Antibody nanoparticle conjugate-based targeted immunotherapy for non-small cell lung cancer.

The use of immune checkpoint inhibitors, which activate T cells, is a paradigm shift in the treatment of non-small cell lung cancer. However, the overall response remains low. To address this limitation, here we describe a novel platform, termed antibody-conjugated drug-loaded nanotherapeutics (ADN), which combines immunotherapy and molecularly targeted therapy. An ADN was designed with an anti-CD47 and anti-programmed death ligand 1 (PDL1) antibody pair on the surface of the nanoparticle and a molecularly targeted inhibitor of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway, PI103, entrapped in the nanoparticle. The anti-CD47-PDL1-ADN exhibited greater antitumor efficacy than current treatment options with a PDL1 inhibitor in vivo in an aggressive lung cancer immunocompetent mouse model. Dual antibody-drug-loaded nanotherapeutics can emerge as an attractive platform to improve outcomes with cancer immunotherapy.

Synthesis of imines from the coupling reaction of alcohols and amines catalyzed by phosphine-free cobalt(II) complexes.

Phosphine-free, air stable cobalt(II) based complexes (1a and 1b) consisting of ligands L1H2 and L2H2 (L1H2 = N,N'-((1,2-phenylenebis(azaneylylidene))bis(methaneylylidene))diphenol and L2H2 = N,N'-bis(4-diethylaminosalicylidene)-4,5-dichloro-1,2-phenylenediamine) were synthesized and utilized as catalysts in the coupling reaction of alcohols with amines into imines following an acceptorless dehydrogenative pathway. The reactions were carried out in the presence of t-BuOK base with low catalyst loading (1 mol%) in an open atmosphere. The corresponding imines were isolated in moderate to excellent yields. The methodology was screened with different substituted alcohols and amines. The proposed mechanistic pathway of this reaction was ascertained through intermediate mass and 1H NMR analyses. Most of the previously reported 3d transition metal catalysts used in imine synthesis reactions have a phosphine ligand environment, and the reactions were performed under inert conditions. Herein we have developed a sustainable route for the synthesis of imines from the coupling reaction of alcohols with amines under aerial reaction conditions using phosphine-free air stable cobalt catalysts.

An inexpensive "do-it-yourself" device for rapid generation of uniform tumor spheroids.

Three-dimensional (3D) cancer cell culture models such as tumor spheroids better recapitulate in vivo tumors than conventional two-dimensional (2D) models. However, two major challenges limit the routine use of 3D tumor spheroids. Firstly, most existing methods of generating tumor spheroids are not high-throughput. Secondly, tumor spheroids generated using current methods are highly variable in dimension. Here, we describe a simple 'Do-It-Yourself (DIY)' device that can be assembled for less than $7 of parts and generate uniform tumor spheroids in a high-throughput manner. We used a simple phone coin vibrating motor to superimpose the vibration for breaking a laminar jet of cell-loaded alginate solution into equally sized spherical beads. We generated 3,970 tumor spheroids/min, which exhibited a hypoxic core recapitulating in vivo tumors and could be used to test the diffusion efficacy of anticancer drugs. Such low-cost, easy-to-fabricate, simple-to-operate systems with high-throughput outcomes are essential to democratize and standardize cancer research.

An architecturally rational hemostat for rapid stopping of massive bleeding on anticoagulation therapy.

Hemostatic devices are critical for managing emergent severe bleeding. With the increased use of anticoagulant therapy, there is a need for next-generation hemostats. We rationalized that a hemostat with an architecture designed to increase contact with blood, and engineered from a material that activates a distinct and undrugged coagulation pathway can address the emerging need. Inspired by lung alveolar architecture, here, we describe the engineering of a next-generation single-phase chitosan hemostat with a tortuous spherical microporous design that enables rapid blood absorption and concentrated platelets and fibrin microthrombi in localized regions, a phenomenon less observed with other classical hemostats without structural optimization. The interaction between blood components and the porous hemostat was further amplified based on the charged surface of chitosan. Contrary to the dogma that chitosan does not directly affect physiological clotting mechanism, the hemostat induced coagulation via a direct activation of platelet Toll-like receptor 2. Our engineered porous hemostat effectively stopped the bleeding from murine liver wounds, swine liver and carotid artery injuries, and the human radial artery puncture site within a few minutes with significantly reduced blood loss, even under the anticoagulant treatment. The integration of engineering design principles with an understanding of the molecular mechanisms can lead to hemostats with improved functions to address emerging medical needs.

Utilizing coordination chemistry through formation of a CuII-quinalizarin complex to manipulate cell biology: An in vitro, in silico approach.

Quinalizarin, an analogue of anthracycline anticancer agents, is an anticancer agent itself. A CuII complex was prepared and characterized by elemental analysis, UV-Vis & IR spectroscopy, mass spectrometry, EPR and DFT. The intention behind the preparation of the complex was to increase cellular uptake, compare its binding with DNA against that of quinalizarin, modulation of semiquinone formation, realization of human DNA topoisomerase I & human DNA topoisomerase II inhibition and observation of anticancer activity. While the first two attributes of complex formation lead to increased efficacy, decrease in semiquinone generation could results in a compromise with efficacy. Inhibition of human DNA topoisomerase makes up this envisaged compromise in free radical activity since the complex shows remarkable ability to disrupt activities of human DNA topoisomerase I and II. The complex unlike quinalizarin, does not catalyze flow of electrons from NADH to O2 to the extent known for quinalizarin. Hence, decrease in semiquinone or superoxide radical anion could make modified quinalizarin [as CuII complex] less efficient in free radical pathway. However, it would be less cardiotoxic and that would be advantageous to qualify it as a better anticancer agent. Although binding to calf thymus DNA was comparable to quinalizarin, it was weaker than anthracyclines. Low cost of quinalizarin could justify consideration as a substitute for anthracyclines but the study revealed IC50 of quinalizarin/CuII-quinalizarin was much higher than anthracyclines or their complexes. Even then, there is a possibility that CuII-quinalizarin could be an improved and less costly form of quinalizarin as anticancer agent.

An insight into the mixed quantum mechanical-molecular dynamics simulation of a ZnII-Curcumin complex with a chosen DNA sequence that supports experimental DNA binding investigations.

An important aspect of research pertaining to Curcumin (HCur) is the need to arrest its degradation in aqueous solution and in biological milieu. This may be achieved through complex formation with metal ions. For this reason, a complex of HCur was prepared with ZnII, that is not likely to be active in redox pathways, minimizing further complications. The complex is monomeric, tetrahedral, with one HCur, an acetate and a molecule of water bound to ZnII. It arrests degradation of HCur to a considerable extent that was realized by taking it in phosphate buffer and in biological milieu. The structure was obtained by DFT calculations. Stable adduct formation was identified between optimized structures of HCur and [Zn(Cur)] with DNA (PDB ID: 1BNA) through experiments validated with multiscale modeling approach. Molecular docking studies provide 2D and 3D representations of binding of HCur and [Zn(Cur)] through different non-covalent interactions with the nucleotides of the chosen DNA. Through molecular dynamics simulation, a detailed understanding of binding pattern and key structural characteristics of the generated DNA-complex was obtained following analysis by RMSD, RMSF, radius of gyration, SASA and aspects like formation of hydrogen bonds. Experimental studies provide binding constants for [Zn(Cur)] with calf thymus DNA at 25 °C that effectively helps one to realize its high affinity towards DNA. In the absence of an experimental binding study of HCur with DNA, owing to its tendency to degrade in solution, a theoretical analysis of the binding of HCur to DNA is extremely helpful. Besides, both experimental and simulated binding of [Zn(Cur)] to DNA may be considered as a case of pseudo-binding of HCur to DNA. In a way, such studies on interaction with DNA helps one to identify HCur's affinity for cellular target DNA, not realized through experiments. The entire investigation is an understanding of experimental and theoretical approaches that has been compared continuously, being particularly useful when a molecule's interaction with a biological target cannot be realized experimentally.

A potent antibiotic-loaded bone-cement implant against staphylococcal bone infections.

New antibiotics should ideally exhibit activity against drug-resistant bacteria, delay the development of bacterial resistance to them and be suitable for local delivery at desired sites of infection. Here, we report the rational design, via molecular-docking simulations, of a library of 17 candidate antibiotics against bone infection by wild-type and mutated bacterial targets. We screened this library for activity against multidrug-resistant clinical isolates and identified an antibiotic that exhibits potent activity against resistant strains and the formation of biofilms, decreases the chances of bacterial resistance and is compatible with local delivery via a bone-cement matrix. The antibiotic-loaded bone cement exhibited greater efficacy than currently used antibiotic-loaded bone cements against staphylococcal bone infections in rats. Potent and locally delivered antibiotic-eluting polymers may help address antimicrobial resistance.

Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer.

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90.

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood-brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays >2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate >350% relative cell killing is achieved in SCI-101-treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.

Nanotherapeutic approaches to overcome distinct drug resistance barriers in models of breast cancer.

Targeted delivery of drugs to tumor cells, which circumvent resistance mechanisms and induce cell killing, is a lingering challenge that requires innovative solutions. Here, we provide two bioengineered strategies in which nanotechnology is blended with cancer medicine to preferentially target distinct mechanisms of drug resistance. In the first 'case study', we demonstrate the use of lipid-drug conjugates that target molecular signaling pathways, which result from taxane-induced drug tolerance via cell surface lipid raft accumulations. Through a small molecule drug screen, we identify a kinase inhibitor that optimally destroys drug tolerant cancer cells and conjugate it to a rationally-chosen lipid scaffold, which enhances anticancer efficacy in vitro and in vivo. In the second 'case study', we address resistance mechanisms that can occur through exocytosis of nanomedicines. Using adenocarcinoma HeLa and MCF-7 cells, we describe the use of gold nanorod and nanoporous vehicles integrated with an optical antenna for on-demand, photoactivation at ~650 nm enabling release of payloads into cells including cytotoxic anthracyclines. Together, these provide two approaches, which exploit engineering strategies capable of circumventing distinct resistance barriers and induce killing by multimodal, including nanophotonic mechanisms.

Inhibition of Tunneling Nanotubes between Cancer Cell and the Endothelium Alters the Metastatic Phenotype.

The interaction of tumor cells with blood vessels is one of the key steps during cancer metastasis. Metastatic cancer cells exhibit phenotypic state changes during this interaction: (1) they form tunneling nanotubes (TNTs) with endothelial cells, which act as a conduit for intercellular communication; and (2) metastatic cancer cells change in order to acquire an elongated phenotype, instead of the classical cellular aggregates or mammosphere-like structures, which it forms in three-dimensional cultures. Here, we demonstrate mechanistically that a siRNA-based knockdown of the exocyst complex protein Sec3 inhibits TNT formation. Furthermore, a set of pharmacological inhibitors for Rho GTPase-exocyst complex-mediated cytoskeletal remodeling is introduced, which inhibits TNT formation, and induces the reversal of the more invasive phenotype of cancer cell (spindle-like) into a less invasive phenotype (cellular aggregates or mammosphere). Our results offer mechanistic insights into this nanoscale communication and shift of phenotypic state during cancer-endothelial interactions.

Enhanced efficacy of a Cu2+ complex of curcumin against Gram-positive and Gram-negative bacteria: Attributes of complex formation.

Curcumin is a tantalizing molecule with multifaceted therapeutic potentials. However, its therapeutic applications are severely hampered because of poor bioavailability, attributed to its instability and aqueous insolubility. In an attempt to overcome this inherent limitation and develop curcumin-based antibacterials, we had earlier synthesized and characterized a metal complex of Cu(II) with curcumin, having the formula [Cu(Curcumin)(OCOCH3)(H2O)], hereafter referred to as Cu(Cur). In this study, the complex, i.e., Cu(Cur), was investigated for its stability and antibacterial activity along with its possible mechanism of action in comparison to the parent molecule, curcumin. Complex formation resulted in improved stability as Cu(Cur) was found to be highly stable under different physiological conditions. Such improved stability was verified with the help of UV-Vis spectroscopy and HPLC. With improved stability, Cu(Cur) exhibited potent and significantly enhanced activity over curcumin against both E. coli and S. aureus. Calcein leakage assay revealed that the complex triggered immediate membrane permeabilization in S. aureus. This membrane disruptive mode of action was further corroborated by microscopic visualization. The excellent potency of the complex was augmented by its safe toxicological profile as it was non-hemolytic and non-cytotoxic towards mammalian cells, making it a suitable candidate for in vivo investigations. Altogether, this investigation is a critical appraisal that advocates the antibacterial potential of this stable, membrane-targeting and non-toxic complex, thereby presenting new perspectives for its therapeutic application against bacterial infections.

Nanoengineered Disruption of Heat Shock Protein 90 Targets Drug-Induced Resistance and Relieves Natural Killer Cell Suppression in Breast Cancer.

Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate "disabled" NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a "chimeric" nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically approved agents. These findings highlight a convergence between drug-induced resistance, the tumor immune contexture, and engineered approaches that consider the tumor and microenvironment to improve the success of combinatorial therapy. SIGNIFICANCE: This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects.

Multitargeting Antibacterial Activity of a Synthesized Mn2+ Complex of Curcumin on Gram-Positive and Gram-Negative Bacterial Strains.

Curcumin is an important molecule with a plethora of pharmacological activities and therapeutic potentials. Despite its efficacy, it remained a potential drug candidate owing to hydrolytic instability and poor aqueous solubility. To overcome the limitations related to low solubility, low bioavailability, and the fact that curcumin is never present in solution as a "single unit", its complex was prepared with MnII with the idea that binding to a metal ion might help to resolve these issues. The complex was characterized by elemental and spectral analysis. The structure of the complex was determined by density functional theory calculations. The complex was stable at physiological buffer conditions, unlike curcumin. It did not have any detrimental effect on mammalian cells. There was a significant enhancement in the antibacterial activity of the complex compared to curcumin against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. It showed a strong affinity for deoxyribonucleic acid (DNA) evident from a high binding constant value with calf thymus DNA and also from the retarded electrophoretic mobility of bacterial plasmid DNA. The complex showed "superoxide dismutase-like" activity leading to the generation of reactive oxygen species (ROS). The complex caused bacterial membrane perturbation evident from calcein leakage assay, which was further corroborated by scanning and transmission electron microscopic experiments. Overall, the present study shows improved stability and antibacterial potency of a nontoxic complex over curcumin. Its multitargeting mode of action such as ROS-production, effective binding with DNA, and permeabilization of bacterial membrane together allows it to be an effective antibacterial agent that could be taken further for therapeutic use against bacterial infections.

Sugar-derived oxazolone pseudotetrapeptide as γ-turn inducer and anion-selective transporter.

The intramolecular cyclization of a C-3-tetrasubstituted furanoid sugar amino acid-derived linear tetrapeptide afforded an oxazolone pseudo-peptide with the formation of an oxazole ring at the C-terminus. A conformational study of the oxazolone pseudo-peptide showed intramolecular C=O···HN(II) hydrogen bonding in a seven-membered ring leading to a γ-turn conformation. This fact was supported by a solution-state NMR and molecular modeling studies. The oxazolone pseudotetrapeptide was found to be a better Cl--selective transporter for which an anion-anion antiport mechanism was established.

Self-Assembly of Fluorinated Sugar Amino Acid Derived α,γ-Cyclic Peptides into Transmembrane Anion Transport.

Syntheses of fluorinated sugar amino acid derived α,γ-cyclic tetra- and hexapeptides are reported. The IR, NMR, ESI-MS, CD, and molecular modeling studies of cyclic tetra- and hexapeptides showed C2 and C3 symmetric flat oval- and triangular-ring shaped ß-strand conformations, respectively, which appear to self-assemble into nanotubes. The α,γ-cyclic hexapeptide (EC50 = 2.14 µM) is found to be a more efficient ion transporter than α,γ-cyclic tetrapeptide (EC50 = 14.75 µM). The anion selectivity and recognition of α,γ-cyclic hexapeptide with NO3- ion is investigated.

Chloride Transport through Supramolecular Barrel-Rosette Ion Channels: Lipophilic Control and Apoptosis-Inducing Activity.

Despite the great interest in artificial ion channel design, only a small number of channel-forming molecules are currently available for addressing challenging problems, particularly in the biological systems. Recent advances in chloride-mediated cell death, aided by synthetic ion carriers, encouraged us to develop chloride selective supramolecular ion channels. The present work describes vicinal diols, tethered to a rigid 1,3-diethynylbenzene core, as pivotal moieties for the barrel-rosette ion channel formation, and the activity of such channels was tuned by controlling the lipophilicity of designed monomers. Selective transport of chloride ions via an antiport mechanism and channel formation in the lipid bilayer membranes were confirmed for the most active molecule. A theoretical model of the supramolecular barrel-rosette, favored by a network of intermolecular hydrogen bonding, has been proposed. The artificial ion-channel-mediated transport of chloride into cells and subsequent disruption of cellular ionic homeostasis were evident. Perturbation of chloride homeostasis in cells instigates cell death by inducing the caspase-mediated intrinsic pathway of apoptosis.

Chloride-Mediated Apoptosis-Inducing Activity of Bis(sulfonamide) Anionophores.

Transmembrane anion transport modality is enjoying a renewed interest because of recent advances toward anticancer therapy. Here we show bis(sulfonamides) as efficient receptors for selective Cl(-) ion binding and transport across lipid bilayer membranes. Anion-binding studies by (1)H NMR indicate a logical correlation between the acidity of sulfonamide N-H proton and binding strength. Such recognition is influenced further by the lipophilicity of a receptor during the ion-transport process. The anion-binding and transport activity of a bis(sulfonamide) system are far superior compared to those of the corresponding bis(carboxylic amide) derivative. Fluorescent-based assays confirm the Cl(-)/anion antiport as the operational mechanism of the ion transport by bis(sulfonamides). Disruption of ionic homeostasis by the transported Cl(-) ion, via bis(sulfonamide), is found to impose cell death. Induction of a caspase-dependent intrinsic pathway of apoptosis is confirmed by monitoring the changes in mitrochondrial membrane potential, cytochrome c leakage, activation of family of caspases, and nuclear fragmentation studies.

Trimodal Control of Ion-Transport Activity on Cyclo-oligo-(1→6)-ß-D-glucosamine-Based Artificial Ion-Transport Systems.

Cyclo-oligo-(1→6)-ß-D-glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion-transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion-transport activity increased from di- to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion-transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion-selectivity sequence: Cl(-) >Br(-) >I(-) . From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed.

Lysosome targeting fluorescence probe for imaging intracellular thiols.

A BODIPY-based fluorescence turn-on probe, exhibiting high selectivity and sensitivity towards intracellular thiols with excellent lysosomal localization is reported. The probe displayed fast response towards biothiols in aqueous solution. Localization of the probe in lysosome was demonstrated by intracellular colocalization studies with the aid of LysoSensor Green.