Multifunctional nanoplatform for tumor chemodynamic and self-amplified photodynamic cascade therapy

Graphical abstract

Highlights

• •Developed 5-ALA@Zn-CuTz@PM nanoparticles with platelet membrane modification, enabling efficient breast cancer targeting and combined PDT/CDT for enhanced tumor inhibition.
• •Zn²⁺-induced Zn-PpIX formation inhibits HO-1, increases intratumoral PpIX, while Cu⁺ generates hydroxyl radicals from H₂​O₂​, boosting PDT/CDT synergy.
• •Luminescence tracking confirmed tumor-specific accumulation and biodistribution of nanodrugs, highlighting excellent targeting efficiency.
• •Demonstrated enhanced PDT/CDT effects with strong biocompatibility and negligible toxicity to major organs.
• •H&E staining and biomarker quantification (HO-1, heme, bilirubin) revealed therapeutic mechanisms and ensured safety at the tissue level.

Abstract

Introduction

5-Aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) has demonstrated considerable potential in breast cancer treatment. However, its efficacy is limited by low tissue selectivity and the rapid conversion of 5-ALA to non-photosensitive heme in tumor tissues, reducing its therapeutic effectiveness.

Objectives

This study aims to develop a multifunctional nanomedicine to enhance 5-ALA’s PDT efficacy while introducing chemodynamic therapy (CDT) for synergistic tumor inhibition. By designing a zinc-ion-doped cuprous metal–organic framework (MOF) nanocarrier loaded with 5-ALA (5-ALA@Zn-CuTz), we seek to improve tumor targeting, prolong photosensitizer retention, and enhance therapeutic outcomes.

Methods

To enhance biocompatibility and active tumor targeting, the surface of 5-ALA@Zn-CuTz nanoparticles (NPs) was modified with a platelet membrane (PM), forming 5-ALA@Zn-CuTz@PM NPs. The therapeutic efficacy was evaluated in vitro and in vivo using mice breast cancer models. Cellular uptake, reactive oxygen species (ROS) generation, and tumor inhibition efficiency were analyzed through fluorescence imaging, biochemical assays, and histological analysis.

Results

Upon intravenous administration, 5-ALA@Zn-CuTz@PM NPs selectively accumulated in breast cancer cells. Within the tumor, Zn²⁺ bound to intracellular protoporphyrin IX (PpIX) to form PpIX-Zn, inhibiting heme oxygenase-1 (HO-1) activity and preventing the conversion of PpIX into heme. This increased the effective intracellular concentration of the photosensitizer, thereby enhancing PDT. Additionally, Cu⁺ catalyzed the decomposition of excess H₂O₂ in the tumor microenvironment, generating oxygen and hydroxyl radicals, which alleviated hypoxia and activated CDT. The synergistic PDT/CDT effect significantly enhanced tumor growth inhibition in vitro and in vivo.

Conclusion

5-ALA@Zn-CuTz@PM NPs effectively enhance PDT efficacy through selective tumor targeting and HO-1 inhibition while simultaneously leveraging CDT for additional tumor suppression. The combined PDT/CDT strategy demonstrated superior therapeutic outcomes, highlighting the potential of this nanoplatform as a promising approach for breast cancer treatment.

Introduction

Cancer is the second leading cause of mortality worldwide, representing a substantial threat to human health and survival[1], [2], [3]. According to the “World Cancer Report”, approximately 2.26 million new cases of breast cancer were recorded globally in 2020, accounting for 11.7 % of all new cancer cases[4], [5]. Notably, this represents the first instance in which breast cancer has overtaken lung cancer as the most prevalent malignancy globally in terms of incidence. This disease imposes significant psychological and physical burdens on patients, and its high rates of metastasis and recurrence contribute to adverse prognoses[6], [7]. The rapid advancement of nanoscience and technology has brought new opportunities for cancer diagnosis and therapy[8], [9], [10], [11], [12], [13]. Despite the progress achieved through various treatment modalities and strategies[14], [15], [16], [17], [18], the emergence of treatment resistance and associated side effects continue to pose substantial challenges and limitations in the pursuit of an effective breast cancer cure[19], [20].

In recent years, 5-Aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) has been extensively investigated and applied in treating melanoma, upper gastrointestinal malignancies, and oral squamous cell carcinoma, demonstrating considerable clinical potential[21], [22], [23]. PDT operates through the activation of a photosensitizer by light, which in the presence of oxygen, generates reactive oxygen species (ROS) to destroy diseased tissues selectively[24]. Due to its minimally invasive nature, reduced side effects, and potential for repeated administration, PDT presents a safe and effective therapeutic approach for breast cancer. Nevertheless, the clinical application of 5-ALA is currently hampered by several limitations, including poor tissue selectivity, instability in aqueous solutions, and rapid metabolic degradation, which restrict its accumulation within tumor tissues[25], [26]. Consequently, the effective drug concentration in the tumor microenvironment remains suboptimal, significantly impairing the therapeutic outcomes of PDT in breast cancer. Moreover, the high levels of glutathione (GSH) and the hypoxic conditions characteristic of tumor tissues both hinder the efficiency of ROS generation in conventional PDT. These factors collectively diminish the therapeutic efficacy of 5-ALA-mediated PDT.

After entering the biological system, 5-ALA is metabolized into protoporphyrin IX (PpIX), a potent photosensitizer[27]. However, PpIX rapidly chelates with intracellular ferrous ions (Fe²⁺), forming heme, a non-photosensitive compound, thereby significantly attenuating the therapeutic efficacy of 5-ALA-mediated photodynamic therapy[28], [29]. Moreover, the overexpression of heme oxygenase-1 (HO-1) in tumor tissues not only promotes tumor growth and invasion, and induces chemoresistance, but also catalyzes the degradation of heme, releasing Fe²⁺ and further facilitating the inactivation of PpIX’s photodynamic activity[30]. Thus, targeting the inhibition of HO-1 activity may enhance the efficacy of existing therapeutic modalities and improve clinical outcomes. Recent studies have demonstrated that the complex formed by PpIX and divalent zinc ions (PpIX-Zn) not only effectively inhibits HO-1 activity, reducing its capacity to degrade heme, but also preserves the photodynamic efficacy of PpIX[31], [32]. Additionally, PpIX-Zn has been shown to mitigate tumor chemoresistance, enhancing tumor sensitivity to chemotherapy.

Inspired by the aforementioned research background, we aimed to design an advanced therapeutic strategy that integrates photodynamic and chemodynamic therapies to overcome the limitations associated with conventional 5-ALA-mediated photodynamic therapy. As depicted in Scheme 1, considering the environmental sensitivity and rapid inactivation of 5-ALA in vivo, we employed the porous architecture of metal–organic framework (MOF) nanomaterials to encapsulate 5-ALA within an acid-responsive zinc-doped copper(I) MOF nanomaterial (Zn-CuTz), thereby constructing a nano phototherapeutic system (5-ALA@Zn-CuTz NPs)[33], [34]. This strategy effectively shields 5-ALA, preserving its bioactivity within the biological milieu[35]. To further improve the biocompatibility and targeted delivery of the system, we functionalized the surface with platelet membranes (PM)[36], [37], yielding platelet membrane-coated nano phototherapeutics (5-ALA@Zn-CuTz@PM NPs). Upon intravenous administration, these nanocarriers can accumulate within breast cancer tissues via both passive tumor-targeting mechanisms and the active targeting facilitated by P-selectin on the platelet membrane, significantly enhancing the intratumoral concentration of 5-ALA. The internalization of nanoparticles by tumor cells typically occurs through various cellular uptake mechanisms, including macropinocytosis, receptor-mediated endocytosis, phagocytosis, and passive diffusion. In this study, the PM coating of the nanomedicine facilitates enhanced tumor cell uptake through immune evasion, targeted tumor accumulation, and receptor-mediated endocytosis. This biomimetic strategy not only improves therapeutic efficacy but also mitigates off-target effects, contributing to the precision and safety of the nanoparticle-based treatment. Upon cellular uptake by tumor cells, the acidic intracellular environment triggers the degradation of the Zn-CuTz framework, resulting in the controlled release of 5-ALA, Cu⁺, and Zn²⁺. Intracellularly, 5-ALA is converted to the PpIX, while Zn²⁺ binds to PpIX, forming PpIX-Zn complexes. This inhibits HO-1 activity, decreases HO-1-mediated heme degradation, and reduces intracellular Fe²⁺ concentrations, thus preventing the conversion of PpIX into non-photosensitive heme and elevating intracellular PpIX levels. Moreover, the inhibition of HO-1 activity sensitizes breast cancer cells to PDT. This system not only blocks the transformation of PpIX into non-photosensitive heme but also induces the formation of PpIX-Zn complexes in situ, further suppressing HO-1 activity and amplifying the therapeutic efficacy of 5-ALA-mediated PDT. Furthermore, the degradation of the Zn-Cu-MOF framework releases Cu⁺, which reacts with hydrogen peroxide (H₂O₂) via a Fenton-like reaction, generating cytotoxic hydroxyl radicals and Cu²⁺, thereby enabling chemodynamic therapy (CDT)[38]. Cu²⁺ also catalyzes the decomposition of excess H₂O₂ within the tumor microenvironment, producing oxygen and alleviating tumor hypoxia. This hypoxia relief enhances ROS generation by photosensitizers, further augmenting PDT efficacy. Additionally, Cu²⁺ facilitates the oxidation of intracellular GSH to glutathione disulfide (GSSG)[39], thereby reducing the reductive environment within the tumor and minimizing ROS scavenging, which further boosts the effectiveness of PDT.

Scheme 1.

Schematic Illustration of the Main Synthesis Procedures of 5-ALA@Zn-CuTz@PM NPs and the mechanism of 5-ALA@Zn-CuTz@PM NPs in synergistic PDT/CDT antitumor activity.

Collectively, this study presents a rationally designed MOF-based intelligent drug delivery nanoplatform, integrating photodynamic and chemodynamic therapies to synergistically enhance antitumor efficacy. This approach not only preserves the photosensitizing activity of 5-ALA but also introduces additional CDT to amplify therapeutic outcomes, offering a novel and potentially more effective platform for breast cancer treatment.

Experimental Section

Chemicals

Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, AR), was purchased from J&K Science (Beijing, China). Methanol (AR), Copper(I) tetraacetonitrile tetrafluoroborate (AR) and 3,5-diphenyl-1-H-1,2,4-triazole (AR) were obtained from Aladdin (China), and 5-aminolevulinic acid hydrochloride (AR) was sourced from Suzhou Namtai (China)Biotechnology Co., Ltd. The platelet isolation kit was obtained from Hefei Bomei Biotechnology Co., Ltd. Cell Counting Kit-8 (CCK-8) kit was supplied by Dojindo Molecular Technologies Inc. The total bilirubin (TBIL) content detection kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the mouse HO-1 ELISA kit was obtained from Jiangsu Feiya Biotechnology Co., Ltd. Anti-mouse CD62P antibody (ab255822) was purchased from Abcam (Shanghai, China). All other reagents were used as received and without further purification.

Cell Lines

Human umbilical vein endothelial cells (HUVEC) and mouse breast cancer cells (4T1) were supplied by Shanghai Institute of Cell Biology (Shanghai, China). Roswell Park Memorial Institute 1640 (RPMI-1640) medium and phosphate buffer saline (PBS) were purchased from KeyGen BioTech (Nanjing, China). Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). BODIPY^581/591-C11 were purchased from Thermo Fisher Scientific Incorporated (America). The oxidative stress detection kit was obtained from Enzo Life Sciences (New York, United States). All cells used in this work were incubated in RPMI-1640 supplemented with 10 % FBS at 37 °C with 5 % CO₂.

Instrument

The morphology of these nanoparticles was observed by JEOL JEM-2100 high-resolution transmission electron microscope and ZEISS ULTRA 55 scanning electron microscope. Crystalline phases of these materials were measured by XRD (λ = 1.54056 Å, Bruker Co., Ltd., Germany). The UV–vis absorbance spectra of samples were detected on UV–vis spectrophotometry (UV3100, Shimadzu, Japan). The ζ-potential and Hydrodynamic size distribution of the nanoparticles in water were obtained on a Zetasizer 3000HS analyzer. The FV3000 confocal laser scanning microscope (Olympus, Japan) was used to acquired confocal fluorescence images. ICP-MS analysis was performed using an inductively coupled plasma mass spectrometer (NexION 300 D, PerkinElmer Corporation, America). Electron spin resonance (ESR) measurements were conducted using an electron spin resonance spectrometer (EMXplus-10/12, Bruker Co., Ltd., Germany). The 635 nm laser was provided by LEO PTICS Technology Co., Ltd. All the tissue sections were prepared by Nanjing KeyGen BioTech Company and observed by fluorescence microscope DMI8(LEICA, Germany).

Collection and purification of platelet membranes

In this study, platelet membranes for modifying nanoparticle were obtained by isolating platelets from SPF-grade SD rats, followed by purification and extraction steps. Following anesthesia induced by intraperitoneal injection of a 10 % sodium pentobarbital solution at 0.3 mg/kg body weight, blood was collected aseptically using vacuum blood collection tubes. For initial processing, blood samples were maintained at room temperature and subjected to low-speed centrifugation at 100 g for 20 min, a step primarily aimed at removing erythrocytes and leukocytes, leaving platelets within the supernatant. A second round of centrifugation under the same conditions was then performed to enhance platelet purity. To avoid inadvertent platelet activation, we added a phosphate-buffered saline (PBS) solution containing 1 mM EDTA and 2 mM prostaglandin E1 (PGE1, purchased from Sigma-Aldrich) to the platelet suspension. EDTA effectively chelated divalent cations, thus inhibiting platelet aggregation, while PGE1—an inhibitor of platelet activation—enhanced intracellular cyclic AMP (cAMP) levels, thereby reducing platelet activity and preventing premature aggregation. The platelet suspension was subsequently centrifuged at 800 g for 20 min at room temperature to facilitate effective platelet sedimentation, allowing for isolation of intact platelet structures. After discarding the supernatant, the platelet pellet was resuspended in PBS with 1 mM EDTA to further minimize coagulation risk. To maintain protein stability during membrane extraction, we introduced a protease inhibitor cocktail (from Pierce), thus protecting the membrane proteins from degradation. To disrupt the platelets and release membrane fractions, the platelet suspension underwent five freeze–thaw cycles, alternating between −80 °C and 25 °C. This process effectively lysed the platelets, yielding the desired membrane components. Final platelet membrane was collected by high-speed centrifugation and stored at −80 °C for nanoparticle formulation and characterization.

Synthesis of Zn-CuTz NPs

To prepare zinc-copper triazole nanoparticles (Zn-CuTz NPs) with varying particle sizes and compositions, starting with the synthesis of pure copper triazole (CuTz) as a reference sample, zinc with varying ratios at 10 %, 20 %, 30 %, 40 %, and 50 % were introduced. To synthesize CuTz NPs, 33 mg of 3,5-diphenyl-1-H-1,2,4-triazole (Tzh) and 75.36 mg of copper(I) tetrafluoroborate-tetraacetonitrile (Cu(NCCH₃)₄ BF₄) were precisely weighed and each dissolved in 3 mL of methanol. Ultrasonication was used to ensure thorough dissolution, forming homogenous solutions. The Tzh solution was then transferred into a 50 mL flask, and methanol was added to reach a final volume of 17 mL. While stirring at 400 rpm to maintain solution uniformity, the Cu(NCCH₃) ₄BF₄ solution was quickly introduced to the flask containing Tzh, ensuring complete mixing of both solutions. This reaction proceeded at room temperature for 4 h. After the reaction, CuTz NPs were collected by centrifugation and the supernatant was removed. The nanoparticles were washed three times with methanol to eliminate residual impurities and by-products, and then were freeze-dried to remove any remaining solvent, yielding a dry CuTz powder. Zn-CuTz NPs were synthesized by similar procedure only by adding varying zinc content to the stating solution. Adjusting zinc content would tailor Zn-CuTz NPs with specific structural and functional characteristics.

Quantitative analysis of Zn doping in Zn-CuTz NPs

To accurately quantify the zinc content in Zn-CuTz NPs, we employed a dual-method approach combining Energy Dispersive X-ray Spectroscopy (EDS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for robust and complementary data. For the ICP-MS measurement, samples were digested in aqua regia t and diluted with ultrapure water to optimize the analyte concentration for ICP-MS measurement.

Synthesis of 5-ALA@Zn-CuTz NPs

In order to prepare Zn-CuTz NPs loaded with 5-ALA, 30 mg of Zn-CuTz NPs were first dispersed in 5 mL of methanol and subjected to ultrasonic treatment to ensure thorough dispersion. Concurrently, 10 mg of 5-ALA was dissolved in 3 mL of methanol. The Zn-CuTz NPs solution was then transferred to a 50 mL round-bottom flask, and an additional volume of methanol was added to achieve a total volume of 17 mL. The solution was stirred at 400 rpm to ensure homogeneity, after which the 5-ALA solution was quickly introduced into the flask. To prevent the degradation of the photosensitive 5-ALA, the entire procedure was conducted under dark conditions. The reaction was maintained at room temperature for 4 h to facilitate the loading of 5-ALA onto the Zn-CuTz NPs. Upon completion of the reaction, the product was isolated by centrifugation, and the supernatant was discarded. The nanoparticles were subsequently washed three times with methanol to remove any unreacted precursors, by-products, and surface-adsorbed impurities, ensuring a high degree of purity of the resulting nanoparticles. Finally, the obtained 5-ALA@Zn-CuTz NPs were dried under vacuum to remove any residual solvent, yielding the 5-ALA@Zn-CuTz NPs powder, which was then prepared for further application and characterization.

Synthesis of 5-ALA@Zn-CuTz@PM NPs

To prepare platelet membrane-enclosed 5-ALA@Zn-CuTz NPs, 5 mg of 5-ALA@Zn-CuTz NPs was added to a platelet membrane buffer solution. This step ensures that the ALA@Zn-CuTz NPs were uniformly encapsulated by the platelet membrane, thereby enhancing their biocompatibility and tumor-targeting properties. The mixture was subjected to ultrasonic treatment under ice-cold conditions. Each sonication step lasted for 30 s, followed by short cooling intervals to prevent overheating of the sample. This process was repeated five times. After ultrasonic treatment, the mixture was separated by high-speed centrifugation to remove any unencapsulated platelet membrane fragments and other potential impurities. The supernatant was carefully aspirated and discarded, leaving behind the precipitate containing the 5-ALA@Zn-CuTz@PM NPs. Finally, the obtained precipitate was subjected to lyophilization at −80 °C to remove the solvent and yield a dried powder of 5-ALA@Zn-CuTz@PM NPs.

Drug loading and encapsulation efficiency of 5-ALA@Zn-CuTz@PM NPs

To accurately determine the drug loading content of 5-ALA in 5-ALA@Zn-CuTz@PM NPs, we employed high-performance liquid chromatography (HPLC) for quantitative analysis. During the nanoparticle synthesis, all washings were collected for subsequent analysis to measure the residual 5-ALA. Considering the potential degradation of 5-ALA in the absence of a catalyst, HPLC was utilized for precise determination to ensure the accuracy of the measurements and exclude potential interference from by-products. The HPLC analysis was performed using a C18 column, with the column temperature maintained at 25 °C to ensure stability and reproducibility of the chromatographic analysis. The mobile phase was prepared by mixing a 50 mmol/L solution of ammonium dihydrogen phosphate (pH approximately 2.2) with methanol in a ratio of 98:2, ensuring good separation efficiency and compatibility. All reagents used were of chromatographic grade to ensure high precision and low background interference. Before testing the samples, a series of standard 5-ALA solutions were prepared at concentrations ranging from 0 to 2 mg/mL for the construction of a 5-ALA standard curve. The absorbance of the standard solutions was measured at a wavelength of 265 nm, and the standard curve was obtained, providing a necessary reference for sample analysis. The drug loading content (DLC) and encapsulation efficiency (EE) were calculated base on following formulas:

$$\mathit{DLC}=\left(\mathit{Weight}\mathit{of}\mathit{drug}\mathit{loaded}\mathit{in}\mathit{nanoparticles}/\mathit{Total}\mathit{weight}\mathit{of}\mathit{nanoparticles}\right)\times 100\%$$

$$\mathit{EE}=\left(\mathit{Weight}\mathit{of}\mathit{drug}\mathit{loaded}\mathit{in}\mathit{nanoparticles}/\mathit{Initial}\mathit{weight}\mathit{of}\mathit{drug}\mathit{added}\right)\times 100\%$$

Characterization of platelet membrane proteins

To confirm the successful coating of platelet membrane on 5-ALA@Zn-CuTz@PM NPs, Western Blot analysis was performed to detect the expression of P-selectin (CD62P) protein in 5-ALA@Zn-CuTz@PM samples. P-selectin, a key marker protein on platelet membranes, serves as a direct indicator of platelet membrane. Specifically, the samples were centrifuged at 12,000 g for 20 min, and the resulting pellet was transferred into precooled RIPA buffer to dissolve and incubate for 30 min, aiming to lyse cell membranes and release intracellular proteins. After centrifugation, the protein-rich supernatant was collected, mixed with protein loading buffer, and then heated in a boiling water bath for 5 min to denature the proteins, ensuring effective separation during electrophoresis. The denatured proteins were then separated via SDS-PAGE and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. To prevent nonspecific protein binding, the PVDF membrane was blocked with 5 % bovine serum albumin (BSA) for approximately 1 h. Following blocking, the membrane was incubated with a primary antibody specific to CD62P, a platelet membrane protein, at room temperature for 2 h to allow efficient binding to the target protein. After incubation, the membrane was washed three times with Tris-buffered saline containing 0.02 % Tween 20 (TBST) to remove unbound primary antibodies and other potential contaminants. The membrane was then incubated with an HRP-conjugated secondary antibody for 1 h to enable signal generation through the HRP tag, allowing visualization of the target protein during subsequent detection. In the final stage, imaging analysis was conducted using the Bio-Rad ChemiDoc Touch imaging system. This system accurately captures and records the chemiluminescent signals produced by HRP (horseradish peroxidase) labeling, facilitating both qualitative and quantitative analysis of the target protein in the samples.

In vitro assay for GSH consumption

Prior to the experiment, a fixed concentration of GSH standard solution was prepared to ensure a consistent starting concentration of GSH across all experimental groups. These GSH solutions were then distributed into a 96-well plate, with each well receiving a different concentration of 5-ALA@Zn-CuTz@PM NPs solution (0.2 mg/mL, 0.4 mg/mL, and 0.8 mg/mL), and PBS buffer-only wells were set up as control. The experiment was conducted over a period of 7 h at 37 °C and 5 % CO₂, with sampling every hour. To measure GSH consumption, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was used as Ellman’s reagent. GSH reacts with DTNB to produce a yellow product with a characteristic absorbance peak at 412 nm. At each sampling point, absorbance was measured using a UV–visible spectrophotometer for both the treatment and control groups to monitor GSH depletion. By comparing absorbance data across different time points, the trend in GSH concentration over time was observed, allowing us to assess the impact of 5-ALA@Zn-CuTz@PM NPs on GSH consumption.

Electron spin resonance (ESR) analysis

To investigate whether samples can induce reactive oxygen species (ROS) generation in vivo and to identify the specific types of ROS generated, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin trap, and electron spin resonance (ESR) was employed for detection. The experimental steps are as follows:

First, samples were uniformly dispersed in deionized water. Then, an appropriate amount of hydrogen peroxide (H₂O₂) was added to the solution to simulate an oxidative stress environment similar to that in vivo. Next, 10 μL of DMPO solution was added and thoroughly mixed to ensure that DMPO could effectively capture the radicals catalytically generated by samples. The mixture was then transferred to a capillary tube, which was subsequently placed in the ESR spectrometer for measurement. By analyzing the ESR spectra, the types and quantities of generated ROS were identified and quantified.

Detection of intracellular ROS

DCFH-DA fluorescence probe was selected for the quantitative analysis of reactive oxygen species (ROS) generation within 4T1 cells. First, 4T1 cells were seeded in confocal microscope-specific culture dishes and incubated at 37 °C with 5 % CO₂ until near-confluence. After removing the old medium, the cells were gently washed twice with saline to remove residual substances. A new medium containing the test samples was then added to the cells, followed by an additional 8-hour incubation to allow the drug to fully interact with the cells. Subsequently, the cells were exposed to laser irradiation at a wavelength of 635 nm for 5 min to stimulate intracellular ROS production. Following laser treatment, 50 μL of 10 μM DCFH-DA solution was added to the culture dish, and the cells were co-incubated with the probe for 30 min, allowing DCFH-DA to penetrate the cells and be hydrolyzed to form DCFH, a compound that reacts with ROS. After incubation, the cells were washed with PBS to remove any DCFH-DA that had not entered the cells or unreacted DCFH-DA. Finally, a laser confocal microscope with an excitation wavelength of 488 nm was used to observe the cells. By measuring fluorescence emission intensity at 525 nm, the level of ROS within the cells was evaluated.

Detection of lipid peroxidation in cell membranes

To evaluate the oxidative stress effects of nanoparticles on cells, particularly in promoting lipid peroxidation, the BODIPY^581/591-C11 probe was employed. First, 4T1 cells were seeded in confocal culture dishes. Cells were then treated with same procedure for intracellular ROS detection. After the treatment, cells were thoroughly washed with phosphate-buffered saline (PBS), and serum-free medium containing 5 μM BODIPY^581/591-C11 probe was added to the cells to label intracellular lipid peroxides. The cells were incubated at 37 °C for 20 min. Finally, confocal laser scanning microscopy was used to image the lipid peroxidation levels.

In vitro cytotoxicity assay

Cell Counting Kit-8 (CCK-8) assay was applied to assess the cytotoxic effects of the samples. First, breast cancer cell line 4T1 and human umbilical vein endothelial cells (HUVEC) were seeded in 96-well plates at approximately 5,000 cells per well and incubated overnight at 37 °C with 5 % CO₂ to allow for cell attachment. Subsequently, sample-containing medium was added into wells and incubated for an additional 8-hour. For specific experimental groups requiring laser irradiation, each well was exposed to a 635 nm laser at 600 mA for 5 min to simulate photodynamic therapy conditions. After incubation, 10 μL of CCK-8 solution was added to each well. The plates were then incubated for an additional 2 h to ensure adequate reaction time. Finally, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader.

Detection of intracellular PpIX and Zn-PpIX concentrations

To quantify the intracellular concentrations of PpIX and Zn-PpIX, a combination of cell biology and spectroscopic methods was employed. Cells were initially cultured in 6-well plates until reaching approximately 80 % confluency. Then cells were treated with trypsin for detachment and collection. The cell membranes were then disrupted by sonication to release intracellular contents, followed by centrifugation to separate the supernatant containing PpIX and Zn-PpIX. For quantitative analysis, the supernatant was diluted appropriately to suit spectroscopic requirements. Fluorescence spectrometry was then used, with the excitation wavelength for PpIX set to 400 nm and the emission wavelength to 635 nm. For Zn-PpIX, the excitation wavelength was set to 416 nm and the emission wavelength to 588 nm. By comparing the fluorescence intensities between treated and control groups, a quantitative analysis of the relative intracellular concentrations of PpIX and Zn-PpIX was conducted, allowing for assessment of the nanoparticles’ effects on the synthesis and stability of these compounds.

Intracellular quantification of HO-1, Heme, and bilirubin

To investigate the inhibitory effects of zinc protoporphyrin (Zn-PpIX) on heme oxygenase-1 (HO-1) activity within cells, 4T1 cells pre-seeded in 6-well plates were cultured at 37 °C for 24 h before treatment with different sample solutions (200 µL, 0.5 mg/mL in culture medium) for another 24 h. After treatment, the cells were lysed with Triton X-100 lysis buffer, followed by ultrasonic homogenization to ensure complete cell disruption. The levels of HO-1, heme, and bilirubin were then quantified using the respective assay kits. Based on the measured concentrations in untreated control cells, we calculated the relative percentages of HO-1, heme, and bilirubin levels within the treated cells, allowing for comparison and evaluation of nanoparticle-induced modulation of these components.

Animal models and Ethics approval

Balb/c mice (female, 6–8 weeks) were supplied by the Comparative Medicine Centre of Yangzhou University and raised in specific pathogen-free facility. To set up the breast tumor model, these mice were inoculated subcutaneously with 4T1 cell line (1 × 10⁶ cells per mouse). All animal experiments were reviewed and approved by the Committee on Animals at Nanjing University (IACUC——D2402094).

In vivo antitumor study

When tumor volume reached approximately 100 mm³, the animals were randomized into six groups, each containing five mice. The groups were as follows: a PBS group (control), a 5-ALA group, a Zn-CuTz group, a 5-ALA@Zn-CuTz@PM group, a 5-ALA combined with 635 nm laser irradiation group (5-ALA + Laser), and a 5-ALA@Zn-CuTz@PM combined with 635 nm laser irradiation group (5-ALA@Zn-CuTz@PM + Laser). Each group received intravenous injections, with a dosing volume of 100 μL at a concentration of 1 mg/mL using PBS as the solvent. Treatments were administered every three days for a total of three injections over a 14-day experimental period. Tumor volume, body weight, and survival were monitored bi-daily to thoroughly assess tumor progression, physiological impact, and overall treatment efficacy across groups. At the end of the experiment, euthanasia was conducted in accordance with ethical standards, followed by excision of tumors and major organs (heart, liver, spleen, lungs, and kidneys) for detailed histopathological and biomarker analysis.

In vivo Luminescence imaging

To track and evaluate the in vivo biodistribution and accumulation of the samples, ICG-labeled 5-ALA@Zn-CuTz@PM NPs were dissolved in PBS to prepare a solution at a concentration of 1 mg/mL. Each mouse bearing a 4T1 tumor received a 200 μL intravenous injection of this solution. Post-injection, mice were dissected at predefined time points, and major organs (heart, liver, spleen, lungs, kidneys) along with tumor tissue were harvested for Luminescence imaging analysis.

Histopathological analysis

At the end of the experimental period, comprehensive histopathological evaluation was performed on the cardiac, hepatic, splenic, pulmonary, renal, and tumoral tissues to thoroughly assess the treatment’s systemic and localized effects in murine models. The collected tissue specimens were immediately fixed in 4 % paraformaldehyde to maintain their cellular architecture. Following fixation, samples underwent standard processing protocols. Thin sections were obtained for histological examination. Hematoxylin and eosin (H&E) staining was employed to enhance visualization of cellular morphology.

Statistical analysis

The results were presented as mean ± SD and the statistical significance was calculated by the one-way ANOVA analysis. **p < 0.01, and ***p < 0.001 were defined as statistical significances.

Result and Discussion

Synthesis and characterization of Zn-CuTz NPs

In this study, we successfully synthesized CuTz and a series of Zn-CuTz NPs with varied Zn²⁺ doping ratios. Their morphology was characterized by SEM, as illustrated in Fig. 1. Images A-F show Zn-CuTz NPs with Zn²⁺ doping ratios ranging from 0 % to 50 %. The results indicate that, with increasing Zn²⁺ content, particle size progressively increased and morphology shifted toward an ellipsoidal shape. At a Zn²⁺ doping level of 20 %, we observed a stable average particle diameter of approximately 150–200 nm, coupled with a roughly ellipsoidal structure and a relatively coarse surface texture, displaying a consistent particle size distribution. Such a size and shape profiles facilitate efficient penetration of biological barriers and provides an enlarged surface area for drug loading and release. Given Zn²⁺’s essential role in this study—its ability to bind with PpIX, to form a functional Zn-PpIX complex, alongside its potential impact on nanoparticle properties and drug release kinetics, we opted for a Zn²⁺ doping level of 20 %.

Fig. 1.

SEM images of nanoparticles synthesized with different zinc feeding ratios. (A) shows CuTz nanoparticles, while (B) to (F) correspond to Zn-CuTz nanoparticle samples with zinc contents of 10%, 20%, 30%, 40%, and 50%, respectively.

Further structural characterization of CuTz and Zn-CuTz NPs was conducted using TEM (Fig. 2A & C) and DLS (Fig. 2B & D). These results aligned well with SEM findings, indicating that both CuTz and Zn-CuTz NPs possess small, defined sizes, with particle size distributions centered around 91 nm and 162 nm, respectively. The increase in particle size and shape transformation in Zn-CuTz NPs is likely attributable to Zn²⁺ doping, which may have influenced the coordination between Cu⁺ and Tzh. Having established the morphology and size distribution of Zn-CuTz NPs, we conducted additional elemental analyses using energy-dispersive X-ray spectroscopy (EDS) and inductively coupled plasma mass spectrometry (ICP-MS) to quantify Zn²⁺ doping levels within the nanoparticles. As shown in Fig. 2E, EDS mapping images provide a clear visual distribution of Cu and Zn elements within the nanoparticles. Quantification of mass percentages, presented in Fig. 2F, displayed distinct peaks for Zn and Cu, with an area ratio of approximately 1:12, suggesting a Zn to Cu mass percentage ratio close to 1:12 in the nanoparticles. Further quantification via ICP-MS confirmed these findings, yielding a Zn ratio of approximately 1:11.75 (Fig. 2G), corroborating the EDS data and verifying the effective incorporation of Zn²⁺ into the CuTz framework. These comprehensive results collectively confirm the successful synthesis of Zn-CuTz NPs with controlled Zn²⁺ doping.

Fig. 2.

(A) TEM image of CuTz NPs. (B) Hydrodynamic size distribution of CuTz NPs. (C) TEM image of Zn-CuTz NPs. (D) Hydrodynamic size distribution of Zn-CuTz NPs. (E) Energy-dispersive X-ray spectroscopy (EDS) images of Zn-CuTz NPs. (F) Overall elemental distribution spectrum of Zn-CuTz NPs. (G) ICP-MS of Zn-CuTz NPs.

Synthesis and characterization of 5-ALA@Zn-CuTz NPs

After obtaining the ideal Zn doping ratio for Zn-CuTz NPs, we successfully encapsulated 5-ALA within the Zn-CuTz framework, yielding the 5-ALA@Zn-CuTz NPs. TEM and DLS analysis, as presented in Fig. 3A & B, revealed that 5-ALA@Zn-CuTz NPs retained a well-defined ellipsoidal morphology with a particle size distribution centered around 162 nm. This uniform size and shape distribution suggest that the loading of 5-ALA had negligible impact on the structural integrity of Zn-CuTz NPs, preserving their stability and homogeneity for potential in vivo applications. Subsequent structural characterization by XRD (Fig. 3C) and UV–Vis (Fig. 3D) spectroscopy further confirmed the successful incorporation of 5-ALA. The XRD pattern in Fig. 3C displays characteristic diffraction peaks associated with Zn-CuTz NPs at 8.27° and 20.48°, with an additional peak at 21.18°, corresponding to the presence of 5-ALA. This result demonstrates that 5-ALA was successfully loaded without altering the crystalline structure of Zn-CuTz NPs. The UV analysis in Fig. 3D reveals a blue shift in the characteristic absorption peak of Zn-CuTz and CuTz at 266 nm upon 5-ALA loading, while the original 5-ALA absorption peak at 232 nm shifts to a new peak at 243 nm. This shift likely results from the electron-withdrawing effect of the carbonyl group in 5-ALA, which modifies the electronic distribution within Zn-CuTz NPs. Additionally, intermolecular hydrogen bonding within 5-ALA could further influence the electronic structure of Zn-CuTz NPs, contributing to these observed shifts in UV absorption. These observations substantiate the successful loading of 5-ALA and provide insights into the electronic modifications conferred by its encapsulation. These findings demonstrate the successful synthesis of structurally stable and size-uniform 5-ALA@Zn-CuTz NPs, with promising characteristics for subsequent biomedical applications.

Fig. 3.

(A) TEM image of 5-ALA@Zn-CuTz NPs. (B) Hydrodynamic size distribution of 5-ALA@Zn-CuTz NPs. (C) XRD pattern and (D) UV–vis spectrometry of CuTz, 5-ALA, Zn-CuTz and 5-ALA@Zn-CuTz NPs.

Synthesis and characterization of 5-ALA@Zn-CuTz@PM NPs

Building on the successful synthesis of 5-ALA@Zn-CuTz NPs, platelet membrane (PM) was coated onto their surface to enhance biocompatibility and provide active tumor-targeting capability, culminating in the formation of 5-ALA@Zn-CuTz@PM NPs. As depicted by TEM imaging (Fig. 4A), the particle size of 5-ALA@Zn-CuTz@PM NPs remained largely unaffected post-PM coating. However, moderate aggregation was observed, which is likely attributable to the fluidity of phospholipids and cholesterol in the PM. Upon close proximity, these lipids can rearrange and fuse, leading to nanoparticle clustering. Although aggregation may influence the particles' in vivo application, the sufficiently small size of Zn-CuTz@5-ALA@PM NPs mitigates these effects, as evidenced by subsequent in vitro and in vivo assays, where therapeutic efficacy remained unaffected. Quantitative analysis of 5-ALA loading within Zn-CuTz@5-ALA@PM NPs was performed using HPLC. A standard curve was established based on absorbance at 265 nm for various 5-ALA concentrations (Fig. 4B), achieving a high linear correlation coefficient (R² = 0.9981), indicating a robust linear relationship between 5-ALA concentration and absorbance within the tested range. The encapsulation efficiency and drug loading of 5-ALA were calculated as 11.95 % and 6.17 %, respectively, yielding a suitable loading level that promotes uniform 5-ALA distribution within Zn-CuTz NPs, which is conducive to controlled release kinetics and prolonged therapeutic efficacy in biological systems. To confirm the presence of PM on 5-ALA@Zn-CuTz NPs, Western blot analysis was conducted for platelet-specific marker P-selectin (CD62P). As shown in Fig. 4C, the expression of CD62P protein on 5-ALA@Zn-CuTz@PM NPs indicates successful PM coating. Additionally, Zeta potential measurements confirmed PM attachment, with the surface potential of 5-ALA@Zn-CuTz NPs decreasing from 20.5 mV to 4.9 mV post-coating, further validating the successful PM functionalization. The PM coating not only enhances biocompatibility but also facilitates active targeting to tumor tissues, thus increasing effective drug concentration at the tumor site and enhancing therapeutic efficacy. To evaluate the CDT potential of Cu⁺ in 5-ALA@Zn-CuTz@PM NPs, ESR spectroscopy was performed, as shown in Fig. 4E. Results indicate a prominent ROS signal, corresponding to the characteristic ·OH peak, verifying the nanoparticles' efficient ·OH generation and consequent oxidative damage to lipids, proteins, and DNA within tumor cells, thus conferring significant CDT efficacy. The acid-responsive characteristics of 5-ALA@Zn-CuTz@PM NPs were further investigated. As demonstrated in Fig. 4F, the nanoparticles exhibited significant morphological degradation after a 2-hour exposure to acidic conditions, reflecting their excellent acid-responsive properties. Such a characteristic enables precise degradation within the acidic tumor microenvironment, promoting localized release of 5-ALA and Cu⁺ to facilitate synergistic PDT and CDT at tumor sites. In summary, we successfully synthesized 5-ALA@Zn-CuTz@PM NPs, which allow for targeted co-delivery of therapeutic agents and photosensitizers to tumor regions. This designed system harnesses tumor-specific microenvironmental characteristics to enhance drug release kinetics and therapeutic efficacy, while concurrently minimizing collateral damage to healthy cells.

Fig. 4.

(A) TEM image of 5-ALA@Zn-CuTz@PM NPs. (B) Standard curve for 5-ALA concentration. (C) Western blot analysis images for P-selectin (CD62P) and @5-ALA@Zn-CuTz @PM. (D) ζ potentials of different samples. (E) Electron spin resonance (ESR) spectroscopy of 5-ALA@Zn-CuTz@PM NPs. (F) TEM images of 5-ALA@Zn-CuTz@PM NPs under acidic degradation.

Evaluation of enhanced PDT and synergistic CDT efficacy of 5-ALA@Zn-CuTz@PM NPs

We successfully synthesized 5-ALA@Zn-CuTz@PM NPs and conducted a comprehensive characterization of their structure and physicochemical properties. Here, we further assess their anti-tumor efficacy in vitro. We initially evaluated the ability of 5-ALA, as a precursor photosensitizer, to convert into the active photosensitizer PpIX within tumor cells. Specifically, we analyzed the impact of a fixed concentration (100 μg/mL) of 5-ALA on intracellular PpIX production over different time intervals. As shown in Fig. 5A, the amount of PpIX produced within the cells increased with time, suggesting that 5-ALA can efficiently convert to PpIX in biological systems, and that its production is positively correlated with time. We then examined the effect of varying 5-ALA concentrations on PpIX production over a fixed incubation period (4 h). The results, depicted in Fig. 5B, indicate that within the specified time, PpIX production increased as the 5-ALA concentration rise, plateauing around 75 μg/mL, above which PpIX generation showed minimal variation, suggesting saturation within the cellular environment. Intriguingly, at a concentration of 600 μg/mL, PpIX levels declined, likely due to feedback inhibition mechanisms triggered by the high 5-ALA concentration, such as increased GSH expression to mitigate oxidative stress. This adaptive response could influence the heme synthesis pathway, thereby indirectly reducing PpIX levels. Following this, we evaluated the capacity of 5-ALA@Zn-CuTz@PM NPs to facilitate the intracellular conversion of 5-ALA to PpIX within 4T1 tumor cells over a 2-hour period. As depicted in Fig. 5C, PpIX levels in 4T1 cells increased with nanoparticle concentration, demonstrating that 5-ALA@Zn-CuTz@PM NPs effectively enhance the intracellular conversion of 5-ALA to PpIX. For comparative purposes, we established a control concentration of free 5-ALA at 37.5 μg/mL and calculated an equivalent nanoparticle concentration of 600 μg/mL based on a 6.17 % drug-loading efficiency (Fig. 5D). Notably, the nanoparticle system induced significantly higher PpIX production than free 5-ALA alone, likely due to the active targeting capability of 5-ALA@Zn-CuTz@PM NPs, which enhances 5-ALA accumulation at tumor sites, thereby increasing its effective concentration. Additionally, the presence of Cu⁺ in the nanoparticles disrupts GSH protection, increasing 5-ALA uptake and enhancing its metabolism to PpIX. Collectively, these findings indicate that 5-ALA@Zn-CuTz@PM NPs not only facilitate efficient intracellular conversion of 5-ALA to PpIX but also enhance drug bioavailability and therapeutic potential, demonstrating substantial promise for application in PDT.

Fig. 5.

(A) The temporal variation of intracellular PpIX concentration. (B) Accumulation of PpIX in 4T1 cells after 4 h of treatment with different concentrations of 5-ALA. (C) Accumulation of PpIX in 4T1 cells after 2 h of treatment with different concentrations of 5-ALA and 5-ALA@Zn-CuTz@PM NPs. (D) Accumulation of PpIX in 4T1 cells after 2 h of incubation with nanoparticles at a fixed 5-ALA concentration (37.5 μg/mL). (E) Detection of intracellular Zn-PpIX content under different treatments. (F) Comparison of HO-1 expression levels in 4T1 cells under different treatment conditions. (G) Hemoglobin and (H) Bilirubin expression levels in 4T1 cells under different treatment conditions. (I) The effect of different concentrations of 5-ALA and 5-ALA@Zn-CuTz@PM NPs on GSH content. **P < 0.01, ***P < 0.001.

Subsequently, we assessed the ability of 5-ALA@Zn-CuTz@PM NPs to facilitate the formation of Zn-PpIX within 4T1 cells. As illustrated in Fig. 5E, Zn-PpIX was detected both in intact cells and in the supernatant from sonicated cell lysates, demonstrating the effective intracellular release of Zn²⁺ from 5-ALA@Zn-CuTz@PM NPs, which then bind with PpIX to form Zn-PpIX. This Zn-PpIX formation plays a critical role in inhibiting HO-1 activity, thereby significantly reducing heme catabolism and the subsequent release of ferrous ions within tumor cells. By limiting the conversion of PpIX into the non-photosensitizing heme, this approach preserves PpIX’s photosensitizing capacity, thus providing a robust foundation for enhancing the photodynamic therapeutic efficacy of 5-ALA. In addition, we further characterized HO-1 expression levels and activity to elucidate the mechanistic enhancement of PDT efficacy by 5-ALA@Zn-CuTz@PM NPs. As depicted in Fig. 5F, HO-1 expression was upregulated in 4T1 cells following both 5-ALA@CuTz and 5-ALA@Zn-CuTz@PM NPs treatment, with a more pronounced 38.3 % increase in the 5-ALA@Zn-CuTz@PM NPs group. This elevated HO-1 expression can be attributed to Cu⁺-mediated oxidative stress within the nanomedicine system. As a crucial protein in cellular redox regulation, HO-1 is upregulated in response to elevated oxidative stress levels as a compensatory mechanism to mitigate oxidative damage. Notably, however, the generation of Zn-PpIX did not impact HO-1 expression levels significantly, indicating that the inhibitory effect of Zn-PpIX on HO-1 is not exerted at the expression level.

To probe the inhibitory mechanism of Zn-PpIX on HO-1 activity, we analyzed cellular heme content and its metabolite, bilirubin, following nanoparticle treatment. As shown in Fig. 5G and H, treatment with 5-ALA@CuTz resulted in a notable reduction in heme levels and a corresponding elevation in bilirubin levels. This trend, coupled with the findings in Fig. 5F, suggests that the increase in HO-1 expression stimulates heme catabolism, resulting in reduced heme concentrations and a corresponding increase in bilirubin as a catabolic byproduct. Conversely, treatment with 5-ALA@Zn-CuTz@PM NPs led to elevated HO-1 expression without concurrent reductions in heme content, which instead showed a slight increase, while bilirubin levels remained relatively unchanged. This observation suggests that Zn-PpIX effectively inhibits HO-1 enzymatic activity, thereby preventing heme degradation and Fe²⁺ release. Consequently, the metabolic conversion of PpIX to non-photoactive heme is curtailed, ensuring sustained intracellular PpIX concentrations and bolstering the PDT efficacy of 5-ALA@Zn-CuTz@PM NPs. As a highly expressed reductive agent within tumor cells, GSH not only maintains redox homeostasis but also directly scavenges free radicals, stabilizing cellular structure and function in tumor cells. Thus, we evaluated the GSH depletion effect of Cu⁺ in 5-ALA@Zn-CuTz@PM NPs to validate its efficacy in CDT. As shown in Fig. 5I, following a 6-hour co-incubation with varying concentrations of 5-ALA@Zn-CuTz@PM NPs, a decrease in GSH levels was observed across all samples, with GSH depletion increasing proportionally to nanoparticle concentration. At a concentration of 0.8 mg/mL, GSH levels declined by 8.3 %, indicating that 5-ALA@Zn-CuTz@PM NPs exhibit strong GSH depletion capabilities. This effective reduction in tumor cells' antioxidant defenses heightens oxidative stress, leading to cellular damage and apoptosis, thus underscoring its promising CDT therapeutic potential. Collectively, these results indicate that 5-ALA@Zn-CuTz@PM NPs can inhibit HO-1 activity, prevent heme degradation, and maintain PpIX levels, thereby enhancing PDT efficacy. Additionally, the NPs exhibit significant GSH depletion, exacerbating oxidative stress and weakening cellular antioxidant defenses, underscoring their significant therapeutic potential in both PDT and CDT applications.

Antitumor effect of 5-ALA@Zn-CuTz@PM in vitro

After confirming that 5-ALA@Zn-CuTz@PM NPs exhibit substantial CDT potential and can effectively enhance PDT, we further evaluated their therapeutic efficacy at the cellular level. First, confocal microscopy was used to observe ROS generation and lipid peroxidation in different treatment groups. As shown in Fig. 6A, both the 5-ALA irradiation group and the Zn-CuTz group displayed significant ROS fluorescence signals. This effect arises because, after cellular uptake, 5-ALA is converted to the photosensitizer PpIX, which generates ROS upon light exposure, while Cu⁺ in Zn-CuTz catalyzes the decomposition of the highly expressed H₂O₂ in tumor cells to produce hydroxyl radicals, contributing to cytotoxicity. Notably, 5-ALA@Zn-CuTz@PM NPs displayed even stronger fluorescence signals, indicating that this nanoparticle formulation induced the highest ROS production and thus significant oxidative stress, underscoring their potential for PDT and CDT synergistic therapy. In addition, lipid peroxidation assays revealed that, while the 5-ALA irradiation group caused some cellular membrane damage, it was relatively mild, suggesting that 5-ALA-induced ROS production was insufficient to deplete membrane-protective GSH significantly. However, in the Zn-CuTz group, stronger fluorescence signals were observed, indicating that the Cu⁺-mediated Fenton-like reaction generates hydroxyl radicals that effectively deplete GSH, leading to lipid peroxidation and subsequent tumor cell damage. Notably, the 5-ALA@Zn-CuTz@PM NPs group exhibited the highest fluorescence signal intensity, likely due to the active targeting capability of the NPs, which facilitates greater delivery of Cu⁺ to tumor cells. This leads to increased OH· production, GSH depletion, and lipid peroxidation, causing significant cell membrane disruption. Such membrane damage may further sensitize tumor cells to ROS generated via 5-ALA-mediated PDT, enhancing lipid peroxidation and maximizing cytotoxic effects on tumor cell membranes. These findings collectively indicate that 5-ALA@Zn-CuTz@PM NPs possess robust ROS generation capacity and hold considerable promise for combined PDT and CDT applications.

Fig. 6.

(A) Fluorescence images of cellular ROS production and lipid peroxidation under different drug treatments. Scale bars: 30 µm. (B) Cytotoxicity of different concentrations of 5-ALA@Zn-CuTz@PM NPs on HUVEC and 4T1 cells. (C) Cytotoxicity of different concentrations of 5-ALA@Zn-CuTz and 5-ALA@Zn-CuTz@PM NPs on 4T1 cells. (D) Cytotoxicity of different concentrations of 5-ALA@Zn-CuTz@PM NPs on 4T1 cells with and without laser exposure. **P < 0.01, ***P < 0.001.

We subsequently evaluated the biosafety and cytotoxicity of 5-ALA@Zn-CuTz@PM NPs in both normal and tumor cells, with results shown in Fig. 6B. At lower concentrations (5 μg/mL and below), normal HUVEC cells maintained viability above 100 %, indicating negligible toxicity toward normal cells. In contrast, at a concentration of 5 μg/mL, 5-ALA@Zn-CuTz@PM NPs exerted notable cytotoxic effects on 4T1 tumor cells, reducing their viability to below 79.25 %. This cytotoxic effect was further amplified at a concentration of 20 μg/mL, where 4T1 cell viability decreased to 43 %, demonstrating the effective cytotoxic potential of 5-ALA@Zn-CuTz@PM NPs against tumor cells.

To further elucidate the impact of PM encapsulation on the therapeutic efficacy of 5-ALA@Zn-CuTz@PM NPs, we compared the cytotoxic effects on 4T1 cells of the NPs with and without PM coating. As illustrated in Fig. 6C, at equivalent concentrations, the 5-ALA@Zn-CuTz@PM NPs consistently showed superior cytotoxicity against 4T1 cells compared to 5-ALA@Zn-CuTz NPs. Specifically, at 20 μg/mL, the 4T1 cell viability for the 5-ALA@Zn-CuTz NPs group was 53 %, while the viability for the 5-ALA@Zn-CuTz@PM NPs group dropped to 43 %, a 10 % improvement. This enhancement is likely due to the PM coating, which facilitates enhanced tumor cell targeting and increases the effective intracellular concentrations of Cu⁺ and 5-ALA, thereby intensifying the cytotoxic effects.

Additionally, we assessed the influence of light irradiation on the therapeutic efficacy of 5-ALA@Zn-CuTz@PM NPs. As shown in Fig. 6D, at a concentration of 12.5 μg/mL, the viability of 4T1 cells in the non-irradiated group was 61.25 %, which decreased to 40.66 % after irradiation. These results indicate that Cu⁺ within the 5-ALA@Zn-CuTz@PM NPs alone provides substantial CDT effects that induce tumor cell damage, while the light-triggered PDT effect of 5-ALA further augments tumor cell cytotoxicity in a synergistic manner, thereby achieving an enhanced therapeutic outcome. At a concentration of 50 μg/mL, the 5-ALA@Zn-CuTz@PM NPs with light irradiation (5-ALA@Zn-CuTz@PM NPs + L) group exhibited a cell viability rate below 12 %, This outcome highlights the nanoparticles' exceptional cytotoxic efficacy against 4T1 tumor cells. Collectively, these findings demonstrate that 5-ALA@Zn-CuTz@PM NPs exhibit pronounced PDT and CDT efficacy at the cellular level, indicating considerable potential for inhibiting 4T1 tumor progression.

Antitumor evaluation of 5-ALA@Zn-CuTz@PM NPs in vivo

Following comprehensive in vitro studies on the mechanisms and antitumor efficacy of 5-ALA@Zn-CuTz@PM NPs, we proceeded to evaluate their therapeutic potential in vivo using a BALB/c mouse model with subcutaneous 4T1 tumors. Once tumors reached a volume of 100 mm³, mice were randomly assigned to six groups for various treatment regimens, as shown in Fig. 7A. To investigate the biodistribution and tumor-targeting capacity of the nanoparticles, we performed in vivo fluorescence imaging (Fig. 7 B). Results indicated a progressive accumulation of 5-ALA@Zn-CuTz@PM NPs at the tumor site within two hours, with fluorescence intensity peaking at four hours. This enhanced accumulation is attributed to the synergistic effect of passive EPR-mediated targeting and active tumor targeting by the PM coating, facilitating rapid and efficient nanoparticle enrichment at the tumor site. Notably, even after 24 hours, the fluorescence intensity remained strong at the tumor site, indicating prolonged retention and sustained therapeutic presence in the tumor. Subsequently, we assessed the biocompatibility of the nanoparticles in vivo. As shown in Fig. 7C, no significant variations in body weight were observed across all treatment groups during the 14-day study, indicating favorable biocompatibility and minimal systemic toxicity of the nanoparticles in vivo. The tumor growth inhibition efficacy of the nanoparticles was further evaluated. Changes in tumor volumes over 14 days are shown in Fig. 7E. In the PBS control group, tumor volumes grew exponentially. Tumors in the 5-ALA and 5-ALA+Laser groups also displayed rapid growth, with no significant impact observed from light irradiation. This limited efficacy is likely because systemically administered 5-ALA does not have specific tumor targeting and is readily metabolized and cleared, resulting in minimal drug accumulation at the tumor site. In contrast, the Zn-CuTz and 5-ALA@Zn-CuTz@PM groups demonstrated notable tumor growth inhibition, attributed to the EPR effect of Zn-CuTz NPs enabling passive tumor targeting. Cu⁺ in the nanoparticles effectively exerted CDT effects, causing tumor cell damage. The PM coating on 5-ALA@Zn-CuTz@PM NPs further enhanced active targeting, increasing drug accumulation and effective concentration at the tumor site, thereby enhancing the inhibitory effect on tumor growth. The best therapeutic efficacy was observed in the 5-ALA@Zn-CuTz@PM+Laser group. Beyond the CDT effect of Cu⁺, light irradiation activated PDT from 5-ALA, achieving a synergistic effect of CDT and PDT, which maximally suppressed tumor growth. Optical images of tumor tissues (Fig. 7D) further supported these findings. Additionally, we performed histological analysis on tumor tissue sections using H&E staining (Fig. 7F). Tumors in the Zn-CuTz and 5-ALA@Zn-CuTz@PM groups showed varying degrees of irregular morphology, fragmentation, and nuclear staining reduction. Tumor tissues in the 5-ALA@Zn-CuTz@PM+Laser group exhibited more severe nuclear condensation and cytoplasmic disruption, indicating that Zn-CuTz and 5-ALA@Zn-CuTz@PM caused some structural damage to the tumor tissue, with light irradiation significantly enhancing the therapeutic effects of 5-ALA@Zn-CuTz@PM. Lastly, we evaluated the histopathological toxicity in major organs (heart, liver, spleen, kidneys, and lungs) using H&E staining, with no significant pathological damage observed across any treatment group (Fig. 8). These findings collectively validate the excellent biosafety and potent in vivo antitumor efficacy of 5-ALA@Zn-CuTz@PM NPs, underscoring their promise as a robust therapeutic platform for targeted cancer treatment.

Fig. 7.

(A) Schematic diagram of the animal experiment design. (B) Fluorescence images of organs harvested at different time points post-injection. (C) Statistical analysis of body weight changes in mice after different treatments. (D) Photos of tumor dissection. (E) Relative tumor volume growth curve. (F) The Optical microscopy images of H&E-stained tumor sections after different treatment. Scale bar: 100 µm ***P < 0.001.

Fig. 8.

H&E-stained sections of major excised organs from mice after 14 days of different drug treatments. Scale bar: 100 µm.

Study limitations and Future challenges Outlook

• 1Potential Off-Target Effects

Although 5-ALA@Zn-CuTz@PM NPs exhibit strong tumor-targeting capabilities, potential off-target effects remain a concern. For instance, nanoparticles may undergo nonspecific uptake by organs rich in the reticuloendothelial system (RES), such as the liver and spleen, leading to accumulation in normal tissues. Additionally, hydroxyl radicals (•OH) generated during the CDT process have extremely high reactivity and may cause damage to surrounding normal cells. Therefore, further optimization of the nanoparticle surface modification strategy is needed to enhance tumor specificity while reducing nonspecific uptake by healthy tissues. Moreover, the use of responsive nanocarriers (e.g., redox-sensitive, or enzyme-sensitive materials) could improve selective release at the tumor site, minimizing systemic toxicity.

• 2Long-Term Stability of Nanoparticles

The physicochemical stability of nanoparticles is crucial for their in vivo applications. In physiological environments, nanoparticles may face challenges such as aggregation, degradation, or leakage of components, which could affect their pharmacokinetics and therapeutic efficacy. For example, during storage, nanoparticles may aggregate, altering their particle size and influencing their biodistribution and cellular uptake. Additionally, in circulation, plasma proteins may adsorb onto the nanoparticle surface, forming a “protein corona” that can alter their metabolic pathways. Therefore, before clinical application, it is essential to conduct in-depth studies on the storage stability, biological stability, and degradation mechanisms of nanoparticles in vivo. Optimizing surface modifications can help extend circulation half-life while maintaining their original activity.

• 3Challenges Before Clinical Application

Before 5-ALA@Zn-CuTz@PM NPs can be translated into clinical applications, several key challenges need to be addressed:

Pharmacokinetics and In Vivo Distribution: Further research is required to investigate the absorption, distribution, metabolism, and excretion (ADME) properties of nanoparticles to ensure appropriate circulation time and safety in vivo.

Immunogenicity and Biocompatibility: Although experimental results indicate good biocompatibility of these nanoparticles, long-term toxicological evaluations are necessary to confirm that they do not trigger immune responses or accumulate over time, leading to adverse effects.

Scalability and Quality Control: The reproducibility and industrial-scale production of nanomedicines remain significant bottlenecks in clinical translation. Optimizing the synthesis process is essential to ensure that nanoparticles maintain stable physicochemical properties and biological performance across different batches.

Animal Models and Preclinical Validation: Current studies are primarily based on mouse models, but the tumor microenvironment and metabolic characteristics of humans differ significantly from those of mice. Therefore, before advancing to clinical trials, further validation in large animal models (such as dogs or primates) is necessary to assess therapeutic efficacy and safety.

Conclusion

In this study, we developed multifunctional nanoparticles, 5-ALA@Zn-CuTz@PM NPs, capable of actively targeting breast cancer cells to improve the low tissue selectivity of 5-ALA and significantly increase the effective drug concentration at the tumor site. The Zn²⁺ in the carrier complex with PpIX to form PpIX-Zn, thereby inhibiting the HO-1 activity that contributes to treatment resistance in tumors. This mechanism enhances intracellular levels of active photosensitizers, amplifying the PDT efficacy of 5-ALA. Additionally, the Cu⁺ within the carrier react with the overexpressed H₂O₂ in tumor cells, generating cytotoxic hydroxyl radicals to exert CDT effects. Experimental validation demonstrated that 5-ALA@Zn-CuTz@PM NPs exhibit robust PDT-enhancing capabilities and achieve a powerful synergistic anticancer effect through the combined action of PDT and CDT therapies. Moreover, 5-ALA@Zn-CuTz@PM NPs showed excellent in vivo biocompatibility, underscoring their potential as a novel approach for combined breast cancer therapy. Although 5-ALA@Zn-CuTz@PM NPs have demonstrated excellent antitumor effects in both in vitro and in vivo experiments, further studies are needed to investigate their off-target effects, long-term stability, and preclinical challenges. Future research should focus on optimizing targeting strategies, enhancing nanoparticle stability, conducting long-term toxicological evaluations, and developing scalable manufacturing processes to facilitate their clinical translation.

Ethics approval

All animal experiments were reviewed and approved by the Committee on Animals at Nanjing University (IACUC——D2402094). All mice were housed in SPF-grade animal facilities.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The authors confirm that the data supporting the findings of this study are available either within the article or in the Supporting Information. Additional data is available from the corresponding authors upon reasonable request.