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Extracellular vesicles (EVs) are a class of cell-derived lipid-bilayer membrane vesicles secreted by almost all mammalian cells and involved in intercellular communication by shuttling various biological cargoes. Over the last decade, EVs - namely exosomes and microvesicles - have been extensively explored as next-generation nanoscale drug delivery systems (DDSs). This is in large due to their endogenous origin, which enables EVs to circumvent some of the limitations associated with existing cancer therapy approaches (i.e. by preventing recognition by the immune system and improving selectivity towards tumor tissue). However, successful translation of these cell-derived vesicles into clinical applications has been hindered by several factors, among which the loading of exogenous therapeutic molecules still represents a great challenge. In order to address this issue and to further advance these biologically-derived systems as drug carriers, EV-biohybrid nano-DDSs, obtained through the fusion of EVs with conventional synthetic nano-DDSs, have recently been proposed as a valuable alternative as DDSs. Building on the idea of "combining the best of both worlds", a combination of these two unique entities aims to harness the beneficial properties associated with both EVs and conventional nano-DDSs, while overcoming the flaws of the individual components. These biohybrid systems also provide a unique opportunity for exploitation of new synergisms, often leading to improved therapeutic outcomes, thus paving the way for advancements in cancer therapy. This review aims to describe the recent developments of EV-biohybrid nano-DDSs in cancer therapy, to highlight the most promising results and breakthroughs, as well as to provide a glimpse on the possible intrinsic targeting mechanisms of EVs that can be bequeathed to their hybrid systems. Finally, we also provide some insights in the future perspectives of EV-hybrid DDSs.

Microfluidics has been used to process self-assembling liposomal systems that are commonly considered for drug delivery applications. However, it has been found that the parameters of the process are not universally suited for all lipid types. We hypothesize here that size aggregation and instability of microfluidic liposomes are a direct consequence of the presence of interdigitation in these liposomes. Interdigitation refers to the phenomenon where two opposing leaflets of a bilayer interpenetrate into one another and form a single layer. When this happens, aggregation results as the single layer is not thermodynamically stable. Such interdigitation can be induced by pressure, chemicals or by the type of lipid structure. In this study, we systematically investigate the role of lipid composition on membrane interdigitation in order to understand the dependency of lipid interdigitation on liposome formation by microfluidics. By doing so, we use nano DSC and SAXS to probe the extent of lipid interdigitation by measuring the changes in thermodynamics and membrane thickness of the lipid bilayers. Our results show that microfluidic-fabricated liposomes undergo chemical interdigitation in the presence of ethanol, in particular saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Strategies to prevent interdigitation is to either remove ethanol above the lipid's main transition temperature (Tm), preventing the formation of interdigitated structures and subsequent aggregated states or by the incorporation of the inhibiting additives, such as cholesterol. The author thanks Liew Wen Jie Melvin for the contribution to the cryo-EM images. Funding was kindly provided by NTUNorthwestern Institute for Nanomedicine.

The technology of drug delivery systems (DDS) has expanded into many applications, such as for treating neurological disorders. Nanoparticle DDS offer a unique strategy for targeted transport and improved outcomes of therapeutics. Stroke is likely to benefit from the emergence of this technology though clinical breakthroughs are yet to manifest. This review explores the recent advances in this field and provides insight on the trends, prospects and challenges of translating this technology to clinical application. Carriers of diverse material compositions are presented, with special focus on the surface properties and emphasis on the similarities and inconsistencies among in vivo experimental paradigms. Research attention is scattered among various nanoparticle DDS and various routes of drug administration, which expresses the lack of consistency among studies. Analysis of current literature reveals lipid- and polymer-based DDS as forerunners of DDS for stroke; however, cell membrane-derived vesicles (CMVs) possess the competitive edge due to their innate biocompatibility and superior efficacy. Conversely, inorganic and carbon-based DDS offer different functionalities as well as varied capacity for loading but suffer mainly from poor safety and general lack of investigation in this area. This review supports the existing literature by systematizing presently available data and accounting for the differences in drugs of choice, carrier types, animal models, intervention strategies and outcome parameters.

A hybrid gene delivery platform, micro Cell Vesicle Technology (mCVT), produced from the fusion of plasma membranes and cationic lipids, is presently used to improve the transfection efficiency of hard-to-transfect (HTT) cells. The plasma membrane components of mCVTs impart specificity in cellular uptake and reduce cytotoxicity in the transfection process, while the cationic lipids complex with the genetic material and provide structural integrity to mCVTs. This journal is

Saw et al. show via the combinatorial deletion of Timp family members in mice that metalloprotease regulation of FGF-2 is a crucial event in the chondrocyte maturation program, underlying the growth plate development and bone elongation responsible for attaining proper body stature., Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia. We identify TIMP/FGF-2/IHH as a novel nexus underlying bone lengthening where TIMPs negatively regulate the release of FGF-2 from chondrocytes to allow IHH expression. Using a knock-in approach that combines MMP-resistant or ADAMTS-resistant aggrecans with TIMP deficiency, we uncouple growth plate activity in axial and appendicular bones. Thus, natural metalloprotease inhibitors are crucial regulators of chondrocyte maturation program, growth plate integrity, and skeletal proportionality. Furthermore, individual and combinatorial TIMP-deficient mice demonstrate the redundancy of metalloprotease inhibitor function in embryonic and postnatal development.

A novel and highly efficient dual‐targeting platform was designed to ensure targeted in vivo delivery of dual‐action PtIV prodrugs. The dual targeting was established by liposomal encapsulation of PtIV complexes, thereby utilizing the enhanced permeability and retention (EPR) effect as the first stage of targeting to attain a high accumulation of the drug‐loaded liposomes in the tumor. After the release of the PtIV prodrug inside cancer cells, a second stage of targeting directed a portion of the PtIV prodrugs to the mitochondria. Upon intracellular reduction, these PtIV prodrugs released two bioactive molecules, acting both on the mitochondrial and on the nuclear DNA. Our PtIV system showed excellent activity in vitro and in vivo, characterized by a cytotoxicity in a low micromolar range and complete tumor remission, respectively. Notably, marked in vivo activity was accompanied by reduced kidney toxicity, highlighting the unique therapeutic potential of our novel dual‐targeting dual‐action platform. Ministry of Education (MOE) Accepted version G.P. acknowledges the National University of Singapore (NUS) for financial support (grant C-141-000-097-001). W.H.A. acknowledges the Ministry of Education Singapore (MOE) for financial support (grant R143000680114). D.G acknowledges the support of the Israel Science Foundation (grant 1611/14) and the support of the Alex Grass Center for Drug Design and Synthesis.Y.Z. acknowledges the Depart- ment of Pharmacy (NUS) and Hebrew University of Jerusa- lem (HUJ) for the research scholarship for ajoint PhD degree.M.V.B.acknowledges E. Fasolko and T. Hajsz for generating the artwork.

Cardiovascular disease remains the leading cause of morbidity and mortality globally. Extracellular vesicles (EVs), a group of heterogeneous nanosized cell-derived vesicles, have attracted great interest as liquid biopsy material for biomarker discovery in a variety of diseases including cardiovascular disease. Because EVs inherit bioactive components from parent cells and are able to transfer their contents to recipient cells, EVs hold great promise as potential cell-free therapeutics and drug delivery systems. However, the development of EV-based diagnostics, therapeutics or drug delivery systems has been challenging due to the heterogenicity of EVs in biogenesis, size and cellular origin, the lack of standardized isolation and purification methods as well as the low production yield. In this review, we will provide an overview of the recent advances in EV-based biomarker discovery, highlight the potential usefulness of EVs and EV mimetics for therapeutic treatment and drug delivery in cardiovascular disease. In view of the fast development in this field, we will also discuss the challenges of current methodologies for isolation, purification and fabrication of EVs and potential alternatives.

Cardiovascular disease remains the leading cause of morbidity and mortality globally. Extracellular vesicles (EVs), a group of heterogeneous nanosized cell-derived vesicles, have attracted great interest as liquid biopsy material for biomarker discovery in a variety of diseases including cardiovascular disease. Because EVs inherit bioactive components from parent cells and are able to transfer their contents to recipient cells, EVs hold great promise as potential cell-free therapeutics and drug delivery systems. However, the development of EV-based diagnostics, therapeutics or drug delivery systems has been challenging due to the heterogenicity of EVs in biogenesis, size and cellular origin, the lack of standardized isolation and purification methods as well as the low production yield. In this review, we will provide an overview of the recent advances in EV-based biomarker discovery, highlight the potential usefulness of EVs and EV mimetics for therapeutic treatment and drug delivery in cardiovascular disease. In view of the fast development in this field, we will also discuss the challenges of current methodologies for isolation, purification and fabrication of EVs and potential alternatives.

Cell-derived Drug Delivery Systems (DDSs), particularly exosomes, have grown in popularity and have been increasingly explored as novel DDSs, due to their intrinsic targeting capabilities. However, clinical translation of exosomes is impeded by the tedious isolation procedures and poor yield. Cell-derived nanovesicles (CDNs) have recently been produced and proposed as exosome-mimetics. Various methods for producing exosome-mimetics have been developed. In this chapter, we present a simple, efficient, and cost-effective CDNs production method that uses common laboratory equipment (microcentrifuge) and spin cups. Through a series of extrusion and size exclusion steps, CDNs are produced from in vitro cell culture and are found to highly resemble the endogenous exosomes. Thus, we envision that this strategy holds great potential as a viable alternative to exosomes in the development of ideal DDS.

Cell-derived Drug Delivery Systems (DDSs), particularly exosomes, have grown in popularity and have been increasingly explored as novel DDSs, due to their intrinsic targeting capabilities. However, clinical translation of exosomes is impeded by the tedious isolation procedures and poor yield. Cell-derived nanovesicles (CDNs) have recently been produced and proposed as exosome-mimetics. Various methods for producing exosome-mimetics have been developed. In this chapter, we present a simple, efficient, and cost-effective CDNs production method that uses common laboratory equipment (microcentrifuge) and spin cups. Through a series of extrusion and size exclusion steps, CDNs are produced from in vitro cell culture and are found to highly resemble the endogenous exosomes. Thus, we envision that this strategy holds great potential as a viable alternative to exosomes in the development of ideal DDS.

Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection., Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa. This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.

A stretchable and self-healable conductive material with high conductivity is critical to high-performance wearable electronics and integrated devices for applications where large mechanical deformation is involved. While there has been great progress in developing stretchable and self-healable conducting materials, it remains challenging to concurrently maintain and recover such functionalities before and after healing. Here, we report a highly stretchable and autonomic self-healable conducting film consisting of a conducting polymer (poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS) and a soft-polymer (poly(2-acrylamido-2-methyl-1-propanesulfonic acid, PAAMPSA). The optimal film exhibits outstanding stretchability as high as 630% and high electrical conductivity of 320 S/cm, while possessing the ability to repair both mechanical and electrical breakdowns when undergoing severe damage at ambient conditions. We further utilized this polymer composite film in a tactile sensor, which exhibits good pressure sensitivity of 164.5 kPa⁻1, near hysteresis-free, an ultrafast response time of 19 ms and excellent endurance over 1500 consecutive presses. Additionally, we successfully demonstrated an integrated 5 × 4 stretchable and self-healable organic electrochemical transistor (OECT) array with great device performance (including high transconductance of 12.95 ± 1.85 mS and good operational stability). The developed stretchable and autonomic self-healable conducting film significantly increases the practicality and shelf life of wearable electronics, which in turn, reduce maintenance costs and build-up of electronic waste. Submitted/Accepted version W.L.L would like to acknowledge funding support from National Robotics Programme (W1925d0106).

Wei Jiang Goh,1,2 Choon Keong Lee,2 Shui Zou,2 Esther CY Woon,2 Bertrand Czarny,2,3 Giorgia Pastorin1,2,4 1NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), 2Department of Pharmacy, National University of Singapore, 3School of Materials Science and Engineering (MSE) & Lee Kong Chian School of Medicine, Nanyang Technological University, 4NUSNNI-NanoCore, National University of Singapore, T-Lab, Singapore, Singapore Abstract: Cell-derived nanovesicles (CDNs) are an emerging class of biological drug delivery systems (DDS) that retain the characteristics of the cells they were derived from, without the need for further surface functionalization. CDNs are also biocompatible, being derived from natural sources and also take advantage of the enhanced permeability and retention effect due to their nanodimensions. Furthermore, CDNs derived from monocytes were shown to have an in vivo targeting effect, accumulating at the tumor site in a previous study conducted in a mouse tumor model. Here, we report a systematic approach pertaining to various loading methods of the chemotherapeutic drug doxorubicin into our CDNs and examine the differential cellular uptake of drug-loaded CDNs in cancerous (HeLa) and healthy (HEK293) cell lines. Lastly, we proved that the addition of doxorubicin-loaded CDNs to the HeLa and HEK293 co-cultures showed a clear discrimination toward cancer cells at the cellular level. Our results further reinforce the intriguing potential of CDNs as an alternative targeted strategy for anticancer therapy. Keywords: cell-derived nanovesicles, cell targeting, doxorubicin, antitumor therapy, extracellular vesicles, biomimetic, bionanotechnology, antitumor strategies

The most exploited strategy to develop potent zinc-metalloprotease inhibitors relies on a core zinc chelator and a peptidic or nonpeptidic scaffold that provides supplementary interactions for optimized potency and selectivity. Applied to matrix metalloproteases (MMPs) with highly conserved catalytic domains, this strategy failed to identify inhibitors with the desired selectivity profiles. To question the precise role of the zinc-binding group (ZBG), we have carried out a study on MMP-12 inhibitors with a common peptidic core but different ZBGs. We find that exchanging the ZBG modifies inhibitor positioning and affects its dynamics and selectivity. The binding properties of these compounds were compared through biochemical, structural, and calorimetric studies, showing a complex interplay between cooperative interactions and dynamics dictated by the ZBG. Improving selectivity will require expanding the ZBG repertoire within inhibitor libraries, since relying on a single ZBG significantly decreases our chance to identify effective inhibitors.

A hybrid drug delivery platform involving the fusion of cell membranes from U937 monocytes and synthetic lipids to create nano-cell vesicle technology systems (nCVTs) is designed. nCVTs are engineered for a targeted approach towards tumour sites by preserving key surface proteins from U937 monocytes, while being amendable to functionalization and loading due to their liposomal components. MOE (Min. of Education, S’pore)

Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted exosomes. Here, we show the first demonstration of a simple and rapid production method of CDNs using spin cups via a cell shearing approach, which offers clear advantages in terms of yield and cost-effectiveness over both traditional exosomes isolation, and also existing CDNs fabrication techniques. The CDNs obtained were of a higher protein yield and showed similarities in terms of physical characterization, protein and lipid analysis to both exosomes and CDNs previously reported in the literature. In addition, we investigated the mechanisms of cellular uptake of CDNs in vitro and their biodistribution in an in vivo mouse tumour model. Colocalization of the CDNs at the tumour site in a cancer mouse model was demonstrated, highlighting the potential for CDNs as anti-cancer strategy. Taken together, the results suggest that CDNs could provide a cost-effective alternative to exosomes as an ideal drug nanocarrier.

Cell-derived nanovesicles (CDNs) have been recently investigated as novel drug delivery systems (DDSs), due to the preservation of key features from the cell membrane of their precursor cells, which are responsible for an efficient cellular uptake by target cells. However, CDNs suffer from low drug loading efficiencies as well as challenges in functionalization compared to conventional DDS like liposomes. Here, we describe the first study proposing the fusion of CDNs with liposomes to form EXOPLEXs. We report the preservation of cell membranes from precursor cells similarly to CDNs, as well as high loading efficiencies of more than 65% with doxorubicin hydrochloride, a model chemotherapeutic drug. The doxorubicin-loaded EXOPLEXs (DOX-EXO) also demonstrated a higher in vitro cell killing effect than liposomes, while EXOPLEXs alone did not show any remarkable cytotoxicity. Taken together, these results illustrate the potential of EXOPLEXs as a novel DDS for targeted delivery of chemotherapeutics.

Matrix metalloproteinases like MMP-13 cleave and remodel the extracellular matrix and thereby play a crucial role in tumor progression in vivo. Using a highly selective inhibitor to block MMP-13 protein activity, we demonstrate a striking inhibitory effect on invasive tumor growth and vascularization in murine skin squamous cell carcinoma (SCC). Therapy outcome critically depends on animal age in C57Bl/6 mice and was successful in old female but not in young female mice. Treatment success was recovered by ovariectomy in young and abolished by 17s-estradiol supplementation in old mice, suggesting a hormone dependent inhibitor effect. Responsiveness of the tumorigenic keratinocytes BDVII and fibroblasts to 17s-estradiol was confirmed in vitro, where MMP-13 inhibitor treatment led to a reduction of cell invasion and vascular endothelial growth factor (VEGF) release. This correlated well with a less invasive and vascularized tumor in treated mice in vivo. 17s-estradiol supplementation also reduced invasion and VEGF release in vitro with no additional reduction on MMP-13 inhibitor treatment. This suggests that low 17s-estradiol levels in old mice in vivo lead to enhanced MMP-13 levels and VEGF release, allowing a more effective inhibitor treatment compared to young mice. In our study, we present a strong link between lower estrogen levels in old female mice, an elevated MMP-13 level, which results in a more effective MMP-13 inhibitor treatment in fibroblasts and SCC cells in vitro and in vivo.

SummaryMatrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases that catalyze cleavage of extracellular matrix and nonmatrix proteins. MMPs play a role in tissue remodeling, and their uncontrolled activity is associated with number of diseases, including tumor metastasis. Thus, there is a need to develop methods to monitor MMP activity, and number of probes has been previously described. The key problem many probes encounter is the issue of selectivity, since 23 human MMPs, despite playing different physiological roles, have structurally similar active sites. Here, we introduce the halogen bonding concept into the probe design and show that the probe containing iodine exhibits an unprecedented selectivity for MMP-9. We provide structure-based explanation for the selectivity, confirming that it is due to formation of the halogen bond that supports catalysis, and we highlight the value of exploring halogen bonding in the context of selective probe design.

An efficient crystallization screening method is important in drug design to yield high resolution crystallographic structures of protein–ligand complexes to understand inhibitor selectivity and potency for various members of an enzyme family. The strategy starts with a single condition for each protein–ligand complex, and more trials encompassing all polymorph crystallization conditions are included later, eventually defaulting to a more extensive screening for difficult cases. The polymorph screening approach relies on an intrinsic positive feedback mechanism. New polymorphs are constantly discovered since certain ligands favor variant lattices. The new best diffracting polymorph is selected for single-conditions testing, ensuring that as more forms are discovered, the resolution of the structures obtained improves. Continual optimization of the conditions for all crystal forms yields new solutions that become increasingly effective in protein–ligand crystallization trials. More polymorphs imply more la...

The molecular determinants responsible for the potency of the RXP470.1 phosphinic peptide inhibitor toward matrix metalloprotease-12 (MMP-12) remain elusive. To address this issue, structure-activity study, X-ray crystallography, and isothermal titration calorimetry (ITC) experiments were performed. The crystal structure of MMP-12/inhibitor complex (1.15 Å) reveals that the inhibitor establishes multiple interactions with the MMP-12 active site, with its long P(1)' side chain filling most of the S(1)' deep cavity. ITC experiments indicate that the binding of this inhibitor to MMP-12 is mostly entropy driven (ΔG° = -13.1 kcal/mol, ΔH° = -2.53 kcal/mol, and -TΔS° = -10.60 kcal/mol) and involves a proton uptake from the buffer. Comparing phosphinic versus hydroxamate inhibitors reveals that the chelation of the zinc ion is slightly different, leading the inhibitor backbone to adopt a position in which the hydrogen bonding with the MMP-12 active site is less favorable in phosphinic inhibitor while maintaining high affinity.

Matrix metalloproteases (MMPs) have attracted considerable attention as critical mediators of pathological tissue remodeling processes. However it remains an unresolved challenge to detect their active forms in biological samples. To prove the efficacy of a recently developed MMP activity-based probe, we examined the content in MMP active forms of bronchoalveolar lavage fluids (BALf) from male C57BL/6 mice exposed to ultrafine carbon black nanoparticles, a model of chronic obstructive pulmonary disease. This probe was shown to label proteins, mostly expressed in BALf of mice exposed to nanoparticles. Using competition assays with a selective MMP-12 inhibitor as well as MMP-12 knock-out mice, one of these proteins was identified as the active form of the catalytic domain of MMP-12. This new probe can detect the active form of MMP-12 down to a threshold of 1 fmol. Radioactive counting showed the concentration of the active form of MMP-12 to be around 1 fmol/μl in BALf from nanoparticle-treated mice. A less sensitive probe would therefore not have detected MMP-12. As the probe can detect other MMPs in the femtomolar range, it is a potentially powerful tool for monitoring the levels of MMP active forms in various diseases.

A photoaffinity probe based on the scaffold of a potent broad-spectrum phosphinic peptide inhibitor of matrix metalloproteinases (MMPs) has been developed. A photolabile diazirine group for covalent modification of MMP active forms was incorporated at the P(1) ' position, and a tritium radioactive label for the sensitive detection of MMP covalent adducts by radioimaging was attached. The probe was characterized on seven catalytic domains of human MMPs (MMP-2, -3, -8, -9, -12, -13 and -14) and was found to display nanomolar affinities towards this set of MMPs, covalently modifying them with crosslinking yields varying from 12 to 58 %, thus leading to highly sensitive detection of these MMPs. In a complex proteome complemented with four recombinant MMPs (MMP-2, -9, -12 and -13), this probe enabled their simultaneous detection with a threshold of few femtomoles and low background labelling. Those properties should make this new pan-activity-based MMP probe a valuable tool for the detection of MMP active forms from biological fluids or tissue extracts.

Purpose Given the progress of fluorescence diffuse optical tomography (fDOT) technology, here, we study the additional benefits provided by multimodal PET/fDOT imaging by comparing the biodistribution of 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) in tumors with three fluorescent probes: a glucose analog, a protease activatable optical probe, and a ligand of αvβ3 integrin. Procedures Sequential fDOT/PET/computed tomography (CT) imaging of mice was performed with a custom multimodal mouse support that allows the subject to be transferred between the fDOT and the PET/CT scanners. Experiments were performed in xenografted tumor models derived from the human breast cancer line MDA-MB 231 and compared to ex vivo analysis. Results The three-dimensional signals showed that the fluorescent glucose analog is not colocalized with [18F]FDG, raising questions about its use as a surrogate probe of the PET tracer. Fusion of [18F]FDG with the other fluorescent probes showed evidence of high variability both for the protease activity and the αvβ3 integrin expression during tumor growth. Conclusion The added value of hybrid PET/fDOT over the two modalities was demonstrated for cross-validation of probes and for better characterization of tumor models.

A series of pseudo-peptides with general formula X-l-Glu-NH2 (with X corresponding to an acyl moiety with a long aryl-alkyl side chain) have been synthesized, evaluated as inhibitors of matrix metalloproteases (MMPs), and found to display remarkable nanomolar affinity. The loss in potency associated with a substitution of the P2′ l-glutamate by a l-glutamine corroborates the importance of a carboxylate at this position. The binding mode of some of these inhibitors was characterized in solution and by x-ray crystallography in complex with various MMPs. The x-ray crystal structures reveal an unusual binding mode with the glutamate side chain chelating the active site zinc ion. Competition experiments between these inhibitors and acetohydroxamic acid, a small zinc-binding molecule, are in accord with the crystallographic results. One of these pseudo-dipeptides displays potency and selectivity toward MMP-12 similar to the best MMP-12 inhibitors reported to date. This novel family of pseudo peptides opens new opportunities to develop potent and selective inhibitors for several metzincins.

Glycerol is often used to protect proteins while stored frozen, to enhance their solubility and as a cryosolvent in cryocrystallography, but less so in protein crystallization, probably because it presents problems during screening. Understanding how glycerol affects protein crystallization is important for manual and for high throughput operations, and it will permit greater control of equilibration by vapor diffusion. Certain synthetic inhibitors of matrix metalloproteinase 12 (MMP-12) on mixing with the enzyme cause precipitation and induce nucleation, resulting in unusable microcrystal showers. Glycerol has been used to reduce this effect. We detail the practical problems and advantages in the use of glycerol with MMP-12 and how these observations may be extended to a larger range of problems, focusing on its use by the vapor diffusion method. Glycerol appears to be more effective when coupled with alternative and complementary methods that can similarly reduce excessive nucleation.

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P1′ residue with the unusual R configuration which, surprisingly, is accommodated by the large S2′ pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi–pi stacking interactions with the amino benzoyl group of the first molecule locking them in a ‘hand-shake’ conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

Objective— Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. Methods and Results— A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. Conclusion— Our data demonstrate that a selective MMP-12 inhibitor retards atherosclerosis development and results in a more fibrous plaque phenotype in mice. Our study provides proof of principle to motivate translational work on MMP-12 inhibitor therapy in humans.

The parallel oxorhenium-mediated assembly of 288 noncyclic RGD analogues is reported. All complexes contain a NS(2) +S chelating motif that enables the unambiguous coordination of the oxorhenium and oxotechnetium cores. In this study, "modules S" contain a variety of pending guanidinium groups whereas the "NS(2) modules" are made of a series of N-acylated amino acids. Combination of sets of "NS(2) " and "S modules" together with tetrabutylammonium tetrachlorooxorhenate gave the corresponding oxorhenium complexes in good yields and satisfactory purities. Evaluation of these metalloconstructs towards integrins α(V) β(3) , α(IIb) β(3) , and α(V) β(5) led to the identification of micromolar and submicromolar antagonists of theses integrins. These compounds exhibit interesting selectivities and promise attractive applications for the molecular imaging of integrin-dependent pathologies.

Following the disappointment of clinical trials with early broad-spectrum synthetic inhibitors of matrix metalloproteases (MMPs), the field is now resurging with a new focus on the development of more selective inhibitors. Compounds able to fully discriminate between different members of the MMP family are sorely needed for therapeutic applications. Chemical efforts over the past years have led to very few selective inhibitors of MMPs. The over-exploitation of the hydroxamate function, or other strong zinc-binding groups, might be responsible for this failure. By resorting to weaker zinc-chelating groups, like phosphoryl or carboxylic groups, inhibitors with improved selectivity profiles have been developed. However, the most encouraging results have been obtained with compounds that avoid targeting the zinc but gain their affinity from plunging deeper into the MMP S1′ cavity. Analyses of the crystal structures of MMP-13 and MMP-8 complexes with such compounds provide novel insights for the design of more selective inhibitors for other members of the MMP family.

While physically crosslinked polycarbonate hydrogels are effective drug delivery platforms, their hydrophobic nature and lack of side chain functionality or affinity ligands for controlled release of hydrophilic drugs underscore the importance of their chemical compositions. This study evaluates an array of anionic hydrogel systems of phenylboronic acid functionalized triblock copolymers prepared via reversible physical interactions. Variation of key chemical functionalities while maintaining similar core structural features demonstrates the influence of the substitution position and protection of the boronic acid functionality on gel viscoelasticity and mechanical strength at physiological pH. The optimum gel systems obtained from the meta-substituted copolymers (m-PAP) are stable at physiological pH and nontoxic to mammalian dermal cells. The polymyxin B loaded m-PAP hydrogels demonstrate controlled in vitro drug release kinetics and in vitro antimicrobial activity against Pseudomonas aeruginosa over 48 h. In vivo antimicrobial efficacy of the drug loaded hydrogels further corroborates the in vitro results, demonstrating sustained antimicrobial activity against P. aeruginosa burn wound infections. The current strategy described in this study demonstrates a straightforward approach in designing physiologically relevant boronic acid hydrogel systems for controlled release of cationic antimicrobials for future clinical applications.

International audience; The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo.

Jian Li,1 Sia Lee Yoong,2 Wei Jiang Goh,2 Bertrand Czarny,1 Zhi Yang,1 Kingshuk Poddar,2,3 Michal M Dykas,2,3 Abhijeet Patra,2,3 T Venkatesan,2,3 Tomasz Panczyk,4 Chengkuo Lee,5 Giorgia Pastorin1–3 1Department of Pharmacy, National University of Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), 3NUSNNI-NanoCore, National University of Singapore, Singapore; 4Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Cracow, Poland; 5Department of Electrical and Computer Engineering, National University of Singapore, Singapore Abstract: Carbon nanotubes’ (CNTs) hollow interior space has been explored for biomedical applications, such as drug repository against undesirable inactivation. To further devise CNTs as smart material for controlled release of cargo molecules, we propose the concept of “gold-carbon nanobottles”. After encapsulating cis-diammineplatinum(II) dichloride (cisplatin, CDDP) in CNTs, we covalently attached gold nanoparticles (AuNPs) at the open-tips of CNTs via different cleavable linkages, namely hydrazine, ester, and disulfide-containing linkages. Compared with our previous study in which more than 80% of CDDP leaked from CNTs in 2 hours, AuNPs were found to significantly decrease such spontaneous release to

Dendritic polyglycerol sulfate (dPGS) is a biocompatible, bioactive polymer which exhibits anti-inflammatory activity in vivo and thus represents a promising candidate for therapeutic and diagnostic applications. To investigate the in vivo pharmacokinetics in detail, dPGS with a molecular weight of ca. 10 kDa was radiolabeled with 3H and 64Cu, and evaluated by performing biodistribution studies and small animal positron emission tomography (PET). 3H-labeling was accomplished by an oxidation-reduction process with sodium periodate and [3H]-borohydride. 64Cu-labeling was achieved by conjugation of isothiocyanate- or maleimide-functionalized copper(II)-chelating ligands based on 1,4-bis(2-pyridinylmethyl)-1,4,7-triazacyclononane (DMPTACN) to an amino functionalized dPGS scaffold, followed by reaction with an aqueous solution containing 64CuCl2. Independent biodistribution by radioimaging and PET imaging studies with healthy mice and rats showed that the neutral dPG was quantitatively renally eliminated, whereas the polysulfated analogs accumulated mainly in the liver and spleen. Small amounts of the dPGS derivatives were slowly excreted via the kidneys. The degree of uptake by the reticuloendothelial system (RES) was similar for dPGS with 40% or 85% sulfation, and surface modification of the scaffold with the DMPTACN chelator did not appear to significantly affect the biodistribution profile. On the basis of our data, the applicability of bioactive dPGS as a therapeutic agent might be limited due to organ accumulation even after 3 weeks. The inert characteristics and clearance of the neutral polymer, however, underlines the potential of dPG as a multifunctional scaffold for various nanomedical applications.

Inflammation is a known mediator of adverse ventricular remodeling after myocardial infarction (MI) that may lead to reduction of ejection fraction and subsequent heart failure. Berberine is a isoquinoline quarternary alkaloid from plants that has been associated with anti-inflammatory, anti-oxidative, and cardioprotective properties. Its poor solubility in aqueous buffers and its short half-life in the circulation upon injection, however, have been hampering the extensive usage of this natural product. We hypothesized that encapsulation of berberine into long circulating liposomes could improve its therapeutic availability and efficacy by protecting cardiac function against MI in vivo. Berberine-loaded liposomes were prepared by ethanol injection and characterized. They contained 0.3 mg/mL of the drug and were 0.11 mu m in diameter. Subsequently they were tested for IL-6 secretion inhibition in RAW 264.7 macrophages and for cardiac function protection against adverse remodeling after MI in C57BL/6J mice. In vitro, free berberine significantly inhibited IL-6 secretion (IC50 = 10.4 mu M), whereas encapsulated berberine did not as it was not released from the formulation in the time frame of the in vitro study. In vivo, berberine-loaded liposomes significantly preserved the cardiac ejection fraction at day 28 after MI by 64% as compared to control liposomes and free berberine. In conclusion, liposomal encapsulation enhanced the solubility of berberine in buffer and preserves ejection fraction after MI. This shows that delivery of berberine-loaded liposomes significantly improves its therapeutic availability and identifies berberine-loaded liposomes as potential treatment of adverse remodeling after MI. (C) 2017 Elsevier B.V. All rights reserved.

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.

Matrix metalloproteinases like MMP-13 cleave and remodel the extracellular matrix and thereby play a crucial role in tumor progression in vivo. Using a highly selective inhibitor to block MMP-13 protein activity, we demonstrate a striking inhibitory effect on invasive tumor growth and vascularization in murine skin squamous cell carcinoma (SCC). Therapy outcome critically depends on animal age in C57Bl/6 mice and was successful in old female but not in young female mice. Treatment success was recovered by ovariectomy in young and abolished by 17ß-estradiol supplementation in old mice, suggesting a hormone dependent inhibitor effect. Responsiveness of the tumorigenic keratinocytes BDVII and fibroblasts to 17ß-estradiol was confirmed in vitro, where MMP-13 inhibitor treatment led to a reduction of cell invasion and vascular endothelial growth factor (VEGF) release. This correlated well with a less invasive and vascularized tumor in treated mice in vivo. 17ß-estradiol supplementation also reduced invasion and VEGF release in vitro with no additional reduction on MMP-13 inhibitor treatment. This suggests that low 17ß-estradiol levels in old mice in vivo lead to enhanced MMP-13 levels and VEGF release, allowing a more effective inhibitor treatment compared to young mice. In our study, we present a strong link between lower estrogen levels in old female mice, an elevated MMP-13 level, which results in a more effective MMP-13 inhibitor treatment in fibroblasts and SCC cells in vitro and in vivo.

A new triazole oxotechnetium chelating agent was synthesized via a 'Click-to-Chelate' strategy. In vivo evaluation of the corresponding (99m)Tc complex shows that the tracer exhibits very interesting properties for molecular imaging.

A series of pseudo-peptides with general formula X-l-Glu-NH(2) (with X corresponding to an acyl moiety with a long aryl-alkyl side chain) have been synthesized, evaluated as inhibitors of matrix metalloproteases (MMPs), and found to display remarkable nanomolar affinity. The loss in potency associated with a substitution of the P(2)' l-glutamate by a l-glutamine corroborates the importance of a carboxylate at this position. The binding mode of some of these inhibitors was characterized in solution and by x-ray crystallography in complex with various MMPs. The x-ray crystal structures reveal an unusual binding mode with the glutamate side chain chelating the active site zinc ion. Competition experiments between these inhibitors and acetohydroxamic acid, a small zinc-binding molecule, are in accord with the crystallographic results. One of these pseudo-dipeptides displays potency and selectivity toward MMP-12 similar to the best MMP-12 inhibitors reported to date. This novel family of pseudo peptides opens new opportunities to develop potent and selective inhibitors for several metzincins.

International audience; Environmental contamination with carbon nanotubes would lead to plant exposure and particularly exposure of agricultural crops. The only quantitative exposure data available to date which can be used for risk assessment comes from computer modeling. The aim of this study was to provide quantitative data relative to multi-walled carbon nanotube (MWCNT) uptake and distribution in agricultural crops, and to correlate accumulation data with impact on plant development and physiology. Roots of wheat and rapeseed were exposed in hydroponics to uniformly C-14-radiolabeled MWCNTs. Radioimaging, transmission electron microscopy and raman spectroscopy were used to identify CNT distribution. Radioactivity counting made it possible absolute quantification of CNT accumulation in plant leaves. Impact of CNTs on seed germination, root elongation, plant biomass, evapotranspiration, chlorophyll, thiobarbituric acid reactive species and H2O2 contents was evaluated. We demonstrate that less than 0.005% of the applied MWCNT dose is taken up by plant roots and translocated to the leaves. This accumulation does not impact plant development and physiology. In addition, it does not induce any modifications in photosynthetic activity nor cause oxidative stress in plant leaves. Our results suggest that if environmental contamination occurs and MWCNTs are in the same physicochemical state than the ones used in the present article, MWCNT transfer to the food chain via food crops would be very low.