Reassembly of 89Zr‐Labeled Cancer Cell Membranes into Multicompartment Membrane‐Derived Liposomes for PET‐Trackable Tumor‐Targeted Theranostics

Nanoengineering of cell membranes holds great potential to revolutionize tumor‐targeted theranostics, owing to their innate biocompatibility and ability to escape from the immune and reticuloendothelial systems. However, tailoring and integrating cell membranes with drug and imaging agents into one versatile nanoparticle are still challenging. Here, multicompartment membrane‐derived liposomes (MCLs) are developed by reassembling cancer cell membranes with Tween‐80, and are used to conjugate ⁸⁹Zr via deferoxamine chelator and load tetrakis(4‐carboxyphenyl) porphyrin for in vivo noninvasive quantitative tracing by positron emission tomography imaging and photodynamic therapy (PDT), respectively. Radiolabeled constructs, ⁸⁹Zr‐Df‐MCLs, demonstrate excellent radiochemical stability in vivo, target 4T1 tumors by the enhanced permeability and retention effect, and are retained long‐term for efficient and effective PDT while clearing gradually from the reticuloendothelial system via hepatobiliary excretion. Toxicity evaluation confirms that the MCLs do not impose acute or chronic toxicity in intravenously injected mice. Additionally, ⁸⁹Zr‐labeled MCLs can execute rapid and highly sensitive lymph node mapping, even for deep‐seated sentinel lymph nodes. The as‐developed cell membrane reassembling route to MCLs could be extended to other cell types, providing a versatile platform for disease theranostics by facilely and efficiently integrating various multifunctional agents.     

Activatable Hybrid Nanotheranostics for Tetramodal Imaging and Synergistic Photothermal/Photodynamic Therapy

A multifunctional core–satellite nanoconstruct is designed by assembling copper sulfide (CuS) nanoparticles on the surface of [⁸⁹Zr]‐labeled hollow mesoporous silica nanoshells filled with porphyrin molecules, for effective cancer imaging and therapy. The hybrid nanotheranostic demonstrates three significant features: (1) simple and robust construction from biocompatible building blocks, demonstrating prolonged blood retention, enhanced tumor accumulation, and minimal long‐term systemic toxicity, (2) rationally selected functional moieties that interact together to enable simultaneous tetramodal (positron emission tomography/fluorescence/Cerenkov luminescence/Cerenkov radiation energy transfer) imaging for rapid and accurate delineation of tumors and multimodal image‐guided therapy in vivo, and (3) synergistic interaction between CuS‐mediated photothermal therapy and porphyrin‐mediated photodynamic therapy which results in complete tumor elimination within a day of treatment with no visible recurrence or side effects. Overall, this proof‐of‐concept study illustrates an efficient, generalized approach to design high‐performance core–satellite nanohybrids that can be easily tailored to combine a wide variety of imaging and therapeutic modalities for improved and personalized cancer theranostics in the future.     

Efficient Radioisotope Energy Transfer by Gold Nanoclusters for Molecular Imaging

Beta‐emitting isotopes Fluorine‐18 and Yttrium‐90 are tested for their potential to stimulate gold nanoclusters conjugated with blood serum proteins (AuNCs). AuNCs excited by either medical radioisotope are found to be highly effective ionizing radiation energy transfer mediators, suitable for in vivo optical imaging. AuNCs synthesized with protein templates convert beta‐decaying radioisotope energy into tissue‐penetrating optical signals between 620 and 800 nm. Optical signals are not detected from AuNCs incubated with Technetium‐99m, a pure gamma emitter that is used as a control. Optical emission from AuNCs is not proportional to Cerenkov radiation, indicating that the energy transfer between the radionuclide and AuNC is only partially mediated by Cerenkov photons. A direct Coulombic interaction is proposed as a novel and significant mechanism of energy transfer between decaying radionuclides and AuNCs.     

Zwitterionic‐Coated “Stealth” Nanoparticles for Biomedical Applications: Recent Advances in Countering Biomolecular Corona Formation and Uptake by the Mononuclear Phagocyte System

Nanoparticles represent highly promising platforms for the development of imaging and therapeutic agents, including those that can either be detected via more than one imaging technique (multi‐modal imaging agents) or used for both diagnosis and therapy (theranostics). A major obstacle to their medical application and translation to the clinic, however, is the fact that many accumulate in the liver and spleen as a result of opsonization and scavenging by the mononuclear phagocyte system. This focused review summarizes recent efforts to develop zwitterionic‐coatings to counter this issue and render nanoparticles more biocompatible. Such coatings have been found to greatly reduce the rate and/or extent of non‐specific adsorption of proteins and lipids to the nanoparticle surface, thereby inhibiting production of the “biomolecular corona” that is proposed to be a universal feature of nanoparticles within a biological environment. Additionally, in vivo studies have demonstrated that larger‐sized nanoparticles with a zwitterionic coating have extended circulatory lifetimes, while those with hydrodynamic diameters of ≤5 nm exhibit small‐molecule‐like pharmacokinetics, remaining sufficiently small to pass through the fenestrae and slit pores during glomerular filtration within the kidneys, and enabling efficient excretion via the urine. The larger particles represent ideal candidates for use as blood pool imaging agents, whilst the small ones provide a highly promising platform for the future development of theranostics with reduced side effect profiles and superior dose delivery and image contrast capabilities.     

In Vivo Repeatedly Activated Persistent Luminescence Nanoparticles by Radiopharmaceuticals for Long‐Lasting Tumor Optical Imaging

Persistent luminescence nanoparticles (PLNPs) with rechargeable near‐infrared afterglow properties attract much attention for tumor diagnosis in living animals since they can avoid tissue autofluorescence and greatly improve the signal‐to‐background ratio. Using UV, visible light, or X‐ray as excitation sources to power up persistent luminescence (PL) faces the challenges such as limited tissue penetration, inefficient charging capability, or tissue damage caused by irradiation. Here, it is proved that radiopharmaceuticals can efficiently excite ZnGa₂O₄:Cr³⁺ nanoparticles (ZGCs) for both fluorescence and afterglow luminescence via Cerenkov resonance energy transfer as well as ionizing radiation. ¹⁸F‐FDG, a clinically approved tumor‐imaging radiopharmaceutical with a short decay half‐life around 110 min, is successfully used as the internal light source to in vivo excite intravenously injected ZGCs for tumor luminescence imaging over 3 h. The luminescence with similar decay time can be re‐obtained for multiple times upon injection of ¹⁸F‐FDG at any time needed with no health concern. It is believed this strategy can not only provide tumor luminescence imaging with high sensitivity, high contrast, and long decay time at desired time, but also guarantee the patients much less radiation exposure, greatly benefiting image‐guided surgery in the future.     

Hybrid Protein Nano‐Reactors Enable Simultaneous Increments of Tumor Oxygenation and Iodine‐131 Delivery for Enhanced Radionuclide Therapy

It is hard for current radionuclide therapy to render solid tumors desirable therapeutic efficacy owing to insufficient tumor‐targeted delivery of radionuclides and severe tumor hypoxia. In this study, a biocompatible hybrid protein nanoreactor composed of human serum albumin (HSA) and catalase (CAT) molecules is constructed via glutaraldehyde‐mediated crosslinking. The obtained HSA‐CAT nanoreactors (NRs) show retained and well‐protected enzyme stability in catalyzing the decomposition of H₂O₂ and enable efficient labeling of therapeutic radionuclide iodine‐131 (¹³¹I). Then, it is uncovered that such HSA‐CAT NRs after being intravenously injected into tumor‐bearing mice exhibit efficient passive tumor accumulation as vividly visualized under the fluorescence imaging system and gamma camera. As the result, such HSA‐CAT NRs upon tumor accumulation would significantly attenuate tumor hypoxia by decomposing endogenous H₂O₂ produced by cancer cells to molecular oxygen, and thereby remarkably improve the therapeutic efficacy of radionuclide ¹³¹I. This study highlights the concise preparation of biocompatible protein nanoreactors with efficient tumor homing and hypoxia attenuation capacities, thus enabling greatly improved tumor radionuclide therapy with promising potential for future clinical translation.     

Facile Preparation of Multifunctional WS2/WOx Nanodots for Chelator‐Free 89Zr‐Labeling and In Vivo PET Imaging

While position emission tomography (PET) is an important molecular imaging technique for both preclinical research and clinical disease diagnosis/prognosis, chelator‐free radiolabeling has emerged as a promising alternative approach to label biomolecules or nanoprobes in a facile way. Herein, starting from bottom‐up synthesized WS₂ nanoflakes, this study fabricates a unique type of WS₂/WO_x nanodots, which can function as inherent hard oxygen donor for stable radiolabeling with Zirconium‐89 isotope (⁸⁹Zr). Upon simply mixing, ⁸⁹Zr can be anchored on the surface of polyethylene glycol (PEG) modified WS₂/WO_x (WS₂/WO_x‐PEG) nanodots via a chelator‐free method with surprisingly high labeling yield and great stability. A higher degree of oxidation in the WS₂/WO_x‐PEG sample (WS₂/WO_x (0.4)) produces more electron pairs, which would be beneficial for chelator‐free labeling of ⁸⁹Zr with higher yields, suggesting the importance of surface chemistry and particle composition to the efficiency of chelator‐free radiolabeling. Such ⁸⁹Zr‐WS₂/WO_x (0.4)‐PEG nanodots are found to be an excellent PET contrast agent for in vivo imaging of tumors upon intravenous administration, or mapping of draining lymph nodes after local injection.     

Highly Efficient Photosensitizers with Far‐Red/Near‐Infrared Aggregation‐Induced Emission for In Vitro and In Vivo Cancer Theranostics

Fluorescence‐imaging‐guided photodynamic therapy has emerged as a promising protocol for cancer theranostics. However, facile preparation of such a theranostic material for simultaneously achieving bright emission with long wavelength, high‐performance reactive oxygen species (ROS) generation, and good targeting‐specificity of cancer cells, is highly desirable but remains challenging. In this study, a novel type of far‐red/near‐infrared‐emissive fluorescent molecules with aggregation‐induced emission (AIE) characteristics is synthesized through a few steps reaction. These AIE luminogens (AIEgens) possess simple structures, excellent photostabilities, large Stokes shifts, bright emission, and good biocompatibilities. Meanwhile, their ROS generation is extremely efficient with up to 90.7% of ROS quantum yield, which is far superior to that of some popularly used photosensitizers. Importantly, these AIEgens are able to selectively target and ablate cancer cells over normal cells without the aid of any extra targeting ligands. Rather than using laser light, one of the presented AIEgens (MeTTPy) shows a remarkable tumor‐targeting photodynamic therapeutic effect by using an ultralow‐power lamp light (18 mW cm^?2). This study thus not only extends the applications scope of AIEgens, but also offers useful insights into designing a new generation of cancer theranostics.     

Core–Satellite Polydopamine–Gadolinium‐Metallofullerene Nanotheranostics for Multimodal Imaging Guided Combination Cancer Therapy

Integration of magnetic resonance imaging (MRI) and other imaging modalities is promising to furnish complementary information for accurate cancer diagnosis and imaging‐guided therapy. However, most gadolinium (Gd)–chelator MR contrast agents are limited by their relatively low relaxivity and high risk of released‐Gd‐ions‐associated toxicity. Herein, a radionuclide‐⁶⁴Cu‐labeled doxorubicin‐loaded polydopamine (PDA)–gadolinium‐metallofullerene core–satellite nanotheranostic agent (denoted as CDPGM) is developed for MR/photoacoustic (PA)/positron emission tomography (PET) multimodal imaging‐guided combination cancer therapy. In this system, the near‐infrared (NIR)‐absorbing PDA acts as a platform for the assembly of different moieties; Gd₃N@C₈₀, a kind of gadolinium metallofullerene with three Gd ions in one carbon cage, acts as a satellite anchoring on the surface of PDA. The as‐prepared CDPGM NPs show good biocompatibility, strong NIR absorption, high relaxivity (r ₁ = 14.06 mM^?1 s^?1), low risk of release of Gd ions, and NIR‐triggered drug release. In vivo MR/PA/PET multimodal imaging confirms effective tumor accumulation of the CDPGM NPs. Moreover, upon NIR laser irradiation, the tumor is completely eliminated with combined chemo‐photothermal therapy. These results suggest that the CDPGM NPs hold great promise for cancer theranostics.     

A Tumor‐Microenvironment‐Activated Nanozyme‐Mediated Theranostic Nanoreactor for Imaging‐Guided Combined Tumor Therapy

Activatable theranostic agents that can be activated by tumor microenvironment possess higher specificity and sensitivity. Here, activatable nanozyme‐mediated 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) loaded ABTS@MIL‐100/poly(vinylpyrrolidine) (AMP) nanoreactors (NRs) are developed for imaging‐guided combined tumor therapy. The as‐constructed AMP NRs can be specifically activated by the tumor microenvironment through a nanozyme‐mediated “two‐step rocket‐launching‐like” process to turn on its photoacoustic imaging signal and photothermal therapy (PTT) function. In addition, simultaneously producing hydroxyl radicals in response to the high H₂O₂ level of the tumor microenvironment and disrupting intracellular glutathione (GSH) endows the AMP NRs with the ability of enhanced chemodynamic therapy (ECDT), thereby leading to more efficient therapeutic outcome in combination with tumor‐triggered PTT. More importantly, the H₂O₂‐activated and acid‐enhanced properties enable the AMP NRs to be specific to tumors, leaving the normal tissues unharmed. These remarkable features of AMP NRs may open a new avenue to explore nanozyme‐involved nanoreactors for intelligent, accurate, and noninvasive cancer theranostics.     

Toxic Reactive Oxygen Species Enhanced Synergistic Combination Therapy by Self‐Assembled Metal‐Phenolic Network Nanoparticles

Engineering functional nanomaterials with high therapeutic efficacy and minimum side effects has increasingly become a promising strategy for cancer treatment. Herein, a reactive oxygen species (ROS) enhanced combination chemotherapy platform is designed via a biocompatible metal‐polyphenol networks self‐assembly process by encapsulating doxorubicin (DOX) and platinum prodrugs in nanoparticles. Both DOX and platinum drugs can activate nicotinamide adenine dinucleotide phosphate oxidases, generating superoxide radicals (O₂^??). The superoxide dismutase‐like activity of polyphenols can catalyze H₂O₂ generation from O₂^??. Finally, the highly toxic HO^? free radicals are generated by a Fenton reaction. The ROS HO^? can synergize the chemotherapy by a cascade of bioreactions. Positron emission tomography imaging of ⁸⁹Zr‐labeled as‐prepared DOX@Pt prodrug Fe³⁺ nanoparticles (DPPF NPs) shows prolonged blood circulation and high tumor accumulation. Furthermore, the DPPF NPs can effectively inhibit tumor growth and reduce the side effects of anticancer drugs. This study establishes a novel ROS promoted synergistic nanomedicine platform for cancer therapy.     

Tumor pH‐Responsive Albumin/Polyaniline Assemblies for Amplified Photoacoustic Imaging and Augmented Photothermal Therapy

Tumor‐microenvironment‐responsive theranostics have great potential for precision diagnosis and effective treatment of cancer. Polyaniline (PANI) is the first reported pH‐responsive organic photothermal agent and is widely used as a theranostic agent. However, tumor pH‐responsive PANI‐based theranostic agents are not explored, mainly because the conversion from the emeraldine base (EB) to emeraldine salt (ES) state of PANI requires pH < 4, which is lower than tumor acidic microenvironment. Herein, a tumor pH‐responsive PANI‐based theranostic agent is designed and prepared for amplified photoacoustic imaging guided augmented photothermal therapy (PTT), through intermolecular acid–base reactions between carboxyl groups of bovine serum albumin (BSA) and imine moieties of PANI. The albumin/PANI assemblies (BSA–PANI) can convert from the EB to ES state at pH < 7, accompanied by the absorbance redshift from visible to near‐infrared region. Both in vitro and in vivo results demonstrate that tumor acidic microenvironment can trigger both the photoacoustic imaging (PAI) signal amplification and the PTT efficacy enhancement of BSA–PANI assemblies. This work not only highlights that BSA–PANI assemblies overcome the limitation of low‐pH protonation, but also provides a facile assembly strategy for a tumor pH‐responsive PANI‐based nanoplatform for cancer theranostics.     

A Versatile Theranostic Nanoemulsion for Architecture‐Dependent Multimodal Imaging and Dually Augmented Photodynamic Therapy

To design a clinically translatable nanomedicine for photodynamic theranostics, the ingredients should be carefully considered. A high content of nanocarriers may cause extra toxicity in metabolism, and multiple theranostic agents would complicate the preparation process. These issues would be of less concern if the nanocarrier itself has most of the theranostic functions. In this work, a poly(ethylene glycol)‐boron dipyrromethene amphiphile (PEG‐F₅₄‐BODIPY) with 54 fluorine‐19 (¹⁹F) is synthesized and employed to emulsify perfluorohexane (PFH) into a theranostic nanoemulsion (PFH@PEG‐F₅₄‐BODIPY). The as‐prepared PFH@PEG‐F₅₄‐BODIPY can perform architecture‐dependent fluorescence/photoacoustic/¹⁹F magnetic resonance multimodal imaging, providing more information about the in vivo structure evolution of nanomedicine. Importantly, this nanoemulsion significantly enhances the therapeutic effect of BODIPY through both the high oxygen dissolving capability and less self‐quenching of BODIPY molecules. More interestingly, PFH@PEG‐F₅₄‐BODIPY shows high level of tumor accumulation and long tumor retention time, allowing a repeated light irradiation after a single‐dose intravenous injection. The “all‐in‐one” photodynamic theranostic nanoemulsion has simple composition, remarkable theranostic efficacy, and novel treatment pattern, and thus presents an intriguing avenue to developing clinically translatable theranostic agents.     

NIR‐II‐Excited Intravital Two‐Photon Microscopy Distinguishes Deep Cerebral and Tumor Vasculatures with an Ultrabright NIR‐I AIE Luminogen

Intravital fluorescence imaging of vasculature morphology and dynamics in the brain and in tumors with large penetration depth and high signal‐to‐background ratio (SBR) is highly desirable for the study and theranostics of vascular‐related diseases and cancers. Herein, a highly bright fluorophore (BTPETQ) with long‐wavelength absorption and aggregation‐induced near‐infrared (NIR) emission (maximum at ≈700 nm) is designed for intravital two‐photon fluorescence (2PF) imaging of a mouse brain and tumor vasculatures under NIR‐II light (1200 nm) excitation. BTPETQ dots fabricated via nanoprecipitation show uniform size of around 42 nm and a high quantum yield of 19 ± 1% in aqueous media. The 2PF imaging of the mouse brain vasculatures labeled by BTPETQ dots reveals a 3D blood vessel network with an ultradeep depth of 924 µm. In addition, BTPETQ dots show enhanced 2PF in tumor vasculatures due to their unique leaky structures, which facilitates the differentiation of normal blood vessels from tumor vessels with high SBR in deep tumor tissues. Moreover, the extravasation and accumulation of BTPETQ dots in deep tumor (more than 900 µm) is visualized under NIR‐II excitation. This study highlights the importance of developing NIR‐II light excitable efficient NIR fluorophores for in vivo deep tissue and high contrast tumor imaging.     

Cross‐Linking of Thiolated Paclitaxel–Oligo(p‐phenylene vinylene) Conjugates Aggregates inside Tumor Cells Leads to “Chemical Locks” That Increase Drug Efficacy

How to reduce the resistance of certain tumor cells to paclitaxel (PTX) and related taxoid anticancer drugs is a major challenge for improving cure rates. An oligo(p‐phenylenevinylene) unit with thiol groups and a PTX unit (OPV‐S‐PTX), which enhances drug efficacy and reverses resistance is thus designed. The mechanism involves diffusion of OPV‐S‐PTX into the cell, where π–π interactions lead to aggregation. Cross‐linking of the aggregates via oxidation of thiol groups is favored in tumor cells because of the higher reactive oxygen species (ROS) concentration. Cross‐linked aggregates “chemically lock” the multichromophore particle for a more persistent effect. The IC₅₀ of OPV‐S‐PTX for tumor cell line A549 is reduced down to 0.33 × 10^?9m from that observed for PTX itself (41 × 10^?9m ). Enhanced efficacy by OPV‐S‐PTX is proposed to proceed via acceleration of microtubule bundle formation. A549/T‐inoculated xenograft mice experiments reveal suppression of tumor growth upon OPV‐S‐PTX treatment. Altogether, these results show that the internal cross‐linking of OPV‐S‐PTX through ROS provides a means to discriminate between tumor and healthy cells and the formation of the chemically locked particles enhances drug efficacy and helps in reducing resistance.     

Molecular Imaging Using Nanoparticle Quenchers of Cerenkov Luminescence

Cerenkov luminescence (CL) imaging is an emerging technique that collects the visible photons produced by radioisotopes. Here, molecular imaging strategies are investigated that switch the CL signal off. The noninvasive molecularly specific detection of cancer is demonstrated utilizing a combination of clinically approved agents, and their analogues. CL is modulated in vitro in a dose dependent manner using approved small molecules (Lymphazurin), as well as the clinically approved Feraheme and other preclinical superparamagnetic iron oxide nanoparticles (SPIO). To evaluate the quenching of CL in vivo, two strategies are pursued. [¹⁸F]‐FDG is imaged by PET and CL in tumors prior to and following accumulation of nanoparticles. Initially, non‐targeted particles are administered to mice bearing tumors in order to attenuate CL. For targeted imaging, a dual tumor model (expressing the human somatostatin receptor subtype‐2 (hSSTr2) and a control negative cell line) is used. Targeting hSSTr2 with octreotate‐conjugated SPIO, quenched CL enabling non‐invasive distinction between tumors' molecular expression profiles is demonstrated. In this work, the quenching of Cerenkov emissions is demonstrated in several proof of principle models using a combination of approved agents and nanoparticle platforms to provide disease relevant information including tumor vascularity and specific antigen expression.     

Efficient Polysulfide‐Based Nanotheranostics for Triple‐Negative Breast Cancer: Ratiometric Photoacoustics Monitored Tumor Microenvironment‐Initiated H2S Therapy

The incidence of triple‐negative breast cancer (TNBC) is difficult to predict, and TNBC has a high mortality rate among women worldwide. In this study, a theranostics approach is developed for TNBC with ratiometric photoacoustic monitored thiol‐initiated hydrogen sulfide (H₂S) therapy. The ratiometric photoacoustic (PA) probe (CY) with a thiol‐initiated H₂S donor (PSD) to form a nanosystem (CY‐PSD nanoparticles) is integrated. In this theranostics approach, H₂S generated from PSD is sensed by CY based on ratiometric PA signals, which simultaneously pinpoints the tumor region. Additionally, H₂S is cytotoxic toward TNBC cells (MDA‐MB 231), showing a tumor inhibition rate of 63%. To further verify its pharmacological mechanism, proteomics analysis is performed on tumors treated with CY‐PSD nanoparticles. Cells are killed by the significant mitochondrial dysfunction via supressed energy supply and apoptosis initiation. Besides, the observed inhibition of oxidative stress also generates the cytotoxicity. Significant Kyoto Encyclopedia of Genes Genomes pathways related to TNBC are found to be inhibited. This H₂S theranostics approach updates the current anticancer therapies which brings promise for women suffering malignant breast cancer.     

An Organic Afterglow Protheranostic Nanoassembly

Cancer theranostics holds potential promise for precision medicine; however, most existing theranostic nanoagents are simply developed by doping both therapeutic agents and imaging agent into one particle entity, and thus have an “always‐on” pharmaceutical effect and imaging signals regardless of their in vivo location. Herein, the development of an organic afterglow protheranostic nanoassembly (APtN) that specifically activates both the pharmaceutical effect and diagnostic signals in response to a tumor‐associated chemical mediator (hydrogen peroxide, H₂O₂) is reported. APtN comprises an amphiphilic macromolecule and a near‐infrared (NIR) dye acting as the H₂O₂‐responsive afterglow prodrug and the afterglow initiator, respectively. Such a molecular architecture allows APtN to passively target tumors in living mice, specifically release the anticancer drug in the tumor, and spontaneously generate the uncaged afterglow substrate. Upon NIR light preirradiation, the afterglow initiator generates singlet oxygen to react and subsequently transform the uncaged afterglow substrate into an active self‐luminescent form. Thus, the intensity of generated afterglow luminescence is correlated with the drug release status, permitting real‐time in vivo monitoring of prodrug activation. This study proposes a background‐free design strategy toward activatable cancer theranostics.     

In vivo Cerenkov luminescence imaging: a new tool for molecular imaging

Cerenkov radiation is a phenomenon where optical photons are emitted when a charged particle moves faster than the speed of light for the medium in which it travels. Recently, we and others have discovered that measurable visible light due to the Cerenkov effect is produced in vivo following the administration of β-emitting radionuclides to small animals. Furthermore, the amounts of injected activity required to produce a detectable signal are consistent with small-animal molecular imaging applications. This surprising observation has led to the development of a new hybrid molecular imaging modality known as Cerenkov luminescence imaging (CLI), which allows the spatial distribution of biomolecules labelled with β-emitting radionuclides to be imaged in vivo using sensitive charge-coupled device cameras. We review the physics of Cerenkov radiation as it relates to molecular imaging, present simulation results for light intensity and spatial distribution, and show an example of CLI in a mouse cancer model. CLI allows many common radiotracers to be imaged in widely available in vivo optical imaging systems, and, more importantly, provides a pathway for directly imaging β(-)-emitting radionuclides that are being developed for therapeutic applications in cancer and that are not readily imaged by existing methods.