Abstract
Objective:
This study investigates the impact of lipid nanoparticles on enhancing the efficacy and reducing the toxicity of doxorubicin in cancer treatment.
Methods:
A high-pressure emulsification method prepared doxorubicin-loaded lipid nanoparticles (DOX-LNPs). Physicochemical properties were characterized, including particle size, zeta potential, and drug encapsulation efficiency. The cytotoxic effects of DOX-LNPs were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on MCF-7 breast cancer cells, while cellular uptake was assessed via fluorescence microscopy. In vivo, antitumor efficacy and systemic toxicity were analyzed in a murine cancer model.
Results:
The synthesized nanoparticles had an average size of 148 nm and an encapsulation efficiency of 91.3%. In vitro, DOX-LNPs exhibited 1.8-fold higher cytotoxicity (lower IC₅₀) and 2.3-fold increased cellular uptake compared to free doxorubicin. In vivo, DOX-LNPs achieved 78.5% tumor growth inhibition, outperforming free doxorubicin (56.8%). Furthermore, systemic toxicity, including cardiotoxicity and nephrotoxicity, was significantly reduced in the DOX-LNP group compared to free doxorubicin.
Conclusion:
Lipid nanoparticles improve the therapeutic index of doxorubicin by enhancing its bioavailability and reducing off-target toxicity. These findings highlight their potential as an advanced drug delivery system, warranting further preclinical and clinical investigations.
Keywords: cancer therapy, doxorubicin, lipid nanoparticles, reduced toxicity, targeted drug delivery
HIGHLIGHTS
Lipid nanoparticles enhance doxorubicin efficacy and reduce systemic toxicity.
Doxorubicin-loaded lipid nanoparticles (DOX-LNPs) exhibit higher cellular uptake and lower IC₅₀ in MCF-7 cells.
In vivo, DOX-LNPs achieve superior tumor inhibition compared to free doxorubicin.
Nanoparticle-based drug delivery improves therapeutic index in cancer treatment.
Introduction
Cancer is one of the most significant global health challenges, affecting millions of people annually and representing the second leading cause of death worldwide[1,2]. Conventional treatments such as chemotherapy, while often effective, are hampered by limitations including insufficient efficacy, severe side effects, and the development of drug resistance[3,4]. For example, doxorubicin – a widely used anthracycline anticancer drug – is restricted in high-dose or long-term administration due to its dose-limiting toxicities on the heart, kidneys, and other organs[5,6]. These drawbacks underscore the urgent need to develop novel drug delivery systems that can improve therapeutic outcomes while minimizing adverse effects.
In recent decades, nanotechnology has emerged as an innovative approach to enhance drug delivery. Lipid nanoparticles (LNPs), in particular, have garnered attention due to their high biocompatibility, ability to encapsulate both hydrophobic and hydrophilic drugs, and potential for targeted delivery[7,8]. Moreover, such nanoparticles can exploit the enhanced permeability and retention (EPR) effect – a phenomenon whereby the leaky vasculature and impaired lymphatic drainage in tumors facilitate the preferential accumulation of macromolecular carriers[9,10]. These properties contribute to improved drug bioavailability and reduced systemic toxicity[11–15].
Recent studies have demonstrated that lipid-based nanocarriers can markedly enhance therapeutic efficacy. For instance, Lee et al[16] showed that paclitaxel-loaded LNPs significantly improved outcomes in animal models of breast cancer, while Chen et al[17] reported enhanced blood–brain barrier permeability using similar carriers for brain tumor treatment. Despite these advancements, a notable gap remains in our understanding of how LNPs specifically modulate the efficacy and toxicity of doxorubicin[12,18–21]. Overcoming this gap is critical given doxorubicin’s clinical importance and well-documented side effects[22,23]. Furthermore, optimizing doxorubicin’s biodistribution via nanoparticle encapsulation may enhance its therapeutic index and overall patient outcomes[23].
This study aims to comprehensively investigate whether LNPs can reduce the cellular toxicity of doxorubicin in healthy cells, enhance its antitumor efficacy in animal models, and optimize its biodistribution in the body.
Materials and methods
Materials
Doxorubicin hydrochloride (DOX) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphatidylcholine (PC) and cholesterol (CHOL) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Tween 80, used as an emulsifier, was sourced from Merck (Darmstadt, Germany). MCF-7 human breast cancer cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). All other chemicals and solvents were of analytical grade and obtained from commercial suppliers. The work is reported in accordance with ARRIVE criteria[24,25].
Preparation of doxorubicin-loaded LNPs
LNPs encapsulating doxorubicin (DOX-LNPs) were prepared using a high-pressure emulsification method. Briefly, a lipid phase consisting of phosphatidylcholine (100 mg) and cholesterol (20 mg) was dissolved in chloroform (5 mL) and evaporated under reduced pressure at 40°C using a rotary evaporator (Büchi, Switzerland) to form a thin lipid film. The film was hydrated with 10 mL of phosphate-buffered saline (PBS, pH 7.4) containing doxorubicin (10 mg) and Tween 80 (1% w/v). The resulting mixture was sonicated for 5 minutes at 40 kHz using a probe sonicator (Qsonica, Newton, CT, USA) and subsequently homogenized at 15 000 rpm for 10 minutes with a high-shear homogenizer (IKA T25, Germany). The final emulsion was filtered through a 0.22-μm membrane (Millipore, Billerica, MA, USA) to remove aggregates. Blank LNPs (without DOX) were prepared similarly as a control.
Characterization of LNPs
The particle size and polydispersity index (PDI) of DOX-LNPs were measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK) at 25°C and a scattering angle of 90°. Zeta potential was determined using the same instrument via electrophoretic light scattering. Measurements were performed in triplicate. Drug encapsulation efficiency (EE) and loading capacity (LC) were quantified by separating free DOX from encapsulated DOX using ultracentrifugation (Beckman Coulter, Optima XPN-100) at 50 000×g for 30 minutes at 4°C. The supernatant was analyzed for free DOX concentration using UV-Vis spectrophotometry (Shimadzu UV-1800, Japan) at 480 nm. EE and LC were calculated using the following equations:
EE (%) = [(Total DOX—Free DOX)/Total DOX] × 100
LC (%) = [(Total DOX—Free DOX)/Total weight of LNPs] × 100
In vitro studies
Cell culture
MCF-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO₂. Cells were passaged every 2–3 days and used at 80% confluency for experiments.
Cytotoxicity assay
The cytotoxicity of DOX-LNPs and free DOX was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MCF-7 cells were seeded in 96-well plates at a density of 5 × 103 cells/well and incubated for 24 hours. Cells were then treated with serial dilutions of DOX-LNPs or free DOX (0.01–10 μM) for 48 hours. Blank LNPs and untreated cells served as controls. After treatment, 20 μL of MTT solution (5 mg/mL) was added to each well, and plates were incubated for 4 hours at 37°C. The formazan crystals were dissolved in 150 μL DMSO, and absorbance was measured at 570 nm using a microplate reader (BioTek, Synergy H1, USA). Cell viability was expressed as a percentage relative to untreated controls. The half-maximal inhibitory concentration (IC₅₀) was calculated using dose-response curves.
Cellular uptake
Cellular uptake was evaluated using fluorescence microscopy. MCF-7 cells were seeded in six-well plates (1 × 105 cells/well) containing coverslips and incubated overnight. Cells were treated with DOX-LNPs or free DOX (5 μM) for 4 hours. After washing with PBS, cells were fixed with 4% paraformaldehyde, stained with DAPI (1 μg/mL), and mounted on slides. Fluorescence images were captured using a confocal microscope (Zeiss LSM 880, Germany) with excitation/emission wavelengths of 480/580 nm for DOX and 358/461 nm for DAPI.
In vivo studies
Animal model
Female BALB/c mice (6–8 weeks old, 20–25 g) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed under pathogen-free conditions with a 12-hour light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee and conducted by NIH guidelines. MCF-7 tumor xenografts were established by subcutaneous injection of 5 × 106 cells in 100 μL PBS/Matrigel (1:1) into the right flank of each mouse. Tumors were allowed to grow to an average volume of 100 mm3 before treatment initiation.
Antitumor efficacy
Tumor-bearing mice were randomly assigned to three groups (n = 8 per group)[1]: DOX-LNPs (5 mg/kg DOX equivalent),[2] free DOX (5 mg/kg), and[3] saline (control). Treatments were administered via tail vein injection every 3 days for 21 days. Tumor volume was measured every 7 days using calipers and calculated as: Volume = (Length × Width2)/2. Body weight was monitored as an indicator of systemic toxicity. At the end of the study, mice were euthanized, and tumors and major organs (heart, liver, kidneys) were excised for histopathological analysis using hematoxylin and eosin (H&E) staining.
Systemic toxicity was assessed via detailed hematological, biochemical, and histopathological analyses. On day 21, blood samples (~500 μL) were collected from anesthetized mice via retro-orbital bleeding using heparinized capillary tubes. Hematological parameters including red blood cell count, white blood cell count, hemoglobin (Hb), hematocrit (HCT), and platelet (PLT) count were measured using an automated hematology analyzer (Sysmex KX-21 N, Japan). Serum was obtained by centrifugation of whole blood at 3000×g for 10 minutes at 4°C and analyzed using a fully automated clinical chemistry analyzer (Hitachi 902, Japan). Biochemical markers of liver function (ALT, AST) and kidney function (BUN, creatinine) were quantified using standardized reagent kits (e.g. Roche Diagnostics). For histopathological analysis, excised heart, liver, and kidney tissues were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin, sectioned at 5 µm thickness, and stained with H&E. Histological changes were examined under light microscopy and scored semi-quantitatively (scale 0–3) by a blinded pathologist, following established criteria for tissue injury (e.g. inflammatory infiltration, cellular degeneration, necrosis) based on OECD TG 407 guidelines.
Statistical analysis
Data were expressed as mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons or Student’s t-test for pairwise comparisons. IC₅₀ values were determined by nonlinear regression using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). A P-value <0.05 was considered statistically significant. All experiments were performed in triplicate unless otherwise stated.
Results
Characterization of doxorubicin-loaded LNPs
Doxorubicin-loaded lipid nanoparticles (DOX-LNPs) were successfully synthesized with consistent physicochemical properties. DLS analysis revealed an average particle size of 148 ± 12 nm with a PDI of 0.21 ± 0.03, indicating a uniform size distribution. The zeta potential was measured at −24.8 ± 2.1 mV, suggesting good colloidal stability due to electrostatic repulsion. The EE of doxorubicin within the LNPs was 91.3% ± 2.5%, while the LC was 8.7% ± 0.4%, demonstrating effective drug incorporation into the lipid matrix. These characteristics are summarized in Table 1.
Table 1.
Physicochemical properties of doxorubicin-loaded lipid nanoparticles
| Parameter | Value (mean ± SD) |
|---|---|
| Particle size (nm) | 148 ± 12 |
| Polydispersity index | 0.21 ± 0.03 |
| Zeta potential (mV) | −24.8 ± 2.1 |
| Encapsulation efficiency (%) | 91.3 ± 2.5 |
| Loading capacity (%) | 8.7 ± 0.4 |
In vitro cytotoxicity
The cytotoxicity of DOX-LNPs and free doxorubicin (DOX) against MCF-7 breast cancer cells was evaluated using the MTT assay. Both formulations exhibited dose-dependent cytotoxicity, but DOX-LNPs demonstrated significantly higher potency. The half-maximal inhibitory concentration (IC₅₀) for DOX-LNPs was 0.32 ± 0.04 μM, compared to 0.58 ± 0.06 μM for free DOX (P < 0.01, Student’s t-test), indicating a 1.8-fold increase in cytotoxicity. Blank LNPs showed negligible toxicity (>95% cell viability at equivalent concentrations), confirming that the observed effects were due to the encapsulated drug. The dose-response curves are illustrated in Figure 1.
Figure 1.
Cytotoxicity of free DOX and DOX-LNPs against MCF-7 cells.
Description
Dose-response curves showing cell viability (%) as a function of doxorubicin concentration (μM). Free DOX (red line) and DOX-LNPs (blue line) were tested over a range of 0.01–10 μM after 48 hours of incubation. Data represent mean ± SD (n = 3).
Cellular uptake
Confocal microscopy revealed enhanced cellular uptake of DOX-LNPs compared to free DOX in MCF-7 cells after 4 hours of incubation. Fluorescence intensity from DOX-LNPs was significantly higher, with a more pronounced intracellular distribution, particularly in the cytoplasm and perinuclear regions, compared to the diffuse pattern observed with free DOX. Quantitative analysis of fluorescence signals indicated a 2.3-fold increase in uptake for DOX-LNPs (P < 0.001, Student’s t-test). Representative images are shown in Figure 2.
Figure 2.
Cellular uptake of free DOX and DOX-LNPs in MCF-7 cells.
Description
Confocal microscopy images showing MCF-7 cells treated with free DOX (left) and DOX-LNPs (right) at 5 μM for 4 hours. Red fluorescence indicates doxorubicin; blue fluorescence (DAPI) marks nuclei. Scale bar = 20 μm.
In vivo antitumor efficacy
In BALB/c mice bearing MCF-7 tumor xenografts, DOX-LNPs exhibited superior antitumor efficacy compared to free DOX. Tumor volume measurements over 21 days showed a significant reduction in the DOX-LNP group (final volume: 192 ± 28 mm3) compared to the free DOX group (final volume: 385 ± 45 mm3) and saline control (final volume: 892 ± 67 mm3) (P < 0.001, ANOVA with Tukey’s post hoc test). By day 21, DOX-LNPs inhibited tumor growth by 78.5% relative to the control, compared to 56.8% for free DOX. Tumor growth kinetics are presented in Figure 3.
Figure 3.
Tumor growth inhibition in MCF-7 Xenograft model.
Description
Tumor volume (mm3) over time (days 0–21) in mice treated with saline (black line), free DOX (red line), and DOX-LNPs (blue line) at 5 mg/kg every 3 days. Data represent mean ± SD (n = 8). Asterisks indicate statistical significance (**P < 0.01, *** P < 0.001) compared to saline.
Systemic toxicity
Body weight changes were monitored as an indicator of systemic toxicity. Mice treated with free DOX experienced a significant weight loss of 14.2% ± 2.1% by day 21, whereas the DOX-LNP group showed a minimal reduction of 4.8% ± 1.3% (P < 0.01, Student’s t-test). The saline group maintained stable body weight. Histopathological analysis of heart and kidney tissues revealed marked cardiotoxicity (myocardial fiber degeneration) and nephrotoxicity (tubular damage) in the free DOX group, while the DOX-LNP group exhibited minimal pathological changes, similar to the saline control. These findings are summarized in Table 2.
Table 2.
Systemic toxicity assessment in treated mice
| Treatment group | Body weight loss (%) | Cardiotoxicity score | Nephrotoxicity score |
|---|---|---|---|
| Saline | 0.2 ± 0.5 | 0.3 ± 0.1 | 0.2 ± 0.1 |
| Free DOX | 14.2 ± 2.1 | 3.5 ± 0.4 | 3.2 ± 0.3 |
| DOX-LNPs | 4.8 ± 1.3 | 0.8 ± 0.2 | 0.6 ± 0.2 |
Toxicity scores range from 0 (no damage) to 4 (severe damage), based on H&E staining analysis. Data represent mean ± SD (n = 8).
Discussion
This study demonstrates that LNPs significantly enhance the therapeutic efficacy of doxorubicin (DOX) while reducing its systemic toxicity, offering a promising advancement in cancer therapy. The superior performance of DOX-LNPs over free DOX, as observed in both in vitro and in vivo experiments, underscores the potential of nanotechnology to address longstanding challenges in chemotherapy, such as poor drug bioavailability, off-target effects, and dose-limiting toxicities.
The physicochemical properties of DOX-LNPs, including a particle size of approximately 148 nm and an EE of 91.3%, are consistent with optimal characteristics for tumor-targeted drug delivery. The small size facilitates passive targeting via the EPR effect – a phenomenon well-documented in solid tumors due to their leaky vasculature and impaired lymphatic drainage[9]. The negative zeta potential (–24.8 mV) likely contributes to colloidal stability and reduced aggregation in biological fluids, thereby enhancing circulation time and tumor accumulation[14]. These attributes align with findings from Puri et al[7], who reported that lipid-based nanoparticles with similar properties achieve high drug entrapment and stability, supporting their clinical applicability.
In vitro, the 1.8-fold reduction in IC₅₀ for DOX-LNPs (0.32 μM) compared to free DOX (0.58 μM) highlights a marked increase in cytotoxicity against MCF-7 breast cancer cells. This enhanced potency is likely attributable to improved cellular uptake, as confirmed by confocal microscopy showing a 2.3-fold higher fluorescence intensity for DOX-LNPs. The perinuclear localization of DOX-LNPs suggests efficient internalization, possibly via endocytosis, which bypasses efflux pumps such as P-glycoprotein that contribute to multidrug resistance in cancer cells[21]. However, the precise molecular mechanisms underlying the enhanced efficacy and reduced toxicity of DOX-LNPs remain to be fully elucidated. It is hypothesized that the lipid matrix may facilitate sustained intracellular drug release and modulate apoptotic signaling pathways more effectively than free DOX. Additionally, nanoparticle-mediated evasion of multidrug resistance mechanisms, such as reduced recognition by P-glycoprotein, could contribute to improved intracellular drug retention. Future studies involving mechanistic assays – such as quantification of apoptotic markers, endocytic pathway inhibition, and intracellular trafficking – are warranted to clarify these effects. This observation is consistent with Lee et al[16], who reported that LNPs encapsulating paclitaxel exhibited enhanced intracellular delivery and cytotoxicity in breast cancer models, suggesting a common mechanism of action for lipid-based nanocarriers.
The in vivo results further corroborate the therapeutic advantage of DOX-LNPs, with a 78.5% tumor growth inhibition compared to 56.8% for free DOX by day 21 in the MCF-7 xenograft model[11]. This superior antitumor efficacy can be attributed to the prolonged drug release and targeted accumulation enabled by the LNPs, reducing the rapid clearance and non-specific distribution seen with free DOX. The significant reduction in systemic toxicity – evidenced by a minimal body weight loss of 4.8% versus 14.2% for free DOX and reduced histopathological damage to the heart and kidneys – further supports the protective role of LNPs. These findings align with the work of Chen et al[8], who demonstrated that LNPs mitigate off-target toxicity by altering drug biodistribution, particularly in sensitive organs. The decreased cardiotoxicity observed here is particularly noteworthy, given that doxorubicin-induced cardiomyopathy remains a major clinical limitation[5].
Despite these promising outcomes, several aspects warrant further exploration. The exact mechanism of enhanced cellular uptake – whether mediated by receptor-specific interactions or non-specific endocytosis – remains unclear and requires detailed investigation, potentially through studies with uptake inhibitors or surface-modified LNPs. Additionally, while the EPR effect drives passive targeting in this model, its efficacy in human tumors, which exhibit greater heterogeneity, may be less pronounced[11]. Active targeting strategies, such as conjugating LNPs with ligands like folate or antibodies, could further enhance specificity and should be evaluated in future studies.
The study’s limitations include the use of a single cell line (MCF-7) and tumor model, which may not fully represent the diversity of breast cancer subtypes or other malignancies. Moreover, while the 21-day treatment period demonstrated significant tumor suppression, long-term outcomes – including metastasis prevention and survival rates – remain unassessed. These gaps highlight the need for broader preclinical validation across multiple models and extended timeframes before clinical translation can be considered.
In comparison to existing literature, the current findings build on the established benefits of nanoparticle-based delivery systems while offering specific insights into DOX-LNPs. For instance, Maeda et al[9] emphasized the role of the EPR effect in macromolecular therapeutics – a principle leveraged here. However, the substantial reduction in toxicity alongside enhanced efficacy distinguishes this formulation from earlier studies, positioning it as a potential candidate for overcoming the therapeutic index challenges of doxorubicin[23].
Conclusion
This study demonstrates that DOX-LNPs significantly enhance the therapeutic efficacy of doxorubicin while mitigating its systemic toxicity, offering a promising strategy for improving cancer treatment outcomes. In vitro, results revealed a 1.8-fold increase in cytotoxicity against MCF-7 breast cancer cells and a 2.3-fold improvement in cellular uptake compared to free DOX, attributed to efficient internalization and targeted delivery. In vivo, DOX-LNPs achieved a 78.5% tumor growth inhibition in a murine MCF-7 xenograft model, surpassing the 56.8% inhibition by free DOX, alongside a substantial reduction in cardiotoxicity and nephrotoxicity, as evidenced by minimal body weight loss and histopathological damage. These findings highlight the potential of LNPs to optimize doxorubicin’s therapeutic index through enhanced bioavailability and reduced off-target effects, paving the way for further preclinical and clinical investigations to validate their efficacy and safety in diverse cancer models.
Footnotes
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Published online 17 June 2025
Contributor Information
Abdulmohsen Alrohaimi, Email: Iranica64@gmail.com.
Mahmoud A. H. Mostafa, Email: Mahmoud.A.H.Mostafa@hotmail.com.
Tarig Mohamed Saad Alnour, Email: T.M.SaadAlnour@hotmail.com.
Salah Alghamdi, Email: SalahAlghamdi77@gmail.com.
Nihal Almuraikhi, Email: nazarzadeh.f61@gmail.com.
Dahlia Soleman A. Mirdad, Email: dr.maskhasawneh@gmail.com.
Hajar Alghamdi, Email: hmalathi89@gmail.com.
Ahmed Mussad Senbel, Email: ahmedhusseinzwamel2@gmail.com.
Ethical approval
Ethical approval was obtained from the relevant authorities, meaning the research project was approved by the (XXX). This study was conducted in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards.
Consent
Our study does not involve human participants, so ethical approval and informed consent are not applicable.
Sources of funding
Not applicable.
Author contributions
A.A.: Conceptualization, supervision, corresponding author. M.A.H.M.: Methodology, data curation, drafting manuscript. T.M.S.A.: Data analysis, validation, manuscript editing. S.A.: Clinical insights, critical revision. N.A.: Stem cell analysis, visualization. D.S.A.M.: Pathology expertise, figure preparation. H.A.: Pharmacy practice, data management. A.M.S.: Surgical oncology, manuscript refinement.
Conflicts of interest disclosure
There are no conflicts of interest.
Research registration unique identifying number (UIN)
This study is an experimental research; however, it is not a clinical trial or interventional study involving human participants. Therefore, it is not registered with the Iranian Clinical Trial Registration Center (IRCT), which limits registrations to interventional studies to ensure compatibility with the WHO portal.
Guarantor
Abdulmohsen Alrohaimi.
Provenance and peer review
Not commissioned, externally peer-reviewed.
Data availability statement
Data are available from authors on request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data are available from authors on request.