Abstract Recently, mesoporous nanomaterials with widespread applications have attracted great interest in the field of drug delivery due to their unique structure and good physiochemical properties. As a biomimetic nanomaterial, mesoporous polydopamine (MPDA) possesses both a superior nature and good compatibility, endowing it with good clinical transformation prospects compared with other inorganic mesoporous nanocarriers. However, the subacute toxicity and underlying mechanisms of biomimetic mesoporous polydopamine nanoparticles remain uncertain. Herein, we prepared MPDAs by a soft template method and evaluated their primary physiochemical properties and metabolite toxicity, as well as potential mechanisms. The results demonstrated that MPDA injection at low (3.61 mg/kg) and medium doses (10.87 mg/kg) did not significantly change the body weight, organ index or routine blood parameters. In contrast, high-dose MPDA injection (78.57 mg/kg) is associated with disturbances in the gut microbiota, activation of inflammatory pathways through the abnormal metabolism of bile acids and unsaturated fatty acids, and potential oxidative stress injury. In sum, the MPDA dose applied should be controlled during the treatment. This study first provides a systematic evaluation of metabolite toxicity and related mechanisms for MPDA-based nanoparticles, filling the gap between their research and clinical transformation as a drug delivery nanoplatform. Supplementary Information The online version contains supplementary material available at 10.1186/s12989-023-00548-4. Keywords: Mesoporous polydopamine, Systematic evaluation, Subacute toxicity, Metabolite, Gut microbiota Introduction With the booming development of nanotechnology, porous nanomaterials have become a hot topic in the field of drug delivery due to their unique structure and good physicochemical properties [[43]1]. For example, mesoporous silica, silver, gold, and manganese dioxide nanoparticles have been widely studied for their homogeneous and adjustable mesopore pore size [[44]2–[45]4], stable skeleton structure, large specific surface area and modifiable inner surface [[46]5]. However, a large number of studies have demonstrated that the fabrication of porous nanomaterials remains complicated with multistep operations, including template preparation, template-directed synthesis, and template removal. Too many steps are prone to the generation of secondary synthesis products and residual organic reagents. They are not only potentially damaging to the environment but also have some acute or subacute toxicity to the organism. Thus, there is an urgent imperative to develop biomimetic nanomaterials that combine simplicity of fabrication, environmentally friendly biodegradation, and improved biocompatibility. Drawing inspiration from mussels, Jing Tang et al. [[47]6] successfully synthesized biomimetic mesoporous polydopamine (MPDA) nanoparticles. They employed the high molecular weight block copolymer, polystyrene-block-poly (ethylene oxide) or PS-b-PEO, as a soft template. In mildly alkaline conditions (pH ~ 8.5), dopamine (DA) underwent spontaneous oxidation, and was deposited on the surface of self-assembled PS-b-PEO micelles [[48]7]. This synthesis process yielded nanoscale particles with a dimension of approximately 200 nm and a mesoporous structure approximately 16 nm in diameter. These aggregates exhibited augmented stability in near-neutral pH conditions (7 − 11) compared to strongly alkaline conditions (pH > 11) [[49]8]. In the creation of mesoporous structures in PDA, hard and soft templating methods are utilized. While the hard templating method allows greater control over nanomaterial morphology, it’s complex, time-consuming, and unsuitable for mass production. Alternatively, soft templates provide simpler construction, diverse morphology, and cause less environmental pollution, though they encounter issues such as low yields and limited availability of template agents. The surfaces and mesopores of MPDA nanoparticles abound with phenyl, amino, and hydroxyl groups. These features endow them the superior capability to load various chemical drugs, such as DOX (doxorubicin) and SN38 (7-ethyl-10-hydroxycamptothecin), via mechanisms like π-π stacking and/or hydrogen bond interactions [[50]9]. Additionally, MPDA particles have unique physicochemical properties, including metal ion chelation, photothermal conversion, environmental (pH or laser)-triggered drug release, high chemical reactivity and facile modification capabilities. Upon incorporating a targeting moiety, these nanoparticles can achieve targeted delivery to specific lesions, making MPDA-based nanodevices a focus of intensive studies for various therapeutic applications, including cancer theranostics, antibacterial strategies, and antifibrotic therapies. In terms of biosafety, MPDA have shown great biocompatibility with various cells, and no organ changes were detected in rats post-injection [[51]10]. However, the processes by which MPDA is metabolized or degraded in vivo, as well as the potential toxicity of its degradation products or metabolites, remain poorly understood. Existing research suggested that the pH-responsive properties of PDA could facilitate its degradation at lower pH levels, a typical characteristic of tumor microenvironments [[52]11]. Furthermore, PDA-based nanoparticles are also reportedly degradable by H[2]O[2] and free radicals present in the body. Moreover, Jin et al. [[53]12, [54]13] identified PDA degradation products in the human acute monocytic leukemia cell line THP-1 using high-performance liquid chromatography (HPLC) and speculated that the most dominant compounds in PDA degradation products were dopamine and some intermediate products, such as quinine and PDA segments. None of these components affected cell viability, reaffirming PDA compatibility. Given the existing knowledge gap regarding full biocompatibility, there is a pressing need for systematic investigation into the subacute toxicity and underlying mechanisms of biomimetic mesoporous polydopamine nanoparticles [[55]4]. In this regard, MPDAs were prepared by the soft template method, and different doses of MPDAs were injected into the tail vein of healthy male mice in this study. After 7 days of treatment, the changes in body weight, organ index, pathology and routine blood tests were analyzed to evaluate subacute toxicity. The possible mechanism was determined based on the microbiome and metabolomics of intestinal content and metabolomics measurements for serum. This study will supplement the toxicity data of MPDA and provide a theoretical basis for its clinical translation. Materials and methods Materials Dopamine hydrochloride, doxorubicin hydrochloride and ammonia (NH[3]·H[2]O, 25–28%) were purchased from Shanghai Macklin Biochemical Technology Co. Limited (Shanghai, China). NH[2]-PEG-NH[2] (Mw = 5000) was purchased from Shanghai ToYongBio Technology Co., Ltd. (Shanghai, China). Pluronic F127, Pluronic 123, and 1,3,5-trimethylbenzene (TMB) were provided by Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of analytical or chromatographic grade. The mice distributed by Institute of Cancer Research (ICR mice) aged 6–8 weeks and weighing 20 g were obtained from Shanghai Silaike Laboratory Animal Limited Liability Company, and given adequate food and water. The animal experiments were conducted according to the National Institutes of Health (NIH, USA) guidelines for the care and use of laboratory animals, with surgical procedures approved by the Committee for Animal Experiments of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (PZSHUTCM2212120003). Preparation of mesoporous polydopamine Biomimetic mesoporous polydopamine (MPDA) nanoparticles were prepared by the soft template method as previously reported [[56]14]. Briefly, P-123 (0.030 g), F-127 (0.075 g), and DA (0.15 g) were uniformly dispersed in 40% ethanol solution (20 mL) under the condition of water bath ultrasound (BC-3B, Shanghai Benting Instrument, China). After that, TMB (0.4 mL) was added to the above solution and sonicated for 10 min in a 40% probe ultrasonic instrument (XM-1500T, Xiaomei ultrasonic instrument, China) at a frequency of 3 s and intermittent 2 s. Then, MPDA nanoparticles were obtained by adding ammonia water (0.375 mL) to the mixed solution and mechanically stirring for 4 h at room temperature. To remove the excess water-soluble agents, the mixed solution was separated in a centrifuge (9500 rpm, 5 °C, 15 min). Then, the precipitates were dispersed in ultrapure water with sonication. The washing process was repeated twice. The crude products were further dispersed in absolute ethanol and washed three times. Subsequently, 0.05 g of NH[2]-PEG-NH[2] was added to the prepared nano-system above and subjected to magnetic stirring for 8 h to ensure that MPDA was adequately modified by NH[2]-PEG-NH[2]. This modification serves to enhance the stability of MPDA nanoparticles. Finally, PEG-modified MPDA was obtained by centrifugation. All tests concerning MPDA nanoparticles in this paper refer to those that are PEG-functionalized. Size distribution and morphology The size distribution of MPDA nanoparticles was analyzed using dynamic light scattering (DLS) with a Zetasizer from Malvern Co., UK. Morphological examinations were carried out using transmission electron microscopy (TEM) with a JEOL JEM-1230 from Japan. Photothermal effect Laser irradiation of MPDA at different concentrations under the same power One milliliter of aqueous solutions of MPDA nanomaterials with different concentrations (1 mg/mL, 0.2 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.625 mg/mL, and purified water) were placed in a 96-well plate at room temperature (24 °C). The nanomaterials were laser irradiated for 300 s with a fixed power (1.68 A, 1.5 W) of the laser irradiator. Afterwards, the temperature of the aqueous solution of MPDA nanomaterials was measured by the thermometer at an interval of 30 s each time (n = 5). Laser irradiation of MPDA at the same concentration under different powers Four aliquots of MPDA nanomaterial aqueous solution (1.5 mg/mL, 1 mL) were placed in a 96-well plate at room temperature (24 °C). The nanomaterials were laser irradiated for 300 s with a fixed power (1.68 A 1.5 W, 0.54 A 0.33 W, 0.75 A 0.5 W, 1.31 A 1 W, 1.86 A 1.5 W) of the laser irradiator. Afterwards, the temperature of the aqueous solution of the MPDA nanomaterial was measured by the thermometer at an interval of 30 s each time (n = 5). Thermal stability experiment of MPDA MPDA nanomaterials (1.5 mg/mL, 1 mL) were placed into a 96-well plate at room temperature (24 °C). After that, the laser irradiated the nanomaterials with the laser irradiator (laser wavelength 808 nm, laser spot 0.09 mm^2) at fixed power (1.68 A, 1.5 W). Then, the temperature of the dispersion naturally returned to room temperature. The operation was repeated twice. The temperature of the aqueous solution of MPDA nanomaterials after irradiation was measured by the thermometer at an interval of 30 s each time (n = 3). Drug loading experiment To study the drug loading property of MPDA nanomaterials, doxorubicin hydrochloride (1 mg/mL, 1 mL) was added to different concentrations of MPDA nanomaterials (0.05 mg/mL 1 mL, 0.1 mg/mL 1 mL, 0.2 mg/mL 1 mL). The mixture was stirred overnight and then centrifuged (9500 rpm, 15 min). After centrifugation, 100 µL of the supernatant was added dropwise to a black shaded 96-well plate, and the absorbance was measured by a fluorescence photometer (SpectraMax i3x, Molecular Devices, USA, doxorubicin hydrochloride, Ex = 504 nm, Em = 550 nm). To prepare the standard curve, aqueous solutions of doxorubicin hydrochloride at different concentrations (0.1 mg/mL, 0.2 mg/ml, 0.4 mg/mL, 0.8 mg/mL, 1 mg/mL) were diluted and measured by a fluorescence photometer (SpectraMax i3x, Molecular Devices, USA, doxorubicin hydrochloride, Ex = 504 nm, Em = 550 nm). The drug loading rate of MPDA nanomaterials was calculated according to the following formula: graphic file with name M1.gif graphic file with name M2.gif Toxicity experiments with MPDA Body weight Twenty-eight mice were randomly divided into 4 groups (n = 7). The initial body weights of the mice were recorded before the experiment, and subsequently, the mice were given saline (control group), 78.57 mg/kg MPDA (H-MPDA group), 10.87 mg/kg MPDA (M-MPDA group), and 3.61 mg/kg MPDA (L-MPDA group) by tail vein injection. Mice were weighed every 2 days after administration to assess MPDA nanoparticle toxicity. The dosage calculation process was listed as below. 1. Based on the drug dosage, the feeding ratio of drug and MPDA, as well as the mice bodyweight documented by previous studies (Table [57]1), we were able to calculate the amount of MPDA to administer to each mouse. Our calculations determined that the lowest amount required was 0.1 mg (low group) or 0.3 mg (median group). In our study, the mice in the median group had a bodyweight of 27.5875 g, while those in the low group weighed 27.6625 g. Accordingly, the dosage for the low group was set at 3.61 mg/kg, while that for the median group was set at 10.87 mg/kg. 2. The dosage for the high group was determined based on the saturated solubility of MPDA as prepared in our research. This was done with the intention of evaluating whether administering MPDA at the highest concentration would cause any acute or subacute damage to the body. It is worth noting that this dosage falls well below the maximum dosage given in previous treatment. Table 1. The amount of PDA given in previous studies PDA dosage Drug dosage Feeding ratio of drug and PDA Mice bodyweight Amount of PDA References