Targeted bismuth-based materials for cancer

Abstract

The use of bismuth and its compounds in biomedicine has developed rapidly in recent years. Due to their unique properties, there are great opportunities for the development of new non-invasive strategies for the early diagnosis and effective treatment of cancers. This perspective highlights key fabrication methods to generate well-defined and clinically relevant bismuth materials of varying characteristics. On the one hand, this opens up a wide range of possibilities for unimodal and multimodal imaging. On the other hand, effective treatment strategies, which are increasingly based on combinatorial therapies, are given a great deal of attention. One of the biggest challenges remains the selective tumour targeting, whether active or passive. Here we present an overview on new developments of bismuth based materials moving forward from a simple enrichment at the tumour site via uptake by the mononuclear phagocytic system (MPS) to a more active tumour specific targeting via covalent modification with tumour-seeking molecules based on either small or antibody-derived molecules.

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Amna Batool

Amna Batool is a doctoral researcher at the School of Science, Monash University specialising in nanobiotechnology and cancer theranostics. Her work focuses on the development and application of functionalised nanomaterials for biomedical use. Through the MHELTHERA collaboration at HZDR (Germany), she has engineered nanoparticles to investigate their potential in tumour-targeted diagnostics and therapy. Passionate about bridging nanotechnology and immunotherapy, Amna's research seeks to advance novel nanotechnology-based strategies for clinically relevant cancer research.

Ina Kopp

Ina Kopp studied chemistry at the University of Paderborn, wrote her thesis at the University of Innsbruck in 2021and is now a doctoral researcher at the Institute of Radiopharmaceutical Cancer Research at Helmholtz-Zentrum Dresden-Rossendorf (HZDR). Her work focuses on the development of novel bispidine chelators for radiopharmaceutical applications and multimodal imaging. As part of the MHELTHERA project, she was working on the detection of activated platelets at the Alfred Hospital and Monash Biomedical Imaging (MBI) in Melbourne, Australia.

Manja Kubeil

Manja Kubeil studied chemistry and received her Ph.D. degree from the Technische Universitat Dresden in 2014. In 2015, she was awarded with a Marie Curie International Outgoing Fellowship to undertake her postdoctoral research at the Monash University in Australia. In 2019, she became topic group leader at the Institute of Radiopharmaceutical Cancer Research at Helmholtz-Zentrum Dresden-Rossendorf (HZDR) in Germany. Since 2025, she is the head of Radiation Research on Biological Systems department at the Institute of Resource Ecology at HZDR. Her research focuses on radiopharmaceuticals for theranostic applications and radiation research to study the effects of radionuclides in living systems.

Michael Bachmann

Michael Bachmann Ph.D. in pharmacy (summa cum laude, Johannes-Gutenberg-University, Mainz, Germany), Professor of Physiological Chemistry, member of the Arthritis and Rheumatism Program (OMRF, OKC, U.S.A.), University Professor Medical Faculty (Technische Universität, Dresden, Germany), (i) tumourimmunology, (ii) Translational Radiopharmacology, and (Managing) Director, of the Institute of Radiopharmaceutical Cancer Research, at the Helmholtz Zentrum Dresden Rossendorf. Selected prizes: Boehringer-Ingelheim, Cell Biology, Bruno-Schuler (Rheumatology), Robert Feulgen (Int. Society of Cyto- and Histochemistry). Research focus: Radio-immunotheranostics for modular targeting systems (UniCAR, RevCAR, UniMAb). Current Research projects: Monash-Helmholtz Laboratory for Radio-Immuno-Theranostics (MHELTHERA), Ph.D. Networks: Oncoprotools, and Tolerate (European Union's Horizon Research and Innovation program).

Philip C. Andrews

Phil Andrews is the Head of the School of Chemistry at Monash University, Australia. After completing his PhD in 1992 at the University of Strathclyde, Glasgow, he held prestigious fellowships, including a Royal Society Fellowship at Griffith University and an Alexander von Humboldt Fellowship at Philipps Universität, Marburg. Joining Monash in 1995 he received an ARC QEII Fellowship, and is a Fellow of the Royal Society of Chemistry and the Royal Australian Chemical Institute. A founding member of the Monash Centre to Impact AMR, his research focuses on the discovery of metal compounds and materials as antimicrobials and anti-cancer agents.

Holger Stephan

Holger Stephan studied chemistry and received his PhD degree from the Technical University Bergakademie Freiberg in 1989. He held positions in industry and worked as Senior Scientist at the Technical University Dresden. In 1997, he moved to the Research Centre Rossendorf and was head of the working group “Nanoscalic Systems” at the Institute of Radiopharmaceutical Cancer Research of the Helmholtz-Zentrum Dresden-Rossendorf. He is currently working as the coordinator of the Monash-Helmholtz Laboratory for Radio-Immuno-Theranostics (MHELTHERA). His research focuses on the development of radiometal complexes, polynuclear metal compounds with photosensitizing and radiation-sensitizing properties, nanomaterials for multifunctional imaging and treatment of cancer.

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1. Introduction

Bismuth and many of its compounds are of great interest for medical applications due to their low toxicity.¹ This behaviour is particularly influenced by the stabilising ligands, the dosage form and the administered concentration.² The solubility of most bismuth compounds is low even in the acidic environment of the stomach and thus an almost complete elimination via the gastrointestinal tract is considered a crucial factor for low toxicity in vivo.³ This has driven forward, in particular, the development of antimicrobial and antiparasitic metallodrugs, such as bismuth subsalicylate, colloidal bismuth subcitrate or ranitidine bismuth citrate.^4–6 Interactions with iron-containing proteins such as transferrin and lactoferrin as well as with various enzymes such as ureases, hydrogenases and ATPase are discussed as the underlying mechanisms of the biological activity of bismuth compounds.⁷ The specific interactions of bismuth compounds with biologically active molecules has also initiated studies of the anticancer activity of both small and nanoscale materials.^8–12 With regard to small bismuth-based compounds, complexes with halide, nitrate, carboxylate and dithiocarbamate ligands as well as various heterocyclic compounds dominate. The majority of the investigations are limited to in vitro studies on cell toxicity.^13–27 In contrast, in vivo studies in connection with bismuth complexes that show anticancer activity are only just beginning.^28,29

This also applies to the structural elucidation of the bismuth complexes used in solution. For example, bismuth complexes with oxido ligands tend to form larger clusters, linking them to nanoscale bismuth-based systems, which are being actively investigated for cancer imaging and therapy.^2,30–34 These new materials reveal great prospects in terms of improved imaging based on X-ray computed tomography (CT),³⁵ photoacoustic imaging (PA),³⁶ and infrared thermography (IRT),^37,38 as well as efficient treatment options using in particular photothermal therapy (PTT),³⁶ photodynamic therapy (PDT)³⁹ and radiotherapy (RT).^40,41 With regard to the latter, i.e. the application of external ionising radiation, there are promising developments to improve the treatment effectiveness of tumours with the help of bismuth-containing radiosensitisers.⁴² In contrast, radionuclide therapy (internal radiotherapy) using radioactive bismuth nuclides is still in its infancy.^43,44 The increasing availability of the alpha-emitting nuclides ²¹²Bi and ²¹³Bi opens up a wide range of possibilities. This includes the use of radioactively labelled bifunctional chelating agents equipped with biological vector molecules and antibodies (BCFAs). There are new BFCAs with improved properties as well as selective and efficient coupling strategies, which opens up new opportunities for active targeting and the use of new radio-immunotherapeutic approaches. In addition, there is a rapid development in the field of bismuth-containing nanomaterials that are suitable for use in the combination therapy of cancer. This requires very defined particles in terms of size and morphology as well as the use of highly stable dispersions.

In this perspective article, suitable synthetic strategies for bismuth-containing materials will be discussed, which may allow a transfer to clinical use (Fig. 1). Furthermore, the state-of-the-art in the development of bismuth chelators for targeted alpha therapy is summarised and selected examples of radiolabelled target-seeking bioconjugates and nanomaterials are presented. New immunotherapeutic strategies are discussed that allow, on the one hand, effective recognition of tumours at an early stage and, on the other hand, various types of combination therapy for tumour treatment.

Fig. 1 Development and characterisation of bismuth materials for imaging and treating cancers with a view to clinical use.

2. Bismuth materials: synthesis strategies and cancer applications

The synthesis of defined bismuth materials with high stability is difficult and challenging. In general, three classes of bismuth-containing materials can be distinguished, namely metallic nanoparticles (NPs), bismuth chalcogenides and bismuth-containing hybrid materials. With regard to clinical use, materials with high biocompatibility and suitable surface functionalisation are required for biological targeting. In this perspective, the following chapter summarises various synthesis strategies for clinically relevant bismuth compounds and discusses selected examples of their use in cancer imaging and therapy.

2.1. Metallic bismuth nanoparticles

Owing to their unique properties, metallic bismuth particles have become very important.^45–48 The core characteristics of these particles are highly dependent on their crystal structure, size, shape and morphology, which can be tailored via various synthesis techniques.^49–51 These are essentially reduction methods, laser ablation, and thermal decomposition techniques. These methods can lead to particles with different size distributions and must be combined with suitable coating strategies for stabilisation in the complex biological environment.^52–54

Synthesis strategies that use chemical reducing agents are the most commonly used methods for obtaining metallic bismuth materials. Reductive synthesis methods in the presence of stabilising molecules usually result in well-defined particles. For example, the reduction of Bi(NO₃)₃ using sodium borohydride in the presence of the biomacromolecules gelatin (GEL), bovine serum albumin (BSA) or human serum albumin (HSA) at 37 °C, was reported to form stable spherical particles with a narrow particle distribution, i.e., the size range was 15.6 ± 3.1 nm for Bi@GEL, 17.4 ± 3.3 nm for Bi@BSA and 18.9 ± 3.8 nm for Bi@HSA, respectively.⁵⁵ Further, the cytotoxic anticancer drug doxorubicin (DOX) was incorporated, which allows for chemotherapeutic application. These biomacromolecule-stabilised metallic Bi-NPs have appropriate properties for theranostic applications, i.e., they are suitable for CT and infrared thermal imaging (IRI) as well as for combination therapy based on chemo- and photothermal therapy.⁵⁵ Metallic Bi-NPs can also be stabilised with cellulose derivatives.^56,57 Chemical reduction using NaBH₄ yields very small particles (2–10 nm), while reduction using UV light results in larger particles 25–55 nm. The use of reducing bacterial cultures such as Delftia also enables the production of metallic Bi-NPs, albeit with relatively large particle sizes (40–120 nm).⁵⁸ Synthesis processes based on green chemistry are becoming increasingly important.^59,60 In this context, a promising way is to use biocompatible compounds as reducing agents. Ouyang et al. fabricated Bi-NPs from BiCl₃ using ethylene glycol as the reducing agent.⁶¹ The metallic Bi-NPs obtained were coated with gold and doxorubicin was incorporated. These Bi@Au particles exhibit excellent blood compatibility, a high photoconversion efficiency (η = 46.6%) and are very effective in synergistic chemo-photothermal therapy proven in an A549 tumour-mice model. However, these particles are quite large at >100 nm. The use of viscous solvents like ethylene glycol slows the diffusion of reactants, which can lead to uneven nucleation and growth of particles.^62,63 There are a number of other Bi-NPs with sizes above 100 nm that exhibit good biocompatibility due to suitable surface functionalisation such as PEGylation, phospholipids and silica shells.^64,65 Smaller particles are better suited for biomedical applications, especially for clinical use.⁶⁶ One very interesting approach is the use of 45 nm-sized Bi-NPs, which have a SiO₂ shell for the incorporation of an H₂O₂-responsive prodrug.⁶⁷ These particles have very good photothermal properties with a high photoconversion efficiency (η = 46.3%) using near IR light at 808 nm. After intra-tumoural injection of these particles, photothermal ablation in combination with sequential X-ray irradiation allows for an improved therapeutic efficacy of >97%. Most of the Bi-containing particles currently used take advantage of passive targeting based on the enhanced permeability retention (EPR) effect.^68–70 The utilisation of active targeting with the help of biological vector molecules is only just beginning. In particular, specific peptides such as AR (androgen receptor) and LyP-1 as well as folate ligands are used for tumour targeting.^65,71–73 An interesting approach is to use the cancer cell membranes for targeting.⁷⁴

To achieve ultrasmall particles, i.e. with sizes <6 nm, chemical reduction methods are also suitable if the appropriate reaction conditions are applied.^75,76 Lei et al. report a very fast (1 min) preparation method using Bi(NO₃)₃ as starting material, NaBH₄ and polyvinylpyrrolidone (PVP) as stabiliser, resulting in rapid generation of ultrasmall uniformly sized spherical Bi-NPs with a narrow size distribution (2.7 ± 1.1 nm).⁷⁵ It is worth mentioning that these Bi nanodots produce a higher CT contrast than the iodine-based contrast agent iobitridol. In vivo dual-modal CT/photothermal-imaging-guided therapy was demonstrated in a U14 tumour-mice model.⁷⁵ Similarly, very small Bi-NPs (3.6 nm) were obtained by reducing bismuth acetate in a mixture of oleic acid and oleylamine.⁷² These primary particles were stabilised with a PEG and phospholipid shell and the nonapeptide LyP-1 is attached to the surface. The particles had a hydrodynamic diameter of 12 nm in aqueous solution and enabled dual imaging based on CT and PA. Moreover, a combination of NIR-laser and X-ray irradiation caused very effective tumour treatment (Fig. 2).

Fig. 2 Tumour treatment with Bi-NPs attached with the LyP-1 peptide (Bi-LyP-1 NPs). (a) IR images of 4T1 tumour bearing mice injected with Bi-LyP-1-NPs using 1064 nm laser irradiation (0.6 W cm⁻²). (b) Temperature variation curves of tumour surface after NP's injection. (c) Tumour growth in each group under different treatments (d) Recorded images of tumour growth in different groups post 14 days. (e) H&E (upper row) and CD31 staining (bottom row) of tumour sections using different conditions (reprinted with permission from ref. 72, Copyright 2017 American Chemical Society).

An elegant method of obtaining metallic particles is laser ablation, in which a high-intensity laser is used to generate NPs from a solid target.⁷⁷ Bulmahn et al. synthesized colloidal stable and pure Bi-NPs via femtosecond laser ablation of a metallic bismuth target. The results showed that metallic particles were only obtained in organic solvents like acetone, leading to spherical, primary Bi-NPs with narrow size distribution (28 ± 4 nm). In contrast, synthesis in water provides rather large nanosheets (455 ± 50 nm), consisting of bismuth subcarbonate. Colloidal stable aqueous solutions of metallic bismuth particles can be obtained in the presence of Pluronic® F68 as stabilising agent. However, DLS experiments show a broad particle distribution in the range of 50 to 150 nm. The metallic Bi-NPs showed efficient production of heat after irradiation with 808 nm NIR light, demonstrating their potential as promising photothermal agents for biomedical applications. Overall, laser ablation is a promising method for producing metallic bismuth particles, with the optimisation of various parameters such as laser energy, choice of solvent and stabilising compounds, allowing a wide range of particle size adjustment.⁷⁸

The thermal decomposition of suitable bismuth compounds is another preparation route to produce metallic Bi-NPs. It should be noted that reduction processes also occur simultaneously with the solvents used. The decomposition methods at high temperatures have several benefits, including precise control over NP size and shape by suitable temperature regimes, which allows for the adjustment of a well-defined crystal structure and fabrication of particles with narrow size distribution.⁷⁹ Thus, mono-dispersed spherical Bi-NPs (size ∼ 4 nm) were obtained with high yield in a one-pot synthesis approach by using bismuth neodecanoate as precursor in octadecene/oleic acid solution at 260 °C.⁷⁹ These Bi-NPs can be stabilised with PEG-NH₂ in phosphate buffered solution, giving narrow-sized particles smaller than 10 nm. The Bi@PEG-NPs were biocompatible as demonstrated by their haemolytic tolerance and cell-viability assessment on L929 cells. Furthermore, these metallic particles show promising contrast (CT) and fluorescence properties that predestine them for in vivo imaging studies. Biodistribution experiments using ICP-MS analysis in BALB/c mice revealed accumulation in large intestine, liver, spleen and lungs accompanied by a rather slow clearance over 7 days. This behaviour is rather atypical for such ultrasmall particles and indicates a lack of stability in vivo.⁸⁰

The table below summarises the composition of metallic bismuth nanoparticles, the corresponding manufacturing methods and conditions, as well as sizes, and provides an overview of possible applications in the imaging and therapy of cancers (Table 1).

Table 1 Summary of metallic Bi-NPs with varying surface functionalization, their synthesis methods and corresponding characterisation and applications

NPs composition	Synthesis method	System	Size [nm]	Application	Ref.
BSA, bovine serum albumin; CT, computed tomography imaging; DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; DOX, doxorubicin; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; HSA, human serum albumin; IRT, infrared thermography; PA, photoacoustic imaging; PEG, polyethylene glycol; Pluronic®F68, polyoxyethylene/polyoxypropylene block copolymer; PTT, photothermal therapy; RBC, red blood cell; TGA, thioglycolic acid; UV, ultraviolet.
Bi@gelatin	Reduction	Bi nitrate	15.6 ± 3.1	CT/IRT imaging	55
Bi@BSA Bi@HSA		NaBH4	17.4 ± 3.3	Chemo/PTT (tumour-bearing nude mice)
		Doxorubicin	18.9 ± 3.8 (TEM)
Bi@nanocellulose	Reduction	Bi nitrate	2–10 (TEM)	RT (4T1 tumour-bearing mice)	56
		NaBH4
		Nanocellulose-COOH
Bi@cellulose	Reduction	Bi/K citrate	25–55 (TEM/DLS)	CT/PA imaging	57
		UV irrediation		Chemo/PTT (4T1 tumour-bearing mice)
		Carboxymethyl-cellulose
		Doxorubicin
Bi@delftia	Reduction	Bi nitrate	40–120 (TEM)	In vitro cytotoxicity (HT29 cell line)	58
		Delftia bacteria
Bi@Au	Reduction	BiCl3	>100 (DLS)	Chemo/PTT (A549 tumour-bearing mice)	61
		Ethylene glycol
		Doxorubicin
Bi@PPy-PEG	Reduction	Bi nitrate	100–200 (TEM)	CT/PA imaging	64
		Morpholine borane	140–160 (DLS)	PTT (tumour-bearing BALB/c mice)
		Polypyrrole (PPy)/PEG-dopamine coating
Bi@SiO2	Reduction	Bi nitrate	65 (TEM)	CT imaging	65
		Dodecanethiol	160–250 (DLS)	Targeted chemo/PTT therapy (MCF-7 tumour-bearing mice)
		Silica coating
		Doxorubicin
		AR peptide
Bi@SiO2	Reduction	Bi dodecanethiolate	∼25 (TEM)	IRT imaging	67
		Tri-octylphosphine	45 (DLS)	PTT/RT (4T1 tumour-bearing mice)
		Silica coating
		Redox-active prodrug
Bi@Ag-PEG	Reduction	Bi nitrate	>100 (TEM)	IRT imaging	71
		Ag@PVP		Chemo/PTT (A549 tumour-bearing mice)
		HS-PEG-folate
		Doxorubicine
Bi@DSPE-PEG	Reduction	Bi acetate	3.6 (TEM)	CT/PA imaging	72
		Oleic acid	12 (DLS)	Targeted PTT/RT (4T1 tumour-bearing mice)
		Oleyl amine
		DSPE-PEG coating
		LyP-1 peptide
Bi@DSPE-PEG	Reduction	Bi nitrate	40 (TEM)	Targeted RT (4T1 tumour-bearing mice)	73
		Morpholine borane	56 (DLS)
		PEG2000-DSPE-folate coating
Bi@DSPE-PEG	Reduction	Bi dodecanethiolate	42 ± 2 (TEM)	Targeted PTT (CT26 tumour-bearing mice)	74
		Oleyl amine
		Octadecene
		CT26 cancer cell membrane-coating
Bi@PVP	Reduction	Bi nitrate	2.7 ± 1.1 (TEM)	CT/IRT imaging	75
		NaBH4	10.8 (DLS)	PTT (U14 tumour-bearing mice)
		PVP
Bi@DLPC	Reduction	Bi acetate	47 ± 3 (TEM)	CT/PA imaging	81
		Dodedanethiol	162 (DLS)	PTT (MDA-MB 231 tumour-bearing mice)
		Oleic acid
		Trioctylphospine
		DLPC coating
Bi@RBC	Reduction	Bi nitrate	196 ± 4 (DLS)	RT/PTT (LLC tumour bearing mice)	82
		HNO3
		PVP
		NaBH4
		RBC membrane vesicles
		DSPE-PEG2000
Bi@Pluronic®F68	Laser ablation	Bi target in acetone	28 ± 4 (TEM)	Photothermal properties	77
			50–150 (DLS)
Bi@PEG-NH2	Thermal decomposition	Bi-neodecanate	4 (TEM)	CT/fluorescence imaging	79
		Octadecene	<10 (DLS)	Biodistribution in BALB/c mice
		Oleic acid

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2.2. Bismuth chalcogenides

Bismuth chalcogenides (Bi₂X₃) constitute unique layered structures composed of bismuth (Bi) with chalcogen elements (X) such as sulfur (S), oxygen (O), selenium (Se), or tellurium (Te).⁸³ It represents a class of materials, which are generally non-toxic in nature, inexpensive and highly stable.⁸⁴ Due to their diverse features such as unique optical, thermal and electronic properties,^85,86 bismuth chalcogenides have been exploited for a variety of applications like thermo-responsive electronics, photovoltaics, (bio)catalysis, sensing as well as cancer imaging and therapy.^83,87–89 With regard to the latter, bismuth chalcogenides combine their structural diversity with a high degree of biocompatibility, efficient light-to-heat conversion in the NIR region and radio-enhancement abilities, leading to exceptional image-guided dual theranostic modalities.^34,90,91 Moreover, suitable surface functionalisation and the introduction of biological vector molecules can lead to improved biocompatibility and increased tumour targeting, making them interesting for a wide range of biomedical applications.^91,92

Hydrothermal methods are the most frequently used processes for producing stable Bi₂X₃ nanomaterials. In this process, bismuth precursors are reacted with chalcogen-containing compounds in an aqueous medium at elevated temperatures and suitable pressure. Depending on the conditions used, different sizes and shapes are obtained. Bi₂S₃-NPs are the most frequently pre-clinical studied bismuth chalcogenides in the imaging and therapy of cancers. A few selected examples are discussed below. The reaction of Bi₂O₃ precursors with thioacetamide (TAA) under pressure at elevated temperature and subsequent PEGylation leads to rather large, colloidal stable particles (>200 nm), which can be loaded with 3-bromopyruvate for enhanced radiosensitising. The resulting particles were shown to serve as efficient bimodal contrast agents for CT/PA imaging and demonstrate excellent X-ray sensitisation and NIR optical absorption properties, leading to high therapeutic efficacy against 4T1 breast tumours.⁹³ An interesting approach is described by Poudel et al., which results in larger particles (173.5 ± 2.8 nm). Starting from Bi₂O₃ nanoshells, a hydrothermal synthesis under pressure in the presence of TAA is used to convert them into Bi₂S₃-based ‘nano-urchins’, which are loaded with indocyanine green (ICG). PEGylation is used for colloidal stabilisation and to introduce the anticancer drug methotrexate and folate units for targeting. In this way redox and photo-responsive targeted Bi₂S₃-NPs can be produced for simultaneous use in chemotherapy, PDT and PTT.⁹⁴ Bi₂S₃ nanorods (60 × 130 nm) were produced by hydrothermal synthesis and stabilised in an aqueous environment using PVP. These particles were then coated with mesoporous silica, loaded with doxorubicin and equipped with the antibody trastuzumab for targeting. The nanorods have excellent contrast and photothermal properties and enable the application of targeted, effective cancer therapy based on chemo- and PTT.⁹⁵ Overall, colloidally stabilised Bi₂S₃-NPs of this size are subject to the problem of a long residence time in the body.

Bi₂O₃, Bi₂Se₃ and Bi₂Te₃-NPs are also accessible via hydrothermal routes and exhibit interesting photothermal and radiosensitising properties.^96,97 80 nm-sized Bi₂O₃-NPs are obtained by hydrothermal synthesis in the presence of Triton X and chitosan as a coating agent. These particles are loaded with the photosensitiser 5-aminolevulinic acid (5-ALA) and the anticancer agent curcumin. These particles can be used for CT imaging. Initial investigations into the therapeutic efficacy of chemotherapy and radiotherapy show effects, but these still need to be optimised.⁹¹ Very small 6 nm-sized bismuth ferrite NPs were synthesised via a facile hydrothermal method. After coating with poly(acrylic acid), these particles can be applied for PTT/PDT combination therapy.⁹⁸ An elegant method that, in the presence of selenocysteine, leads to well-defined, 15 nm-sized Bi₂Se₃-NPs, is described by Du et al.⁹⁹ These selenocysteine-modified NPs coated with PVP are highly biocompatible and show high therapeutic efficacy in vivo when radiotherapy and PTT are used simultaneously. It is worth mentioning that selenium can enter the bloodstream in parts and stimulate the immune system, as well as achieve a radioprotective effect throughout the body (Fig. 3).

Fig. 3 Schematic illustration of the therapeutic principle based on Bi₂Se₃@PVP nanoparticles, containing selenocystein (Sec) for simultaneously synergistic photothermal/radiotherapy of tumour and enhancement of the immunity to reduce the side effects of radiation. (Reprinted with permission from ref. 99, Copyright 2017, WILEY-VCH Verlag GmbH & Co.)

The production of bismuth chalcogenides in organic solvents (solvothermal synthesis) instead of water usually results in smaller particles. Various shapes are available in this way. Bi₂S₃ nanorods,^90,100–104 Bi₂S₃ nanosheets,¹⁰⁵ Bi₂O₃ spherical particles,^106,107 Bi₂Te₃ nanosheets¹⁰⁸ and Bi₂Te₃ spherical particles¹⁰⁹ have been generated using the solvothermal technique. The so-called polyol process yields relatively small homogeneous particles, for example Bi₂O₃ (40–50 nm), which can be stabilised with hyaluronic acid.¹¹⁰ These particles exhibit excellent contrast and radiosensitising properties and are being discussed for use in CT imaging-guided radiotherapy. Small Bi₂O₃-NPs (19.2 ± 6.5 nm) were produced in propylene glycol and then coated with a silica shell, which has terminal NH₂ groups for conjugation of folic acid and the photosensitiser 5-ALA. These particles show high in vitro cell toxicity after UV irradiation and are being discussed for use in PDT.¹⁰⁶ Silica-coated 100 nm large Bi₂S₃-NPs were produced via a surfactant-assisted solvothermal synthesis.¹¹¹ The NPs were loaded with doxorubicin and RGD peptides were incorporated as biological vector molecules using a PEG linker. These particles can be used for dual imaging (CT/IRT) and show a high therapeutic efficacy based on combined chemotherapy and PTT. Bi₂S₃ nanorods were prepared in organic solution, then coated with a thermally retracted poly(acrylamide) based polymer, where the photosensitiser zinc protoporphyrin X has been linked. This system shows promising contrast (CT), photodynamic and photothermal properties, enabling the combination of PDT and PTT.¹⁰⁴ Oleyl amine-capped 36 nm-sized Bi₂Te₃-NPs have been fabricated, stabilised in biological environment by phospholipid-PEG coating and then loaded with a Pt(IV) prodrug. These particles show a very high photoconversion efficiency (η = 48.7%), are suitable for dual imaging (PA/IRI) and enable effective combination therapy based on chemotherapy and PTT.¹⁰⁹ Chen et al. report on an immunotherapeutic approach using sulphur-doped Bi₂O₃-NPs, which were produced in ethylene glycol and then stabilised with PVP.¹¹² These NPs are loaded with the drug Ivermectin and induce a highly effective immunogenic cell death of tumours by combining PTT and a sonocatalytic therapy. Bi₂Se₃-NPs are also obtained by solvothermal synthesis and stabilised using PVP.¹¹³ These particles are then coated with polydopamine, loaded with doxorubicin and stabilised with HSA. These particles can be used for dual imaging (CT/IRT) and show a synergistic therapeutic effect when PTT and chemotherapy are applied in a tumour mouse model.

Over the last 10 years, biomineralisation methods have been used to produce bismuth chalcogenides. This method has been reported to generate Bi₂S₃ spherical particles with sizes generally below 50 nm, possessing good stability in complex biological environment and high biocompatibility.^{114–122,123} The treatment of bismuth precursors in the presence of BSA leads to defined Bi₂S₃-NPs and was first described by Wang et al. in 2016.¹²⁰ The primary particles are smaller than 10 nm (TEM). However, DLS measurements show a size of around 40 nm. Accordingly, the accumulation of Bi₂S₃-NPs occurs not only in the tumour but also in the liver, spleen, kidneys and lungs, as shown by biodistribution studies with ⁹⁹Tc-labelled particles. These particles are suitable for imaging based on CT and IRT. In vivo studies in a tumour mouse model show that a combination of radiotherapy and PTT can achieve complete tumour remission. In terms of a therapeutic application, BSA-stabilised Bi₂S₃-NPs are mainly used in radio-, chemo- and immunotherapy, or a combination of these.^{114,122,124,125} Curcumin, specific peptides such as triptorelin, folic acid and the immunoactive ganoderma lucidum polysaccharide (GLP) are used to increase the effectiveness of the therapy and for targeting. While the primary particles Bi₂S₃@BSA are rather small, with sizes around 10 nm, larger associates of around 100 nm are often found in the biological environment. An interesting approach for in vivo applications is the use of 3D-printed alginate scaffolds with Bi₂S₃@BSA-NPs.¹²³ These implanted scaffolds show a high therapeutic efficacy in a tumour mouse model after X-ray irradiation and are discussed for post-surgical use to remove residual tumour cells in breast cancer patients. Ultrasmall Bi₂Se₃-NPs (3 nm) were obtained by biomineralisation and encapsulated in tumour cell-derived microparticles together with doxorubicin.¹²⁶ These particles are ideal for image-guided tumour targeting with subsequent combination of chemotherapy and PTT.

The table below summarises the composition of bismuth chalcogenide-based nanoparticles, the corresponding manufacturing methods and conditions, as well as sizes, and provides an overview of possible applications in the imaging and therapy of cancers (Table 2).

Table 2 Summary of Bi₂X₃-NPs with varying surface functionalization, their synthesis methods and corresponding characterisation and applications

NPs composition	Synthesis method	System	Size [nm]	Application	Ref.
5-ALA, 5-aminolevulinic acid; APTES, 3-aminopropyltriethoxysilane; BFO, bismuth ferrite; BSA, bovine serum albumin; CT, computed tomography imaging; CTAB, cetyltrimethylammonium bromide; Cur, curcumin; DOX, doxorubicin; FA, folic acid; GLP, ganoderma lucidum polysaccharide; HSA, human serum albumin; ICG, indocyanine green; IRT, infrared thermography; mPS, mesoporous silica; MTX, methotrexate; PA, photoacoustic imaging; PDA, polydopamine; PDT, photodynamic therapy; PEG, polyethylene glycol; PM, platelet membrane; Pt, platinum; PTT, photothermal therapy; TEOS, tetraethyl orthosilicate; US, ultrasound.
Bi2O3@Chitosan	Hydrothermal	Bi nitrate	80 (TEM)	CT imaging Chemo/RT (4T1 tumour bearing mice)	91
		Triton-X	204 (DLS)
		Chitosan
		5-ALA and curcumin loading
Bi2S3@PEG-SH	Hydrothermal	Bi2O3	260 (DLS)	CT/PA imaging	93
		Thioacetamide		RT (4T1 tumour-bearing mice)
		PEG-SH coating
		3-Bromopyruvate loading
Bi2S3@(ICG)-PEG-S-S-MTX	Hydrothermal	Bi2O3 nanoshells	173.5 ± 2.8 (DLS)	Targeted Chemo/PTT/PDT (HCT116 tumour bearing mice)	94
		Thioacetamide
		ICG loading
		SH-PEG-S-S-MTX
Bi2S3@PVP(SiO2)	Hydrothermal	Bi nitrate	Diameter 26	CT imaging	95
		Ethylene glycol	Length 100 (TEM)	Targeted Chemo/PTT (SKBR-3 tumour bearing mice)
		Sodium sulfide
		PVP (SiO2)
		Trastuzumab
		DOX loading
Bi2O3@Fe-PAA	Hydrothermal	Bi nitrate	6 (TEM)	PTT/PDT (HepG2 tumour-bearing mice)	98
		Fe nitrate	69.6 ± 1.6 (DLS)
		Poly(acrylic acid)
Bi2Se3@PVP	Hydrothermal	Bi nitrate	15 ± 3 (TEM)	PTT	99
		PVP	29.2 (DLS)	RT (Bel-7402 tumour-bearing mice)
		L-Selenocystine
Bi2S3@PAA	Solvothermal	Bi neodecanoate	∅ 13 ± 2	CT/IRT imaging	104
		Thioacetamide	l = 40 ± 5 (TEM)	PTT/PDT therapy (4T1 tumour bearing mice)
		Oleic acid
		Oleyl amine
		PAA coating
		Protoporphyrin IX
Bi2O3@SiO2-NH2	Solvothermal	Bi nitrate	19.2 ± 6.5 (TEM)	PDT (KB cell line)	106
		Propylene glycol
		APTMS functionalization
		5-ALA and folic acid conjugation
Bi2Te3@DSPE-PEG	Solvothermal	Bi neodecanoate	36 (TEM)	PA/IRT imaging	109
		TrioctylP-Te		Chemo/PTT (4T1 tumour bearing mice)
		DSPE-PEG-CH3 coating
		Pt(IV) prodrug loading
Bi2O3@HA	Solvothermal	Bi chloride	45 ± 0.6 (TEM)	CT imaging	110
		NaOH	47.4 (DLS)	RT (tumour bearing mice)
		Diethylene glycol
		Hyaluronic acid
Bi2S3@SiO2-NH2	Solvothermal	Bi chloride	100 (TEM)	CT/IRT imaging	111
		Na2S	120 (DLS)	Targeted Chemo/PTT (UMR-106 tumour bearing mice)
		Ethylene glycol
		CTAC
		APTES
		DOX loading
		RGD peptide
Bi2O3−xSx@PVP	Solvothermal	Bi nitrate	30 (TEM)	PTT	112
		Ethylene glycol	130 (DLS)	Sonocatalytic therapy (4T1 Balb/c female mice)
		NH4F, Na2S
		PVP
		Ivermectin
Bi2Se3@PDA	Solvothermal	Bi nitrate	104 (TEM) > 400 (DLS)	CT/IRT imaging	113
		Na2SeO3		Chemo/PTT (HeLa tumour bearing mice)
		Ethylene glycol
		PVP, PDA
		DOX loading HSA
Bi2S3@BSA	Biomineralisation	Bi nitrate	<10 (TEM)	CT imaging	114
		BSA	170 (DLS)	RT (4T1 tumour bearing mice)
		FA coupling
		Curcumin loading
		FITC labeling
Bi2S3@BSA	Biomineralisation	Bi nitrate	6.1 ± 0.9 (TEM)	CT/IRT imaging	120
		BSA	40 (DLS)	PTT/RT (4T1 tumour bearing mice)
		HNO3
		NaOH
Bi2S3@BSA	Biomineralisation	Bi nitrate	15 ± 4 (TEM)	In vitro Chemo/RT (HT-29 cells)	122
		HNO3	100 (DLS)
		NaOH
		BSA
		Curcumin
Bi2S3@BSA	Biomineralisation	Bi nitrate	8.5 ± 1.5 (TEM)	In vitro RT (MCF-7 cells)	124
		BSA	17 ± 2 (DLS)
		Triptorelin
Bi2S3@BSA	Biomineralisation	Bi nitrate	10 ± 3 (TEM)	RT/immuotherapy (4T1 tumour-bearing BALB/c mice)	125
		HNO3	>130 (DLS)
		NaOH
		BSA
		GLP-SH
Bi2S3@BSA	Biomineralisation	Bi nitrate	21.6 ± 5.7 (TEM)	RT (4T1 tumour bearing mice)	123
		BSA	55 (DLS)
		Alginate
Bi2Se3@BSA	Biomineralisation	Bismuth chloride	3.0 ± 0.4 (TEM)	CT/PA imaging	126
		Selenium powder	13.5 ± 1.5 (DLS)	Targeted Chemo/PTT (H22-tumour-bearing mice)
		Ethylenediamine
		Mercaptoethanol
		BSA
		Doxorubicin
		H22 tumour cell-derived microparticles

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2.3. Bismuth hybrid materials and nanocomposites

Bismuth-based hybrid materials and nanocomposites that consist of other inorganic or organic compounds and are equipped with target-seeking units are becoming increasingly important. Bismuth and bismuth compounds, when combined with other metals and metal compounds, organic materials such as stimuli-response polymers or carbon-based structures, provide novel functionalities and improved performances due to the synergistic interplay of the components, rendering nanohybrids/nanocomposites novel versatile functionalities.^127–129 In the field of cancer research, on the one hand improved contrast properties can be achieved in imaging, while on the other, the range of treatment options for cancer can be expanded. Concerning the latter, emerging technologies for optimisation of targeted PTT, PDT, RT, hypoxia modulation, and combinatorial therapeutic regimes are in a state of rapid development.^130–132

In the following, there are a few examples that show the diversity in this field and provide an insight into new fields of application. The combination of bismuth in its metallic form or various bismuth chalcogenides with metals such as Ag, Au, Pt and Pd leads in particular to improved contrast and radiosensitising properties, harnessing narrow band gaps and enables the use of highly effective sonodynamic therapy.^133–137 By mixing bismuth chalcogenides with metallic Bi-NPs, the bismuth content can be increased compared to the chalcogenides alone, thus significantly boosting both the contrast properties and the effectiveness of PTT.^138–141 Bismuth molybdate- and tungstate-based nanomaterials are gaining importance as sonosensitisers.^142,143 Polyoxometalates (POMs) are an extremely interesting class of substances for cancer therapy.^144,145 POMs have the great advantage that they can be produced in a very defined manner over a wide size range. However, the surface functionalisation is not trivial. Due to their radiosensitising properties, bismuth-containing POMs are of interest for improving the effectiveness of RT.¹⁴⁶ New drug delivery systems based on biocompatible bismuth-containing metal–organic frameworks (MOFs) should also be mentioned.¹⁴⁷ This field of research is only just beginning. However, it offers a wide range of biomedical applications. The blending of bismuth-containing materials with graphene oxide (GO) and reduced graphene oxide (rGO) is used to improve biocompatibility and for drug delivery. In addition, GO/rGO can be used to adjust the electrical and thermal conductivity of hybrid materials very precisely, thus enhancing imaging and radiosensitisation properties.^{127,148–151} In recent years, considerable work has been done on the development of hybrid materials with the addition of manganese oxide, magnetite and gadolinium oxide.^{116,152–158} This provides magnetic resonance tomography (MRT) as an additional imaging technique that is particularly suitable for good soft tissue contrast. With respect to its use in cancer therapy, the efficacy of hyperthermia can be increased and a hypoxia-related radioresistance can be overcome with these materials. An interesting inorganic–organic hybrid material is described by Zhang et al. in which metallic Bi-NPs are coated with silica and then stabilised with an organic polymer to which porphyrin loaded with iron(II) is covalently bound.¹⁵⁹ This material can be used for combination therapy consisting of chemotherapy, PTT and PDT. Core–shell nanocomposites, consisting of ultra-small Bi₂S₃ nanoparticles, cerium oxide and the photosensitiser chlorin e6 have been developed for NIR-triggered PDT.¹⁶⁰ Liu et al. reported a biodegradable and biocompatible core–shell bismuth-based hybrid material coated with a MnO₂ layer and loaded with the anticancer drug docetaxel (DTX) to treat radio-resistant hypoxic tumours. The bismuth/manganese-based radiosensitising material has been stabilised with amphiphilic PEG equipped with folic acid as target-seeking groups (Fig. 4). For this targeted nanocomposite, an efficient CT/MR image-guided combination therapy based on chemotherapy, chemodynamic therapy and radiotherapy is described for a tumour mouse model.¹⁵⁵

Fig. 4 Schematic diagram of the synthesis process of Bi@Mn-DTX-PFA and its synergistic therapy mechanism for hypoxic tumours. (Reprinted with permission from ref. 155, Copyright 2021 John Wiley and Sons.)

3. Radiolabelled bismuth compounds

Radiolabelled compounds enable both imaging and the treatment of cancer. Bismuth offers two isotopes of interest for radiopharmaceutical applications. Both of them partially emit α-particles and decay via short-living daughter nuclides, which is expected to limit off-target accumulation.¹⁶¹ One promising candidate for targeted alpha therapy (TAT) is ²¹²Bi, which decays via a branched pathway to ²⁰⁸Tl (t_1/2 = 3.1 min, 36% α) and ²¹²Po (t_1/2 = 0.3 μs, 64% β⁻) and finally to stable ²⁰⁸Pb. It can be eluted from a ²²⁴Ra/²¹²Pb/²¹²Bi generator system as [²¹²Bi][BiI₅]²⁻/[²¹²Bi][BiI₄]⁻, either selectively or co-eluted with ²¹²Pb. Due to the short half-life of ²¹²Bi (t_1/2 = 60.55 min), co-elution and subsequent use as a ²¹²Pb/²¹²Bi in vivo generator became of interest over the last few years.^162,163 However, one major issue is the high energetic γ-line emitted by ²⁰⁸Tl (E_γ = 2.6 MeV), which can be problematic regarding dosimetry.¹⁶⁴

The second isotope of interest is ²¹³Bi (t_1/2 = 45.6 min), one longer living daughter nuclide of ²²⁵Ac. It decays via²⁰⁹Tl (t_1/2 = 2.2 min, 2% alpha) and ²¹³Po (t_1/2 = 3.7 μs, 98% beta) to ²⁰⁹Pb (t_1/2 = 3.2 h) and ultimately to ²⁰⁹Bi (stable). It can be eluted from the respective ²²⁵Ac/²¹³Bi generator as [²¹³Bi][BiI₅]²⁻/[²¹³Bi][BiI₄]⁻, but the separation from the other daughter nuclides ²²¹Fr (t_1/2 = 4.9 min) and ²¹⁷At (t_1/2 = 32.3 ms) can be challenging. A mixture of 0.1 M HCl/0.1 M HI was found to enable selective elution of ²¹³Bi.¹⁶⁴

The relatively short half-life of ^212/213Bi can be advantageous for radiopharmaceutical applications, but some studies (e.g. biodistribution studies) can require measurements over a longer time. For that purpose, the β⁺-emitter ^205/206Bi (t_1/2 = 14.9 d and 6.2 d) can be used instead. While ²⁰⁶Bi decays directly to stable ²⁰⁶Pb, ²⁰⁵Bi decays via the long living daughter ²⁰⁵Pb (t_1/2 = 1.7 × 10⁷ years) to stable ²⁰⁵Tl.^{165–167205/206}Bi can be obtained by irradiation of ^natPb with a proton beam for up to 7 h and selectively eluted from a Pb-SpecTM resin (18-crown-6-based resin) using HNO₃.¹⁶⁸

To ensure safe delivery of bismuth radioisotopes to the targeted tissue, a wide range of chelators (Scheme 1) have been developed with some already tested in vivo. One class of interest is acyclic chelators, which typically enable fast labelling kinetics under mild conditions. Prominent examples are DTPA (diethylenetriaminepentaacetic acid) and its derivatives, which were conjugated to a variety of proteins.^40,169,170 However, some of the derivatives were found to be unstable, which led to the development of CHX-A′′-DTPA, in which a cyclohexyl moiety was installed in the backbone to provide rigidity and improve the stability.¹⁷⁰ Other promising acyclic chelators such as H₄Neunpa and H₂ampa showed favourable labelling kinetics, high molar activities and provided high stabilities (pM = 26 for [Bi(ampa)]⁺).^161,171

Scheme 1 Chemical structures of acyclic and macrocyclic chelators for bismuth(III) discussed in this article.

Cyclic chelators generally form highly stable complexes, but the formation kinetics of the respective complex can be slow. The gold standard for a broad range of metals is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), a cyclen-based chelator that undergoes continuous optimization to match the chemical requirements of different metals even better. The introduction of “soft” electron donors such as phosphonic acids was found to increase the thermodynamic stability of the complex and lead to higher radiolabelling efficiencies compared to the carboxylic acid bearing DOTA.¹⁷²

Another subclass is based on the crown ether macropa (N,N′-bis((6-carboxy-2-pyridil)methyl)-4,13-diaza-18-crown-6), which is also known to form stable complexes and, in contrast to DOTA, forms complexes at milder reaction conditions. Especially py-macrodipa, a derivative with one oxygen donor replaced by a nitrogen and a pyridine ring attached to the backbone, showed efficient radiolabelling with ²¹³Bi after 5 min at room temperature.¹⁷³ Recently, it was shown that the introduction of sulphur donors into the macropa system reduces both the thermodynamic stability and the kinetic inertness of (radio)bismuth complexes.¹⁷⁴

Characteristics of both chelator classes are combined in bispidines (3,7-diazabicylco[3.3.1]nonanes), which have a rigid backbone but flexible side arms to form stable complexes under mild conditions. Some octa- and nonadentate bispidines (L¹–L⁵) were investigated and especially the nonadentate ones were found to be promising candidates for TAT.^175,176

3.1 Radiolabelled small molecules, peptides and proteins

Targeted radiobismuth therapies have been applied with small molecules, peptides, antibody fragments, monoclonal antibodies, or any other kind of immunoproteins. They have shown promising results in pre-clinical and clinical studies in various tumour entities especially in small-volume or micro-metastasis such as breast cancer,^177–179 bladder cancer,¹⁸⁰ colon cancer,^181,182 glioma,^183,184 melanoma,^{165,171,185–188} multiple myeloma,^189–191 non-Hodgkin lymphoma,^192,193 ovarian cancer,^194–196 pancreatic cancer and neuroendocrine tumours^197–202 and prostate cancer.²⁰³ Of note, the first radiobismuth immunoconjugate (²¹²Bi-CHIP) was studied in human pancreatic carcinoma cells (SHAW) in 1988.²⁰⁰ To this end, the results are summarized in other reviews,^40,169,170 in which TAT showed success with locoregional and systemic administration. Herein, we only focus on recently published pre-clinical studies and highlight the clinical translations of radiolabelled bismuth conjugates.

3.1.1 Bladder cancer. In a recent pilot bacillus Calmette Guèrin (BCG) therapy study, 12 patients with carcinoma in situ (CIS) were treated with [²¹³Bi]Bi-CHX-A′′-DTPA-anti-EGFR-mAb by injecting 366–821 MBq via transurethral catheter. The pilot study proved that no severe side effects were observed in all patients. However, the authors addressed several strategies to improve therapeutic efficacy (3 out of 12 patients showed complete eradication after treatment).¹⁶⁹

3.1.2 Glioma. In an initial study, five patients with critically located gliomas were treated with the neurokinin type-1-receptor (NK-1) targeting conjugate [²¹³Bi]Bi-DOTA-SP (substance P). Substance P, a small peptide with a weight of 1.8 kDa, showed high NK-1 affinity in the nanomolar range, which in turn was found to be selectively overexpressed in all malignant glioblastomas. Biodistribution studies were performed by co-injection of the ⁶⁸Ga-labelled conjugate via an intratumoural catheter. One patient received 7.36 GBq of the conjugate over 4 therapeutic cycles, the rest was treated in one cycle with 1.07–2.00 GBq of [²¹³Bi]Bi-DOTA-SP. All patients showed radionecrotic effects on the tumour, however the patient with the lowest dose administered showed only 50% reduction in tumour volume. No severe local or systemic toxicity was observed in any of the patients.²⁰⁴

A subsequent study with 50 patients was performed with [²¹³Bi]Bi-DOTA-SP, where nine patients were diagnosed with secondary glioblastomas and the rest with different types of malignant gliomas. The median total dose administered over 1–6 cycles was 5.8 GBq and ranged between 1.4–9.7 GBq. The overall survival of patients who received [²¹³Bi]Bi-DOTA-SP was extended to ten months in comparison to patients who underwent surgical resection only (overall survival of two months).²⁰⁵

In the latest glioma study, 20 patients with recurrent glioblastoma were treated with [²¹³Bi]Bi-DOTA-SP (substance P) with a median injected activity of 3.3 GBq in total. The median overall survival of treated patients increased to 10.9 months after diagnosis of recurrence.¹⁸³

3.1.3 Melanoma. For metastatic uveal melanoma, [²¹³Bi](BiNeunpa-Ph-Pip-Nle-CycMSH_hex) targeting melanocortin 1 receptor (MC1R) was studied in vivo. Labelling of the conjugate was performed at room temperature at pH 5, achieving molar activities up to 21 MBq nmol⁻¹ after 5 min of incubation. The novel bismuth complex showed high stability (pM = 27.0) as well as high chemical inertness in human serum over 2 h. The conjugate showed high tumour uptake in B16-F10 tumour xenografted mice (5.91 %ID g⁻¹ 1 h p.i.).¹⁷¹

The biodistribution of [^205/206Bi]Bi-DOTA-IPB-NAPamide in B16-F10 tumour bearing mice was investigated with and without the albumin binder 4-(p-iodo-phenyl)butyryl (IPB) to prolong the circulation time and therefore increase the tumour uptake. Higher tumour uptake was observed for the IPB containing conjugate (4.50 %ID g⁻¹vs. 3.14 %ID g⁻¹), but high off-target accumulation in kidneys (8.78 %ID g⁻¹) as well. Therefore, application of albumin binders for short-living radionuclides was found to be disadvantageous.¹⁶⁵

In a first initial patient trial the efficacy of a radiobismuth labelled monoclonal antibody targeting melanoma receptors (melanoma-associated chondroitin sulfate proteoglycan) was conducted in 2005.¹⁸⁶ Since the [²¹³Bi]Bi-cDTPA-9.2.27 was injected intralesional, high doses of up to 49.95 MBq (1350 μCi) were tolerable with no accumulation found in the kidneys. The author highlighted that intralesional TAT can be used for inoperable secondary melanoma or primary ocular melanoma.

In 2007, 22 patients with stage IV melanoma/in-transit metastasis were treated intravenously with the same radiolabelled bismuth immunoconjugate (55–947 MBq) to establish an effective dose.¹⁸⁷ A tolerable dose of 592 MBq with no renal damage was found. 20% of the patients showed partial or complete response (6% complete and 14% partially).

Building on the results gained from the phase I trial, another phase I study with 38 patients with end-stage metastatic melanoma was conducted by the same group in 2011.¹⁸⁸ The study extended the factors which may influence overall survival by investigating the melanoma-inhibitory activity protein, age, gender, disease stage and lactate dehydrogenase. The maximum injected does of 925 MBq did not cause any severe effects and thus, the maximum tolerance could not be determined. The phase I trial showed an overall 50% response rate and has the potential to be used as a treatment option for end-stage metastatic melanoma cancer.

The group of E. Dadachova injected intraperitoneally [²¹³Bi]Bi-CHX-A′′-DTPA labelled (3.7 MBq μL⁻¹) mAb (8C3) targeting melanin alone and in combination with a standard drug for melanoma treatment (anti-CTLA4 mAb 9D9; 100 μg μL⁻¹), into tumour-bearing melanoma C57BL6 mice.¹⁸⁵ As an outcome, there was no synergistic effect observed when RIT was used in combination with immunotherapy. The authors justified it by the lack of anti-CTLA4 mAb efficacy in the murine model.

3.1.4 Myeloid leukemia. A systemic α immunotherapy study with 18 patients suffering from myeloid leukemia were treated with 10.36–37.0 MBq kg⁻¹ [²¹³Bi]Bi-CHX-A′′-DTPA-lintuzumab (anti-CD33).²⁰⁶ Although an anti-leukemic effect could be measured in most of the patients, complete remission was not observed.

Eight years later the same group performed a phase I/II trial in 31 patients in combination with cytoreductive chemotherapy to test the maximum tolerated dose and antileukemia effect of [²¹³Bi]Bi-CHX-A′′-DTPA-anti-CD33 (18.5–46.25 MBq kg⁻¹).²⁰⁷ The authors claimed in both trials to administer alpha emitters with longer half-life to significantly enhance the therapeutic outcome.

3.1.5 Ovarian cancer. For the treatment of metastatic ovarian cancer, a radiolabelled HER2-targeting single domain antibody fragment (a2Rs15d) was investigated. The overall survival rate was studied in SKOV-3 xenografted mice by intravenously injection of fractionated administration of [²¹³Bi]Bi-DTPA-a2Rs15d (0.5–2 MBq) alone or in combination with trastuzumab.²⁰⁸ Synergistic effects led to an increased median survival by applying three times 0.5 MBq in combination with trastuzumab compared to the control group. Biodistribution data was gained from Cherenkov radiation (from β decay and β decay of its daughters) and SPECT/CT imaging. High accumulation was not only found in the tumour (4.9 ± 0.05 %ID g⁻¹ after 15 min p.i.), but also in the kidneys (59.9 ± 5.1 %ID g⁻¹ after 60 min p.i.). By co-injection of gelofusine the uptake was reduced by 50%. However, the severe kidney uptake currently hinders translation into the clinic.

3.1.6 Neuroendocrine tumours (NETs) and pancreatic cancer. The conjugate [^205/206Bi][Bi(AAZTA-C4-TATE)]⁻ was obtained quantitatively with a chelator concentration of 10⁻⁵ M at pH 3 at room temperature after 5 min of incubation. The conjugate was found to be stable in PBS, 10 mM DTPA and human plasma for 21 h. In vivo experiments in AR42 tumour bearing mice showed higher tumour uptake compared to [²¹³Bi][Bi(DOTA-TATE)]⁻ (9.32 vs. 6.5 %ID g⁻¹) and reduced kidney uptake (10.1 vs. 17.4 %ID g⁻¹).¹⁹⁷ One initial clinical trial was performed using [²¹³Bi]Bi-DOTATOC in seven patients with NET pre-treated with ¹⁷⁷Lu and ⁹⁰Y and one with bone marrow carcinosis (Fig. 5). Successful treatment of all patients was achieved with even progression free survival up to 30 months after treatment.¹⁹⁹

Fig. 5 (a) [⁶⁸Ga]Ga-DOTATOC-PET image of a patient with a tumour in the liver and metastases in the bone marrow as shown in coronal and sagittal maximum intensity projections. (B) Reduced tumour burden in the liver after treatment with 10.5 GBq [²¹³Bi]Bi-DOTATOC, as well as reduction of the bone metastases 6 month after treatment. (Reprinted with permission from ref. 199 Copyright 2014 Springer.)

Novel proteins belonging to the family of Cancer/Testis Antigen (CTA) were developed to address the highly aggressive and to this stage incurable pancreatic ductal adenocarcinoma.²⁰¹ [²¹³Bi]Bi-CHX-A′′-DTPA-labelled antibodies targeting CETN1 (centrin1) showed a tumour-specific uptake and effective treatment of intraperitoneally injected [²¹³Bi]Bi-CHX-A′′-DTPA-labelled antibodies (single dose of 3.7 and 7.4 MBq) in MiaPaCa tumour-bearing mice. In comparison to the ¹⁷⁷Lu-labelled injected counterpart (same dose), only the alpha labelled mAb showed a reduced tumour growth rate pointing out that RIT is more effective than radionuclide therapy (RNT).

3.1.7 Prostate cancer. Initial dose calculations for [²¹³Bi]Bi-DOTA-PSMA617 were performed based on [⁶⁸Ga]Ga-DOTA-TATE PET images from three patients. The dosimetry was found to be reasonable, but off-target accumulation, especially in the kidneys, was found to be even higher than for the respective ²²⁵Ac or ¹⁷⁷Lu conjugates.²⁰⁹

However, a first patient with metastatic castration resistant prostate cancer (mCRPC) was treated with 592 MBq [²¹³Bi]Bi-DOTA-PSMA-617 given in two cycles. PSA levels were found to decrease from 237 μg L⁻¹ to 43 μg L⁻¹ eleven months post treatment (Table 3).²¹⁰

Table 3 Overview over preclinically and clinically investigated radiolabelled small molecules and antibodies

Preclinical
Substance	Dose [MBq]	Tumour uptake [%ID g^−1]	Cytotoxic effects [%ID g^−1]	Disease	Ref.
[^205/206Bi]Bi-DOTA-IPB-NAPamide with and without 4-(p-iodo-phenyl)butyryl	2.39	4.50 ± 0.98/3.14 ± 0.32	8.78% ± 3.61 in kidneys	Melanoma	165
[^213Bi](Bi-Neunpa-Ph-Pip-Nle-CycMSHhex)	0.1	5.91 ± 1.33	31.5 ± 4.99 in liver	Melanoma	171
[^213Bi]Bi-CHX-A′′-DTPA-8C3	3.7 + 7.4	N/A	N/A	Melanoma	185
[^205/206Bi][Bi(AAZTA-C4-TATE)]	1.18	9.32 ± 3.96	10.1 in kidneys	NET	197
[^213Bi][Bi(DOTA-TATE)]	16.8 + 33.1	6.5 ± 2.3	17.4 ± 2.2 in kidneys	NET	197
[^213Bi]Bi-CHX-A′′-DTPA-labelled antibodies targeting CETN1	3.7 + 7.4	N/A	N/A	NET	201
[^213Bi]Bi-DTPA-a2Rs15d	8.8 + 10.7	4.9 ± 0.05	59.9 ± 5.1 in kidneys	Ovarian cancer	208

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Clinical
Substance	Total administered dose [MBq]	Cytotoxic effects	Disease	Ref.
[^213Bi]Bi-CHX-A′′-DTPA-anti-EGFR-mAb	366–821	—	Bladder cancer	169
[^213Bi]Bi-DOTA-SP	1400–9700	—	Glioma	183, 204 and 205
[^213Bi]Bi-DOTATOC	3300–20 600	Moderate hematological effects	NET	195
[^213Bi]Bi-cDTPA-9.2.27	592–925	—	Melanoma	186–188
[^213Bi]Bi-CHX-A′′-DTPA-lintuzumab	1195–4755	10% treatment-related death	Myeloid leukemia	206 and 207
		16% liver abnormalities
[^213Bi]Bi-DOTA-PSMA617	592	N/A	Prostate cancer	209 and 210

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3.2 Radiolabelled nanostructures

There are only limited reports on radiolabelled bismuth nanomaterials. The short half-life of ^212/213Bi hinders the feasibility of implementation. An indirect strategy is the use of ²¹²Pb/²¹²Bi as an in vivo generator.¹⁶²

The first study was reported by Diener et al.²¹¹ They synthesised ²¹²Pb-labelled endohedral fullerenes by recoil synthesis following α decay and were further functionalized with malonic acid. The biodistribution behaviour was investigated in non-tumour bearing mice. A high uptake in the liver (53.7 ± 7.1 %ID g⁻¹) and spleen (27.2 ± 3.2 %ID g⁻¹) could be observed 8 h post injection and pointed to slow clearence from the body. The authors claimed that ²¹²Pb was encapsulated otherwise bone marrow uptake would have been observed.

Recently, ultrasmall silver telluride nanoparticles (∼2 nm) were coated with gluthathion and radiolablled with ²¹²Pb without any chelator.²¹² The [²¹²Pb]Pb-GSHAg₂Te showed high stability (∼96% monitored by radio-TLC) in the presence of 1 mM EDTA after 24 h. However, these particles have not been investigated further neither in vitro nor in vivo.

One example of a ²¹²Bi-labelled nanostructure is reported by Kauffman et al.²¹³ They sucessfully labelled FDA-approved macroaggregated albumin (MAA) and investigated the conjugate in vitro and in vivo in breast cancer models based on 4T1 and EO771 cells. Reduction of tumour cell growth could be observed in both cell lines in vitro using clonogenic and survival assays. Biodistribution studies showed that after intratumoural administration, about 90% of the injected dose remains in 4T1 or EO771 orthotopic breast tumours in mice, resulting in a significant reduction in tumour size over a 18 day monitoring period.

4. Novel targeting/therapeutic strategies

Approaches to widen the spectrum of therapeutic conjugates by implementing a variety of molecules and nanomaterials have been provided over the years and attempts to create synergistic effects in combination with other treatment options are still ongoing. A combination of radiation therapies with immunotherapeutic approaches, in particular, appears to be highly promising.²¹⁴ For example, beam irradiation or endogenous radiotherapies may result in the release of tumour associated antigens (TAA) from dying tumour cells.²¹⁵ Uptaken by dendritic cells, TAAs can be processed and presented by the immune system, thereby, leading to the activation of immune effector cells. This may explain the observed vaccination effects of radiotherapies against tumour cells, including in patients. For example, even non-irradiated tumour tissues become recognized by the immune system as a consequence of radiotherapy, also known as abscopal effect.²¹⁶ Besides vaccination, there is experimental evidence that irradiation of tumour tissues including by TAT²¹⁷ can enhance the entry of immune effector cells into tumour tissues, which can modulate the immunosuppressive tumour microenvironment (TME) to more inflammatory conditions, thereby facilitating the recognition of tumour cells by the immune system.^218–220 For these reasons it is expected that the combination of radiotherapy can improve immunotherapies including with bispecific antibodies (BsAb) or adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CAR).^189,221

Bearing in mind, (i) the above summarised potential applications of bismuth-based radiotheranostics on the one hand, and (ii) the promising data related to combined applications of radiotherapeutic strategies with immunotherapies on the other hand, novel bismuth-based immunotheranostics appear highly attractive and should therefore come into the focus of future pre-clinical and clinical evaluation. For these reasons, we include here an overview of such potential future combinatorial applications in the next chapter.

4.1 Immunotheranostics: history and future

Since the description of the magic bullets by Paul Ehrlich at the beginning of the 20^th century,²²² for decades experimental immunotherapeutic approaches were developed for the treatment of tumours. Since the underlying mechanisms were not well understood such treatments were not reliable and difficult to reproduce. Therefore, tumour immunology was even considered as “alternative medicine”. Even the development of monoclonal antibodies (mAbs), and later the detection of cellular immunity mediated by T-, NK and dendritic cells, did not lead to the anticipated major break-through of immunotherapies. The obvious drawback of murine mAbs is their weak interaction with both the human humoral and cellular immune effector mechanisms known as complement derived cytotoxicity (CDC), and antibody derived cellular cytotoxicity (ADCC). ADCC is mediated mainly by NK cells via the interaction of the Fc portion of the antibody with the Fc receptor expressed on immune cells. To overcome the limited cytotoxic capability of murine mAbs a variety of technologies were put forward, including for example the construction of immunodrug conjugates or radiolabelling of antibodies, as mentioned above. Limitations still remain today: the most obvious problem of full-size antibodies is their pharmacology. Antibodies having an Ig format stay within the blood stream for several weeks. Due to the high osmotic pressure, their entry into the tumour tissues is difficult and takes around 4 to 48 h. In addition, ADCC is inhibited by the immunosuppressive environment inside of tumours. One of the next steps to overcome this limitation of full-size natural antibodies was the development of smaller antibody derived fragments such as the single fragment variables (scFvs) using upcoming recombinant antibody technologies. Alternatively, natural occurring small antibodies, so called nanobodies (nbs), from camelides or sharks came in the focus as potential immunotheranostics. However, their foreign immunogenic character leads to neutralization due to an immune response in humans. Besides, the pharmacokinetic of nbs and scFvs is also not favourable for usage as immune drug conjugate, either for imaging or therapy, as they are rapidly eliminated via the kidney, usually within 15 to 30 min after application. For this reason, only a low tumour enrichment can be achieved. So even now, and in spite of the dramatic progress made, there is still significant room for improvement in antibody based humoral or cellular immune therapies.

The first real major breakthrough of antibody-based immunotherapies was the detection of Abs that can release the brakes of the immune system,²²³ which were termed as checkpoint inhibitors. The treatment of tumour patients with checkpoint inhibitors provided for the first-time convincing evidence for the tremendous potential of the humoral and cellular immune components to recognize and destroy tumour cells. In addition, it became obvious that tumour cells must have tricked the immune system and successfully circumvented all the obviously existing highly efficient natural immunological defence barriers when cancer is established in a patient.

Unfortunately, checkpoint inhibitors are only working against tumour cells that have collected manifold mutations during their evolution to become a tumour cell.²²⁴ Therefore, scientists try nowadays to reinstall the capability of the immune system via the development of more efficient immunotherapeutic treatment modalities based on vaccination, immunotheranostic compounds including BsAb, modified advanced nanomaterials, and most recently another major breakthrough using immune cells genetically modified to express chimeric artificial receptors (CARs).²²⁵

4.1.1 Overview on immunotherapeutic approaches.
Bispecific antibodies. Bispecific antibodies (BsAbs) for tumour therapies are usually directed to a tumour associated antigen (TAA, e.g. CD19), which is overexpressed on the surface of a tumour cell, and also to an activating receptor (e.g. CD3) on the cell surface of an immune effector cell. BsAbs can be constructed from the variable domains of the heavy and light chains of two monoclonal antibodies (mAbs). The domains of both mAbs are recombinantly fused to form single chain fragment variables (scFv), which are then fused to form the BsAb. BsAb can cross-link T cells with target cells, which finally leads to the destruction of the tumour cell. A schematic overview is shown in Fig. 6 (upper panel).

Fig. 6 Construction and retargeting of immune cells with BsAbs (upper panel) and conventional CAR T cells (lower panel). ScFvs can be constructed from the variable domains of two mAbs and fused to a BsAb. Cross-linkage via the BsAb leads to the killing of the tumour cell by the redirected T cell. A conventional CAR consists of three portions, the extracellular domain targeting a TAA usually a scFv directed to a TAA, a transmembrane domain and intracellular signalling domains taken from activatory receptors of the T cell receptor complex. The gene encoding such an artificial receptor can be transduced into T cells. The resulting CAR T cells can recognize tumour cells via their extracellular antibody domain. The interaction with the target cell leads to the formation of an artificial immune synapse, which finally triggers the cell death of the tumour cell.

Conventional CARs. Conventional CARs consist of three domains: (i) an extracellular antibody-based domain, usually in the scFv format (ii) a transmembrane domain, and (iii) intracellular signalling domains. Via the extracellular antibody domain CAR T cells can bind to the TAA on the surface of the tumour cell and destroy it (Fig. 6 lower panel).

For CAR T immunotherapy, T cells are isolated from the patient and transduced with the gene encoding the respective CAR construct. The resulting genetically modified CAR T cells are then adoptively transferred back into the patient.

The idea to equip T cells with an antibody based artificial receptor was already described at the end of the 1980s.²²⁶ However, it took decades of development until their more recent successful clinical application and first approvals by the respective legal authorities.

Although the CAR T cell technology remains extremely expensive, an impressive number of patients have been successfully treated with such individualised living drugs. While the positive outcome of CAR T cell treatments have underlined their high potential, their limitations have also become evident: activated CAR T cells strongly proliferate and produce high amounts of cytokines. As a consequence, CAR T cell treatments are associated with a high risk of severe life-threatening side effects, including cytokine release syndrome (CRS). In addition, OFF tumour On target effects can occur as the expression of the targeted TAA is usually not limited to the tumour cell but may also occur on the surface of healthy tissues.

Despite their side effects, the specificity of conventional CAR T cells is limited to just one target, which facilitates the development of tumour cell escape mechanisms.

Adaptor CARs. To overcome these problems, we and others have developed modular CAR T cell treatment platforms, nowadays known as adaptor CAR T cells. Starting from a modular BsAb approach, which we termed the UniMAB system (Fig. 7, left panel),²²⁷ we have established two versions of adaptor CAR platforms which we term the UniCAR and RevCAR system (patents^228–230) (Fig. 7, right panel).^221,225 For both adaptor CAR platforms proof of functionality has not only been shown in pre-clinical in vitro and in vivo studies^231,232 but also in currently running clinical phase 1 trials (UniCAR: NCT04230265; NCT04633148; RevCAR: NCT05949125).

Fig. 7 Originally, to accelerate the development of conventional BsAbs, we established the modular bispecific antibody platform UniMAB. For this purpose, we decided to split the BsAB into two components, an effector module (EM) and a targeting module (TM). The EM is a bispecific antibody, which recognizes on the one hand an activating domain of the immune cell (e.g. CD3) and on the other hand a peptide epitope. This epitope sequence is part of the TM. Thus, EM and TM can form an immune complex with properties similar to conventional BsAbs. This is the same strategy as we later used for our adaptor CAR platform technologies UniCAR and RevCAR. In contrast to conventional CARs, UniCARs do not recognize a TAA directly on the surface of a tumour cell but the same peptide epitope as the EM of the UniMAB system. Consequently, UniCAR T cells can establish an interaction with tumour cells via the same TMs used in the UniMAB system.²²¹ The RevCAR system differs from the UniCAR system as follows: the scFv domain of the UniCAR is replaced by the peptide epitope and vice versa the epitope tag in the TM is replaced by the anti-peptide epitope scFv.

In contrast to conventional CAR T cells, which are permanently active after adoptive transfer of the genetically modified immune cells, the function of adaptor CAR T cells depends on the presence of a bridging molecule (target module, TM), which is required for the cross-linkage of the adaptor CAR T cell with the tumour cell (Fig. 7). This strategy allows a repeated stop and go treatment via regulation of the infusion of the respective TM (Fig. 8).

Fig. 8 UniCAR (and RevCAR T cells) are inactive in the absence of a TM. They can be turned on by adding of the TM and turned OFF by elimination of the TM. In the clinical setting TMs are applied by continuous infusion. In case of side effects, the infusion can be stopped and thereby the function of the UniCAR T cells (RevCAR T cells) can be turned OFF. The adaptor CAR T cells can be restarted by further infusion of the TM. An additional advantage of adaptor CARs is the chance to apply an alternative TM with a different specificity in case tumour escape variants occur.

Obviously, the pharmacokinetics of the respective TM determines the efficiency and safety profile of adapter CAR T cells. TMs that can be rapidly eliminated allow a fast turning OFF of the adaptor CAR T cells. However, the downside is that their application requires a permanent infusion to achieve the TM concentration necessary for efficiency. During treatment, the patient is hospitilized at an intensive care unit, which is obviously not convenient for the patient. Vice versa, a TM having an extended half-life would facilitate the application for the patient but increases the risk of severe site-effects as adaptor CARs targeting TMs based on full size antibodies will behave more or less like non-regulatable conventional CARs.

For the UniCAR and RevCAR platforms, we have therefore developed a series of TMs having different pharmacokinetic profiles, which is achieved by the construction of molecules having different molecular weight.²²¹ A revised treatment modality with UniCAR or RevCAR T cells would start with TMs having a short half live allowing a fast interruption of the T cell function if necessary. Once most of the tumour load has been destroyed and the risk of CRS becomes low, a TM having a long half live could be applied. Although feasible and working in in vitro and in vivo models, the need for different TMs would obviously enhance the cost for the development of the respective GMP grade TMs and would require additional clinical trials.

Conjugation of TMs with Bi-NMs may not only be useful for optimization of the pharmacokinetic properties of TMs but also for imaging of tumours and therapeutic treatments. Furthermore, radioactive versions could help to overcome the immunosuppressive tumour microenvironment.

5. Outlook and conclusion

Recent advances in cancer therapies, integrating metals to precisely target cancer cells for effective imaging and therapy, are transforming the modern therapeutics. Towards this goal, bismuth in its various forms, has garnered much attention as a non-toxic and highly stable metal with many fascinating properties. The clinical application of small bismuth complexes appears to be feasible as only minor side effects, if at all, were observed when these compounds were carefully applied.^2,8 Kidney damage was only seen in the event of an overdose.²³³ Overall, the lower toxicity but also higher cost-effectiveness of bismuth-based materials compared to common precious metal-based anticancer compounds has led to an emerging rush towards commercialization. In the field of bismuth-containing nanomaterials, there are also promising developments that would justify clinical use. For example, the CT contrast of Bi-NMs is significantly higher than that of clinically approved iodine-based contrast agents, the photoconversion efficiency is very high and there are targeted materials available that achieve high tumour accumulation. However, to our knowledge, no clinical studies with Bi-NMs have been conducted to date. In the future, extensive clinical studies will be required here, with scientists, physicians and stakeholders of regulatory authority having to work closely together. In particular, the fabrication of nanomaterials under the conditions of good manufacturing practice (GMP) is a challenge. However, in the meantime, the art of synthesis in this field has progressed considerably.

At the nanoscale, bismuth is known for its effective radiosensitising and strong photothermal conversion properties as well as its remarkable performance as a strong contrast agent for various imaging modalities including CT, IRT, PA and MRI. In the field of imaging, ultrasmall particles (<6 nm) with appropriate surface functionalisation are becoming increasingly important because, they can reach all parts of the body and are quickly excreted by the kidneys.²³⁴ Additionally, Bi-NPs can be functionalised with various targeting agents and coating molecules to generate well-defined and site-targeted Bi-NPs for therapeutic applications on the basis of PTT, PDT and RT. Besides imaging, another highly attractive feature of Bi-based materials is based on the existence of radioactive bismuth compounds. Such radiobismuth compounds conjugated for instance with small molecules like peptides are highly promising radiopharmaceuticals including for endoradionuclide therapy.

With clinical applications in patients in mind, it is noteworthy to mention that the surface of Bi-NPs can be designed to prevent the formation of a protein corona and thus avoid off-target effects.²³⁵ Besides well-known biological vector molecules such as specific peptides and antibodies and their fragments, there is the possibility of using cancer cell membranes for targeting. A major problem with the introduction of new materials into the clinic still remains the dilemma of nanotoxicity.²³⁶ But with no doubt, nanomaterials and especially Bi-NPs may lead to promising novel treatment modalities for the imaging and treatment of otherwise fatal diseases for which global unmet medical needs exists. The currently remaining problems may hopefully be solved in the near future through interdisciplinary collaboration and partnerships across various disciplines.²³⁷

One example of a success story of interdisciplinary cooperation is currently the development of tumours therapies based on combinations of cytostatic drugs, radio-, and immunotherapies. Bearing in mind that local beam or endoradionuclide-based irradiation or the treatment with cytostatic drugs does not only lead to cell death but also alters the tumour microenvironment from immunosuppressive to inflammation, combinations of modern immunotherapeutic approaches based on BsAbs or CAR T cell technologies with well-established conventional tumour therapies appear very promising. Also, combinations with immunotheranostics and/or radiosensitisers based on advanced nanomaterials represent highly promising strategies to further improve the efficacy of future tumour immunotherapies. Due to the above summarized interesting features, bismuth-based nanoparticles may become of special interest and should therefore be tested in the near future in combination with BsAbs, modular BsAbs, CAR T cells, and the adaptor CAR T cell platforms UniCAR and RevCARs. TMs for adaptor CAR T cells could be constructed, consisting of Bi-NPs conjugated with both the antibody domain against a TAA and the peptide epitope recognized by an adaptor CAR T cell (Fig. 9).

Fig. 9 Multimodal (Bi based) NP can be constructed and conjugated with TMs to optimize their pharmacokinetic properties. Radioactively labelled TM versions could be used to modulate the tumour microenvironment from immunosuppressive to inflammation. Besides such Bi based NP could also be used for imaging of tumours and of the therapeutic effects: Bi-NPs based TMs could be established and equipped with additional features, for example via conjugation of small molecules or recombinant antibody derivatives to Bi-NPs, the pharmacokinetic property of a TM could be altered and fine-tuned. Radioactive versions of Bi-NP based TMs could directly modulate the tumour microenvironment from immunosuppressive towards inflammation as a prerequisite for attraction, invasion and activation of adaptor CAR T cells.

In summary, this perspective provides an overview of different types of Bi-NMs, such as metallic Bi-NPs, Bi-chalcogenides and Bi-hybrid structures that are relevant for clinical applications, and discusses key examples of colloidal stable, biocompatible and efficacious anti-cancer Bi-NPs and its related forms. Moreover, an overview of developments in radiobismuth-labelled compounds for targeted alpha therapy are presented, highlighting specific examples of radiolabelled bioconjugates and nanomaterials engineered for selective tumour treatment. A potential construction of Bi-NPs based TMs for multimodal usage in combination with adaptor CAR T cell technologies for cellular immunotherapy approaches of tumours are also presented. Using such Bi-NP based TMs may not only allow an optimisation of the pharmacokinetic properties of TMs but the modulation of the tumour microenvironment towards an improved attraction, invasion and killing efficacy of adaptor CAR T cells and thereby be helpful to overcome current limitations of cellular immunotherapies. These insights into the anti-cancer potential of Bi-materials offer a strong foundation for future research in cancer diagnostics and therapy, with the potential to drive the clinical implementation of Bi-based materials for cancer (immuno)theranostics.

Data availability

This is a perspective article that contains no original data.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We are grateful for the excellent work carried out by co-workers and colleagues mentioned in the references, specifically the groups in the MHELTHERA consortium. For financial support, we acknowledge Helmholtz-Zentrum Dresden-Rossendorf, the Australian Research Council (DP220103632), Monash University, and in particular the Helmholtz Initiative and Networking Fund (Radio-Immuno-Theranostics (MHELTHERA) project ID: InterLabs-0031).

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