Effect of stroma on the behavior of temoporfin-loaded lipid nanovesicles inside the stroma-rich head and neck carcinoma spheroids.

BACKGROUND: Despite the highly expected clinical application of nanoparticles (NPs), the translation of NPs from lab to the clinic has been relatively slow. Co-culture 3D spheroids account for the 3D arrangement of tumor cells and stromal components, e.g., cancer-associated fibroblasts (CAFs) and extracellular matrix, recapitulating microenvironment of head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated how the stroma-rich tumor microenvironment affects the uptake, penetration, and photodynamic efficiency of three lipid-based nanoformulations of approved in EU photosensitizer temoporfin (mTHPC): Foslip® (mTHPC in conventional liposomes), drug-in-cyclodextrin-in-liposomes (mTHPC-DCL) and extracellular vesicles (mTHPC-EVs). RESULTS: Collagen expression in co-culture stroma-rich 3D HNSCC spheroids correlates with the amount of CAFs (MeWo cells) in individual spheroid. The assessment of mTHPC loading demonstrated that Foslip®, mTHPC-DCL and mTHPC-EVs encapsulated 0.05 × 10- 15 g, 0.07 × 10- 15 g, and 1.3 × 10- 15 g of mTHPC per nanovesicle, respectively. The mid-penetration depth of mTHPC NPs in spheroids was 47.8 µm (Foslip®), 87.8 µm (mTHPC-DCL), and 49.7 µm (mTHPC-EVs), irrespective of the percentage of stromal components. The cellular uptake of Foslip® and mTHPC-DCL was significantly higher in stroma-rich co-culture spheroids and was increasing upon the addition of serum in the culture medium. Importantly, we observed no significant difference between PDT effect in monoculture and co-culture spheroids treated with lipid-based NPs. Overall, in all types of spheroids mTHPC-EVs demonstrated outstanding total cellular uptake and PDT efficiency comparable to other NPs. CONCLUSIONS: The stromal microenvironment strongly affects the uptake of NPs, while the penetration and PDT efficacy are less sensitive to the presence of stromal components. mTHPC-EVs outperform other lipid nanovesicles due to the extremely high loading capacity. The results of the present study enlarge our understanding of how stroma components affect the delivery of NPs into the tumors.

Forced- and Self-Rotation of Magnetic Nanorods Assembly at the Cell Membrane: A Biomagnetic Torsion Pendulum.

In order to provide insight into how anisotropic nano-objects interact with living cell membranes, and possibly self-assemble, magnetic nanorods with an average size of around 100 nm × 1 µm are designed by assembling iron oxide nanocubes within a polymeric matrix under a magnetic field. The nano-bio interface at the cell membrane under the influence of a rotating magnetic field is then explored. A complex structuration of the nanorods intertwined with the membranes is observed. Unexpectedly, after a magnetic rotating stimulation, the resulting macrorods are able to rotate freely for multiple rotations, revealing the creation of a biomagnetic torsion pendulum.

Biodegradation by Cancer Cells of Magnetite Nanoflowers with and without Encapsulation in PS-b-PAA Block Copolymer Micelles.

Magnetomicelles were produced by the self-assembly of magnetite iron oxide nanoflowers and the amphiphilic poly(styrene)-b-poly(acrylic acid) block copolymer to deliver a multifunctional theranostic agent. Their bioprocessing by cancer cells was investigated in a three-dimensional spheroid model over a 13-day period and compared with nonencapsulated magnetic nanoflowers. A degradation process was identified and monitored at various scales, exploiting different physicochemical fingerprints. At a collective level, measurements were conducted using magnetic, photothermal, and magnetic resonance imaging techniques. At the nanoscale, transmission electron microscopy was employed to identify the morphological integrity of the structures, and X-ray absorption spectroscopy was used to analyze the degradation at the crystalline phase and chemical levels. All of these measurements converge to demonstrate that the encapsulation of magnetic nanoparticles in micelles effectively mitigates their degradation compared to individual nonencapsulated magnetic nanoflowers. This protective effect consequently resulted in better maintenance of their therapeutic photothermal potential. The structural degradation of magnetomicelles occurred through the formation of an oxidized iron phase in ferritin from the magnetic nanoparticles, leaving behind empty spherical polymeric ghost shells. These results underscore the significance of encapsulation of iron oxides in micelles in preserving nanomaterial integrity and regulating degradation, even under challenging physicochemical conditions within cancer cells.

The role of tumor model in magnetic targeting of magnetosomes and ultramagnetic liposomes.

The combined passive and active targeting of tumoral tissue remains an active and relevant cancer research field. Here, we exploit the properties of two highly magnetic nanomaterials, magnetosomes and ultramagnetic liposomes, in order to magnetically target prostate adenocarcinoma tumors, implanted orthotopically or subcutaneously, to take into account the role of tumor vascularization in the targeting efficiency. Analysis of organ biodistribution in vivo revealed that, for all conditions, both nanomaterials accumulate mostly in the liver and spleen, with an overall low tumor retention. However, both nanomaterials were more readily identified in orthotopic tumors, reflecting their higher tumor vascularization. Additionally, a 2- and 3-fold increase in nanomaterial accumulation was achieved with magnetic targeting. In summary, ultramagnetic nanomaterials show promise mostly in the targeting of highly-vascularized orthotopic murine tumor models.

Ultra magnetic liposomes for MR imaging, targeting, and hyperthermia.

Magnetic liposomes offer opportunities as theranostic systems. The prerequisite for efficient imaging, tissue targeting or hyperthermia is high magnetic load of these vesicles. Here we describe the preparation of Ultra Magnetic Liposomes (UMLs), which may encapsulate iron oxide nanoparticles in a volume fraction of up to 30%. This remarkable magnetic charge provides UMLs with high magnetic mobilities, MRI relaxivities, and heating capacities for magnetic hyperthermia. Moreover, these UMLs are rapidly and efficiently internalized by cultured tumor cells and, when they are administered to mice, they can be vectorized to tumors by an external magnet.

Tuning the load of gold and magnetic nanoparticles in nanogels through their design for enhanced dual magneto-photo-thermia.

We describe a novel synthesis allowing one to enhance the load of magnetic nanoparticles and gold nanorods in nanogels. Two different structures, simple cores and core-shell, were synthesized and their heating properties upon alternating magnetic field or laser exposure are compared. Remarkably, the core-shell structure showed a greater heating capacity in the two modalities.

Immune Reprogramming Precision Photodynamic Therapy of Peritoneal Metastasis by Scalable Stem-Cell-Derived Extracellular Vesicles.

The dissemination of tumor metastasis in the peritoneal cavity, also called peritoneal metastasis (PM) or carcinomatosis, represents a late stage of gastrointestinal and gynecological cancer with very poor prognosis, even when cytoreductive surgery is effective, due to residual microscopic disease. Photodynamic therapy (PDT) in the management of peritoneal metastasis has been clinically limited by the low tumor selectivity of photosensitizers (PS) and important adverse effects. Here, we propose extracellular nanovesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) as the fourth generation of immune active PS vectors that are able to target peritoneal metastasis with superior selectivity, potentiate PDT cytotoxicity at the tumor site without affecting healthy tissues, modulate the tumor microenvironment of immunocompetent colorectal and ovarian carcinomatosis models, and promote an antitumor immune response. A pioneering strategy was developed for high yield, large-scale production of MSC-EVs encapsulating the drug meta(tetrahydroxyphenyl)chlorin (mTHPC) (EVs-mTHPC) that is compatible with requirements of clinical translation and also preserves the topology and integrity of naturally produced EVs. Intraperitoneal injection of EVs-mTHPC showed an impressive enhancement of tumoral selectivity in comparison to the free drug and to the liposomal formulation Foslip (mean ratio of PS in tumors/organs of 40 for EVs-mTHPC versus 1.5 for the free PS and 5.5 for Foslip). PDT mediated by EVs-mTHPC permitted an important tumoral necrosis (55% of necrotic tumoral nodules versus 18% for Foslip (p < 0.0001)) and promoted antitumor immune cell infiltration, mainly proinflammatory M1-like CD80+ and CD8+ T cell effector. Intratumor proliferation was significantly decreased after PDT with EVs-mTHPC. Overall EVs vectorization of mTHPC afforded important tumoral selectivity while overcoming the PDT toxicity of the free drug and prolonged mice survival in the colorectal carcinomatosis model. MSC-EVs produced by our scalable manufacturing method appears like the clinically relevant fourth-generation PDT vehicle to overcome current limitations of PDT in the treatment of peritoneal metastasis and promote a hot tumor immune environment in PM.

Enhancing digestive fistula healing by the off-label use of a thermoresponsive vessel occluder polymer associated with esophageal stent placement: A case report.

This case report relates to the first-in-man use of a vessel occluder gel medical device as a fistula occluder in a repurposing strategy. A patient with chronic colocutaneous fistula received an off-label treatment with a thermoresponsive Poloxamer 407 gel (20%) via percutaneous administration and injected under endoscopic control. Treatment consisted in the association of esophageal stent placement and gel injection. The product was administered just after the stent placement at<20°C in its liquid form, gelling at body temperature to form a fistula plug. However, the stent was removed at day 26 because of major pain and the fistula was still present. Treatment was continued a total of 14 administrations of thermoresponsive Poloxamer 407 gel during 7 weeks via the external fistula orifice. The treatment reduced fistula orifice diameter from 4.0±0.5 to 1mm and fistula daily output decreased from 425±65 to 23±4mL, when comparing the months before and after treatment. Gel administration was not associated with any toxic effects. The therapeutic outcome remained stable 1 year after treatment. The external fistula diameter and the fistula output were similar to what was observed after the last Poloxamer 407 gel administration.

Biological Fate of Magnetic Protein-Specific Molecularly Imprinted Polymers: Toxicity and Degradation.

Magnetic nanoparticles coated with protein-specific molecularly imprinted polymers (MIPs) are receiving increasing attention thanks to their binding abilities, robustness, and easy synthesis compared to their natural analogues also able to target proteins, such as antibodies or aptamers. Acting as tailor-made recognition systems, protein-specific MIPs can be used in many in vivo nanomedicine applications, such as targeted drug delivery, biosensing, and tissue engineering. Nonetheless, studies on their biocompatibility and long-term fate in biological environments are almost nonexistent, although these questions have to be addressed before considering clinical applications. To alleviate this lack of knowledge, we propose here to monitor the effect of a protein-specific MIP coating on the toxicity and biodegradation of magnetic iron oxide nanoparticles, both in a minimal aqueous degradation medium and in a model of cartilage tissue formed by differentiated human mesenchymal stem cells. Degradation of iron oxide nanoparticles with or without the polymer coating was monitored for a month by following their magnetic properties using vibrating sample magnetometry and their morphology by transmission electron microscopy. We showed that the MIP coating of magnetic iron oxide nanoparticles does not affect their biocompatibility or internalization inside cells. Remarkably, the imprinted polymer coating does not hinder the magnetic particle degradation but seems to slow it down, although this effect is more visible when degradation occurs in the buffer medium than in cells. Hence, the results presented in this paper are really encouraging and open up the way to future applications of MIP-coated nanoparticles into the clinic.

Impact of magnetic nanoparticle surface coating on their long-term intracellular biodegradation in stem cells.

Magnetic nanoparticles (MNPs) internalized within stem cells have paved the way for remote magnetic cell manipulation and imaging in regenerative medicine. A full understanding of their interactions with stem cells and of their fate in the intracellular environment is then required, in particular with respect to their surface coatings. Here, we investigated the biological interactions of MNPs composed of an identical magnetic core but coated with different molecules: phosphonoacetic acid, polyethylene glycol phosphonic carboxylic acid, caffeic acid, citric acid, and polyacrylic acid. These coatings vary in the nature of the chelating function, the number of binding sites, and the presence or absence of a polymer. The nanoparticle magnetism was systematically used as an indicator of their internalization within human stem cells and of their structural long-term biodegradation in a 3D stem cell spheroid model. Overall, we evidence that the coating impacts the aggregation status of the nanoparticles and subsequently their uptake within stem cells, but it has little effect on their intracellular degradation. Only a high number of chelating functions (polyacrylic acid) had a significant protective effect. Interestingly, when the nanoparticles aggregated prior to cellular internalization, less degradation was also observed. Finally, for all coatings, a robust dose-dependent intracellular degradation rate was demonstrated, with higher doses of internalized nanoparticles leading to a lower degradation extent.

The effect of magnetic targeting on the uptake of magnetic-fluid-loaded liposomes by human prostatic adenocarcinoma cells.

Interactions of magnetic-fluid-loaded liposomes (MFL) with human adenocarcinoma prostatic cell line PC3 were investigated in vitro. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 180 nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5 mol.% of distearylphosphatidylcholine poly(ethylene glycol)(2000) (DSPE-PEG(2000)) in the vesicle bilayer. The association processes with living cells, including binding and effective internalization, were followed versus time at two levels. On one hand, the lipid vesicles labeled by 1 mol.% of rhodamine-marked phosphatidylethanolamine were imaged by confocal fluorescence microscopy. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. This allowed modeling of MFL uptake kinetics as a two-step process involving first binding adsorption onto the outer cell membrane followed by subsequent internalization. Capture efficiency was significantly improved by guiding MFL in the near vicinity of the cells by means of a 0.29-T external magnet developing a magnetic field gradient close to 30 mT/mm. Double detection of lipids by fluorescence tracking and of iron oxide by magnetophoresis showed excellent correlation. This demonstrated that MFL associate with tumor cells as intact vesicle structures which conserve their internal content.

Modification of Extracellular Vesicles by Fusion with Liposomes for the Design of Personalized Biogenic Drug Delivery Systems.

Extracellular vesicles (EVs) are recognized as nature's own carriers to transport macromolecules throughout the body. Hijacking this endogenous communication system represents an attractive strategy for advanced drug delivery. However, efficient and reproducible loading of EVs with therapeutic or imaging agents still represents a bottleneck for their use as a drug delivery system. Here, we developed a method for modifying cell-derived EVs through their fusion with liposomes containing both membrane and soluble cargoes. The fusion of EVs with functionalized liposomes was triggered by polyethylene glycol (PEG) to create smart biosynthetic hybrid vectors. This versatile method proved to be efficient to enrich EVs with exogenous lipophilic or hydrophilic compounds, while preserving their intrinsic content and biological properties. Hybrid EVs improved cellular delivery efficiency of a chemotherapeutic compound by a factor of 3-4, as compared to the free drug or the drug-loaded liposome precursor. On one side, this method allows the biocamouflage of liposomes by enriching their lipid bilayer and inner compartment with biogenic molecules. On the other side, the proposed fusion strategy enables efficient EV loading, and the pharmaceutical development of EVs with adaptable activity and drug delivery property.

Monitoring the dynamics of cell-derived extracellular vesicles at the nanoscale by liquid-cell transmission electron microscopy.

Cell-derived extracellular vesicles (EVs) circulating in body fluids hold promises as bioactive therapeutic agents and as biomarkers to diagnose a wide range of diseases. However nano-imaging methods are needed to characterize these complex and heterogeneous soft materials in their native wet environment. Herein, we exploit liquid-cell transmission electron microscopy (LCTEM) to characterize the morphology and dynamic behavior of EVs in physiological media with nanometer resolution. The beam-induced controlled growth of Au nanoparticles on bilayer membranes is used as an original in situ staining method to improve the contrast of EVs and artificial liposomes. LCTEM provides information about the size distribution and concentration of EVs that are consistent with Cryo-TEM and nanoparticle tracking analysis measurements. Moreover, LCTEM gives a unique insight into the dynamics of EVs depending on their liquid environment. The size-dependent morphology of EVs is sensitive to osmotic stress which tends to transform their spherical shape to ellipsoidal, stomatocyte or discocyte morphologies. In the liquid-cell, EVs exhibit a sub-diffusive motion due to strong interactions between the Au nanoparticles and the liquid-cell windows. Finally, the high-resolution monitoring of EV aggregation and fusion illustrate that LCTEM opens up a new way to study cell-membrane dynamics.

mTHPC-loaded extracellular vesicles outperform liposomal and free mTHPC formulations by an increased stability, drug delivery efficiency and cytotoxic effect in tridimensional model of tumors.

Efficient photodynamic therapy with meta-tetra(hydroxyphenyl)chlorine requires the application of specific nanoformulations. mTHPC liposomal formulation (Foslip®) demonstrated favorable pharmacokinetics properties. However, rapid liposomes destruction in circulation and rapid mTHPC release impedes Foslip® applications. Alternatively, mTHPC nanovectorization using extracellular vesicles (EVs) could be an attractive option. EVs are naturally secreted by the organism to play a role in intercellular communication due to the capacity to transport proteins and nucleic acids. EVs also possess a natural ability to deliver therapeutic molecules into cancer cells. The aim of the present study was to evaluate photophysical and photobiological properties of mTHPC loaded in endothelial EVs as nanocarriers. We also studied efficiency of nanovectorisation on mTHPC distribution and PDT activity in multicellular tumor spheroids (MCTSs). MCTS is a nonvascularized in vitro 3D model of cells that mimics a similar microenvironment to in vivo situation. mTHPC-EVs were characterized by means of spectroscopic techniques, flow cytometry and nanoparticle tracking analysis. Compared with Foslip®, mTHPC-EVs are stable in murine plasma. Better mTHPC accumulation and penetration (up to 100 µm) in MCTS was observed for mTHPC-EVs compared with liposomal mTHPC. These factors could explain enhanced photodynamic activity of mTHPC-EVs compared with free and liposomal mTHPC. The light dose inducing 50% of cell death with mTHPC-EVs was 4 and 2.5-times lower than that of free and liposomal mTHPC. The obtained results demonstrate that EVs should be considered as perspective nanocarriers for mTHPC-mediated PDT.

Thermoresponsive Gel Embedded with Adipose Stem-Cell-Derived Extracellular Vesicles Promotes Esophageal Fistula Healing in a Thermo-Actuated Delivery Strategy.

Extracellular vesicles (EVs) are increasingly envisioned as the next generation of biological pro-regenerative nanotherapeutic agents, as has already been demonstrated for heart, kidney, liver, and brain tissues; lung injury repair; and skin regeneration. Herein, we explore another potential EV therapeutic application, fistula healing, together with a local minimally invasive delivery strategy. Allogenic extracellular vesicles (EVs) from adipose tissue-derived stromal cells (ASCs) are administered in a porcine fistula model through a thermoresponsive Pluronic F-127 (PF-127) gel, injected locally at 4 °C and gelling at body temperature to retain EVs in the entire fistula tract. Complete fistula healing is reported to be 100% for the gel plus EVs group, 67% for the gel group, and 0% for the control, supporting the therapeutic use of Pluronic F-127 gel alone or combined with EVs. However, only the combination of gel and EVs results in a statistically significant (i) reduction of fibrosis, (ii) decline of inflammatory response, (iii) decrease in the density of myofibroblasts, and (iv) increase of angiogenesis. Overall, we demonstrate that ASC-EV delivery into a PF-127 gel represents a successful local minimally invasive strategy to induce a therapeutic effect in a swine fistula model. Our study presents prospects for EV administration strategies and for the management of post-operative fistulas.

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages.

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.

Combining magnetic hyperthermia and photodynamic therapy for tumor ablation with photoresponsive magnetic liposomes.

The ongoing nanotech revolution has the potential to transform diagnostic and therapeutic methods. Stimuli-triggered nanotherapies based on remotely activated agents have become attractive alternatives to conventional chemotherapy. Herein, we designed an optimized smart nanoplatform based on dually loaded hybrid liposomes to achieve enhanced tumor therapy. The aqueous core was highly loaded with iron oxide nanoparticles, while the lipid bilayer was supplied with a photosensitizer payload. The double cargo translated into double functionality: generation of singlet oxygen under laser excitation and heat production under alternating magnetic field stimulation, coupling photodynamic therapy (PDT) to magnetic hyperthermia (MHT). These liposomes address both therapeutic agents within tumor cells, and the combined PDT/MHT therapy resulted in complete cancer cell death in vitro while total solid-tumor ablation was achieved in an in vivo rodent model.

Heat-generating iron oxide nanocubes: subtle "destructurators" of the tumoral microenvironment.

Several studies propose nanoparticles for tumor treatment, yet little is known about the fate of nanoparticles and intimate interactions with the heterogeneous and ever-evolving tumor environment. The latter, rich in extracellular matrix, is responsible for poor penetration of therapeutics and represents a paramount issue in cancer therapy. Hence new strategies start aiming to modulate the neoplastic stroma. From this perspective, we assessed the efficacy of 19 nm PEG-coated iron oxide nanocubes with optimized magnetic properties to mediate mild tumor magnetic hyperthermia treatment. After injection of a low dose of nanocubes (700 µg of iron) into epidermoid carcinoma xenografts in mice, we monitored the effect of heating nanocubes on tumor environment. In comparison with the long-term fate after intravenous administration, we investigated spatiotemporal patterns of nanocube distribution, evaluated the evolution of cubes magnetic properties, and examined nanoparticle clearance and degradation processes. While inside tumors nanocubes retained their magnetic properties and heating capacity throughout the treatment due to a mainly interstitial extracellular location, the particles became inefficient heaters after cell internalization and transfer to spleen and liver. Our multiscale analysis reveals that collagen-rich tumor extracellular matrix confines the majority of nanocubes. However, nanocube-mediated hyperthermia has the potential to "destructure" this matrix and improve nanoparticle and drug penetration into neoplastic tissue. This study provides insight into dynamic interactions between nanoparticles and tumor components under physical stimulation and suggests that nanoparticle-mediated hyperthermia could be used to locally modify tumor stroma and thus improve drug penetration.

Cell-derived vesicles as a bioplatform for the encapsulation of theranostic nanomaterials.

There is a great deal of interest in the development of nanoplatforms gathering versatility and multifunctionality. The strategy reported herein meets these requirements and further integrates a cell-friendly shell in a bio-inspired approach. By taking advantage of a cell mechanism of biomolecule transport using vesicles, we engineered a hybrid biogenic nanoplatform able to encapsulate a set of nanoparticles regardless of their chemistry or shape. As a proof of versatility, different types of hybrid nanovesicles were produced: magnetic, magnetic-metallic and magnetic-fluorescent vesicles, either a single component or multiple components, combining the advantageous properties of each integrant nanoparticle. These nanoparticle-loaded vesicles can be manipulated, monitored by MRI and/or fluorescence imaging methods, while acting as efficient nano-heaters. The resulting assets for targeting, imaging and therapy converge for the outline of a new generation of nanosystems merging versatility and multifunctionality into a bio-camouflaged and bio-inspired approach.