negative MCF10A cells, treatment with TMX-FL induced modest cytotoxicity, with 8.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55% cell death at 30 \u0026micro;g/mL and 19.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25% at 50 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In stark contrast, HER2-positive BT-474 cells exhibited significantly higher sensitivity to TMX-FL, showing 65.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34% cell death at 30 \u0026micro;g/mL and 90.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10% at 50 \u0026micro;g/mL. Statistical analysis (unpaired t-test with Welch\u0026rsquo;s correction) confirmed that TMX-FL has selective and enhanced cytotoxicity toward HER2-positive BT-474 cells compared to HER2-negative MCF10A cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), highlighting its potential for targeted therapy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.5.2. Cell uptake study of liposomes.\u003c/h2\u003e\u003cp\u003eCellular uptake studies in HER2-positive BT-474 cells revealed that trastuzumab-functionalized liposomes (FITC-FL) were internalized significantly more efficiently than non-targeted FITC-loaded stealth liposomes (FITC-SL) following a 4-hour incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Statistical analysis (one-way ANOVA with Tukey\u0026rsquo;s post hoc test) confirmed highly significant differences in uptake between the groups (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), underscoring the critical role of trastuzumab-mediated targeting in enhancing liposomal delivery to HER2-overexpressing cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.5.3. Biomarkers estimation.\u003c/h2\u003e\u003cp\u003eAfter a 24-hour exposure to standard TMX, TMX-SL, or TMX-FL, cells were harvested for western blot analysis. The results demonstrated a marked downregulation of key proteins, including BCL2, ABCB1, and HER2, with the most pronounced reduction observed in TMX-FL-treated cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Statistical analysis (two-way ANOVA with Bonferroni\u0026rsquo;s post hoc test) confirmed significant differences in protein expression across treatment groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), highlighting the superior efficacy of TMX-FL in modulating these critical therapeutic targets.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTamoxifen (TMX), a selective estrogen receptor modulator, has been a cornerstone in the treatment of hormone receptor-positive breast cancer. However, its clinical application is often limited by poor aqueous solubility, systemic toxicity, and the development of multidrug resistance, particularly in HER2-positive subtypes. To address these challenges, we developed a novel trastuzumab-conjugated liposomal formulation co-loaded with tamoxifen and anti-ABCB1 siRNA (TMX-FL) for targeted therapy against HER2-overexpressing breast cancer.\u003c/p\u003e\u003cp\u003eThe optimized TMX-FL liposomes exhibited a particle size of 97.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 nm and a zeta potential of 16.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mV, which are within the optimal range for enhanced permeability and retention (EPR)-based tumor targeting. The entrapment efficiencies for tamoxifen and siRNA were 79.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46% and 79.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14%, respectively, with a trastuzumab conjugation efficiency of 61.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18%. These values are comparable to our previously reported study by Kumar et al. (2024) [28], who developed trastuzumab-conjugated liposomes co-loaded with paclitaxel and anti-ABCB1 siRNA, achieving similar conjugation efficiency but with a larger particle size (229\u0026thinsp;\u0026plusmn;\u0026thinsp;4 nm), which may limit tumor penetration compared to our smaller-sized TMX-FL in the present study.\u003c/p\u003e\u003cp\u003eScanning electron microscopy confirmed the spherical morphology of TMX-FL, which is favorable for uniform biodistribution and cellular uptake. Drug release studies demonstrated a sustained release profile, with only 53.43\u0026thinsp;\u0026plusmn;\u0026thinsp;8.50% of tamoxifen released over 48 hours, in contrast to the burst release observed with free tamoxifen (99.75\u0026thinsp;\u0026plusmn;\u0026thinsp;9.72% in 12 hours). This controlled release is consistent with findings by Du et al. (2024) [51], who reported similar sustained release behavior in trastuzumab-functionalized liposomes loaded with pyrotinib, reinforcing the advantage of liposomal encapsulation in prolonging drug action.\u003c/p\u003e\u003cp\u003eThe serum stability study showed that siRNA encapsulated in TMX-FL remained protected for up to 12 hours, with 58% intact at 4 hours. This aligns with previous reports, such as those by Mainini and Eccles (2020) [52], which demonstrated that lipid-based nanocarriers can effectively shield siRNA from enzymatic degradation, thereby enhancing gene silencing efficiency.\u003c/p\u003e\u003cp\u003eIn vitro cytotoxicity assays revealed that TMX-FL exhibited significantly higher anticancer activity against HER2-positive BT-474 cells compared to free tamoxifen and non-targeted liposomes (TMX-SL). At 50 \u0026micro;g/mL, TMX-FL induced 86.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50% cell death, outperforming TMX-SL (72.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93%) and free TMX (64.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39%). These results are comparable to those reported by Kumar et al. (2024) [28], where their paclitaxel-based formulation also showed enhanced cytotoxicity in HER2-positive models. However, our study uniquely demonstrates the efficacy of tamoxifen in a similar dual-delivery system, offering a potentially safer and more cost-effective alternative.\u003c/p\u003e\u003cp\u003eImportantly, TMX-FL showed selective cytotoxicity, with minimal toxicity in HER2-negative MCF10A cells, highlighting its targeted therapeutic potential. Cellular uptake studies confirmed that trastuzumab-functionalized liposomes were internalized significantly more efficiently than non-targeted liposomes, consistent with the findings of Du et al. (2024) [51] and Zafar et al. (2024) [53], who emphasized the role of HER2-targeting in enhancing intracellular delivery.\u003c/p\u003e\u003cp\u003eWestern blot analysis further validated the therapeutic efficacy of TMX-FL, showing significant downregulation of HER2, ABCB1, and BCL2 proteins. This multi-target modulation is critical for overcoming drug resistance and inducing apoptosis and is in line with the results of Gao et al. (2022) [20], who demonstrated that co-delivery of chemotherapeutics and siRNA can synergistically suppress oncogenic signaling pathways.\u003c/p\u003e\u003cp\u003eIn summary, our study demonstrates that TMX-FL liposomes offer a promising strategy for the targeted treatment of HER2-positive breast cancer by combining the benefits of controlled drug release, siRNA-mediated gene silencing, and trastuzumab-guided delivery. Compared to recent studies using paclitaxel or pyrotinib, our tamoxifen-based system achieves comparable efficacy with potentially improved safety and cost-effectiveness.\u003c/p\u003e"},{"header":"5. Conclusion and future perspectives","content":"\u003cp\u003eTo achieve targeted delivery of tamoxifen along with anti-ABCB1 siRNA, trastuzumab-decorated multifunctional liposomes (TMX-FL) were successfully developed and evaluated for their HER2-targeting efficiency and anticancer potential against HER2-positive breast cancer. The optimized liposomes met all critical formulation criteria, including appropriate particle size, surface charge, and high entrapment efficiencies for both tamoxifen and siRNA. Trastuzumab conjugation enabled selective targeting of HER2-overexpressing BT-474 cells, as confirmed by cellular uptake studies using flow cytometry. In vitro cytotoxicity assays demonstrated that TMX-FL exhibited significantly enhanced anticancer activity compared to free tamoxifen and non-targeted liposomes, owing to the synergistic effects of tamoxifen, anti-ABCB1 siRNA, and trastuzumab. Furthermore, western blot analysis revealed a marked downregulation of HER2, ABCB1, and BCL2 protein expression in TMX-FL-treated BT-474 cells, indicating effective modulation of key pathways involved in tumor progression and drug resistance. These findings suggest that the trastuzumab-decorated liposomes co-loaded with tamoxifen and siRNA represent a promising nanotherapeutic strategy for the treatment of HER2-positive breast cancer. Future in vivo studies and clinical translation efforts are warranted to further validate the therapeutic potential of this formulation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthical Approval\u003c/h2\u003e\n\u003cp\u003eAll animal experiments were conducted in compliance with the ARRIVE guidelines and the National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals (Eighth Edition). The Institutional Animal Ethics Committee (IAEC) of the Kasturba Medical College, Manipal, India, reviewed and approved all procedures on October 23, 2021, under approval Reference No. IAEC/KMC/92/2021.\u003c/p\u003e\n\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eNo funding was received for this work.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eG.K. wrote the main manuscript. F.B., P.C.G., and P.M. collected and acquired data. K.N., S.M., and C.M.R. guided and reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment\u003c/h2\u003e\n\u003cp\u003eThe authors sincerely acknowledge the Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education (MAHE), Karnataka, India, for providing essential infrastructure, research facilities, and library resources. We are grateful to the All India Council for Technical Education (AICTE), Government of India, New Delhi, for awarding the National Doctoral Fellowship to Gautam Kumar (Ref. No. 53120), which supported this research.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eAdditional data can be provided if required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eF. Bray \u003cem\u003eet al.\u003c/em\u003e, \u0026lsquo;Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries\u0026rsquo;, \u003cem\u003eCA. Cancer J. Clin.\u003c/em\u003e, vol. 74, no. 3, pp. 229\u0026ndash;263, 2024, doi: 10.3322/caac.21834.\u003c/li\u003e\n\u003cli\u003eF. Schettini and A. 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BCR\u003c/em\u003e, vol. 26, no. 1, p. 99, Jun. 2024, doi: 10.1186/s13058-024-01853-2.\u003c/li\u003e\n\u003cli\u003eF. Mainini and M. R. Eccles, \u0026lsquo;Lipid and Polymer-Based Nanoparticle siRNA Delivery Systems for Cancer Therapy\u0026rsquo;, \u003cem\u003eMolecules\u003c/em\u003e, vol. 25, no. 11, Art. no. 11, Jan. 2020, doi: 10.3390/molecules25112692.\u003c/li\u003e\n\u003cli\u003eM. N. Zafar, W. G. Pitt, and G. A. Husseini, \u0026lsquo;Encapsulation and release of calcein from herceptin-conjugated eLiposomes\u0026rsquo;, \u003cem\u003eHeliyon\u003c/em\u003e, vol. 10, no. 6, p. e27882, Mar. 2024, doi: 10.1016/j.heliyon.2024.e27882.\u003c/li\u003e\n\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":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triple-positive breast cancer, Tamoxifen-targeted delivery, anti-ABCB1 siRNA, Trastuzumab-conjugated liposomes","lastPublishedDoi":"10.21203/rs.3.rs-7025151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7025151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHR+/HER2\u0026thinsp;+\u0026thinsp;breast cancer is characterized by the overexpression of hormone receptors (estrogen and progesterone) and the HER2 receptor, leading to aggressive tumor growth and poor prognosis. Conventional treatments often face challenges such as drug resistance and systemic toxicity. This study aims to develop and evaluate trastuzumab-conjugated siRNA and tamoxifen-loaded liposomes for targeted therapy in HR+/HER2\u0026thinsp;+\u0026thinsp;breast cancer. Liposomes were prepared using the thin film hydration. Surface morphology was analyzed using scanning electron microscopy, while drug release profiles and serum stability of siRNA were assessed. In vitro cytotoxicity, cellular uptake, and Western blot analyses were conducted using HR+/HER2+ (BT474) breast cancer cells. Optimized trastuzumab-conjugated liposomes exhibited a particle size of 97.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 nm, a zeta potential of 16.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mV, and high entrapment efficiencies for tamoxifen (79.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46%) and siRNA (79.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14%). Scanning electron microscopy confirmed spherical morphology. Drug release studies indicated controlled and sustained release, enhancing tamoxifen retention and stability. Serum stability tests demonstrated siRNA protection from degradation. In vitro cytotoxicity assays showed superior anticancer activity of TMX-FL compared to simple liposomes and pure tamoxifen. These findings suggest that TMX-FL liposomes offer a promising strategy for targeted cancer therapy, warranting further in vivo evaluations and clinical trials.\u003c/p\u003e","manuscriptTitle":"Enhanced Anticancer Activity in HR+/HER2+ Breast Cancer Cells Using Trastuzumab- Conjugated Liposomes co-loaded with siRNA and Tamoxifen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 07:43:35","doi":"10.21203/rs.3.rs-7025151/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-25T11:06:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-23T08:09:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T05:22:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104758015105441149424966490970822558144","date":"2025-07-16T11:08:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232479917318133414334970692285732180427","date":"2025-07-15T11:35:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272764742969687595345222478378836088590","date":"2025-07-14T08:08:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T07:50:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-07T07:44:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T04:00:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-07-02T04:49:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ae82198d-0748-453f-a063-0bbe031957f6","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T15:58:03+00:00","versionOfRecord":{"articleIdentity":"rs-7025151","link":"https://doi.org/10.1007/s12668-025-02149-1","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2025-09-17 15:56:55","publishedOnDateReadable":"September 17th, 2025"},"versionCreatedAt":"2025-07-10 07:43:35","video":"","vorDoi":"10.1007/s12668-025-02149-1","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02149-1","workflowStages":[]},"version":"v1","identity":"rs-7025151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7025151","identity":"rs-7025151","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}