Methacrylated Cellulose Nanocrystals as Fillers for the Development of Photo-Cross-Linkable Cytocompatible Biosourced Formulations Targeting 3D Printing.

Cellulose nanocrystals (CNCs) from cotton were functionalized in aqueous medium using methacrylic anhydride (MA) to produce methacrylated cellulose nanocrystals (mCNCs) with a degree of methacrylation (DM) up to 12.6 ± 0.50%. Dispersible as-prepared CNCs and mCNCs were then considered as reinforcing fillers for aqueous 3D-printable formulations based on methacrylated carboxymethylcellulose (mCMC). The rheological properties of such photo-cross-linkable aqueous formulations containing nonmodified CNCs or mCNCs at 0.2 or 0.5 wt% in 2 wt% mCMC were fully investigated. The influence of the presence of nanoparticles on the UV-curing kinetics and dimensions of the photo-cross-linked hydrogels was probed and 13C CP-MAS NMR spectroscopy was used to determine the maximum conversion ratio of methacrylates as well as the optimized time required for UV postcuring. The viscoelasticity of cross-linked hydrogels and swollen hydrogels was also studied. The addition of 0.5 wt% mCNC with a DM of 0.83 ± 0.040% to the formulation yielded faster cross-linking kinetics, better resolution, more robust cross-linked hydrogels, and more stable swollen hydrogels than pure mCMC materials. Additionally, the produced cryogels showed no cytotoxicity toward L929 fibroblasts. This biobased formulation could thus be considered for the 3D printing of hydrogels dedicated to biomedical purposes using vat polymerization techniques, such as stereolithography or digital light processing.

Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE-/- mice.

Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

Measuring Particle Size Distribution by Asymmetric Flow Field Flow Fractionation: A Powerful Method for the Preclinical Characterization of Lipid-Based Nanoparticles.

Particle size distribution and stability are key attributes for the evaluation of the safety and efficacy profile of medical nanoparticles (Med-NPs). Measuring particle average size and particle size distribution is a challenging task which requires the combination of orthogonal high-resolution sizing techniques, especially in complex biological media. Unfortunately, despite its limitations, due to its accessibility, low cost, and easy handling, batch mode dynamic light scattering (DLS) is still very often used as the only approach to measure particle size distribution in the nanomedicine field. In this work the use of asymmetric flow field flow fractionation coupled to multiangle light scattering and dynamic light scattering detectors (AF4-MALS-DLS) was evaluated as an alternative to batch mode DLS to measure the physical properties of lipid-based nanoparticles. A robust standard operating procedure (SOPs) developed by the Nanomedicine Characterization Laboratory (EUNCL) was presented and tested to assess size stability, batch to batch consistency, and the behavior of the lipid-based nanoparticles in plasma. Orthogonal sizing techniques, such as transmission electron microscopy (TEM) and particle tracking analysis (PTA) measurements, were performed to support the results. While batch mode DLS could be applied as a fast and simple method to provide a preliminary insight into the integrity and polydispersity of samples, it was unsuitable to resolve small modifications of the particle size distribution. The introduction of nanoparticle sorting by field-flow fractionation coupled to online DLS and MALS allowed assessment of batch to batch variability and changes in the size of the lipid nanoparticles induced by the interaction with serum proteins, which are critical for quality control and regulatory aspects. In conclusion, if a robust SOP is followed, AF4-MALS-DLS is a powerful method for the preclinical characterization of lipid-based nanoparticles.

Quantification of Surface GalNAc Ligands Decorating Nanostructured Lipid Carriers by UPLC-ELSD.

Nanoparticles have been extensively studied for drug delivery and targeting to specific organs. The functionalization of the nanoparticle surface by site-specific ligands (antibodies, peptides, saccharides) can ensure efficient recognition and binding with relevant biological targets. One of the main challenges in the development of these decorated nanocarriers is the accurate quantification of the amount of ligands on the nanoparticle surface. In this study, nanostructured lipid carriers (NLC) were functionalized with N-acetyl-D-galactosamine (GalNAc) units, known to target the asialoglycoprotein receptor (ASGPR). Different molar percentages of GalNAc-functionalized surfactant (0%, 2%, 5%, and 14%) were used in the formulation. Based on ultra-high-performance liquid chromatography separation and evaporative light-scattering detection (UPLC-ELSD), an analytical method was developed to specifically quantify the amount of GalNAc units present at the NLC surface. This method allowed the accurate quantification of GalNAc surfactant and therefore gave some insights into the structural parameters of these multivalent ligand systems. Our data show that the GalNAc decorated NLC possess large numbers of ligands at their surface and suitable distances between them for efficient multivalent interaction with the ASGPR, and therefore promising liver-targeting efficiency.

Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about -5 - -10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation.

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.

FRET imaging approaches for in vitro and in vivo characterization of synthetic lipid nanoparticles.

DiI and DiD, two fluorophores able to interact by FRET (Förster resonance energy transfer), were coencapsulated in the core of lipid nanocapsules (LNCs) and nanoemulsions (LNEs), lipophilic reservoirs for the delivery of drugs. The ability of FRET imaging to provide information on the kinetics of dissociation of the nanoparticles in the presence of bovine serum albumin (BSA) or whole serum, or after incubation with cancer cells, and after systemic administration in tumor-bearing mice, was studied. Both microscopic and macroscopic imaging was performed to determine the behavior of the nanostructures in a biological environment. When 2 mg/mL FRET LNEs or LNCs were dispersed in buffer, in the presence of unloaded nanoparticles, BSA, or in whole serum, the presence of serum was the most active in destroying the particles. This occurred immediately with a diminution of 20% of FRET, then slowly, ending up with still 30% intact nanoparticles at 24 h. LNCs were internalized rapidly in cultured cells with the FRET signal decreasing within the first minutes of incubation, and then a plateau was reached and LNCs remained intact during 3 h. In contrast, LNEs were poorly internalized and were rapidly dissociated after internalization. Following their iv injection, LNCs appeared very stable in subcutaneous tumors implanted in mice. Intact particles were found using microscopic FRET determination on tumor sections 24 h after injection, that correlated well with the 8% calculated noninvasively on live animals. FRET investigations showed the potential to determine valid and reliable information about in vitro and in vivo behavior of nanoparticles.

Resorbable conductive materials for optimally interfacing medical devices with the living.

Implantable and wearable bioelectronic systems are arising growing interest in the medical field. Linking the microelectronic (electronic conductivity) and biological (ionic conductivity) worlds, the biocompatible conductive materials at the electrode/tissue interface are key components in these systems. We herein focus more particularly on resorbable bioelectronic systems, which can safely degrade in the biological environment once they have completed their purpose, namely, stimulating or sensing biological activity in the tissues. Resorbable conductive materials are also explored in the fields of tissue engineering and 3D cell culture. After a short description of polymer-based substrates and scaffolds, and resorbable electrical conductors, we review how they can be combined to design resorbable conductive materials. Although these materials are still emerging, various medical and biomedical applications are already taking shape that can profoundly modify post-operative and wound healing follow-up. Future challenges and perspectives in the field are proposed.

Influence of size, surface coating and fine chemical composition on the in vitro reactivity and in vivo biodistribution of lipid nanocapsules versus lipid nanoemulsions in cancer models.

Lipid nanocapsules (LNCs) and lipid nanoemulsions (LNEs) are biomimetic synthetic nanocarriers. Their in vitro and in vivo performance was evaluated as a function of their size (25, 50 and 100 nm) and the surface PEG chain length. Analysis methods included complement activation test, particle uptake in macrophage and HEK293(ß3) cells and biodistribution studies with tumor-grafted mice by fluorescence imaging. A particular attention was paid to keep the concentration of each nanocarrier and to the amount of fluorescent dye in comparable conditions between the in vitro and in vivo studies. Under these conditions, no significant differences were found among the three tested particle sizes and the two nanocarrier types. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers. Passive accumulation of LNCs and LNEs in different tumor types depended on the degree of tumor vascularization. FROM THE CLINICAL EDITOR: This study of lipid nanocapsules and lipid nanoemulsions compares their vitro and in vivo performance as a function of size and surface PEG chain length, demonstrating no significant difference among the tested particle sizes. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers.

Design of hyaluronan-based dopant for conductive and resorbable PEDOT ink.

Conformable biocompatible conductive materials are increasingly sought for the development of bioelectronics. If additionally resorbable, they could serve for the design of transient implantable electronic devices, opening the way to new healthcare applications. Hyaluronan (HA) derivatives including sulfate and aminophenylboronic acid (PBA) groups (HAS-PBA) were therefore designed to serve as dopants of poly(3,4-ethylenedioxy)thiophene (PEDOT). The optimized HA sulfation protocol allowed good control on polymer sulfation degree while minimizing polymer chain degradation. Sulfated HA was shown to be degradable in physiological conditions. A synergy was observed between the sulfate negative charges and the PBA aromatic groups promoting hydrophobic interactions and π-stacking between PEDOT and HAS-PBA, to boost the material conductivity that reached 1.6 ± 0.2 S/cm in physiological conditions. Moreover the PEDOT:HAS-PBA material was not cytotoxic and could be formulated for easy processing by inkjet printing, appearing as promising candidate for the design of soft transient electronics for in vivo applications.

Fluorescent nanoprobes dedicated to in vivo imaging: from preclinical validations to clinical translation.

With the fast development, in the last ten years, of a large choice of set-ups dedicated to routine in vivo measurements in rodents, fluorescence imaging techniques are becoming essential tools in preclinical studies. Human clinical uses for diagnostic and image-guided surgery are also emerging. In comparison to low-molecular weight organic dyes, the use of fluorescent nanoprobes can improve both the signal sensitivity (better in vivo optical properties) and the fluorescence biodistribution (passive "nano" uptake in tumours for instance). A wide range of fluorescent nanoprobes have been designed and tested in preclinical studies for the last few years. They will be reviewed and discussed considering the obstacles that need to be overcome for their potential everyday use in clinics. The conjugation of fluorescence imaging with the benefits of nanotechnology should open the way to new medical applications in the near future.

A multistep in vitro hemocompatibility testing protocol recapitulating the foreign body reaction to nanocarriers.

The development of drug nanocarriers based on polymeric, lipid and ceramic biomaterials has been paving the way to precision medicine, where the delivery of poorly soluble active compounds and personalized doses are made possible. However, the nano-size character of these carriers has been demonstrated to have the potential to elicit pathways of the host response different from those of the same biomaterials when engineered as larger size implants and of the drugs when administered without a carrier. Therefore, a specific regulatory framework needs to be made available that can offer robust scientific insights and provide safety data by reliable tests of these novel nano-devices. In this context, the present work presents a multistep protocol for the in vitro assessment of the hemocompatibility of nanocarriers of different physicochemical properties. Poly (ethyl butyl cyanoacrylate) nanoparticles and lipid-based (LipImage™ 815) nanoparticles of comparable hydrodynamic diameter were tested through a battery of assays using human peripheral blood samples and recapitulating the main pathways of the host response upon systemic administration; i.e., protein interactions, fibrinogen-platelet binding, cytotoxicity, and inflammatory response. The data showed the sensitivity and reproducibility of the methods adopted that were also demonstrated to determine individual variability as well as to discriminate between activation of pathways of inflammation and unintended release of inflammatory signaling caused by loss of cell integrity. Therefore, this multistep testing is proposed as a reliable protocol for nanoparticle development and emerging regulatory frameworks.

A comparative biodistribution study of polymeric and lipid-based nanoparticles.

Biodistribution of nanoencapsulated bioactive compounds is primarily determined by the size, shape, chemical composition and surface properties of the encapsulating nanoparticle, and, thus, less dependent on the physicochemical properties of the active pharmaceutical ingredient encapsulated. In the current work, we aimed to investigate the impact of formulation type on biodistribution profile for two clinically relevant nanoformulations. We performed a comparative study of biodistribution in healthy rats at several dose levels and durations up to 14-day post-injection. The studied nanoformulations were nanostructured lipid carriers incorporating the fluorescent dye IR780-oleyl, and polymeric nanoparticles containing the anticancer agent cabazitaxel. The biodistribution was approximated by quantification of the cargo in blood and relevant organs. Several clear and systematic differences in biodistribution were observed, with the most pronounced being a much higher (more than 50-fold) measured concentration ratio between cabazitaxel in all organs vs. blood, as compared to IR780-oleyl. Normalized dose linearity largely showed opposite trends between the two compounds after injection. Cabazitaxel showed a higher brain accumulation than IR780-oleyl with increasing dose injected. Interestingly, cabazitaxel showed a notable and prolonged accumulation in lung tissue compared to other organs. The latter observations could warrant further studies towards a possible therapeutic indication within lung and conceivably brain cancer for nanoformulations of this highly antineoplastic compound, for which off-target toxicity is currently dose-limiting in the clinic.

Luminescence of polyethylene glycol coated CdSeTe/ZnS and InP/ZnS nanoparticles in the presence of copper cations.

The use of click chemistry for quantum dot (QD) functionalization could be very promising for the development of bioconjugates dedicated to in vivo applications. Alkyne-azide ligation usually requires copper(I) catalysis. The luminescence response of CdSeTe/ZnS nanoparticles coated with polyethylene glycol (PEG) is studied in the presence of copper cations, and compared to that of InP/ZnS QDs coated with mercaptoundecanoic acid (MUA). The quenching mechanisms appear different. Luminescence quenching occurs without any wavelength shift in the absorption and emission spectra for the CdSeTe/ZnS/PEG nanocrystals. In this case, the presence of copper in the ZnS shell is evidenced by energy-filtered transmission electron microscopy (EF-TEM). By contrast, in the case of InP/ZnS/MUA nanocrystals, a redshift of the excitation and emission spectra, accompanied by an increase in absorbance and a decrease in photoluminescence, is observed. For CdSeTe/ZnS/PEG nanocrystals, PL quenching is enhanced for QDs with 1) smaller inorganic-core diameter, 2) thinner PEG shell, and 3) hydroxyl terminal groups. Whereas copper-induced PL quenching can be interesting for the design of sensitive cation sensors, copper-free click reactions should be used for the efficient functionalization of nanocrystals dedicated to bioapplications, in order to achieve highly luminescent QD bioconjugates.

How to prepare and stabilize very small nanoemulsions.

Practical and theoretical considerations that apply when aiming to formulate by ultrasonication very small nanoemulsions (particle diameter up to 150 nm) with very high stability are presented and discussed. The droplet size evolution during sonication can be described by a monoexponential function of the sonication time, the characteristic time scale depending essentially on the applied power. A unique master curve is obtained when plotting the mean diameter size evolution as a function of sonication energy. We then show that Ostwald ripening remains the main destabilization mechanism whereas coalescence can be easily prevented due to the nanometric size of droplets. The incorporation of "trapped species" within the droplet interior is able to counteract Ostwald ripening, and this concept can be extended to the membrane compartment. We finally clarify that nanoemulsions are not thermodynamically stable systems, even in the case where their composition lies very close to the demixing line of a thermodynamically stable microemulsion domain. However, as exemplified in the present work, nanoemulsion systems can present very long-term kinetic stability.

Development, formulation, and cellular mechanism of a lipophilic copper chelator for the treatment of Wilson's disease.

Copper homeostasis is finely regulated in human to avoid any detrimental impact of free intracellular copper ions. Upon copper accumulation, biliary excretion is triggered in liver thanks to trafficking of the ATP7B copper transporter to bile canaliculi. However, in Wilson's disease this protein is mutated leading to copper accumulation. Current therapy uses Cu chelators acting extracellularly and requiring a life-long treatment with side effects. Herein, a new Cu(I) pro-chelator was encapsulated in long-term stable nanostructured lipid carriers. Cellular assays revealed that the pro-chelator protects hepatocytes against Cu-induced cell death. Besides, the cellular stresses induced by moderate copper concentrations, including protein unfolding, are counteracted by the pro-chelator. These data showed the pro-chelator efficiency to deliver intracellularly an active chelator that copes with copper stress and surpasses current and under development chelators. Although its biological activity is more mitigated, the pro-chelator nanolipid formulation led to promising results. This innovative approach is of outmost importance in the quest of better treatments for Wilson's disease.

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.

Copper-free click chemistry for highly luminescent quantum dot conjugates: application to in vivo metabolic imaging.

Quantum dots (QD) are inorganic nanocrystals with outstanding optical properties, specially suited for biological imaging applications. Their attachment to biomolecules in mild aqueous conditions for the design of bioconjugates is therefore highly desirable. 1,3-dipolar [3 + 2] cycloaddition between azides and terminal alkynes ("click chemistry") could represent an attractive QD functionalization method. Unfortunately, the use of the popular Cu(I)-catalyzed version of this reaction is not applicable for achieving this goal, since the presence of copper dramatically alters the luminescence properties of QD dispersions. We demonstrate here that copper-free click chemistry, between strained cyclooctyne functionalized QD and azido-biomolecules, leads to highly luminescent conjugates. In addition, we show that QD-cyclooctyne can be used at previously unreported low concentration (250 nM) for imaging the incorporation of azido-modified sialic acid in cell membrane glycoproteins.

In vivo imaging of quantum dots.

Noninvasive whole-body near-infrared fluorescence imaging is now acknowledged as a powerful method for the molecular mapping of biological events in live small animals such as mouse models. With outstanding optical properties such as high fluorescence quantum yields and low photobleaching rates, quantum dots (QDs) are labels of choice in the near-infrared domain. The main applications described in the literature for in vivo imaging of mice after injection of QDs encompass imaging of lymph nodes and tumors and cell tracking. Standard methods for the preparation, the purification, and the in vivo fluorescence whole-body imaging of QDs in the live mouse are described. Nanoparticles coated by PEG chains of different sizes and terminal groups are prepared using 705-nm-emitting commercial QDs. Their biodistribution after intravenous or intradermal injections in tumor-bearing mice is reported here.

Verteporfin-Loaded Lipid Nanoparticles Improve Ovarian Cancer Photodynamic Therapy In Vitro and In Vivo.

Advanced ovarian cancer is the most lethal gynecological cancer, with a high rate of chemoresistance and relapse. Photodynamic therapy offers new prospects for ovarian cancer treatment, but current photosensitizers lack tumor specificity, resulting in low efficacy and significant side-effects. In the present work, the clinically approved photosensitizer verteporfin was encapsulated within nanostructured lipid carriers (NLC) for targeted photodynamic therapy of ovarian cancer. Cellular uptake and phototoxicity of free verteporfin and NLC-verteporfin were studied in vitro in human ovarian cancer cell lines cultured in 2D and 3D-spheroids, and biodistribution and photodynamic therapy were evaluated in vivo in mice. Both molecules were internalized in ovarian cancer cells and strongly inhibited tumor cells viability when exposed to laser light only. In vivo biodistribution and pharmacokinetic studies evidenced a long circulation time of NLC associated with efficient tumor uptake. Administration of 2 mg.kg-1 free verteporfin induced severe phototoxic adverse effects leading to the death of 5 out of 8 mice. In contrast, laser light exposure of tumors after intravenous administration of NLC-verteporfin (8 mg.kg-1) significantly inhibited tumor growth without visible toxicity. NLC-verteporfin thus led to efficient verteporfin vectorization to the tumor site and protection from side-effects, providing promising therapeutic prospects for photodynamic therapy of cancer.