In 2014, the synthesis of Bi NPs coated with d-glucose (Bi@d-glucose) was described [13]. These Bi NPs contain around 6 million bismuth atoms per nanoparticle and are characterized by a very dense bismuth core that constitutes the majority (~60%) of the particle volume. Quantitative computed tomography (CT) using phantoms has demonstrated that Bi@d-glucose NPs have greater X-ray opacity than clinical, iodinated contrast agents (iopamidol, a marketed iodinated XCA) at the same concentration. Moreover, Bi NP attenuation is relatively insensitive to the range of tube voltages used in clinical CT scanners (80 to 140 kV), which is advantageous because the same contrast is produced using any CT imaging protocol. The imaging of cells (HeLa cells and murine macrophages) incubated with different Bi NP concentrations enabled an in vitro quantitative analysis of CT attenuation. CT imaging also revealed that the uptake by both types of cells had a linear correlation with XCA concentration, indicating a nonspecific uptake process. As expected, this uptake was more pronounced with the murine macrophage line, which is consistent with its greater phagocytic activity.

The preparation of highly monodisperse aqueous Bi NPs coated with polymerized d-glucose was reported in 2016 [14]. The CT contrast efficiency of these Bi NPs was evaluated in comparison with that of a BaSO₄ suspension in vitro. The Bi NPs produced much higher CT contrast per unit of mass concentration than did BaSO₄, regardless of the CT scanner operating voltage (80 kV and 120 kV). The high stability of these Bi NPs allowed for their oral or rectal administration to mice to achieve CT imaging of the gastrointestinal (GI) tract. After oral administration of these Bi NPs, the upper GI tract and the arrangement of the small intestinal loops were visualized with high contrast. Rectal administration enabled the visualization of the lower GI tract (the rectum and descending colon).

Chakravarty et al. studied two kinds of Bi NPs obtained by complex procedures: Bi Ganex (BiG) nanocrystals encapsulated by poly (DL-lactic-co-glycolic acid) (BiG@PLGA) and by a SiO₂ coating (BiG@SiO₂) [15]. These two kinds of Bi NPs were dispersed in a 0.5% agarose gel. These phantoms were imaged by a CT scanner operating at a tube voltage of 80 kV and compared with those generated with iopamidol (a marketed iodinated XCA) at various concentrations (0 to 80 mM). The CT contrast enhancement of both Bi NPs was threefold that of Iopamidol (300 mg/mL) at isoconcentration, as demonstrated by the Hounsfield units (HU) quantified with respect to the bismuth or iodine concentration.

Swy et al. used poly (DL-lactic-co-glycolic acid) (PLGA) to encapsulate the Bi NPs [16]. The resulting encapsulated NP had a diameter of 120 nm. After 24 h in an acidic solution imitating the lysosomal medium, the Bi@PLGA NPs showed nearly 70% degradation, whereas in cytosol- and extracellular fluid-imitating media, they remained completely stable. Both a clinical imager and a μCT imager detected these Bi NPs in vitro. The rate of attenuation was higher using μCT because the low-energy component of the μCT X-ray beam was greater than that of the clinical CT system. The ability to detect Bi NPs ex vivo by CT and μCT was also demonstrated by injecting Bi NPs into a chicken wing forearm.

Bovine serum albumin (BSA)-coated Bi NPs (hydrodynamic diameter of 62 nm) were imaged by CT imaging, operating at a tube voltage of 60 kV [17]. Bi NPs were intravenously injected in mice subcutaneously transplanted with mammary carcinoma tumour cell line 4T1. One hour later, in vivo CT imaging of the mice showed an enhanced contrast of tumour due to Bi NP accumulation, because of the high permeability and retention effect of the tumour.

The theranostic potential of Bi NPs was demonstrated by evaluating both their diagnostic efficiency in X-ray imaging and their therapeutic efficacy in PTT and RT [29]. For these purposes, uniform and stable lipophilic Bi NPs (TEM diameter: 40 nm) coated with 1-dodecanethiol ligands and encapsulated by amphiphilic PEGylated phospholipid (DSPE-PEG) were prepared and named Bi@SR-PEG NPs. HU quantification demonstrated that these Bi@SR-PEG NPs provided better CT imaging signals than did iobitridol (a marketed iodinated XCA). Subcutaneous 4T1 tumour-bearing mice were intravenously (IV) injected with Bi@SR-PEG NPs (200 mL, 2.0 g L⁻¹). CT imaging enabled the detection of the tumour area with a maximum contrast obtained 1 h post-injection, before the washed-out phase. A long-lasting liver contrast enhancement was observed, reflecting the phagocytosis of Bi@SR-PEG NPs by Kupffer cells. Then, Bi@SR-PEG was evaluated first in vitro for its performance in NIR-PTT and RT applications. High NIR absorbance and photothermal efficiency were observed under irradiation with an 808-nm laser (1.0 W cm⁻²) for 10 min. The NIR-PTT potential was confirmed in vitro in the 4T1 cells. After the 4T1 cells were incubated with Bi@SR-PEG NPs (40 mg L⁻¹) and exposed to an 808-nm laser (intensity of 1.0 W cm⁻² for 10 min), a negligible number of stained living cells were observed. The in vitro efficiency of Bi@SR-PEG NPs as X-ray radiosensitizers was demonstrated by using a clonogenic survival assay. After incubation of 4T1 cells with Bi@SR-PEG NPs (40 mg L⁻¹) and exposure to X-ray irradiation (4 Gy), the survival fraction of the 4T1 cells was determined to be 0.27 and 0.57 in the presence and absence of Bi-SR-PEG, respectively. The sensitization effect was similar, and these promising in vitro results encouraged the authors to test the synergistic effect of Bi@SR-PEG NPs in NIR PTT and RT in vivo. Subcutaneous 4T1 tumour-bearing mice were intravenously injected with Bi@SR-PEG NPs (200 mL, 2 g L⁻¹) and then exposed, successively, to an 808-nm laser with an intensity of 1.0 W cm⁻² for 10 min and to X-ray irradiation (4 Gy). After 14 days, the mice were sacrificed. This synergistic dual treatment induced the inhibition of tumour growth (determined by measuring the average tumour volume) and cell apoptosis (determined by cell morphology and haematoxylin and eosin staining). Individual therapy (NIR-PTT or RT) had a limited therapeutic effect compared with the synergistic effect of the combined NIR-PTT and RT treatment, as demonstrated in this publication.

Yu et al. described the synthesis and biological evaluation of a multifunctional theranostic agent based on a peptide (LyP-1)-labelled Bi NP (Bi@LyP-1 NP) [21]. The ability to absorb both ionizing radiation and laser radiation ensured that Bi@LyP-1 NPs could be used for dual-modal computed tomography and photoacoustic imaging and the synergistic NIR-photothermal and radiotherapy treatment of tumours. By evaluating the HU values, it was demonstrated that the CT signal enhancement capability of Bi@LyP-1 NPs in vitro was superior to that of iohexol (a marketed iodinated XCA). This was further confirmed in vivo by CT imaging after the intratumoural injection of Bi@LyP-1 NPs into 4T1 tumour-bearing mice. As Bi@LyP-1 NPs enable strong and broad NIR absorption, it was possible to study the potential of the Bi NPs with PAI. After the injection of Bi@LyP-1 NPs intratumorally or intravenously into mice bearing subcutaneous 4T1 cell tumours, high photoacoustic signal intensity was observed in the tumours under a wide range of NIR wavelengths (from 700 to 900 nm). The in vitro performance of the Bi@LyP-1 NPs in NIR PTT and RT was then assessed. The Bi@LyP-1 NPs exhibited a broad absorption, between 200 and 2000 nm, and were capable of acting as NIR PTT agents when irradiated with a near-infrared (NIR-II) laser. The use of an NIR-II laser enabled deeper penetration and higher maximum permissible exposure, which increased the photothermal efficacy, especially in humans in the clinic. The in vitro performance was demonstrated when irradiation of a 200 ppm Bi NP solution with a 1064-nm laser produced a hyperthermic effect, with a temperature increase in approximately 17.2 °C, in comparison to that of pure water, which caused a temperature increase in only approximately 2.8 °C. An in vitro clonogenic survival assay demonstrated the ability of the Bi@LyP-1 NPs to enhance the RT efficacy, even at low concentrations and low doses of radiation. The fraction of the 4T1 cells incubated in a 10 ppm Bi@LyP-1 NP solution that survived was dependent on X-ray irradiation doses. The combined PTT and RT effects were studied in vivo after Bi@LyP-1 NPs were IV injected. The surface temperature of the tumours after laser irradiation of 0.6 W cm⁻² with a 1064-nm laser was monitored by NIR imaging. After Bi@LyP-1 NP injection, the tumour temperature rapidly increased to approximately 45 °C and then stabilized, while after a PBS injection, the temperature was found to increase slowly and only to 39.5 °C. This mild photothermal effect resulted only in a delay in tumour growth. However, after irradiation by X-ray and laser irradiation, tumour growth was completely inhibited, demonstrating strong synergistic effects.

Multifunctional Bi NPs were developed as theranostic agents for PA/CT imaging and NIR-PTT [23]. The preparation of 1,2-dilauroyl-sn-glycero-3 phosphocholine (DLPC)-coated spherical Bi NPs (Bi@DLPC NPs) was described, and these Bi NPs were characterized by a TEM-determined size of 47 nm and a hydrodynamic size of 162 nm. The photothermal effect was demonstrated in vitro. The temperature of a Bi@DLPC NP (500 μg mL⁻¹) aqueous solution increased by 37 °C after 10 min of irradiation with an NIR laser (880 nm, power density of 1 W cm²). This kind of irradiation also induced mitochondrial damage by hyperthermia and MDA-MB-231 cell death in vitro, which was likely due to changes in cell membrane permeability.

PA and CT imaging were performed in vitro and in vivo. In vitro, the PA signal at 880 nm increased linearly with the Bi@DLPC NP concentration. A peak PA signal was observed in the MDA-MB-231 tumour cell-bearing mice 6 h after the intravenous injection of the Bi@DLPC NPs. In vitro, the CT signal intensity also linearly increased with the Bi@DLPC NP concentration.

The CT signal was observed in vivo after intratumoural injection of Bi@DLPC NPs (100 μL, 4 mg mL⁻¹) into MDA-MB-231 tumour cell-bearing mice and PTT. Then, an in vivo photothermal effect was demonstrated with MDA-MB-231 tumour cell-bearing mice intravenously injected with Bi@DLPC NPs (1 mg mL⁻¹) and irradiated with an NIR laser (880 nm, 1 W cm²) for 10 min. The thermic infrared images demonstrated that the surface temperature of the tumour area dramatically increased, from 37.4 to 57.7 °C. Cancer cell growth was abrogated by hyperthermia 14 days after treatment.

Physiologically stable PEG-modified polypyrrole-coated Bi Nanohybrids (Bi@PPy-PEG NHs) were recently prepared by Yang Sisi et al. [22]. The Bi@PPy-PEG NH aqueous dispersion revealed a high photothermal conversion capacity and stability after five repeated irradiation cycles with an 808-nm laser (2.0 W cm⁻²) completed within 10 min. The high efficiency was attributed to the contribution of PPy, which is known for its excellent photothermal effect. Consequently, highly effective photothermal ablation of cancer cells (4T1 cells) was achieved in vitro. At a concentration of 100 μg mL⁻¹, the Bi@PPy-PEG NHs killed more than 90% of the 4T1 cells in 3 min after NIR 808 nm laser irradiation (2.0 W cm⁻²). Then, the PTT effect was determined in vivo with tumour-bearing BALB/c mice subjected to NIR 808-nm laser irradiation (2.0 W cm⁻²) twenty-four hours after the Bi@PPy-PEG NHs were IV injected.

Follow-up tumour volume measurements demonstrated that tumour growth was efficiently inhibited after irradiation. Furthermore, high-contrast CT/PA dual-modal imaging was demonstrated both in vitro and in vivo (intratumoural injection of Bi@PPy-PEG NHs in tumour-bearing BALB/c mice), showing great potential to provide accurate diagnostic information for antitumour treatment.

Lu et al. recently prepared Bi NPs coated with PEG-2000-2-distearoyl-sn-glycero-3-phosphoethanolamine (Bi@DSPE-PEG NPs), which were used as CT and PAI contrast agents and PTT agents [32]. The performance of the Bi@DSPE-PEG NPs as CT and PAI contrast agents was demonstrated with phantoms (HU measurement and PA signals as functions of Bi concentration) and in vivo by measurement of PA and CT signals after the Bi NPs were IV injected into the C6 glioma tumour-bearing mice. Photothermal therapeutic performance was demonstrated. The temperature of the Bi@DSPE-PEG NP solution (1 mg mL⁻¹) rapidly increased to 70 °C within 4 min under 808-nm laser irradiation at a power density of 1.0 W cm⁻². The same laser irradiation conditions were used to confirm this photothermal in vivo with C6 glioma tumour-bearing mice with Bi@DSPE-PEG NPs (200 μL of 10 mg mL⁻¹ Bi NPs IV injected). Indeed, the tumour volume was significantly reduced 12 days after this PTT treatment.

Several Bi NPs have been developed with coatings containing natural biomolecules such as gelatin GEL (BGPs) [33]. These Bi NPs showed CT/infrared thermal (IRT) imaging capabilities as well as antitumor effects under CPTT in vitro and in vivo. After 15 min irradiation with an 808-nm laser (1.3 W cm⁻²), a strong increase in temperature is observed, up to 78.6 °C for a BGP suspension of 500 µg mL⁻¹, showing the significant hyperthermia induced by Bi NPs compared to a deionized water solution. After five cycles of on–off irradiation, BGPs demonstrated an excellent photostability. The PTT effect was evaluated in vitro after 5 min irradiation on HepG-2 and HeLa cells with different BGP concentrations. Cell viability decreased below 10% with 500 µg mL⁻¹ of BGPs after irradiation while it remained almost unchanged (90%) without irradiation. CT and IRT imaging were then performed on tumour-bearing mice, after IV injection of BGPs (20 mg kg⁻¹) and exposure to NIR laser (808 nm, 1.3 W cm⁻²) for 5 min. CT imaging demonstrated that BGPs were excellent contrast agents, while IRT imaging measured a rapid temperature rise to 58 °C, demonstrating a favourable photothermal efficiency of BGPs on HepG-2 tumour cells. In vivo, after 24 h of IV injection of a mixture of BGPs and doxorubicin and under NIR laser irradiation, a significant growth inhibition of HepG-2 tumour cells was observed.

Pegylated metallic Bi NPs (Bi@PEG) were used to produce an in vitro and in vivo proof of concept of their use in trimodal imaging (CT, PA and infrared thermal (IRT)) and antitumour PTT [20].

The performance of the Bi NPs in CT imaging was demonstrated in vitro (HU measurements) and in vivo after they were IV injected into HeLa tumour cell-bearing BALB/c mice. After injection, the signal in the tumour increased gradually and considerably, with a burst 6 h post-injection. A linear PA signal was observed with the increasing aqueous concentration of the Bi@PEG NPs, indicating adequate PA imaging ability of the Bi NPs in vitro. PA imaging was investigated in vivo on tumour-bearing Balb/c mice after Bi NPs were IV injected. The PA signal in the tumour was enhanced and lasted up to 24 h, with the strongest signal observed 6 h after injection. The high imaging contrast and favourable residence time in the tumours make Bi NPs excellent contrast agents for both CT and PA imaging in vivo.

The in vitro PTT effect was confirmed by measuring the number of HeLa cancer cells that died after being incubated with Bi@PEG NPs and irradiated with a laser (808 nm, 1.0 W cm⁻²) for 10 min. The majority of the cells were destroyed under these conditions. The in vivo antitumour PTT was investigated in the HeLa tumour-bearing BALB/c mice after the Bi@PEG NPs were IV injected, and they were subjected to irradiation (808 nm, 1.0 W cm⁻²). During irradiation, real-time IRT imaging was used to monitor the tumour temperature change at different times. With laser irradiation, the tumour temperature rapidly increased and reached a plateau of ~51.6 °C. This temperature was sufficient to inhibit further tumour growth and to ablate the tumour after 10 days.

Stable Bi nanoparticles (NPs) coated by mesoporous silica (Bi@mSiO₂@MnO₂/DOX) were synthetized using a stepwise reaction protocol and a further loading with doxorubicin (DOX) [34]. In vitro, after laser irradiation (808 nm, power density, 1 W cm⁻²), Bi@mSiO₂@MnO₂ reached a stable photothermal effect with a high photothermal conversion efficiency of 50%. It was also demonstrated in vitro that MnO₂ incorporated in the coating of these Bi NPs was able to catalyse H₂O₂ decomposition in O₂. These Bi NPs are also efficient contrast agents for X-ray CT imaging of tumours with a high CT value (6.865 HU mM⁻¹). The authors propose a sequential and synergistic mechanism of tumor cell destruction. First, after IV injection, Bi@mSiO₂@MnO₂/DOX accumulate in the tumour site. (NIR) light irradiation of Bi NPs induces hyperthermia and a PTT effect. The generated heat triggers the release of DOX and induces a chemotherapeutic effect. The chemotherapeutic effect is then enhanced due to the generation of O₂ by decomposition of endogeneous H₂O₂ by MnO₂. This production of O₂ consumes glutathione to produce Mn²⁺, which provide a magnetic resonance imaging signal. Moreover, the presence of H₂O₂ and Mn²⁺ in the tumor site also produce toxic hydroxyl radicals, inducing a chemodynamic therapy effect. This clever and rational design of a theranostic Bi NP results in an excellent global performance, as Bi@mSiO₂@MnO₂/DOX-injected mice group treated with laser irradiation for 10 min exhibited a complete tumour elimination after 16 days, without any recurrence.

All these examples show the significant potential of Bi NPs to create versatile multifunctional theranostic nanomedicines. It is possible to design nanoparticles that are visible to several complementary imaging technologies in terms of spatial and temporal resolution and contrast. It is also possible to combine several types of synergistic treatment, in particular in oncology. However, the design of these nanoparticles remains complex and will require significant efforts to make their synthesis reliable in order to comply with cGMP drug manufacturing standards.