Peptidic heterodimer-based radiotracer targeting fibroblast activation protein and integrin αvß3.

PURPOSE: Several studies have demonstrated the advantages of heterodimers over their corresponding monomers due to the multivalency effect. This effect leads to an increased number of effective targeted receptors and, consequently, improved tumor uptake. Fibroblast activation protein (FAP) and integrin αvß3 are found to be overexpressed in different components of the tumor microenvironment. In our pursuit of enhancing tumor uptake and retention, we designed and developed a novel peptidic heterodimer that synergistically targets both FAP and integrin αvß3. METHODS: FAP-RGD was synthesized from FAP-2286 and c(RGDfK) through a multi-step organic synthesis. The dual receptor binding property of 68Ga-FAP-RGD was investigated by cell uptake and competitive binding assays. Preclinical pharmacokinetics were determined in HT1080-FAP and U87MG tumor models using micro-positron emission tomography/computed tomography (micro-PET/CT) and biodistribution studies. The antitumor efficacy of 177Lu-FAP-RGD was assessed in U87MG tumor models. The radiation exposure and clinical diagnostic performance of 68 Ga-FAP-RGD were evaluated in healthy volunteers and cancer patients. RESULTS: Bi-specific radiotracer 68Ga-FAP-RGD exhibited high binding affinity for both FAP and integrin αvß3. In comparison to 68Ga-FAP-2286 and 68Ga-RGDfK, 68Ga-FAP-RGD displayed enhanced tumor uptake and longer tumor retention time in preclinical models. 177Lu-FAP-RGD could efficiently suppress the growth of U87MG tumor in vivo when applied at an activity of 18.5 and 29.6 MBq. The effective dose of 68Ga-FAP-RGD was 1.06 × 10-2 mSv/MBq. 68Ga-FAP-RGD demonstrated low background activity and stable accumulation in most neoplastic lesions up to 3 h. CONCLUSION: Taking the advantages of multivalency effect, the bi-specific radiotracer 68Ga-FAP-RGD showed superior tumor uptake and retention compared to its corresponding monomers. Preclinical studies with 68Ga- or 177Lu-labeled FAP-RGD showed favorable image contrast and effective antitumor responses. Despite the excellent performance of 68Ga-FAP-RGD in clinical diagnosis, experimental efforts are currently underway to optimize the structure of FAP-RGD to increase its potential for clinical application in endoradiotherapy.

Molecular basis for the recognition of 24-(S)-hydroxycholesterol by integrin αvß3.

A growing body of evidence suggests that oxysterols such as 25-hydroxycholesterol (25HC) are biologically active and involved in many physiological and pathological processes. Our previous study demonstrated that 25HC induces an innate immune response during viral infections by activating the integrin-focal adhesion kinase (FAK) pathway. 25HC produced the proinflammatory response by binding directly to integrins at a novel binding site (site II) and triggering the production of proinflammatory mediators such as tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). 24-(S)-hydroxycholesterol (24HC), a structural isomer of 25HC, plays a critical role in cholesterol homeostasis in the human brain and is implicated in multiple inflammatory conditions, including Alzheimer's disease. However, whether 24HC can induce a proinflammatory response like 25HC in non-neuronal cells has not been studied and remains unknown. The aim of this study was to examine whether 24HC produces such an immune response using in silico and in vitro experiments. Our results indicate that despite being a structural isomer of 25HC, 24HC binds at site II in a distinct binding mode, engages in varied residue interactions, and produces significant conformational changes in the specificity-determining loop (SDL). In addition, our surface plasmon resonance (SPR) study reveals that 24HC could directly bind to integrin αvß3, with a binding affinity three-fold lower than 25HC. Furthermore, our in vitro studies with macrophages support the involvement of FAK and NFκB signaling pathways in triggering 24HC-mediated production of TNF. Thus, we have identified 24HC as another oxysterol that binds to integrin αvß3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway.

RGD Peptidomimetic MMAE-Conjugate Addressing Integrin αVß3-Expressing Cells with High Targeting Index.

Small molecule-drug conjugates (SMDCs) mimicking the RGD sequence (-Arg-Gly-Asp-) with a non-peptide moiety require a pharmacophore-independent attachment site. A library of 36 sulfonamide-modified RGD mimetics with nM to pM affinity for integrin αV ß3 was synthesized and analysed via DAD mapping. The best structure of the conjugable RGD mimetic was used and a linker was attached to an aromatic ring by Negishi cross-coupling. The product retained high affinity and selectivity for integrin αV ß3 . The conjugable RGD mimetic was then attached to an enzymatically cleavable GKGEVA linker equipped with a self-immolative PABC and the antimitotic drug monomethyl auristatin E (MMAE). The resulting SMDC preferred binding to integrin αV ß3 over α5 ß1 in a ratio of 1 : 4519 (ELISA) and showed selectivity for αV ß3 -positive WM115 cells over αV ß3 -negative M21-L cells in the in vitro cell adhesion assay as well as in cell viability assays with a targeting index of 134 (M21-L/WM115).

Nuclear αvß3 integrin expression, post translational modifications and regulation in hematological malignancies.

αvß3 integrin, a plasma membrane protein, is amply expressed on an array of tumors. We identified nuclear αvß3 pool in ovarian cancer cells and were interested to explore this phenomenon in two rare and aggressive types of leukemia, T-cell acute lymphoblastic leukemia (T-ALL) and Mast cell leukemia (MCL) using Jurkat and HMC-1 cell lines, respectively. Moreover, we collected primary cells from patients with chronic lymphocytic leukemia (CLL, n = 11), the most common chronic adult leukemia and used human lymphoblastoid cell lines (LCL) generated from normal B cells. Nuclear αvß3 integrin was assessed by Western blots, confocal microscopy, and the ImageStream technology which combines flow-cytometry with microscopy. We further examined post translational modifications (phosphorylation/glycosylation), nuclear trafficking regulation using inhibitors for MAPK (U0126) and PI3K (LY294002), as well as nuclear interactions by performing Co-immunoprecipitation (Co-IP). αvß3 integrin was identified in all cell models within the nucleus and is N-glycosylated. In primary CLL cells the ß3 integrin monomer is tyrosine Y759 phosphorylated, suggesting an active receptor conformation. MAPK and PI3K inhibition in Jurkat and CLL cells led to αvß3 enhancement in the nucleus and a reduction in the membrane. The nuclear αvß3 integrin interacts with ERK, Histone H3 and Lamin B1 in Jurkat, Histone H3 in CLL cells, but not in control LCL cells. To conclude, this observational study provides the identification of nuclear αvß3 in hematological malignancies and lays the basis for novel cancer-relevant actions, which may be independent from the membrane functions.

Discovery of dual targeting PEGylated BG-P1600-TAT to norepinephrine transporter (NET) and thyrointegrin αvß3 in the treatment of neuroblastoma.

Polymer-drug conjugates are growing in interest as novel anticancer agents for targeted cancer therapy. The aim of this study was to synthesize a poly(ethylene glycol) (PEG) conjugated anticancer drug for neuroblastoma, which is the most common extracranial solid tumor of childhood and the deadliest tumor of infancy. In our previous studies, we designed and synthesized a dual targeting agent using benzylguanidine (BG) conjugated with the high affinity thyrointegrin αvß3 antagonist TriAzole Tetraiodothyroacetic acid (TAT) via non-cleavable bonding to PEG400 to make BG-P400-TAT and its derivatives as agents against neuroblastoma. Here, we improved the pharmacodynamic properties and increased the solubility by changing the polymer length to 1600 molecular weight. The TAT group, which acts as an integrin αvß3 antagonist, and the BG group, which can be taken up by neuroblastoma cells through the norepinephrine transporter (NET) system, are conjugated to PEG1600 to make BG-PEG1600-TAT. The binding affinity of BG-PEG1600-TAT was 40-fold higher to integrin αvß3 versus BG-P400-TAT and was associated with greater anticancer activities against neuroblastoma cells (SK-N-F1 and SKNAS) implanted in SCID mice along with broad spectrum anti-angiogenesis activities versus the FDA approved anti-Vascular Endothelial Growth Factor (VEGF) monoclonal antibody Avastin (bevacizumab). In conclusion, our novel dual targeting of NET and αvß3 receptor antagonist, BG-P1600-TAT demonstrated broad spectrum anti-angiogenesis and anti-cancer activities in suppressing neuroblastoma tumor progression and metastasis. Thus, BG-PEG1600-TAT represents a potential clinical candidate for targeted therapy in neuroblastoma management.

Biological Relevance of RGD-Integrin Subtype-Specific Ligands in Cancer.

Integrins are heterodimeric transmembrane proteins able to connect cells with the micro-environment. They represent a family of receptors involved in almost all the hallmarks of cancer. Integrins recognizing the Arg-Gly-Asp (RGD) peptide in their natural extracellular matrix ligands have been particularly investigated as tumoral therapeutic targets. In the last 30 years, intense research has been dedicated to designing specific RGD-like ligands able to discriminate selectively the different RGD-recognizing integrins. Chemists' efforts have led to the proposition of modified peptide or peptidomimetic libraries to be used for tumor targeting and/or tumor imaging. Here we review, from the biological point of view, the rationale underlying the need to clearly delineate each RGD-integrin subtype by selective tools. We describe the complex roles of RGD-integrins (mainly the most studied αvß3 and α5ß1 integrins) in tumors, the steps towards selective ligands and the current usefulness of such ligands. Although the impact of integrins in cancer is well acknowledged, the biological characteristics of each integrin subtype in a specific tumor are far from being completely resolved. Selective ligands might help us to reconsider integrins as therapeutic targets in specific clinical settings.

Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-ß-ACPC) Scaffolds.

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-ß-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvß3 and α5ß1 receptors using biotinylated vitronectin (αvß3) and fibronectin (α5ß1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvß3 over α5ß1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvß3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVß3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

A Peptide Stapling Strategy with Built-In Fluorescence by Direct Late-Stage C(sp2 )-H Olefination of Tryptophan.

Fluorescent stapled peptides are important chemical tools for detecting intracellular distribution, protein-protein interactions, and localization of target proteins. These peptides are usually labeled with bulky sized fluorophores through reactive functional groups, which may alter the physical properties and biological activities of peptides. Herein, a unique strategy is developed for synthesizing new stapled peptides with built-in fluorescence. The stapled peptides were prepared through Rh-catalyzed C(sp2 )-H olefination in tryptophan (Trp) residues by using pyridine/pyrimidine as the directing groups under mild conditions. This method displays good regioselectivity and high efficiency. Furthermore, as a proof of concept for its biological applications, stapled peptides without additional fluorophore 9 a and 9 b were constructed for a cell imaging study. These peptides displayed strong binding affinity toward integrin αvß3 in A549 cells by cell imaging experiments. Notably they demonstrated even better anticancer activity than commercial antagonist cyclic (RGDfK). The method will provide robust tools for the peptide macrocyclization field.

The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin.

Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16-18 nm Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvß3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvß3 integrin.

Side chain effect in the modulation of αvß3/α5ß1 integrin activity via clickable isoxazoline-RGD-mimetics: development of molecular delivery systems.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.

Structure-Activity Relationship of RGD-Containing Cyclic Octapeptide and αvß3 Integrin Allows for Rapid Identification of a New Peptide Antagonist.

The αvß3 integrin, a receptor for many extracellular matrix proteins with RGD-sequence motif, is involved in multiple physiological processes and highly expressed in tumor cells, therefore making it a target for cancer therapy and tumor imaging. Several RGD-containing cyclic octapeptide (named LXW analogs) were screened as αvß3 antagonists with dramatically different binding affinity, and their structure-activity relationship (SAR) remains elusive. We performed systematic SAR studies and optimized LXW analogs to improve antagonistic potency. The NMR structure of LXW64 was determined and docked to the integrin. Structural comparison and docking studies suggested that the hydrophobicity and aromaticity of the X7 amino acid are highly important for LXW analogs binding to the integrin, a potential hydrophobic pocket on the integrin surface was proposed to play a role in stabilizing the peptide binding. To develop a cost-efficient and fast screening method, computational docking was performed on LXW analogs and compared with in vitro screening. A consistency within the results of both methods was found, leading to the continuous optimization and testing of LXW mutants via in silico screening. Several new LXW analogs were predicted as the integrin antagonists, one of which-LXZ2-was validated by in vitro examination. Our study provides new insight into the RGD recognition specificity and valuable clues for rational design of novel αvß3 antagonists.

Multimeric Presentation of RGD Peptidomimetics Enhances Integrin Binding and Tumor Cell Uptake.

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD (Arg-Gly-Asp) peptidomimetics were designed to target αv ß3 integrin-expressing tumor cells. These compounds were prepared by an oxime chemoselective assembly of cyclo(DKP-RGD) ligands and a cyclodecapeptide scaffold, which allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve αv ß3 integrin binding compared with the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo(DKP-RGD) ligands for in vivo imaging and drug delivery.

Integrin Subtypes and Nanoscale Ligand Presentation Influence Drug Sensitivity in Cancer Cells.

Cancer cell-matrix interactions have been shown to enhance cancer cell survival via the activation of pro-survival signaling pathways. These pathways are initiated at the site of interaction, i.e., integrins, and thus, their inhibition has been the target of therapeutic strategies. Individual roles for fibronectin-binding integrin subtypes αvß3 and α5ß1 have been shown for various cellular processes; however, a systematic comparison of their function in adhesion-dependent chemoresistance is lacking. Here, we utilize integrin subtype-specific peptidomimetics for αvß3 and α5ß1, both as blocking agents on fibronectin-coated surfaces and as surface-immobilized adhesion sites, in order to parse out their role in breast cancer cell survival. Block copolymer micelle nanolithography is utilized to immobilize peptidomimetics onto highly ordered gold nanoparticle arrays with biologically relevant interparticle spacings (35, 50, or 70 nm), thereby providing a platform for ascertaining the dependence of ligand spacing in chemoprotection. We show that several cellular properties-morphology, focal adhesion formation, and migration-are intricately linked to both the integrin subtype and their nanospacing. Importantly, we show that chemotherapeutic drug sensitivity is highly dependent on both parameters, with smaller ligand spacing generally hindering survival. Furthermore, we identify ligand type-specific patterns of drug sensitivity, with enhanced chemosurvival when cells engage αvß3 vs α5ß1 on fibronectin; however, this is heavily reliant on nanoscale spacing, as the opposite is observed when ligands are spaced at 70 nm. These data imply that even nanoscale alterations in extracellular matrix properties have profound effects on cancer cell survival and can thus inform future therapies and drug testing platforms.

Synthesis and Biological Characterization of Monomeric and Tetrameric RGD-Cryptophycin Conjugates.

The effective delivery of cytotoxic agents to tumor cells is a key challenge in anticancer therapy. Multivalent integrinspecific ligands are considered a promising tool to increase the binding affinity, selectivity, and internalization efficiency of small-molecule drug conjugates. Herein, we report the synthesis and biological evaluation of a multimeric conjugate containing the high-affinity integrin αv ß3 binding ligand RAFT-c(RGDfK)4 , a lysosomally cleavable Val-Cit linker, and cryptophycin-55 glycinate, a potent inhibitor of tubulin polymerization. In vitro cytotoxicity assays verified that the multimeric RGD-cryptophycin conjugate displays improved potency compared to the monomeric analogue in integrin αv ß3 overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin-negative cell line.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

We have developed a fully synthetic and multifunctional antibody-recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre-immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo-RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and αv ß3 integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.

Zerumbone Induces Apoptosis in Breast Cancer Cells by Targeting αvß3 Integrin upon Co-Administration with TP5-iRGD Peptide.

Cell-penetrating peptides (CPPs) are highly promising tools to deliver therapeutic molecules into tumours. αVß3 integrins are cell-matrix adhesion receptors, and are considered as an attractive target for anticancer therapies owing to their roles in the process of metastasis and angiogenesis. Therefore, this study aims to assess the effect of co-administration of zerumbone (ZER) and ZERencapsulated in hydroxypropyl-ß-cyclodextrin with TP5-iRGD peptide towards cell cytotoxicity, apoptosis induction, and proliferation of normal and cancerous breast cells utilizing in vitro assays, as well as to study the molecular docking of ZER in complex with TP5-iRGD peptide. Cell viability assay findings indicated that ZER and ZERencapsulated in hydroxypropyl-ß-cyclodextrin (ZER-HPßCD) inhibited the growth of estrogen receptor positivebreast cancer cells (ER+ MCF-7) at 72 h treatment with an inhibitory concentration (IC)50 of 7.51 ± 0.2 and 5.08 ± 0.2 µg/mL, respectively, and inhibited the growth of triple negative breast cancer cells (MDA-MB-231) with an IC50 of 14.96 ± 1.52 µg/mL and 12.18 ± 0.7 µg/mL, respectively. On the other hand, TP5-iRGD peptide showed no significant cytotoxicity on both cancer and normal cells. Interestingly, co-administration of TP5-iRGD peptide in MCF-7 cells reduced the IC50 of ZER from 7.51 ± 0.2 µg/mL to 3.13 ± 0.7 µg/mL and reduced the IC50 of ZER-HPßCD from 5.08 ± 0.2 µg/mL to 0.49 ± 0.004 µg/mL, indicating that the co-administration enhances the potency and increases the efficacy of ZER and ZER-HPßCD compounds. Acridine orange (AO)/propidium iodide (PI) staining under fluorescence microscopy showed evidence of early apoptosis after 72 h from the co-administration of ZER or ZER-HPßCD with TP5-iRGD peptide in MCF-7 breast cancer cells. The findings of the computational modelling experiment provide novel insights into the ZER interaction with integrin αvß3 in the presence of TP5-iRGD, and this could explain why ZER has better antitumor activities when co-administered with TP5-iRGD peptide.

Effective treatment of cancer metastasis using a dual-ligand nanoparticle.

Metastasis is responsible for the majority of deaths of breast cancer patients. While cytotoxic drugs are available with high potency to kill breast cancer cells, they are not designed to specifically seek and navigate in the dynamic and continuously changing microenvironment of metastatic disease. To effectively delivery chemotherapeutic agents to metastasis, we designed a dual-ligand nanoparticle loaded with doxorubicin by using two different types of ligands targeting EGFR and αvß3 integrin. Metastatic cancer cells continuously change resulting in heterogeneity even across adjacent micrometastatic regions with variable expression of these targetable receptors. Using a mouse model of breast cancer metastasis, in vivo and ex vivo imaging showed that both EGFR and αvß3 integrin-targeting were required to reliably direct the nanoparticle to metastasis and capture the spread and exact topology of the disease. Survival studies compared the anticancer efficacy of the standard drug, EGFR-targeting nanoparticle, αvß3 integrin-targeting nanoparticle and the dual-ligand nanoparticle. While all the other treatments produced moderate therapeutic outcomes, treatment with the dual-ligand nanoparticle yielded significant improvement and event-free survival in a mouse model of breast cancer metastasis.

Flower-like Mn-Doped Magnetic Nanoparticles Functionalized with αvß3-Integrin-Ligand to Efficiently Induce Intracellular Heat after Alternating Magnetic Field Exposition, Triggering Glioma Cell Death.

Despite the potential of magnetic nanoparticles (NPs) to mediate intracellular hyperthermia when exposed to an alternating magnetic field (AMF), several studies indicate that the intracellular heating capacity of magnetic NPs depends on factors such as cytoplasm viscosity, nanoparticle aggregation within subcellular compartments, and dipolar interactions. In this work, we report the design and synthesis of monodispersed flowerlike superparamagnetic manganese iron oxide NPs with maximized SAR (specific absorption rate) and evaluate their efficacy as intracellular heaters in the human tumor-derived glioblastoma cell line U87MG. Three main strategies to tune the particle anisotropy of the core and the surface to reach the maximum heating efficiency were adopted: (1) varying the crystalline anisotropy by inserting a low amount of Mn2+ in the inverse spinel structure, (2) varying the NP shape to add an additional anisotropy source while keeping the superparamagnetic behavior, and (3) maximizing NP-cell affinity through conjugation with a biological targeting molecule to reach the NP concentration required to increase the temperature within the cell. We investigate possible effects produced by these improved NPs under the AMF (f = 96 kHz, H = 47 kA/m) exposure in the glioblastoma cell line U87MG by monitoring the expression of hsp70 gene and reactive oxygen species (ROS) production, as both effects have been described to be induced by increasing the intracellular temperature. The induced cell responses include cellular membrane permeabilization and rupture with concomitant high ROS appearance and hsp70 expression, followed by cell death. The responses were largely limited to cells that contained the NPs exposed to the AMF. Our results indicate that the developed strategies to optimize particle anisotropy in this work are a promising guidance to improve the heating efficiency of magnetic NPs in the human glioma cell line.

Phosphorylated fibronectin enhances cell attachment and upregulates mechanical cell functions.

A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosphorylation of fibronectin by a casein kinase II (CKII) significantly upregulated cell traction forces and total strain energy generated by fibroblasts on nanopillar arrays, and consequently other elementary cell functions including cell spreading and metabolic activity. Mass spectrometry of plasma fibronectin from healthy human donors then identified a constitutively phosphorylated site in the C-terminus, and numerous other residues that became phosphorylated by the CKII kinase in vitro. Our findings open up novel strategies for translational applications including targeting diseased ECM, or to develop assays that probe the phosphorylation state of the ECM or blood as potential cancer markers.

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVß3 and α5ß1.

Targeting both integrins αVß3 and α5ß1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVß3 and α5ß1, whereas knottin 2.5D is αVß3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVß3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVß3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.