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Minimalistic peptide-based systems that bind sugars in water are challenging to design due to the weakness of interactions and required cooperative contributions from specific amino-acid side chains. Here, we used a bottom-up approach to create peptide-based adaptive glucose-binding networks by mixing glucose with selected sets of input dipeptides (up to 4) in the presence of an amidase to enable in situ reversible peptide elongation, forming mixtures of up to 16 dynamically interacting tetrapeptides. The choice of input dipeptides was based on amino-acid abundance in glucose-binding sites found in the protein data bank, with side chains that can support hydrogen bonding and CH-π interactions. Tetrapeptide sequence amplification patterns, determined through LC-MS analysis, served as a readout for collective interactions and led to the identification of optimized binding networks. Systematic variation of dipeptide input revealed the emergence of two networks of non-covalent hydrogen bonding and CH-π interactions that can co-exist, are cooperative and context-dependent. A cooperative binding mode was determined by studying the binding of the most amplified tetrapeptide (AWAD) with glucose in isolation. Overall, these results demonstrate that the bottom-up design of complex systems can recreate emergent behaviors driven by covalent and non-covalent self-organization that are not observed in reductionist designs and lead to the identification of system-level cooperative binding motifs.
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Dipéptidos , Péptidos , Péptidos/química , Dipéptidos/química , Sitios de Unión , Aminoácidos/química , Glucosa , Enlace de HidrógenoRESUMEN
Distortion of nominally planar phthalocyanine macrocycles affects the excited state dynamics in that most of the excited-state energy decays through internal conversion. A click-type annulation reaction on a perfluorophthalocyanine platform appending a seven-membered ring to the ß-positions on one or more of the isoindoles distorts the macrocycle and modulates solubility. The distorted derivative enables photoacoustic imaging, photothermal effects, and strong surface-enhanced resonance Raman signals.
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The use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as porphyrins provides a promising strategy to improve the selectivity and cell uptake of the photosensitizers, enhancing the overall photosensitizing efficacy. Most porphyrin-carbohydrate conjugates are linked via the carbon-1 position of the carbohydrate because this is the most synthetically accessible approach. Previous studies suggest that carbon-1 galactose derivatives show diminished binding since the hydroxyl group in the carbon-1 position of the sugar is a hydrogen bond acceptor in the galectin-1 sugar binding site. We therefore synthesized two isomeric porphyrin-galactose conjugates using click chemistry: one linked via the carbon-1 of the galactose and one linked via carbon-3. Free base and zinc analogs of both conjugates were synthesized. We assessed the uptake and photodynamic therapeutic (PDT) activity of the two conjugates in both monolayer and spheroidal cell cultures of four different cell lines. For both the monolayer and spheroid models, we observe that the uptake of both conjugates is proportional to the protein levels of galectin-1 and the uptake is suppressed after preincubation with an excess of thiogalactose, as measured by fluorescence spectroscopy. Compared to that of the carbon-1 conjugate, the uptake of the carbon-3 conjugate was greater in cell lines containing high expression levels of galectin-1. After photodynamic activation, MTT and lactate dehydrogenase assays demonstrated that the conjugates induce phototoxicity in both monolayers and spheroids of cancer cells.
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Galactosa/análogos & derivados , Galactosa/farmacología , Neoplasias/tratamiento farmacológico , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Porfirinas/química , Porfirinas/farmacología , Carbono/química , Línea Celular Tumoral , Galactosa/síntesis química , Galactosa/farmacocinética , Humanos , Fotoquimioterapia , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/síntesis química , Porfirinas/farmacocinéticaRESUMEN
Chlorins have unique photophysical properties that are exploited in diverse biological and materials applications. De novo chlorin synthesis with specific exocyclic motifs can be challenging and many are not stable to photobleaching and/or oxidation. A facile approach to a stable synthetic chlorin with a fused N-methyl pyrrolidine uses cyclo addition of a sarcosine-based azomethine ylide on 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)-porphyrin (TPPF20) is reported, but this approach has limitations. We report the synthesis of stable chlorin scaffolds starting with TPPF20 using a new glycine-based N-(hydroxymethyl)- N-methelenemethanideaminium ylide. Careful control of the 1,3-dipolar cycloaddition reaction allows a divergent use of the glycine derived ylide to yield four new chlorins, including the fused NH-pyrrolidine, two dimers, and the same N-methyl chlorin product from the sarcosine ylide reaction. The mechanism begins with the formation of a bis(hydroxymethyl)glycine, which then dehydrates and decarboxylates to form the active N-(hydroxymethyl)- N-methelenemethanideaminium ylide, which then reacts with TPPF20 to form a key N-(hydroxymethyl)-17,18-pyrrolidinyl-chlorin intermediate. Deformylation of this intermediate affords the (17,18-pyrrolidinyl)-chlorin, whereas a Cannizzaro-type reaction promotes a hydride attack to an imine chlorin cation to yield the N-methyl chlorin. The exocyclic NH-pyrrolidine provides a unique mode of attaching chiral moieties that avoids formation of diasteromers at the bridgehead carbons.
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Porfirinas/síntesis química , Compuestos Azo/química , Reacción de Cicloadición , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Porfirinas/química , Espectrofotometría Ultravioleta , Tiosemicarbazonas/químicaRESUMEN
Drug nanoaggregates are particles that can deleteriously cause false positive results during drug screening efforts, but alternatively, they may be used to improve pharmacokinetics when developed for drug delivery purposes. The structural features of molecules that drive nanoaggregate formation remain elusive, however, and the prediction of intracellular aggregation and rational design of nanoaggregate-based carriers are still challenging. We investigate nanoaggregate self-assembly mechanisms using small molecule fragments to identify the critical molecular forces that contribute to self-assembly. We find that aromatic groups and hydrogen bond acceptors/donors are essential for nanoaggregate formation, suggesting that both π-π stacking and hydrogen bonding are drivers of nanoaggregation. We apply structure-assembly-relationship analysis to the drug sorafenib and discover that nanoaggregate formation can be predicted entirely using drug fragment substructures. We also find that drug nanoaggregates are stabilized in an amorphous core-shell structure. These findings demonstrate that rational design can address intracellular aggregation and pharmacologic/delivery challenges in conventional and fragment-based drug development processes.
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Simulación de Dinámica Molecular , Preparaciones FarmacéuticasRESUMEN
Correction for 'Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation' by Chrysafis Andreou et al., Nanoscale Horiz., 2022, 7, 1540-1552, https://doi.org/10.1039/d2nh00331g.
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Cell surface marker expression in tumors dictates the selection of therapeutics, therapy response, and survival. However, biopsies are invasive, sample only a small area of the tumor landscape and may miss significant areas of heterogeneous expression. Here, we investigated the potential of antibody-conjugated surface-enhanced resonance Raman scattering nanoparticles (SERRS-NPs) to depict and quantify high and low tumoral surface marker expression, focusing on the surface markers epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in an intracerebral and peripheral setting with an inter- and intratumoral comparison of Raman signal intensities. Methods: ICR-Prkdc
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Glioblastoma , Nanopartículas , Animales , Modelos Animales de Enfermedad , Glioblastoma/diagnóstico por imagen , Glioblastoma/patología , Ratones , Ratones Endogámicos ICR , Nanopartículas/química , Espectrometría Raman/métodosRESUMEN
Rubber band ligation is a commonly used method for the removal of tissue abnormalities. Most often, rubber band ligation is performed to remove internal hemorrhoids unresponsive to first line treatments to avoid surgery. While the procedure is considered safe, patients experience mild to significant pain and discomfort until the tissue sloughs off. As patients often require multiple bandings and sessions, reducing these side effects can have a considerable effect on patient adherence and quality of life. To reduce pain and discomfort, we developed drug-eluting rubber bands for ligation procedures. We investigated the potential for a band to elute anesthetics and drug combinations to durably manage pain for a period of up to 5 days while exhibiting similar mechanical properties to conventional rubber bands. We show that the rubber bands retain their mechanical properties despite significant drug loading. Lidocaine, released from the bands, successfully altered the calcium dynamics of cardiomyocytes in vitro and modulated heart rate in zebrafish embryos, while the bands exhibited lower cytotoxicity than conventional bands. Ex vivo studies demonstrated substantial local drug release in enteric tissues. These latex-free bands exhibited sufficient mechanical and drug-eluting properties to serve both ligation and local analgesic functions, potentially enabling pain reduction for multiple indications.
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Calidad de Vida , Pez Cebra , Animales , Humanos , Ligadura/efectos adversos , Ligadura/métodos , Dolor/etiología , Resultado del TratamientoRESUMEN
Visualizing the presence and distribution of multiple specific molecular markers within a tumor can reveal the composition of its microenvironment, inform diagnosis, stratify patients, and guide treatment. Raman imaging with multiple molecularly-targeted surface enhanced Raman scattering (SERS) nanoprobes could help investigate emerging cancer treatments preclinically or enable personalized treatment assessment. Here, we report a comprehensive strategy for multiplexed imaging using SERS nanoprobes and machine learning (ML) to monitor the early effects of immune checkpoint blockade (ICB) in tumor-bearing mice. We used antibody-functionalized SERS nanoprobes to visualize 7 + 1 immunotherapy-related targets simultaneously. The multiplexed images were spectrally resolved and then spatially segmented into superpixels based on the unmixed signals. The superpixels were used to train ML models, leading to the successful classification of mice into treated and untreated groups, and identifying tumor regions with variable responses to treatment. This method may help predict treatment efficacy in tumors and identify areas of tumor variability and therapy resistance.
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Neoplasias , Espectrometría Raman , Ratones , Animales , Espectrometría Raman/métodos , Inmunoterapia , Anticuerpos/uso terapéutico , Neoplasias/diagnóstico por imagen , Neoplasias/terapia , Factores Inmunológicos , Imagen Molecular , Microambiente TumoralRESUMEN
Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two 'flavors' of such nanoprobes: an antibody-functionalized one that targets the folate receptor - overexpressed in many ovarian cancers - and a non-targeted control nanoprobe, with distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity - which can be done conveniently during the surgical procedure - can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.
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Receptores de Folato Anclados a GPI/metabolismo , Nanotecnología/métodos , Neoplasias Ováricas/diagnóstico , Espectrometría Raman/métodos , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Recurrencia , Sensibilidad y EspecificidadRESUMEN
Recently, surface-enhanced Raman scattering nanoprobes have shown tremendous potential in oncological imaging owing to the high sensitivity and specificity of their fingerprint-like spectra. As current Raman scanners rely on a slow, point-by-point spectrum acquisition, there is an unmet need for faster imaging to cover a clinically relevant area in real-time. Herein, we report the rational design and optimization of fluorescence-Raman bimodal nanoparticles (FRNPs) that synergistically combine the specificity of Raman spectroscopy with the versatility and speed of fluorescence imaging. DNA-enabled molecular engineering allows the rational design of FRNPs with a detection limit as low as 5 × 10-15 M. FRNPs selectively accumulate in tumor tissue mouse cancer models and enable real-time fluorescence imaging for tumor detection, resection, and subsequent Raman-based verification of clean margins. Furthermore, FRNPs enable highly efficient image-guided photothermal ablation of tumors, widening the scope of the NPs into the therapeutic realm.
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Neoplasias Encefálicas/terapia , ADN/química , Nanopartículas del Metal/química , Imagen Óptica/métodos , Neoplasias Ováricas/terapia , Espectrometría Raman/métodos , Animales , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/cirugía , Línea Celular Tumoral , ADN/metabolismo , Portadores de Fármacos/síntesis química , Portadores de Fármacos/farmacocinética , Femenino , Colorantes Fluorescentes/química , Ingeniería Genética , Humanos , Terapia por Láser/instrumentación , Terapia por Láser/métodos , Límite de Detección , Terapia por Luz de Baja Intensidad/instrumentación , Terapia por Luz de Baja Intensidad/métodos , Nanopartículas del Metal/administración & dosificación , Ratones , Imagen Óptica/instrumentación , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/genética , Neoplasias Ováricas/cirugía , Fantasmas de Imagen , Plata/química , Espectrometría Raman/instrumentación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Photodynamic Therapy (PDT) relies on the use of non-toxic photosensitizers that are locally and selectively activated by light to induce cell death or apoptosis through reactive oxygen species generation. The conjugation of porphyrinoids with sugars that target cancer is increasingly viewed as an effective way to increase the selectivity of PDT. To date, in vitro PDT efficacy is mostly screened using two-dimensional monolayer cultures. Compared to monolayer cultures, three-dimensional spheroid cultures have unique spatial distributions of nutrients, metabolites, oxygen and signalling molecules; therefore better mimic in vivo conditions. We obtained 0.05 mm3 spheroids with four different human tumor cell lines (HCT-116, MCF-7, UM-UC-3 and HeLa) with appropriate sizes for screening PDT agents. We observed that detachment from monolayer culture and growth as tumor spheroids was accompanied by changes in glucose metabolism, endogenous ROS levels, galectin-1 and glucose transporter GLUT1 protein levels. We compared the phototoxic responses of a porphyrin conjugated with four glucose molecules (PorGlu4) in monolayer and spheroid cultures. The uptake and phototoxicity of PorGlu4 is highly dependent on the monolayer versus spheroid model used and on the different levels of GLUT1 protein expressed by these in vitro platforms. This study demonstrates that HCT-116, MCF-7, UM-UC-3 and HeLa spheroids afford a more rational platform for the screening of new glycosylated-photosensitizers compared to monolayer cultures of these cancer cells.
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Técnicas de Cultivo de Célula/métodos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Esferoides Celulares/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Galectina 1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glicosilación , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Porfirinas/química , Especies Reactivas de Oxígeno/metabolismo , Esferoides Celulares/citología , Células Tumorales Cultivadas/citologíaRESUMEN
A multifunctional chlorin platform appended with four short polyethylene glycols and a carboxylate-linker allows rapid conjugation to biotargeting motifs such as proteins and oligonucleotides. The stability and photophysical properties of the chlorin enable development of diagnostics, imaging, molecular tracking, and theranostics.