Uniform trehalose nanogels for glucagon stabilization.

Glucagon is a peptide hormone that acts via receptor-mediated signaling predominantly in the liver to raise glucose levels by hepatic glycogen breakdown or conversion of noncarbohydrate, 3 carbon precursors to glucose by gluconeogenesis. Glucagon is administered to reverse severe hypoglycemia, a clinical complication associated with type 1 diabetes. However, due to low stability and solubility at neutral pH, there are limitations in the current formulations of glucagon. Trehalose methacrylate-based nanoparticles were utilized as the stabilizing and solubilizing moiety in the system reported herein. Glucagon was site-selectively modified to contain a cysteine at amino acid number 24 to covalently attach to the methacrylate-based polymer containing pyridyl disulfide side chains. PEG2000 dithiol was employed as the crosslinker to form uniform nanoparticles. Glucagon nanogels were monitored in Dulbecco's phosphate-buffered saline (DPBS) pH 7.4 at various temperatures to determine its long-term stability in solution. Glucagon nanogels were stable up to at least 5 months by size uniformity when stored at -20 °C and 4 °C, up to 5 days at 25 °C, and less than 12 hours at 37 °C. When glucagon stability was studied by either HPLC or thioflavin T assays, the glucagon was intact for at least 5 months at -20 °C and 4 °C within the nanoparticles at -20 °C and 4 °C and up to 2 days at 25 °C. Additionally, the glucagon nanogels were studied for toxicity and efficacy using various assays in vitro. The findings indicate that the nanogels were nontoxic to fibroblast cells and nonhemolytic to red blood cells. The glucagon in the nanogels was as active as glucagon alone. These results demonstrate the utility of trehalose nanogels towards a glucagon formulation with improved stability and solubility in aqueous solutions, particularly useful for storage at cold temperatures.

Noncovalent Enzyme Nanogels via a Photocleavable Linkage.

Enzyme nanogels (ENGs) offer a convenient method to protect therapeutic proteins from in vivo stressors. Current methodologies to prepare ENGs rely on either covalent modification of surface residues or the noncovalent assembly of monomers at the protein surface. In this study, we report a new method for the preparation of noncovalent ENGs that utilizes a heterobifunctional, photocleavable monomer as a hybrid approach. Initial covalent modification with this monomer established a polymerizable handle at the protein surface, followed by radical polymerization with poly(ethylene glycol) methacrylate monomer and ethylene glycol dimethacrylate crosslinker in solution. Final photoirradiation cleaved the linkage between the polymer and protein to afford the noncovalent ENGs. The enzyme phenylalanine ammonia lyase (PAL) was utilized as a model protein yielding well-defined nanogels 80 nm in size by dynamic light scattering (DLS) and 76 nm by atomic force microscopy. The stability of PAL after exposure to trypsin or low pH was assessed and was found to be more stable in the noncovalent nanogel compared to PAL alone. This approach may be useful for the stabilization of active enzymes.

Synthesis and Application of Trehalose Materials.

Trehalose is a naturally occurring, nonreducing disaccharide that is widely used in the biopharmaceutical, food, and cosmetic industries due to its stabilizing and cryoprotective properties. Over the years, scientists have developed methodologies to synthesize linear polymers with trehalose units either in the polymer backbone or as pendant groups. These macromolecules provide unique properties and characteristics, which often outperform trehalose itself. Additionally, numerous reports have focused on the synthesis and formulation of materials based on trehalose, such as nanoparticles, hydrogels, and thermoset networks. Among many applications, these polymers and materials have been used as protein stabilizers, as gene delivery systems, and to prevent amyloid aggregate formation. In this Perspective, recent developments in the synthesis and application of trehalose-based linear polymers, hydrogels, and nanomaterials are discussed, with a focus on utilization in the biomedical field.

Poly(trehalose methacrylate) as an Excipient for Insulin Stabilization: Mechanism and Safety.

Insulin, the oldest U.S. Food and Drug Administration (FDA)-approved recombinant protein and a World Health Organization (WHO) essential medicine for treating diabetes globally, faces challenges due to its storage instability. One approach to stabilize insulin is the addition of poly(trehalose methacrylate) (pTrMA) as an excipient. The polymer increases the stability of the peptide to heat and mechanical agitation and has a low viscosity suitable for injection and pumps. However, the safety and stabilizing mechanism of pTrMA is not yet known and is required to understand the potential suitability of pTrMA as an insulin excipient. Herein is reported the immune response, biodistribution, and insulin plasma lifetime in mice, as well as investigation into insulin stabilization. pTrMA alone or formulated with ovalbumin did not elicit an antibody response over 3 weeks in mice, and there was no observable cytokine production in response to pTrMA. Micropositron emission tomography/microcomputer tomography of 64Cu-labeled pTrMA showed excretion of 78-79% ID/cc within 24 h and minimal liver accumulation at 6-8% ID/cc when studied out to 120 h. Further, the plasma lifetime of insulin in mice was not altered by added pTrMA. Formulating insulin with 2 mol equiv of pTrMA improved the stability of insulin to standard storage conditions: 46 weeks at 4 °C yielded 87.0% intact insulin with pTrMA present as compared to 7.8% intact insulin without the polymer. The mechanism by which pTrMA-stabilized insulin was revealed to be a combination of inhibiting deamidation of amino acid residues and preventing fibrillation, followed by aggregation of inactive and immunogenic amyloids all without complexing insulin into its hexameric state, which could delay the onset of insulin activity. Based on the data reported here, we suggest that pTrMA stabilizes insulin as an excipient without adverse effects in vivo and is promising to investigate further for the safe formulation of insulin.

Light sheet fluorescence microscopy versus confocal microscopy: in quest of a suitable tool to assess drug and nanomedicine penetration into multicellular tumor spheroids.

We recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the three-dimensional (3D) architecture of solid tumors. In this study, we used these hetero-type spheroids as a tool to assess the penetration of doxorubicin (used as a model drug) through the whole tumor mass either in a free form or loaded into polymer nanoparticles (NPs), and we investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM), would be best to provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to accurately monitor the diffusion of small molecules such as the doxorubicin. Indeed, it only allowed to scan a layer of 100 µm depth and no information on deeper layers could be available because of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type multicellular tumor spheroids (MCTS) was obtained by LSFM and multi-view image fusion which revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm in diameter. However, no doxorubicin-loaded polymer nanoparticles were detected in the spheroids, highlighting the challenge of nanomedicine delivery through biological barriers. Overall, the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in tumors. Accordingly, it could provide useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.

Protein-functionalized nanoparticles derived from end-functional polymers and polymer prodrugs for crossing the blood-brain barrier.

Nanoparticles may provide a viable way for neuroprotective drugs to cross the blood-brain barrier (BBB), which limits the passage of most drugs from the peripheral circulation to the brain. Heterotelechelic polymer prodrugs comprising a neuroprotective model drug (adenosine) and a maleimide functionality were synthesized by the "drug-initiated" approach and subsequent nitroxide exchange reaction. Nanoparticles were obtained by nanoprecipitation and exhibited high colloidal stability with diameters in the 162-185 nm range and narrow size distributions. Nanoparticles were then covalently surface-conjugated to different proteins (albumin, α2-macroglobulin and fetuin A) to test their capability of enhancing BBB translocation. Their performances in terms of endothelial permeability and cellular uptake in an in vitro BBB model were compared to that of similar nanoparticles with surface-adsorbed proteins, functionalized or not with the drug. It was shown that bare NPs (i.e., NPs not surface-functionalized with proteins) without the drug exhibited significant permeability and cellular uptake, which were further enhanced by NP surface functionalization with α2-macroglobulin. However, the presence of the drug at the polymer chain-end prevented efficient passage of all types of NPs through the BBB model, likely due to adecrease in the hydrophobicity of the nanoparticle surface and alteration of the protein binding/coupling, respectively. These results established a new and facile synthetic approach for the surface-functionalization of polymer nanoparticles for brain delivery purposes.

Drug-Initiated Synthesis of Heterotelechelic Polymer Prodrug Nanoparticles for in Vivo Imaging and Cancer Cell Targeting.

" Drug-initiated" nitroxide-mediated synthesis of two well-defined, heterotelechelic polymer prodrugs ( Mn = 1960-5260 g·mol-1, D = 1.31-1.37) was performed by using the newly developed nitroxide exchange reaction. These polymers comprised, at the chain end, gemcitabine (Gem) as anticancer drug and either cyanine 7.5 (Cy7.5) as a near-infrared (NIR) dye suitable for in vivo imaging or biotin (Biot) for cancer cell targeting. These materials were co-nanoprecipitated into fluorescently labeled polymer prodrug nanoparticles of average diameter in the 100-180 nm range with narrow particle size distribution and variable surface amounts of biotin. Nanoparticles containing 15 wt % biotinylated polymer showed superior uptake and the highest cytotoxicity in vitro on A549 human lung cancer cells. In vivo, on A549 tumor bearing mice, biotinylated nanoparticles showed significantly higher efficacy than free Gem and maintained the same anticancer activity than nontargeted nanoparticles without inducing prohibitive body weight loss. Biotinylated polymer prodrug nanoparticles did not result in an improved anticancer activity or significant increase in tumor accumulation, which may be the result of a nonoptimal biotin surface display and/or insufficient affinity toward the target. They however displayed delayed liver accumulation compared to nonbiotinylated counterparts, suggesting the premise of a stealth property likely due to the hydrophilic tetraethylene glycol-Biot positioned at the nanoparticle surface. This work showed for the first time the applicability of this simple construction method to in vivo imaging and cancer cell targeting and might stimulate the design of new functional materials for biomedical applications.

Heterotelechelic polymer prodrug nanoparticles: Adaptability to different drug combinations and influence of the dual functionalization on the cytotoxicity.

Well-defined, heterotelechelic polymer prodrugs for combination therapy were synthesized by using a combination of the "drug-initiated" nitroxide-mediated polymerization from a gemcitabine-alkoxyamine initiator and the nitroxide exchange reaction using TEMPO-bearing drugs to end-cap the drug-polymer chain-end by a second drug. This methodology was successfully applied to two different clinically relevant combinations, gemcitabine/doxorubicin (Gem/Dox) and gemcitabine/lapatinib (Gem/Lap), showing a certain degree of universality of the synthetic methodology. It also represented the first nanocarrier for the co-delivery of Gem and Lap ever reported. Well-controlled, low molar mass heterotelechelic polymers (Mn = 2100-4090 g.mol-1, Ð = 1.18-1.38) with ~1:1 drug ratios and high overall drug loadings up to 40 wt% were obtained. They were formulated into nanoparticles by nanoprecipitation and exhibited average diameters in the 34-154 nm range, with narrow particle size distributions (PSD = 0.01-0.22) and excellent colloidal stability over time. Their biological evaluation in terms of drug release and cytotoxicity was performed and compared to that of different monofunctional polymer prodrug formulations. We showed that heterobifunctional polymer prodrugs induced cytotoxicity to MCF-7 cells, with IC50 values in the 120-300 nM range depending on the combination tested. Interestingly, whereas Gem/Dox combination did not lead to noticeable improvement over monofunctional therapies, co-nanoprecipitation of Gem/Lap prodrugs led to synergistic effect.

Telechelic polymers from reversible-deactivation radical polymerization for biomedical applications.

The main strategies for the design of telechelic polymers synthesized by reversible-activation radical polymerization for biomedical applications are covered spanning from drug delivery and targeting to theranostics and sensing.

Stereochemical Assignment of Strigolactone Analogues Confirms Their Selective Biological Activity.

Strigolactones (SLs) are new plant hormones with various developmental functions. They are also soil signaling chemicals that are required for establishing beneficial mycorrhizal plant/fungus symbiosis. In addition, SLs play an essential role in inducing seed germination in root-parasitic weeds, which are one of the seven most serious biological threats to food security. There are around 20 natural SLs that are produced by plants in very low quantities. Therefore, most of the knowledge on SL signal transduction and associated molecular events is based on the application of synthetic analogues. Stereochemistry plays a crucial role in the structure-activity relationship of SLs, as compounds with an unnatural D-ring configuration may induce biological effects that are unrelated to SLs. We have synthesized a series of strigolactone analogues, whose absolute configuration has been elucidated and related with their biological activity, thus confirming the high specificity of the response. Analogues bearing the R-configured butenolide moiety showed enhanced biological activity, which highlights the importance of this stereochemical motif.