Membrane-Coated Iron Oxide (Fe₃O₄) Nanoparticles Encapsulating Dacarbazine Suppress B16F10 Melanoma Cell Activity in vitro and in a Subcutaneous Mouse Model

Abstract

Background

Malignant melanoma is a highly aggressive skin cancer. Dacarbazine (DTIC) chemotherapy, while historically used, is limited by poor solubility, short half-life, and systemic toxicity. Fe₃O₄ nanoparticles have been explored as drug delivery vectors with potential for photothermal conversion and Fenton-like catalytic activity. Cell membrane-camouflaged platforms may offer improved tumor targeting.

Methods

A biomimetic nanoplatform (Fe₃O₄PD@CCM) was engineered by encapsulating an Fe₃O₄ core within a DTIC-loaded PLGA-PEG2000 matrix, followed by functionalization with B16F10 cancer cell membranes. Physicochemical properties were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), and liquid chromatography–mass spectrometry (LC-MS). Antitumor effects were assessed in B16F10 melanoma cells and in a subcutaneous syngeneic mouse model (n=4 per group).

Results

Fe₃O₄PD@CCM Nanoparticles (NPs) exhibited a hydrodynamic diameter of ~25–30 nm, sustained DTIC release, and retention of membrane proteins. In vitro, the Fe₃O₄PD@CCM + NIR (Near-infrared irradiation) group showed reduced cell viability, increased apoptosis, G2/M phase arrest, and elevated intracellular reactive oxygen species (ROS) levels compared to controls. In vivo, Cy5-labeled Fe₃O₄PD@CCM NPs showed preferential fluorescence accumulation in tumors relative to non-coated NPs, with whole-body fluorescence diminishing within 24 h. In a 12-day efficacy study, Fe₃O₄PD@CCM + NIR treatment was associated with reduced tumor growth compared to control groups. Acute biosafety assessment at 24 h post-injection of a single high dose (200 mg/kg) revealed no overt abnormalities in the evaluated hematological, biochemical, or histopathological parameters.

Conclusion

Fe₃O₄PD@CCM NPs combined with NIR suppressed B16F10 cell activity in vitro and subcutaneous tumor growth in a preliminary mouse model. These findings represent a proof-of-concept demonstration. Further studies—including detailed mechanistic validation, long-term toxicity assessment, and evaluation in more clinically relevant models—are required to assess the translational potential of this platform.

Introduction

Malignant melanoma remains one of the most aggressive and therapy‑resistant malignancies of the skin.^1,2 Its pathogenesis is complex, arising from the interplay between genetic predisposition and environmental exposures.³ In recent years, both the incidence and mortality of melanoma have persisted at unacceptably high levels.^2,4 Conventional chemotherapy—while remaining a cornerstone of systemic treatment—is limited by dose‑related toxicities and inadequate tumor specificity.⁵ Meanwhile, immunotherapy and neoadjuvant chemoradiotherapy continue to be refined and optimized. Advances in nanotechnology have enabled the broad application of nanoparticle‑based drug delivery systems across diverse pathologies, including oncology, chronic inflammatory disorders, and cardiovascular disease.^6–8 Defined as structures with dimensions up to 100 nm, nanoparticles (NPs) possess unique physicochemical properties that, combined with surface functionalization capabilities, render them highly versatile in biomedical applications.⁹ Accumulating clinical evidence supporting the therapeutic utility of various nanoparticle formulations provides a strong rationale for exploring their use in the management of melanoma.^10–12

Iron oxide (ferroferric oxide, Fe₃O₄) nanoparticles have emerged as a prominent research focus in nanobiomedicine owing to their extensive applications in diagnostics and therapeutics.¹³ Distinguished by their superparamagnetic properties, Fe₃O₄ NPs based nanostructures exhibit directional mobility under external magnetic fields, outperforming conventional nanoparticles in terms of spatial controllability.¹⁴ This unique feature facilitates precise magnetic-targeted drug delivery and endows these agents with high efficacy as T₂-weighted magnetic resonance imaging (MRI) contrast enhancers, thereby enabling a seamless integration of diagnosis and therapy.^15,16 Notably, Fe₃O₄ nanoparticles demonstrate exceptional near-infrared photothermal conversion efficiency. Upon irradiation at specific wavelengths, they effectively transduce photon energy into localized hyperthermia, enabling selective ablation of tumor tissues.¹⁷ In addition, these nanoparticles catalyze sequential Fenton-like reactions within the tumor microenvironment, generating cytotoxic reactive oxygen species (ROS)—particularly highly oxidizing hydroxyl radicals (·OH)—which induce apoptosis and necrosis of malignant cells.^18,19 Beyond their direct cytotoxic effects, iron oxide nanoparticles actively modulate the phenotype of tumor-associated macrophages, driving the polarization of pro-tumorigenic phenotypes toward antitumorigenic ones, thus exerting immunoregulatory control over tumor progression.²⁰ Complementarily, abundant evidence supports their favorable biocompatibility and biosafety profiles.^21,22 To further refine targeting specificity and circumvent immunological barriers, recent strategies have incorporated cell membrane coating technologies into nanoparticle engineering. Leveraging the innate capacity of cellular membranes to engage with their microenvironment, nanoparticles functionalized with homologous tumor cell membranes achieve immune evasion and homotypic tumor targeting, significantly enhancing intratumoral drug accumulation while minimizing off-target effects.^23,24

Recent strategies in nanomedicine span multiple cargo and probe paradigms: cyclic acetal-based ionizable Lipid nanoparticle co-formulated with toll-like receptor agonist adjuvant lipids enable spleen-biased mRNA delivery and antigen-presenting cells maturation for tumor immunotherapy;²⁵ activatable organic Near-infrared irradiation (NIR) fluorophores—including boron-dipyrromethene, cyanine, rhodamine, diketopyrrolopyrrole and squaraine scaffolds—support enzyme-, pH- and ROS-responsive turn-on imaging for cervical cancer theragnosis with emerging NIR-II utility;^26,27 transition-metal complexes (Ru(II), Ir(III), Re(I), Mn, Zn, Pt) encapsulated in nanocarriers act as Fenton-like or photosensitizing cores for stimulus-responsive photodynamic therapy (PDT)/photothermal therapy (PTT)/sonodynamic therapy (SDT) with immunogenic cell death;²⁸ and analogous NIR small-molecule probes have been extended to breast cancer for lymph-node metastasis imaging and dual-activation detection.^29,30 Parallel accounts on green-synthesized metal oxide nanoparticles highlight the broad role of nanoparticle-generated oxidative stress across antimicrobial and oncologic contexts.^31–33

Dacarbazine (DTIC), the first chemotherapeutic agent approved for melanoma, remained the clinical standard of care for over three decades.^34,35 Its antitumor activity is primarily mediated by hepatic activation to yield a DNA-alkylating metabolite.³⁶ Extensive preclinical and clinical investigations have corroborated both its cytotoxic and immunomodulatory effects against melanoma.^37,38 Nevertheless, its therapeutic utility is substantially constrained by poor aqueous solubility, a short systemic half-life, and pronounced off-target toxicities.³⁹ To address these limitations and potentiate the efficacy of DTIC against melanoma, we engineered a biomimetic nanoplatform (Fe₃O₄PD@CCM NP). This system comprises a magnetic Fe₃O₄ core encased within a PLGA–PEG2000 matrix for DTIC loading, further cloaked with B16F10 melanoma cell membranes (Scheme 1). The resulting nanoparticles facilitate targeted accumulation within the cutaneous tumor microenvironment and promote selective internalization by melanoma cells. This design markedly diminishes systemic exposure of DTIC to healthy tissues, thereby mitigating adverse effects and safety concerns. Beyond its role as a drug carrier, the Fe₃O₄ core serves as a therapeutic amplifier. Capitalizing on its intrinsic photothermal conversion and Fenton-like catalytic properties,^40–44 Fe₃O₄ enables a transition from mere additive effects to a synergistic amplification of DTIC’s antitumor activity. Collectively, this biomimetic nanoplatform represents a promising and translatable strategy for the safe and effective clinical management of advanced melanoma.

Scheme 1.

Schematic overview of the synthesis and in vivo application of Fe₃O₄PD@CCM NPs. Upward arrows (↑) indicate high expression of these markers (4HNE, C-casp3), while downward arrows (↓) indicate low expression of Ki67.

Materials and Methods

Materials

Dacarbazine hydrochloride (DTIC), tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO) were procured from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-PEG2000) and ferroferric oxide nanoparticles (Fe₃O₄NPs) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cell culture supplies, including 96-, 24-, and 6-well plates, T25 flasks, and centrifuge tubes, were purchased from Servicebio Technology Co., Ltd. (Wuhan, China). Hematoxylin and eosin (H&E) staining kit and immunohistochemistry (IHC) detection system kit were sourced from Dowobio Co., Ltd. (Shanghai, China). Primary antibodies against 4-hydroxynonenal (4-HNE), cleaved caspase-3 (C-casp3), and Ki-67 antigen were acquired from Shenyang Wanlei Biotechnology Co., Ltd. (Shenyang, China). The crystal violet reagent used for cell staining was purchased from Servicebio Technology Co., Ltd. (Wuhan, China). The following primary antibodies were purchased from Wuhan Proteintech Group Biotechnology Co., Ltd. (Wuhan, China): HO-1, Nrf2, Bax, Bcl-2, GAPDH, CD44, CD47, E-cadherin, Na⁺/K⁺-ATPase, and N-cadherin. RIPA lysis buffer, protease inhibitor cocktail, bovine serum albumin (BSA), TBST (Tris buffered saline Tween) buffer, BCA protein assay kit, enhanced chemiluminescence (ECL) reagent, and polyvinylidene fluoride (PVDF) membranes were obtained from Servicebio Technology Co., Ltd. (Wuhan, China). Western blot electrophoresis was conducted using a Bio-Rad system (Bio-Rad, USA), and protein signals were visualized using a Tanon 5200 chemiluminescence imaging system (Tanon, China). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody was obtained from Dowobio Co., Ltd. (Shanghai, China). Cyanine 5 (Cy5) fluorescent dye was purchased from MeilunBio (Dalian, China). Assay kits for cell apoptosis, cell cycle analysis, and reactive oxygen species (ROS) detection were procured from Dowobio Co., Ltd. (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA, 1× phosphate-buffered saline (PBS), and normal saline (NS) were supplied by Servicebio Technology Co., Ltd. (Wuhan, China). SDS-PAGE electrophoresis reagents and Coomassie Brilliant Blue staining solution were purchased from Servicebio Technology Co., Ltd. (Wuhan, China). The Matrigel matrix gel was procured from Corning Incorporated (Corning, NY, USA). An 808 nm near-infrared laser (Taizhu AnFord Laser Co., Ltd., Shenzhen, China) was applied at an energy density (power density) of 1 W/cm². For cellular experiments, each irradiation session lasted 15 min. For in vivo experiments using C57BL/6 mice, animals were irradiated every 2 days with each session lasting 15 min. The physicochemical characteristics of the nanoparticles were systematically characterized using dynamic light scattering (DLS; Zetasizer Ultra, Malvern Panalytical, UK), scanning electron microscopy (SEM; Apreo 2, Thermo Fisher Scientific, USA), and liquid chromatography–mass spectrometry (LC-MS; LCMS-8060, Shimadzu, Japan). Mini-Extruder⁴⁵ (Model No. 610020; Avanti Research, a Croda brand; USA).

Cell Line and Mice

The mouse melanoma cell line B16F10 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. Male C57BL/6 mice (body weight 20–22 g) were obtained from Ensiwei Biotechnology Co., Ltd. (Chongqing, China) and housed in a specific pathogen-free facility under controlled temperature and humidity with a 12-h light/dark cycle (Approval No. AMUWEC20267017).

Preparation and Characterization of Nanoparticles

Fe₃O₄-PLGA-PEG2000-DTIC-membrane nanoparticles (Fe₃O₄PD@CCM NPs) were synthesized utilizing a combined freeze-drying and centrifugal sedimentation method. Briefly, 10 mg of dacarbazine (DTIC) and 20 mg of PLGA-PEG2000 were dissolved in a 4 mL cosolvent mixture of tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) (1:1, v/v). Subsequently, 1 mL of THF containing 50 mg of Fe₃O₄ NPs was introduced into the organic phase. Solvent removal was achieved by lyophilization at −80°C under a vacuum of 0 Pa. The resulting precipitate was rehydrated in 2 mL of normal saline (NS) for 30 min, followed by centrifugation at 3000 rpm and 4°C for 5 min to isolate Fe₃O₄-PLGA-PEG2000 NPs (Fe₃O₄P) and Fe₃O₄-PLGA-PEG2000-DTIC NPs (Fe₃O₄PD). For the fabrication of Fe₃O₄PD@CCM NPs, B16F10 cancer cell membranes (CCM) were harvested via repeated freeze–thaw cycles and density-gradient centrifugation. Thereafter, 50 mg of CCM and 50 mg of Fe₃O₄PD NPs were co-extruded through a mini-extruder in 1 mL of NS to afford Fe₃O₄PD@CCM NPs. The physicochemical characteristics of the nanoparticles were systematically evaluated using dynamic light scattering (DLS), scanning electron microscopy (SEM), and liquid chromatography–mass spectrometry (LC-MS). For fluorescence labeling, 20 μL of Cy5 solution (25 mg/mL) was pre-mixed into the organic solvent phase prior to the addition of Fe₃O₄PD NPs and cell membranes, yielding Cy5-Fe₃O₄PD NPs and Cy5-Fe₃O₄PD@CCM NPs, respectively.

Drug Release and Encapsulation Efficiency

The in vitro release of dacarbazine (DTIC) from nanoparticles was evaluated using a dialysis bag method. Two milliliters of nanoparticle suspension (containing 1 mg DTIC equivalent) were sealed in a pre-activated dialysis membrane (molecular weight cut off 8–14 kDa) (Beyotime, China) and immersed in 20 mL of phosphate-buffered saline (pH 7.4) containing 0.5% Tween-80 at 37 °C with shaking at 100 rpm. At predetermined time points (0–24 h), 1 mL of release medium was withdrawn and replaced with an equal volume of fresh medium. DTIC concentration was determined by LC-MS (λ = 278 nm), and cumulative release was calculated as:

where C_n is the DTIC concentration at the nth sampling, V₀ = 20 mL, V_S = 1 mL, and M_total = 1 mg. Encapsulation efficiency (EE%) and drug loading (DL%) were determined by ultrafiltration centrifugation. One milliliter of nanoparticle suspension was centrifuged (molecular weight cut off 30 kDa, Beyotime, China, 4°C, 5000 g, 20 min), and the filtrate was analyzed for free DTIC concentration (C_free). Another 1 mL aliquot was mixed with 2 mL methanol, sonicated for 30 min, and centrifuged (12000 g, 10 min). The supernatant DTIC concentration (C_sup) was measured, and the total DTIC concentration in the original suspension was calculated as C_total = C_sup × 3. EE% and DL% were calculated as:

,

where M_encapsulated= (C_total − C_free) × 1 mL and M_{nanoparticles} is the dry mass of nanoparticles in 1 mL suspension. All measurements were performed in triplicate.

In vitro Cell Cytotoxicity

Cell Counting Kit-8 (CCK-8) assays were conducted to assess the cytotoxicity of various nanoparticle formulations against B16F10 murine melanoma cells. Cells were seeded into 96-well plates at a density of 3×10⁴ cells per well and allowed to adhere overnight. Subsequently, cells were exposed to serial dilutions of dacarbazine (DTIC), Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs or Fe₃O₄PD@CCM NPs at concentrations ranging from 0 to 1024 μg/mL. Following a 24 h incubation period at 37°C in a humidified atmosphere containing 5% CO₂, the culture medium was aspirated, and 100 μL of CCK-8 working solution (10% v/v in fresh medium) was added to each well. The plates were incubated for an additional 30 min under the same conditions. The absorbance was measured at 450 nm using a microplate reader (Thermo Scientific Multiskan SkyHigh, USA). Relative cell viability was calculated using the following equation: Cell viability (%) = [(ODtreatment− ODblank)/(ODcontrol− ODblank)] × 100%.

In vitro Apoptosis

Apoptosis was quantitatively evaluated by flow cytometry using an Annexin V-FITC/PI Apoptosis Detection Kit. Briefly, B16F10 murine melanoma cells were seeded into 6-well plates at a density of 5×10⁵ cells/well and allowed to adhere overnight. Subsequently, the cells were exposed to complete DMEM supplemented with 200 μg/mL of either dacarbazine (DTIC), Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs or Fe₃O₄PD@CCM NPs for 24 hours. Following treatment, adherent and non-adherent cells were harvested via trypsinization, washed with cold PBS, and resuspended in binding buffer. Cells were then stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s instructions. Data acquisition and analysis were performed using a flow cytometer (Beckman Coulter, Brea, CA, USA).

Cell Cycle Analysis

For cell cycle analysis, B16F10 cells were seeded into 6-well plates at a density of 5 × 10⁵ cells/well and allowed to adhere overnight. After treatment with the indicated nanoparticle formulations for 24 h, cells were harvested by trypsinization, fixed in ice-cold 70% ethanol at 4 °C for ≥12 h, and stained with propidium iodide (PI) for 30 min. Cell cycle distribution was subsequently analyzed using flow cytometry (FCM; Beckman Coulter, Brea, CA, USA).

Intracellular ROS Detection

Intracellular reactive oxygen species (ROS) generation was evaluated using an inverted fluorescence microscope. B16F10 cells were seeded onto sterilized coverslips in 12‑well plates at 3 × 10⁴ cells/well. Following 24 h of exposure to the specified nanoparticles, cells were loaded with 2′,7′‑dichlorodihydrofluorescein diacetate (DCFH‑DA; 1:1000 dilution) for 30 min, fixed with 4% paraformaldehyde, and mounted with an anti‑fluorescence quenching medium. Fluorescent images were acquired using an inverted fluorescence microscope (IFM; Leica Microsystems, Wetzlar, Germany).

Wound-Healing Assay

B16F10 cells were seeded in 6-well plates at 5 × 10⁶ cells/well and cultured until a confluent monolayer was formed. A standardized scratch wound was introduced using a 1 mL sterile pipette tip, and detached cells were removed by two washes with phosphate-buffered saline (PBS). The monolayers were then incubated in serum-free DMEM containing either dacarbazine (DTIC; 200 μg/mL), Fe₃O₄ NPs (200 μg/mL), Fe₃O₄P NPs (200 μg/mL), Fe₃O₄PD NPs (200 μg/mL), or Fe₃O₄PD@CCM NPs (200 μg/mL). A negative control group received no treatment. Wound closure was monitored and imaged at 0 h and 24 h using an inverted fluorescence microscope (IFM; Leica Microsystems, Wetzlar, Germany).

Transwell Assay

Transwell inserts were pre-coated with 50 μL of diluted Matrigel matrix (Matrigel: serum-free DMEM = 1:9) and polymerized overnight at 37 °C. Subsequently, 300 μL of serum-free DMEM containing 4 × 10⁴ B16F10 cells suspended in various drug-containing media was seeded into the upper chambers. The lower chambers were supplemented with 600 μL of complete growth medium. Following a 24-h incubation, the inserts were gently washed with 1× PBS, fixed with 4% paraformaldehyde, and stained with 0.2% crystal violet for 10 min. Invaded cells on the lower membrane surface were imaged using an inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

In vivo Biodistribution in B16F10 Tumor-Bearing Mice

After a 3-day acclimatization period, B16F10 tumor models were established by subcutaneous inoculation of 5 × 10⁶ cells per mouse. Tumor-bearing mice were randomized into experimental groups and intravenously administered Cy5-labeled Fe₃O₄PD@CCM nanoparticles via the tail vein. A control cohort receiving Cy5-Fe₃O₄PD nanoparticles lacking the cell membrane coating served as the negative control. Whole-body fluorescence imaging was performed at predetermined time points (1, 6, 12, 18, and 24 h post-injection) using an in vivo imaging system (RWD Life Science, Shenzhen, China). At 24 h post-administration, animals were humanely euthanized by controlled CO₂ inhalation. Major organs (heart, liver, spleen, lungs, and kidneys) and tumor tissues were harvested for ex vivo Cy5 fluorescence quantification.

In vivo Biosafety Evaluation

Healthy C57BL/6 mice were intravenously administered a single high dose (200 mg/kg) of Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs or Fe₃O₄PD@CCM NPs suspended in normal saline (NS) via the tail vein. Following a 24-h systemic circulation period, peripheral blood was collected via retro-orbital bleeding for hematological and serum biochemical analyses. Hematological parameters, including red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), and platelets (PLT), were quantified. Serum biochemistry was assessed via levels of Alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (UREA). Major organs (heart, liver, spleen, lungs, and kidneys) were harvested for histopathological examination using hematoxylin and eosin (H&E) staining. In addition, to evaluate the hemocompatibility of the nanoparticles, 50 μL of fresh whole blood collected from mice was co-incubated with 2 mg/mL of each nanoparticle suspension. Meanwhile, equal volumes of blood were mixed with double distilled water (DDW) and normal saline (NS), respectively, as controls to verify whether DDW, NS or these nanoparticles induce red blood cell hemolysis. Samples were centrifuged at 1500 rpm for 10 min, and digital images were acquired both pre- and post-centrifugation to evaluate hemolytic activity.

In vivo Antitumor Efficacy Against B16F10 Melanoma

After subcutaneous inoculation of B16F10 melanoma cells, tumor-bearing mice were randomized into six groups (n = 4 per group) and received intravenous injections via the tail vein: (1) NS (Control); (2) free dacarbazine (DTIC); (3) Fe₃O₄ NPs; (4) Fe₃O₄P NPs; (5) Fe₃O₄PD NPs; and (6) Fe₃O₄PD@CCM NPs (20 mg/kg). Treatments were administered every 2 days. Tumors in the nanoparticle-treated groups were irradiated with an infrared laser (NIR) for 15 min every 2 days. Body weights and tumor volumes were measured and recorded at regular intervals. After a 12-day treatment regimen, all mice were euthanized; tumors were excised, weighed, and fixed in 4% paraformaldehyde for subsequent histological analysis. In the volumetric assessment of tumors, the longest axial diameter (maximum value, denoted as Lmax) and the shortest axial diameter perpendicular to it (minimum value, denoted as Lmin) are measured. The tumor volume (V) is then calculated using the modified ellipsoid formula: V = ½ × Lmax × Lmin² (Unit: mm³).

Hematoxylin and Eosin (H&E) and Immunohistochemistry (IHC) Staining

Upon dissection, tumor tissues were fixed and embedded in paraffin. Serial sections (4 μm) were prepared and baked at 60°C for 1 h. Deparaffinization was achieved using xylene, followed by rehydration through a graded ethanol series. For antigen retrieval, sections were processed using a commercial kit according to the manufacturer’s protocol (Servicebio, Wuhan, China). For histological assessment, sections were stained with H&E. For IHC analysis, sections were incubated with primary antibodies targeting 4-hydroxynonenal (4-HNE), cleaved Caspase-3 (C-casp3), and Ki-67, followed by detection using a diaminobenzidine (DAB) substrate kit (Servicebio). All stained slides were examined and photographed using a Leica inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Western Blot

Total protein was extracted from cells using RIPA buffer containing 1× protease inhibitor cocktail, followed by centrifugation at 12,000 × g for 15 min at 4 °C. Protein concentration was determined by BCA assay with BSA as the standard. Equal amounts of protein (20 μg per lane) were denatured in 5× SDS loading buffer at 95 °C for 10 min and resolved by 10% SDS-PAGE, then transferred to a PVDF membrane at 300 mA for 90 min at 4 °C. The membrane was blocked with 5% non-fat dry milk in TBST for 1 h at room temperature, incubated overnight at 4 °C with primary antibody diluted in 5% BSA/TBST, and subsequently probed with an HRP-conjugated secondary antibody (1:5000) for 1 h at room temperature, with three 10-min TBST washes performed between and after incubations. Immunoreactive bands were detected using an ECL substrate on a Tanon 5200 imaging system, and band intensities were quantified with ImageJ (version 1.8.0, National Institutes of Health, USA). GAPDH served as the loading control. All experiments were conducted in triplicate, and data are expressed as mean ± SD.

Statistical Analysis

All quantitative data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Multigroup comparisons were performed using one-way analysis of variance (ANOVA). Pairwise comparisons between two groups were conducted using Student’s t-test. Statistical significance was set at p< 0.05, with significance levels indicated as follows: p< 0.05 (*), p< 0.01 (**), p< 0.001 (***), and p< 0.0001 (****). Statistical analyses were performed using IBM SPSS Statistics (Version 27, IBM Corp., Armonk, NY, USA). Data visualization, including histograms and graphs, was generated using GraphPad Prism (Version 8, GraphPad Software, San Diego, CA, USA) and Origin (Version 2024, OriginLab Corporation, Northampton, MA, USA).

Results

Preparation and Characterization of Fe₃O₄PD@CCM NPs

Fe₃O₄ NPs (∼25 nm; Figure 1A), serving as the magnetic cores, were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). To fabricate Fe₃O₄P NPs, Fe₃O₄ NPs and PLGA-PEG2000 were thoroughly dispersed in tetrahydrofuran (THF) at room temperature. The solution was subsequently frozen at −80 °C under 0 Pa and lyophilized. The resulting black residue was hydrated in normal saline (NS), followed by centrifugation at 1500 rpm for 10 min to obtain purified Fe₃O₄P NPs (Figure 1A). For the synthesis of Fe₃O₄PD NPs, a THF suspension containing Fe₃O₄ NPs and PLGA-PEG2000 was homogeneously mixed with a dimethyl sulfoxide (DMSO) solution of the chemotherapeutic agent dacarbazine (DTIC). Purification was carried out using the procedure described above. Fe₃O₄PD@CCM NPs were engineered by sequentially coating Fe₃O₄PD NPs with B16F10 cell membranes via sonication and repeated extrusion, followed by the same purification protocol. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) analyses revealed that Fe₃O₄, Fe₃O₄P, Fe₃O₄PD, and Fe₃O₄PD@CCM NPs maintained desirable hydrodynamic diameters ranging from 25 to 30 nm (Figure 1A) and exhibited characteristic zeta potential shifts corresponding to surface modification (Figure 1B). In vitro drug release studies demonstrated that both Fe₃O₄PD and Fe₃O₄PD@CCM NPs facilitated the sustained release of DTIC, reaching peak cumulative release within 24 h (Figure 1C). Stability assessments indicated negligible changes in particle size for all formulations upon dispersion in NS over the observation period (Figure 1D). SEM imaging further confirmed that all nanoparticle variants retained intact, well-defined nanostructures (Figure 1E). Quantitative analysis via liquid chromatography–mass spectrometry (LC-MS) determined the DTIC encapsulation efficiencies of Fe₃O₄PD and Fe₃O₄PD@CCM NPs to be approximately 14% and 13%, respectively. Collectively, these results demonstrate the successful fabrication of structurally sound, stable nanocarriers with efficient drug-loading and delivery capabilities. To evaluate whether pivotal membrane proteins remain structurally and functionally intact throughout the fabrication of biomimetic nanoassemblies, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was first performed. Electrophoretic profiling revealed a high degree of concordance between the protein band patterns of native cancer cell membranes (CCM) and Fe₃O₄PD@CCM NPs, indicative of effective and comprehensive protein preservation during the coating process. In stark contrast, unmodified Fe₃O₄, Fe₃O₄P, and Fe₃O₄PD NPs exhibited no discernible protein bands, consistent with their inherently protein-free composition (Figure 1F). To further substantiate the retention of functionally relevant species, Western blot analysis was conducted, which confirmed the successful preservation of key membrane-associated proteins on Fe₃O₄PD@CCM NPs, including the homophilic adhesion molecules CD44, CD47, E-cadherin, and N-cadherin, as well as the housekeeping transmembrane protein Na⁺/K⁺-ATPase (Figure 1G). The faithful retention of these proteins is anticipated to confer prolonged systemic circulation and enhanced tumor-targeted accumulation to the biomimetic nanoassemblies.

Figure 1.

Physicochemical characterization of Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs. (A and B) Diameter distribution and zeta potential measured by dynamic light scattering (DLS; n=3). (C) Cumulative dacarbazine (DTIC) release profile from nanoparticles in phosphate-buffered saline quantified by liquid chromatography–mass spectrometry (LC-MS; n=3). (D) Stability of nanoparticles in NS over 24 h assessed by DLS (n=3). (E) Representative scanning electron microscopy (SEM) images (scale bars = 200 nm). Data are expressed as mean ± standard deviation (SD). (F) SDS-PAGE protein analysis of CCM (Cancer cell membrane), Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs. (G) Western blot analysis for the expression of membrane functional protein markers.

Cytotoxicity of Nanoparticles Combined with Near-Infrared Irradiation (NIR) Against B16F10 Melanoma Cells

The cytotoxic profiles of dacarbazine (DTIC), iron oxide nanoparticles (Fe₃O₄ NPs), polyethylene glycol-modified iron oxide nanoparticles (Fe₃O₄P NPs), DTIC-loaded Fe₃O₄P NPs (Fe₃O₄PD NPs), and Fe₃O₄PD@CCM NPs were systematically evaluated in B16F10 murine melanoma cells, both with and without near-infrared irradiation (NIR). As shown in (Figure 2A), DTIC, Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs exerted a concentration-dependent inhibitory effect on tumor cell proliferation. Notably, NIR irradiation significantly potentiated the antitumor efficacy of Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs compared to their non-irradiated controls (Figure 2A). Based on these quantitative results, a nanoparticle concentration of 200 μg/mL under NIR irradiation was identified as the optimal parameter set and was therefore adopted for all subsequent in vitro experiments. To elucidate the mechanism underlying the observed cytotoxicity, flow cytometry (FCM) was employed to quantify apoptosis and cell cycle distribution following 24 h of treatment. Quantitative apoptosis analysis revealed that while B16F10 cells exhibited a basal level of spontaneous apoptosis, treatment with the nanoparticle formulations markedly elevated the percentage of early and late apoptotic cells. The Fe₃O₄PD@CCM + NIR group demonstrated the most potent pro-apoptotic effect (Figure 2B). Consistent with this, cell cycle profiling indicated that the tested formulations differentially modulated cell cycle progression across the G0/G1, S, and G2/M phases. Specifically, the Fe₃O₄PD@CCM + NIR regimen induced the most significant perturbation of the cell cycle (Figure 2C). Collectively, these findings robustly validate the enhanced anticancer activity conferred by the combination of NIR and Fe₃O₄-based nanoparticle platforms, particularly the Fe₃O₄PD@CCM formulation, in B16F10 cells.

Figure 2.

In vitro cytotoxic effects of nanoparticle formulations combined with near-infrared (NIR) irradiation. (A) Relative viability of B16F10 murine melanoma cells following various treatments, determined by Cell Counting Kit-8 (CCK-8) assay (n=8). (B) Quantitative analysis of apoptosis in B16F10 cells treated with nanoparticles plus NIR, evaluated by flow cytometry (FCM; n=3). (C) Cell cycle distribution of B16F10 cells treated with nanoparticles plus NIR, evaluated by flow cytometry (FCM; n=3). Data are expressed as mean ± standard deviation (SD). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

Impact of Nanoparticle-Mediated Therapy on Cellular Function and Oxidative Stress in B16F10 Cells

Having established the inhibitory effects of the nanoparticles on B16F10 cells, we next investigated the role of reactive oxygen species (ROS) in mediating tumor cell apoptosis. Fluorescence-based assays revealed that all formulations induced ROS generation in B16F10 cells to varying extents; notably, the Fe₃O₄PD@CCM + NIR group exerted the most potent pro-oxidant effect (Figure 3A). The resultant ROS overproduction significantly compromised cellular physiology. Wound healing (Figure 3B) and Transwell invasion (Figure 3C) assays demonstrated that Fe₃O₄PD@CCM + NIR treatment most effectively suppressed the migratory and invasive capacities of B16F10 cells compared with other treatment groups. These findings suggest that Fe₃O₄PD@CCM NP-mediated phototherapy inhibits B16F10 cellular functions predominantly via ROS induction.

Figure 3.

Effect of various nanoparticles combined with NIR irradiation on intracellular ROS generation and functional phenotypes of B16F10 cells in vitro. (A) Intracellular ROS levels in B16F10 cells treated with different nanoparticles plus NIR, detected by immunofluorescence microscopy (IFM; n=3). (B) Migration capability of B16F10 cells inhibited by nanoparticle–NIR treatment, assessed using wound healing assays (n=3). (C) Invasive potential of B16F10 cells suppressed by nanoparticle–NIR treatment, evaluated via Transwell invasion assays (n=3). Data are expressed as mean ± standard deviation (SD). ***p<0.001, ****p<0.0001; ns, not significant.

In vivo Biodistribution and Tumor-Targeting Specificity of Fe₃O₄PD@CCM NPs in B16F10 Melanoma Models

The top row presents bright-field (white-light) images, whereas the bottom row displays the corresponding fluorescence (Cy5) images for each experimental group. Serial fluorescence imaging (bright-field) revealed a marked reduction in systemic circulation signals (Figure 4), indicating efficient metabolic clearance of the nanoparticles. Regarding tissue distribution, both nanoparticle formulations exhibited minimal accumulation in major organs—excluding the liver (Figure 4). Crucially, Fe₃O₄PD@CCM NPs functionalized with homotypic tumor cell membranes demonstrated significantly enhanced melanoma-specific targeting compared to their non-modified counterparts (fluorescence images). These results validate the homotypic targeting strategy as an effective approach for improving the specificity of nanomedicine delivery to malignant melanoma.

Figure 4.

In vivo pharmacokinetics, biodistribution, and tumor-targeting efficiency of Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs. Blood circulation profiles of different nanoparticles at 1, 6, 12, 18, and 24 h post-injection, monitored by laser-based fluorescence imaging (LBFI). Tumor and major organ accumulation of Cy5-conjugated Fe₃O₄PD NPs and Fe₃O₄PD@CCM NPs at 24 h post-injection, visualized by LBFI. The first row shows bright-field (white-light) images and the second row shows the corresponding fluorescence (Cy5) images. Each organ is now marked directly on the images (Heart, Liver, Spleen, Lung, Kidney, Tumor).

In vivo Biosafety Evaluation of Nanoparticle Formulations in C57BL/6 Mice

The systemic biosafety profiles of Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs and Fe₃O₄PD@CCM NPs were comprehensively evaluated in healthy C57BL/6 mice through hematological analysis, hemocompatibility testing, and histopathological assessment. Compared with the control group, hematological examinations revealed no statistically significant alterations in blood routine indices or serum biochemical parameters in the high-dose treatment groups (Figure 5A). Ex vivo hemolysis assays confirmed that these metallic nanoparticles did not induce erythrocyte lysis (Figure 5B). Furthermore, H&E-stained sections exhibited preserved organ architectures without evidence of pathological lesions, inflammatory infiltration, or tissue necrosis (Figure 5C). Collectively, these results demonstrate that the synthesized nanoparticles possess favorable biocompatibility and do not elicit overt systemic toxicity in vivo.

Figure 5.

In Vivo Biosafety Assessment of Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs. (A) Hematological analysis of whole blood samples, including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and platelet (PLT) counts, alongside serum biochemistry profiles for Alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea, quantified using an automated hematology analyzer (n=3). (B) Photographic documentation of erythrocytes following exposure to nanoparticle suspensions, acquired under bright-field to evaluate hemocompatibility. (NS, normal saline; DDW, double distilled water). (C) Representative hematoxylin and eosin (H&E)-stained histological sections of major organs, illustrating preserved tissue architecture (n=3). Data are expressed as mean ± standard deviation (SD). ns, not significant.

Antitumor Efficacy of Nanoparticle Formulations Against B16F10 Melanoma in vivo

To assess the in vivo therapeutic efficacy, male C57BL/6 mice bearing subcutaneous B16F10 melanoma tumors were randomly assigned to receive different nanoparticle formulations. Tumor-bearing mice were imaged at predetermined intervals to monitor disease progression. Quantitative analysis revealed that all treatment groups significantly suppressed tumor growth compared to the control cohort, with the Fe₃O₄PD@CCM + NIR group exhibiting the most potent antitumor activity. Upon study termination, animals were euthanized, and tumors were excised for tumor-weight analysis. Consistent with the volumetric data, tumor weights were markedly reduced across all treatment groups, with the Fe₃O₄PD@CCM + NIR regimen achieving the maximal tumor regression (Figure 6A–D). Histopathological examination further corroborated these findings; H&E staining revealed extensive disruption of the dense tumor parenchyma and inhibition of cellular proliferation within the treatment groups (Figure 6E). Moreover, immunohistochemical (IHC) analysis confirmed a significant upregulation of 4-hydroxynonenal (4-HNE), a lipid peroxidation marker indicative of oxidative stress, and cleaved caspase-3 (C-casp3), a key executor of apoptosis, in the tumor tissues.

Figure 6.

In Vivo Antitumor Efficacy of Fe₃O₄ NPs, Fe₃O₄P NPs, Fe₃O₄PD NPs, and Fe₃O₄PD@CCM NPs Under NIR Irradiation. (A) Representative images of C57BL/6 mice bearing subcutaneous B16F10 melanoma tumors subjected to various nanoparticle treatments combined with near-infrared (NIR) laser irradiation (n= 4). (B) Gross morphology of excised B16F10 tumor xenografts harvested at the experimental endpoint (n= 4). (C) Temporal kinetics of tumor growth, plotted as relative tumor volume over time (n= 4). (D) Quantitative analysis of excised tumor mass, expressed as average tumor weight (n= 4). (E) H&E-stained tumor sections revealing histopathological alterations. Immunohistochemical (IHC) staining of tumor tissues for 4-hydroxynonenal (4HNE), cleaved caspase-3 (C-casp3), and Ki67, indicating oxidative damage, apoptotic induction, and proliferative suppression, respectively. Data are expressed as mean ± standard deviation (SD). **p<0.01, ****p<0.0001, ns, not significant.

Gene Expression Profiling of ROS Metabolism and Apoptosis Markers Across B16F10 Treatment Groups

To systematically evaluate the therapeutic mechanism of our nanoplatform, we analyzed the expression of some key genes spanning ROS metabolism and apoptosis regulation across all six B16F10 treatment groups (Control, DTIC, Fe₃O₄ + NIR, Fe₃O₄P + NIR, Fe₃O₄PD + NIR, and Fe₃O₄PD@CCM + NIR) (Figure 7A–F).

Figure 7.

Western blot analysis revealed metabolism and apoptosis markers across all treatment groups(A). (B–F) The Relative protein expression of Nrf2, HO-1, Bcl-2, Bax and Cleaved caspase-3. Data are expressed as mean ± standard deviation (SD). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

ROS metabolism. The antioxidant transcription factor Nrf2 and its downstream effector HO-1—two central regulators of intracellular ROS scavenging—were most highly expressed in the Control group. Moderate reductions were observed in the DTIC, Fe₃O₄ + NIR, and Fe₃O₄P + NIR groups, while Fe₃O₄PD + NIR further suppressed both markers, and Fe₃O₄PD@CCM + NIR achieved the strongest inhibition, driving Nrf2 and HO-1 to their lowest levels across all groups.

Apoptosis assay. We next quantified the pro-apoptotic markers Bax and cleaved caspase-3. The Control group exhibited minimal expression. Moderate elevations appeared in the Fe₃O₄ + NIR and Fe₃O₄P + NIR groups, with a slightly stronger response in the DTIC group. Fe₃O₄PD + NIR triggered a substantial increase, and Fe₃O₄PD@CCM + NIR drove both markers to their highest levels among all groups. Conversely, the anti-apoptotic factor Bcl-2 followed an inverse trajectory: highest in Control, gradually attenuated through DTIC and the Fe₃O₄-based groups, and most sharply downregulated by Fe₃O₄PD@CCM + NIR, which reduced Bcl-2 to its lowest level.

Discussion

Malignant melanoma remains poorly served by conventional dacarbazine (DTIC) monotherapy because of its poor aqueous solubility, short systemic half-life, and narrow therapeutic window.^39,46,47 In this study, we constructed a biomimetic nanoplatform—Fe₃O₄PD@CCM—by embedding a DTIC-loaded PLGA-PEG2000 matrix onto an Fe₃O₄ core and cloaking it with B16F10 cancer-cell membranes. The resulting particles preserved a hydrodynamic diameter of ~25–30 nm (Figure 1A and E), showed sustained DTIC release peaking within 24 h (Figure 1C), and retained canonical membrane proteins (CD44, CD47, E-cadherin, N-cadherin, Na⁺/K⁺-ATPase) as verified by SDS-PAGE and Western blot^48–50 (Figure 1F and G). Zeta-potential shifts across the fabrication steps further corroborated sequential PLGA2000 coating and membrane functionalization (Figure 1B). These characterization data indicate that the coating step did not strip functionally relevant membrane identity, which is a prerequisite for the homologous-targeting behavior later observed in vivo. It should be noted, however, that the absolute drug-loading efficiency (~13–14%) is modest, suggesting that future formulation optimization—such as adjusting the PLGA2000: DTIC mass ratio or exploring alternative hydrophobic matrices—may be necessary to improve the therapeutic payload per particle.

In vitro, B16F10 cells treated with Fe₃O₄PD@CCM under 808 nm NIR (1 W/cm², 15 min) showed the lowest viability among all groups in the CCK-8 assay, the highest apoptotic fraction by Annexin V/PI flow cytometry, and the most pronounced G2/M arrest (Figure 2). The same group produced the strongest DCFH-DA signal, indicating elevated intracellular ROS (Figure 3A). Because ROS elevation tracked closely with suppressed wound closure and Transwell invasion (Figure 3B and C), the functional phenotype is consistent with, but not proof of, oxidative impairment of motility rather than a separate targeted pathway. The photothermal contribution is supported by the fact that blank Fe₃O₄ and Fe₃O₄P also gained cytotoxicity only when NIR was added (Figure 2A), while DTIC alone lacked this NIR-dependent jump. Thus the in-vitro data support a combined effect of delivered DTIC, Fe₃O₄-mediated photothermal heating, and Fenton-like ROS catalysis,^51,52 with the membrane coat improving potency at equal mass dose rather than creating a new mechanism. A neutral reading of the concentration–response curve (Figure 2A) also reveals that even without NIR, Fe₃O₄PD@CCM exhibited a modest but measurable viability reduction relative to free DTIC at equivalent concentrations—likely reflecting the slow-release profile of the PLGA matrix and partial cellular uptake of the nanoparticle-bound drug. This baseline activity, while not the primary therapeutic driver, may contribute to the overall efficacy in the in vivo setting where NIR penetration depth is a variable.

Mechanistically, Western blot of tumor tissue and treated cells showed that Fe₃O₄PD@CCM + NIR drove Nrf2 and HO-1 to their lowest levels among all groups, while Bax and cleaved caspase-3 rose and Bcl-2 fell most sharply (Figure 7A–F). IHC confirmed upregulated 4-HNE and cleaved caspase-3 and reduced Ki-67 in excised tumors (Figure 6E). Since Nrf2/HO-1 is a canonical antioxidant transcription axis,^3,6,11 its downregulation together with lipid-peroxidation marker 4-HNE upregulation is internally consistent with “excessive oxidative stress overriding antioxidant defense”.^53,54 We did not, however, measure mitochondrial membrane potential, GPX4 depletion, or ferroptosis markers; therefore the manuscript’s claim is correctly limited to apoptosis and oxidative stress, not to ferroptosis or pyroptosis. The observed suppression of the Nrf2/HO-1 axis is also worth interpreting cautiously: while it aligns with the ROS-centric narrative, Nrf2 has complex roles in tumor progression and therapy resistance that extend beyond antioxidant defense, including modulation of autophagy and metabolic reprogramming.^9,55 Whether the downregulation seen here is a cause or a consequence of ROS elevation cannot be definitively resolved from the current data.

In vivo, Cy5-Fe₃O₄PD@CCM exhibited substantially greater accumulation at B16F10 tumor sites at 24 h than the non-coated Cy5-Fe₃O₄PD formulation, with detectable signal limited to liver-associated background (Figure 4). This validates homotypic targeting under the syngeneic C57BL/6 setting but does not demonstrate deep intratumoral penetration or human-relevant biodistribution. The liver signal observed in both formulations is not unexpected given the well-documented hepatic clearance of intravenously administered nanoparticles,^7,14 but quantitative organ-level biodistribution would be needed to fully characterize off-target deposition. Antitumor growth curves and terminal tumor weights (Figure 6A–D) showed the Fe₃O₄PD@CCM + NIR group achieved the greatest inhibition, ahead of Fe₃O₄PD + NIR and free DTIC. Biosafety tests at 200 mg/kg IV–hematology, ALT/AST/CREA/URE.

A, hemolysis, and H&E of major organs–revealed no significant abnormality (Figure 5). The dose is high relative to the 20 mg/kg therapeutic regimen, but the observation window was only 24 h; chronic toxicity, iron clearance, and PLGA degradation remain unaddressed. The hemolysis assay, while negative at the tested concentration (2 mg/mL), used an ex vivo setup that does not capture complement activation or coagulation cascade interactions in live animals—both relevant considerations for clinical translation of membrane-coated nanoparticles.^23,24

From a design perspective, the integration of four components—magnetic core, polymeric drug carrier, small-molecule chemotherapeutic, and cell-membrane cloak—represents a rational layering strategy wherein each element addresses a specific limitation of DTIC monotherapy. The Fe₃O₄ core contributes both diagnostic (MRI contrast) and therapeutic (photothermal + Fenton) functionalities,^15,17,18 the PLGA-PEG2000 shell provides biocompatibility and controlled release,⁷ the DTIC cargo supplies established alkylating activity, and the membrane coating adds homotypic targeting.^23,24 Whether these individual contributions operate truly synergistically or merely additively in vivo is difficult to disentangle from the current experimental design, as no four-way factorial comparison (±membrane, ±NIR, ±DTIC, ±Fe₃O₄) was performed. This is a methodological limitation worth acknowledging in future iterations of the work.

Taken together, the data support a dose- and NIR-dependent combined effect: membrane coating improves tumor accumulation, Fe₃O₄ provides photothermal + catalytic ROS, DTIC adds alkylating stress, and the net result is redox collapse → apoptosis → proliferation arrest. The magnitude of effect is meaningful in the B16F10 subcutaneous model, but the study does not establish superiority over immunotherapy or serine/threonine-protein kinase B-Raf (BRAF)-targeted agents in translational terms. The findings are best positioned as a proof-of-concept demonstration that warrants further de-risking rather than as a near-term clinical candidate.

Conclusions

In this study, we successfully engineered a biomimetic nanoplatform (Fe₃O₄PD@CCM) by encapsulating a dacarbazine (DTIC)-loaded PLGA-PEG2000 matrix within an Fe₃O₄ core, further cloaked with B16F10 cancer cell membranes. Physicochemical characterization confirmed a uniform hydrodynamic diameter (∼25–30 nm), sustained DTIC release peaking within 24 h, colloidal stability in normal saline, and retention of key membrane proteins (CD44, CD47, E‑cadherin, N‑cadherin, Na⁺/K⁺‑ATPase). The DTIC encapsulation efficiency was moderate (∼13–14%). In vitro, the combination of Fe₃O₄PD@CCM and 808 nm NIR irradiation (1 W/cm², 15 min) yielded the most potent antitumor effects among all tested formulations, as evidenced by the lowest B16F10 cell viability, the highest apoptotic rate, pronounced G2/M phase arrest, robust intracellular ROS generation, and the greatest suppression of migration and invasion. In vivo, Cy5‑labeled Fe₃O₄PD@CCM exhibited enhanced homotypic tumor targeting. Comprehensive biosafety evaluations at a high dose (200 mg/kg) revealed no hemolysis, no significant alterations in hematological or biochemical parameters, and no histopathological organ damage. Most importantly, Fe₃O₄PD@CCM + NIR treatment achieved the most pronounced tumor growth inhibition in a syngeneic B16F10 melanoma model, accompanied by upregulated 4‑HNE and cleaved caspase‑3, downregulated Ki‑67, suppressed Nrf2/HO‑1 antioxidant axis, increased Bax, and decreased Bcl‑2. These findings demonstrate that Fe₃O₄PD@CCM + NIR effectively inhibits melanoma growth through integrated chemotherapy, photothermal therapy, with a favorable acute biosafety profile in healthy mice. However, the platform remains at an exploratory preclinical stage. Future studies should prioritize orthotopic or patient‑derived xenograft models, systematic dose‑response and long‑term toxicokinetic assessments, and combination with immune‑checkpoint blockade to fully evaluate its translational potential.

Limitations of the Study

Despite the encouraging therapeutic outcomes, several limitations should be acknowledged. First, the current in vivo evaluation was restricted to a short‑term acute study (24 h for biosafety and 12‑day antitumor treatment), necessitating future longitudinal investigations to assess chronic toxicity, long‑term biodistribution, and the fate of degraded components (eg, iron accumulation in the reticuloendothelial system, PLGA degradation products, and membrane‑protein immunogenicity upon repeated dosing). Second, although the syngeneic B16F10 model in C57BL/6 mice retains immune competence, it does not fully recapitulate the genetic complexity, tumor microenvironment heterogeneity, or anatomical features of human melanoma. Third, the moderate drug‑loading efficiency (∼13–14%) may limit the absolute drug payload per particle, which could pose challenges for dose escalation in clinical translation. Fourth, budgetary constraints precluded the use of advanced models such as genetically engineered mouse models (GEMMs) or patient‑derived organoids, which are critical for validating therapeutic responses in a more clinically relevant context. Fifth, the NIR irradiation protocol (1 W/cm², 15 min, every 2 days) was empirically selected; optimization of irradiation timing relative to nanoparticle accumulation kinetics and evaluation of deeper‑tissue penetration scenarios are needed to strengthen the photothermal rationale. Addressing these gaps in subsequent studies will be essential to advance the translational potential of this nanoplatform.

Abbreviations

NIR, near-infrared irradiation; Fe₃O₄, ferroferric oxide; NPs, Nanoparticles; Fe₃O₄ NP, ferroferric oxide nanoparticle; Fe₃O₄P NP, Fe₃O₄-PLGA-PEG2000 nanoparticle; DTIC, Dacarbazine; Fe₃O₄PD NP, Fe₃O₄-PLGA-PEG2000-DTIC nanoparticle; Fe₃O₄PD@CCM NP, Fe₃O₄-PLGA-PEG2000-DTIC-membrane nanoparticle; THF, Tetrahydrofuran; DMSO, Dimethyl sulfoxide; DLS, dynamic light scattering; SEM, scanning electron microscopy; LC-MS, liquid chromatography–mass spectrometry; HE/H&E, hematoxylin and eosin; IHC, Immunohistochemistry; ROS, Reactive oxygen species; 4HNE, 4-hydroxynonenal; C-casp3, Cleaved caspase-3; Ki-67, Ki-67 antigen; Cy5, Cyanine 5; LBFI, laser-based fluorescence imaging; CCM, Cancer cell membrane; NS, normal saline; DDW, double distilled water; HO-1, Heme oxygenase-1; Nrf2, Nuclear Factor Erythroid 2-Related Factor 2; Bax, BCL2-associated X protein; Bcl-2, B-cell CLL/lymphoma 2; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; BSA, bovine serum albumin; TBST, Tris buffered saline Tween buffer; PVDF, polyvinylidene fluoride.

Ethics Approval

All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (Chongqing, China) under Approval No. AMUWEC20267017. Mice were maintained under specific pathogen–free (SPF) conditions. All procedures strictly adhered to the 3R principles, and every effort was made to minimize pain and distress in the animals.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no competing interests in this work.