The present work involves the development of new organic crystalline nanomedicines, namely, co-nanocrystals (co-NCs), that combine a chemotherapeutic agent with an anti-inflammatory active pharmaceutical ingredient (API), both in their nanocrystalline forms. These mixed systems were formulated to improve their anticancer efficiency. For this purpose, formulations composed of etoposide (ETO) and prednisolone (PRD) NCs were prepared. The co-formulations were optimized with the stabilizing agent poloxamer P407. The average size obtained was 191.9 ± 6.5 nm with a polydispersity index of 0.28 ± 0.09 and a zeta potential of -5.0 ± 0.7 mV for the fresh formulations. The drug loading was determined to be at 71.9% ± 0.6% for PRD and 84.1% ± 3.9% for ETO. The obtained co-NCs presented a long-term stability of at least 70 days after storage at 4 °C. Dialysis experiments showed that both APIs were released from the co-formulations following 1st-order kinetics. In addition, the NCs exhibited stronger antitumoral activities in vitro on Lewis lung carcinoma cells compared to the free ETO formulation or the ETO mono-NCs. Moreover, the anti-angiogenic properties tested on endothelial cells using capillary tube formation and the effect against cell migration analyzed by the scratch healing assay were enhanced by the ETO/PRD NCs co-formulation. The effect of the addition of PRD to the nanocrystalline formulation was shown by the greater reduction in inflammatory cytokine IL-6 levels in vitro when endothelial cells were incubated with the co-NCs as opposed to the mono-NCs. These results open the way for the preparation of stable co-formulations and better-tolerated combined therapies.

Immunogenic cell death (ICD), which converts tumor cells into their own vaccine, plays a pivotal role in the development of novel anti-cancer therapies. Here, a small series of osmium(II) polypyridyl complexes were synthesized and their biological activity in the dark and upon light irradiation against various cancer cell lines was studied. The compound Os2 (bearing two 4,7-diphenyl-1,10-phenanthrolines and one substituted bipyridine ligand) was discovered to be the most effective photosensitizer (PS) for photodynamic therapy (PDT) of this series through the photogeneration of 1O2 and •OH. In addition, Os2 was found to exhibit promising toxicity upon near-infrared (NIR) irradiation under both normoxia and hypoxia. These observations indicate that this PS is working through a mixture of Type-I and Type-II mechanisms. More interestingly, upon 740 nm irradiation, Os2 can stimulate a strong ICD response both in vitro and in vivo. A comprehensive immune analysis showed that mice vaccinated with Os2-treated CT26-luc cells boosted the systemic specific adaptive immune responses, including the activation of CD8+ T cells and reprograming of macrophages, leading to effective inhibition of tumor growth. Os2 is, to the best of our knowledge, the first photoactive osmium-based complex inducing ICD.

Cancer treatment is a crucial area of research and development, as current chemotherapeutic treatments can have severe side effects or poor outcomes. In the constant search for new strategies that are localized and minimally invasive and produce minimal side effects, photodynamic therapy (PDT) is an exciting therapeutic modality that has been gaining attention. The use of theranostics, which combine diagnostic and therapeutic capabilities, can further improve treatment monitoring through image guidance. This study explores the potential of a theranostic agent consisting of four Gd(III) DTTA complexes (DTTA: diethylenetriamine-N,N,N″,N″-tetraacetate) grafted to a meso-tetraphenylporphyrin core for PDT, fluorescence, and magnetic resonance imaging (MRI). The agent was first tested in vitro on both nonmalignant TIB-75 and MRC-5 and tumoral CT26 and HT-29 cell lines and subsequently evaluated in vivo in a preclinical colorectal tumor model. Advanced MRI and optical imaging techniques were employed with engineered quantitative in vivo molecular imaging based on dynamic acquisition sequences to track the biodistribution of agents in the body. With 3D quantitative volume computed by MRI and tumoral cell function assessed by bioluminescence imaging, we could demonstrate a significant impact of the molecular agent on tumor growth following light application. Further exhaustive histological analysis confirmed these promising results, making this theranostic agent a potential drug candidate for cancer.

Locoregional therapies have emerged as key components in the therapeutic arsenal against gastrointestinal cancers, offering minimally invasive options with curative potential. Beyond their direct cytotoxic effects, these interventions can remodel the tumour microenvironment by inducing immunogenic cell death and initiating local and systemic immune responses, including abscopal effects. This review aims to examine how these immunomodulatory properties may support locoregional therapies as an ideal platform for intratumoural immunotherapy, positioning their combination at the core of the evolving field of interventional immuno-oncology. Recent studies demonstrate that locoregional therapies can induce immune responses through modulation of the tumour microenvironment, creating conditions that may be exploited by intratumoural immunotherapy. This review provides a comprehensive overview of current locoregional modalities, including thermal and non-thermal ablative techniques, transarterial therapies, and minimally invasive radiation methods, detailing their mechanisms of action, clinical applications, and immune-related effects. Special attention is given to emerging evidence supporting the combination of these approaches with intratumoural delivery of immunomodulatory agents, to enhance anticancer immune responses. The convergence of locoregional intervention and targeted immunomodulation represents an emerging therapeutic concept in the treatment of gastrointestinal cancers, offering the prospect of personalised, tumour-directed immune activation. This approach holds the potential to extend the benefits of immunotherapy beyond genetically defined subgroups to a broader patient population.

There has been a recent paradigm shift among scientists concerning the development of novel anticancer diagnostic and treatment agents. The interest lies in the utilization of nanomedicine which has triggered significant attention in cancer research. At present, nanomedicine has transformed the current landscape of science through the use of numerous nanoformulations not only in cancer diagnosis but also in treatment. Nanoformulations including but not limited to polymeric, lipidic, metallic, crystal, and quantum dots nanomaterials have been demonstrated to play a pivotal role in circumventing major challenges associated with anticancer medications such as targeting cancer cells and reducing side effects. The use of nanomaterials in cancer is largely attributed to their distinct tunable features such as small size, improved drug loading, high sensitivity, high surface area-to-volume ratio, and site specificity, all of which are key in the development of next-generation antitumoural formulations. Additionally, the versatility of these nanomedicine formulations extends to the overall improvement of the bioavailability of antitumoural medicines. Consequently, researchers are investigating the potential use of nanomaterials in oncology to improve tumor diagnosis and treatment. This article focuses on the potential role of nanomedicines in breast cancer diagnosis and treatment using polymeric, lipidic, metallic, organic, quantum dot nanomaterials which are promising for innovant therapies and personalized nanomedicine.

Microfluidics mixing is the current lab-scale method used for producing mRNA-loaded lipid nanoparticles (mRNA-LNPs) thanks to reproducibility and robustness of microfluidic mixing. Despite these advantages, the production of small LNP volumes is associated with significant material waste. Given the high cost of synthetic mRNA, this waste can be a major limitation, particularly for early-stage screening of formulations. This study proposes alternative methods for mRNA-LNP formulation aiming to improve their stability for both formulation and mRNA screening, while reducing material waste on a research scale. Specifically, we investigated post-encapsulation of mRNA into pre-formed vesicles (PFVs) obtained by microfluidic mixing. These PFVs were complexed with mRNA by: (1) a microfluidic or (2) a manual pipetting method. The resulting mRNA-LNPs produced using these two post-encapsulation methods exhibit similar physicochemical properties and morphologies to those obtained by conventional microfluidic protocol. These mRNA-LNPs were assessed on in vitro and in vivo expression. mRNA-LNPs prepared by our alternative methods showed a similar transfection level compared to the conventional formulation taken as a control. The suitability of post-encapsulation methods to other lipids, mRNAs and microfluidic systems was also confirmed. This work offers robust, simple and economic alternative methods for preparing small volumes of mRNA-LNPs. The versatility of post-encapsulation methods allows to screen mRNA formulations in a wide range of laboratories. These methods could be applied to encapsulate tailored doses of mRNA and various mRNA constructs to achieve an optimal and personalized therapy.

Over the last few years, the success of COVID-19 mRNA vaccines has resulted in the emergence of RNA lipid nanoparticles (LNPs) with promising prospects for the prevention and treatment of various diseases. The context of the SARS-CoV-2 pandemic has led to the rapid development of vaccines with abbreviated non-clinical programs. However, there are currently no official guidelines defining the required standards for global marketing of mRNA based therapeutic products. Nevertheless, to guarantee a well-controlled product, it is essential to characterize both the drug substance and the final product in terms of their structure, composition, formulation, physico-chemical features, potency, and safety. This lack of guidance has resulted in a wide variety of heterogeneous in vitro tests being used to assess the potency and cytotoxicity of RNA-LNP. This review discusses the commonly used in vitro assays, primarily 2D monolayer assays, employed to evaluate the biological properties of RNA-LNP. We then explore novel alternative methods to bridge the gap between in vitro and in vivo results. We summarize (i) co-culture models, (ii) multilayer 3D assays and (iii) in vivo replacement models, exploring their potential applications in assessing the potency and safety of RNA-LNPs. Finally, we discuss the use of in silico and machine learning as models for optimizing and predicting the biological behavior of RNA-LNPs. Graphical Abstract

Persistent luminescence (PersL) nanoparticles emit a signal that lasts long after the excitation has ended, enabling highly sensitive bioimaging without background noise. By using UV light as the excitation source prior to injection, a strong PersL signal can be detected in vivo before disappearing within a few hours. For long-term imaging, we have shown in the past that visible LED can be used to re-excite ZnGa1.995Cr0.005O4 (ZGO:Cr3+) nanoparticles in vivo, producing a signal intensity that is, however, much lower compared to the signal obtained after the UV pre-excitation method. This lower signal intensity of the nanoprobe when using a visible LED may prevent its detection in some cases. Herein, we report an improvement in visible LED excitation efficiency using PersL Zn1.33Ga1.335Cr0.005Sn0.33O4 (ZGSO:Cr3+) nanoparticles. We fully compared ZGSO:Cr3+ and the original ZGO:Cr nanoparticles in terms of structure, optical properties and bio-imaging potential. Co-doping with tin strongly increases persistent luminescence, even at the nanoscale. In vivo imaging results showed that ZGSO:Cr3+ exhibited an approximately 3-fold signal enhancement over ZGO:Cr3+ using UV pre-excitation. More interestingly, when using LED excitation, the signal intensity of ZGSO: Cr3+ is more than 10 times higher than that of ZGO:Cr3+, making ZGSO nanoparticles much easier to detect. ZGSO:Cr nanoparticles can be surface-modified with PEG to produce nanoprobes with much longer residence time in blood. This unique comparison between the two compositions makes ZGSO:Cr3+ a more effective imaging diagnostic probe than the original ZGO:Cr3+ nanoparticles, opening up new applications for in vivo diagnostics.

Background Surgeons may discover perioperative clinical situations associated with an increased risk of peritoneal carcinomatosis recurrence after primary resection. We developed a thermogel that can be used as a drug carrier to deliver an anticancer agent in the peritoneal cavity as a rescue solution. The spatial distribution of the thermogel and pharmacokinetics of chemotherapy have been studied in pigs. The safety of the thermogel was assessed based on the healing of bowel sutures. Methods Nine pigs received gel with oxaliplatin at 130 mg/200 mL (TG-Ox group), and 4 pigs received the gel alone (TG group). Digestive tract and bladder wounds were made and sutured. Pigs were sacrificed at different times after surgery to monitor the distribution of the thermogel and to detect the occurrence of bowel fistulas. Oxaliplatin plasma and tissue concentrations were determined via mass spectrometry. Results After 3 h, 100 % of the regions of interest were covered by the gel, 78 % were covered after 2 days, and 38 % were covered after 4 days. The thermogel delayed the release of oxaliplatin into the systemic circulation and significantly prolonged tissue impregnation. Anastomotic fistulas were observed in the TG-Ox group (10 %) versus 0 % in the TG group (p = 0.31). Conclusions A homogeneous distribution of the thermogel throughout the peritoneal cavity was observed, and the thermogel fulfilled its functions as a drug carrier, including ensuring safety and delaying chemotherapy delivery. Treatment-induced toxicity due to oxaliplatin was identified. The concept of a rescue solution being available in operating rooms was demonstrated.

Radiofrequency ablation (RFA) of cancer induces an anti-tumor immunity, which is insufficient to prevent recurrences. In mice, RFA–intratumoral immunotherapy by granulocyte–macrophage colony-stimulating factor (GM-CSF) and Bacillus Calmette-Guerin resulted in complete metastases regression. Infectious risk in human needs replacement of live vaccines. Intratumoral purified protein derivatives (PPD) have never been tested in digestive cancers, and the safety of intratumoral immunotherapy after RFA has not yet been validated in human models. We investigated the therapeutic efficacy of combined radiofrequency ablation (RFA) and intratumoral immunotherapy (ITI) using an immune-muco-adherent thermogel (IMT) in a mouse model of metastatic colorectal cancer (CRC) and the safety of this approach in a pig model. Intratumoral stability of the immunogel was assessed using magnetic resonance imaging (MRI) and bioluminescent imaging. Seventy-four CT26 tumor-bearing female BALB/c mice were treated with RFA either alone or in combination with intratumoral IMT. Regression of distant metastasis and survival were monitored for 60 days. Six pigs that received liver radiofrequency and intralesional IMT injections were followed for 15 days. Experimental gel embolisms were treated using an intravascular approach. Pertinent rheology of IMT was confirmed in tumors, by the signal stability during 3 days in MRI and 7 days in bioluminescence imaging. In mice, the abscopal effect of RFA–intratumoral immunotherapy resulted in regression of distant lesions completed at day 16 vs. a volume of 350 ± 99.3 mm3 in the RFA group at day 25 and a 10-fold survival rate at 60 days. In pigs, injection of immunogel in the liver RFA area was safe after volume adjustment without clinical, hematological, and liver biology disorder. Flow cytometry showed an early increase in CD3 TCRγδ+T cells at D7 (p < 0.05) and a late decrease in CD29+-CD8 T cells at D15 (p < 0.05), reflecting the inflammation status changes. Systemic GM-CSF release was not detectable. Experimental caval and pulmonary thermogel embolisms were treated by percutaneous catheterism and cold serum infusion. RFA–intratumoral immunotherapy as efficient and safe mini-invasive interventional oncology is able to improve ablative treatment of colorectal liver metastases.

Persistent luminescence (PersL) is widely used for near infrared (NIR-I, 650-950 nm) imaging as they allow getting images without background. Bio-imaging in the second shortwave-infrared region SWIR-II (NIR-II, 1000-1400 nm) is less widespread but is growing as it offers the advantages of low photon scattering, increased in vivo penetration depth, and improved imaging clarity. In this work, the preparation and the complete optical properties of a new material is reported, Zn1.33Ga1.33Ni0.005Cr0.005Sn0.33O3.995 (ZGSO:Cr3+, Ni2+) able of emitting in both deep-red/NIR-I and SWIR (NIR-II) and shows its potential in bioimaging. ZGSO:Cr3+, Ni2+ can be excited using different sources such as X-rays, UV, and visible light to emit persistent signals in dual biological windows (dual-BW). By integrating an energy transfer process from Cr3+ to Ni2+ within this newly synthesized material, the influence of co-dopants on signal intensity and emission wavelengths is sought to explore. PersL at approximate to 700 nm (NIR-I) and approximate to 1300 nm (NIR-II) have been tested in preliminary bioimaging experiments using different protocols, allowing signal detection with good spatial resolution and depth sensitivity. The dual-BW PersL imaging strategy expands the toolbox for highly accurate analysis and has, for the first time, allowed access to accurately high-resolution sensing, and tracing.

Bioluminescence imaging (BLI) is an optical imaging technology used in preclinical oncology to estimate tumor size by spatio-temporal photon counting. It generally requires the biologist to hand-draw the region of interest around tumors to quantify its activity, which can be time-consuming and tricky when the subjects bear several tumors. In the latter context, we here propose to use a non-negative matrix factorization algorithm to separate the kinetics of each tumor, which is furthermore constrained by the prior prior knowledge of the tumors spatial appearance. Such prior is expressed using a sparsity constraint in a transformed wavelet domain. Results are presented on a cohort of 18 real BLIs, showing enhanced estimated spatial repartition of the tumors.

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Maleimide-containing prodrugs can quickly and selectively react with circulating serum albumin following their injection in the bloodstream. The drug-albumin complex then benefits from longer blood circulation times and better tumor accumulation. Herein, we have applied this strategy to a previously reported highly phototoxic Ru polypyridyl complex-based photosensitizer to increase its accumulation at the tumor, reduce off-target cytotoxicity, and therefore improve its pharmacological profile. Specifically, two complexes were synthesized bearing a maleimide group: one complex with the maleimide directly incorporated into the bipyridyl ligand, and the other has a hydrophilic linker between the ligand and the maleimide group. Their interaction with albumin was studied in-depth, revealing their ability to efficiently bind both covalently and noncovalently to the plasma protein. A crucial finding is that the maleimide-functionalized complexes exhibited significantly lower cytotoxicity in noncancerous cells under dark conditions compared to the nonfunctionalized complex, which is a highly desirable property for a photosensitizer. The binding to albumin also led to a decrease in the phototoxicity of the Ru bioconjugates in comparison to the nonfunctionalized complex, probably due to a decreased cellular uptake. Unfortunately, this decrease in phototoxicity was not compensated by a dramatic increase in tumor accumulation, as was demonstrated in a tumor-bearing mouse model using inductively coupled plasma mass spectrometry (ICP-MS) studies. Consequently, this study provides valuable insight into the future design of in situ albumin-binding complexes for photodynamic therapy in order to maximize their effectiveness and realize their full potential.

Local optical properties of a biological tissue, like its absorption coefficient, can yield valuable information for medical diagnosis. Extracting such information from deep inside the tissue is, however, a challenging task, due to strong light scattering, resulting in low-resolution image at depths larger than a few mm. Combining optical imaging with ultrasound (US) - the latter propagating ballistically inside the tissue, is a promising approach to solve this problem. There is indeed the example of photoacoustic imaging - where one collects US generated following light absorption to map the absorption coefficient, with successful clinical trials. Concurrently, ultrasound-modulated optical tomography (UOT) - where one collects the light scattered due to the propagation of US in the tissue (“tagged light”), can also be used to perform deep-tissue imaging. However, due to a poor signal-to-noise ratio (SNR), the latter is yet to be tested on an in vivo subject.

Bioluminescent imaging is used in oncology to measure tumoral size and activity via spatio-temporal photon emission counting. Bioluminescent signal analysis often requires delineating regions of interest around each tumor by hand, which complicates quantification in the case of mice bearing multiple tumors. In this work, we propose to use Non-Negative Matrix Factorization with data-adaptive sparsity constraints to enable automated separation of signals emitted from multiple tumors in mice. Results are presented on a set of 18 long-exposure acquisitions.

The charging of persistent luminescence using an infrared laser as a power source and an upconversion process is a recently proposed trend in the field of persistent luminescence that allows expansion of the potential applications for these materials. In bioimaging at nanoscale, it allows for increased rechargeability capacity, as the excitation wavelength is fully inside the biological window. In this work, a novel approach is proposed to this phenomenon using energy transfer on associated materials. For this purpose, β‐NaGd 0.8 Yb 0.17 Er 0.03 F 4 nanoparticles, known for their efficient upconversion, and Zn 1.33 Ga 1.335 Sn 0.33 Cr 0.005 O 4 nanoparticles, known for their persistent luminescence properties, have been synthesized and associated through a dry impregnation method. The obtained hybrid material is found to present persistent luminescence at 700 nm after charging with a 980 nm laser. A mechanism is proposed to explain this energy transfer process and the capabilities of the hybrid material as rechargeable persistent nanoprobe for in vivo applications are shown.

After excitation in the biological transparency window, chromium-doped zinc gallate nanoparticles (ZGO NPs) emit near-infrared luminescence for more than an hour, allowing long-term imaging to be performed without background autofluorescence. However, these nanoparticles are recognized in just a few minutes by serum proteins and are then trapped in the liver. In this article, we put forth that liver uptake can be delayed when coating the surface of ZGO NPs with zwitterions. We focused on the use of a very small zwitterion molecule of 330 Da derived from sulfobetaine silane (SBS) and its grafting in one step and in water onto zinc gallate nanoparticles, and we compared the colloidal stability, the in vitro interactions with serum proteins, and the biodistribution in mice with PEGylated ZGO NPs (5000 Da) prepared in two steps in organic solvent. In vitro quantification of serum protein adsorption suggests that the similarity between the sulfobetaine and the cell membrane is enough to reduce protein adsorption as much as a PEGylation, despite the difference in coating thickness and molecular weight. This study has also proved that a combination of good protein repulsion and a smaller size compared to PEGylated NPs allows similar circulation times to be obtained in mice with zwitterionic or PEG coatings. Therefore, its use could offer new opportunities for further in vivo application of functionalized ZGO derivative NPs.

Charging persistent luminescence materials using an infrared laser as a power source and an upconversion process allows expanding the potential applications for these materials. In bioimaging at the nanoscale, it allows increasing recharge ability capacity, as the excitation wavelength is fully inside the biological window. In this work, we propose a novel approach to this phenomenon using energy transfer on associated materials. For this purpose, we have synthesized β- NaGd0.8Y0.17Er0.03F4 nanoparticles, known for their efficient upconversion, and Zn1.33Ga1.335Sn0.33Cr0.005O4 nanoparticles known for their persistent luminescence properties, and we have associated them through a dry impregnation method. The obtained hybrid material presents persistent luminescence after charging with a 980 nm laser. A mechanism is proposed to explain the energy transfer process.

Flavonoids have been considered as promising molecules for cancer treatment due to their pleiotropic properties such as anti-carcinogenic, anti-angiogenic or efflux proteins inhibition. However, due to their lipophilic properties and their chemical instability, vectorization seems compulsory to administer flavonoids. Flavonoids have been co-encapsulated with other anti-cancer agents in a broad range of nanocarriers aiming to i) achieve a synergistic/additive effect at the tumor site, ii) delay drug resistance apparition by combining agents with different action mechanisms or iii) administer a lower dose of the anti-cancer drug, reducing its toxicity. However, co-encapsulation could lead to a change in the nanoparticles' diameter and drug-loading, as well as a decrease in their stability during storage. The preparation process should also take into accounts the physicochemical properties of both the flavonoid and the anti-cancer agent. Moreover, the co-encapsulation could affect the release and activity of each drug. This review aims to study the formulation, preparation and characterization strategies of these co-loaded nanomedicines, as well as their stability. The in vitro assays to predict the nanomedicines' behavior in biological fluids, as well as their in vivo efficacy, are also discussed. A special focus concerns the evaluation of their synergistic effect on tumor treatment.