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1.
ACS Appl Mater Interfaces ; 16(8): 9680-9689, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38364813

RESUMO

Nitric oxide (NO) generated within the tumor microenvironment is an established driver of cancer progression and metastasis. Recent efforts have focused on leveraging this feature to target cancer through the development of diagnostic imaging agents and activatable chemotherapeutics. In this context, porphyrins represent an extraordinarily promising class of molecules, owing to their demonstrated use within both modalities. However, the remodeling of a standard porphyrin to afford a responsive chemical that can distinguish elevated NO from physiological levels has remained a significant research challenge. In this study, we employed a photoinduced electron transfer strategy to develop a panel of NO-activatable porphyrin photosensitizers (NOxPorfins) augmented with real-time fluorescence monitoring capabilities. The lead compound, NOxPorfin-1, features an o-phenylenediamine trigger that can effectively capture NO (via N2O3) to yield a triazole product that exhibits a 7.5-fold enhancement and a 70-fold turn-on response in the singlet oxygen quantum yield and fluorescence signal, respectively. Beyond demonstrating excellent in vitro responsiveness and selectivity toward NO, we showcase the potent photodynamic therapy (PDT) effect of NOxPorfin-1 in murine breast cancer and human non-small cellular lung cancer cells. Further, to highlight the in vivo efficacy, two key studies were executed. First, we utilized NOxPorfin-1 to ablate murine breast tumors in a site-selective manner without causing substantial collateral damage to healthy tissue. Second, we established a nascent human lung cancer model to demonstrate the unprecedented ability of NOxPorfin-1 to halt tumor growth and progression completely. The results of the latter study have tremendous implications for applying PDT to target metastatic lesions.


Assuntos
Neoplasias Pulmonares , Fotoquimioterapia , Porfirinas , Humanos , Animais , Camundongos , Óxido Nítrico , Porfirinas/farmacologia , Porfirinas/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/química , Neoplasias Pulmonares/tratamento farmacológico , Linhagem Celular Tumoral , Microambiente Tumoral
2.
J Vis Exp ; (193)2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-37010308

RESUMO

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP<71>) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).


Assuntos
Ambiente Controlado , Infertilidade , Humanos
3.
J Am Chem Soc ; 145(13): 7313-7322, 2023 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-36973171

RESUMO

The attachment of glucose to drugs and imaging agents enables cancer cell targeting via interactions with GLUT1 overexpressed on the cell surface. While an added benefit of this modification is the solubilizing effect of carbohydrates, in the context of imaging agents, aqueous solubility does not guarantee decreased π-stacking or aggregation. The resulting broadening of the absorbance spectrum is a detriment to photoacoustic (PA) imaging since the signal intensity, accuracy, and image quality all rely on reliable spectral unmixing. To address this major limitation and further enhance the tumor-targeting ability of imaging agents, we have taken a biomimetic approach to design a multivalent glucose moiety (mvGlu). We showcase the utility of this new group by developing aza-BODIPY-based contrast agents boasting a significant PA signal enhancement greater than 11-fold after spectral unmixing. Moreover, when applied to targeting cancer cells, effective staining could be achieved with ultra-low dye concentrations (50 nM) and compared to a non-targeted analogue, the signal intensity was >1000-fold higher. Lastly, we employed the mvGlu technology to develop a logic-gated acoustogenic probe to detect intratumoral copper (i.e., Cu(I)), which is an emerging cancer biomarker, in a murine model of breast cancer. This exciting application was not possible using other acoustogenic probes previously developed for copper sensing.


Assuntos
Neoplasias , Técnicas Fotoacústicas , Humanos , Animais , Camundongos , Cobre , Biomimética , Corantes Fluorescentes , Análise Espectral , Técnicas Fotoacústicas/métodos
4.
Angew Chem Int Ed Engl ; 62(13): e202214855, 2023 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-36722146

RESUMO

Shortwave infrared (SWIR) dyes are characterized by their ability to absorb light from 900 to 1400 nm, which is ideal for deep tissue imaging owing to minimized light scattering and interference from endogenous pigments. An approach to access such molecules is to tune the photophysical properties of known near-infrared dyes. Herein, we report the development of a series of easily accessible (three steps) SWIR xanthene dyes based on a dibenzazepine donor conjugated to thiophene (SCR-1), thienothiophene (SCR-2), or bithiophene (SCR-3). We leverage the fact that SCR-1 undergoes a bathochromic shift when aggregated for in vivo studies by developing a ratiometric nanoparticle for NO (rNP-NO), which we employed to successfully visualize pathological levels of nitric oxide in a drug-induced liver injury model via deep tissue SWIR photoacoustic (PA) imaging. Our work demonstrates how easily this dye series can be utilized as a component in nanosensor designs for imaging studies.


Assuntos
Óxido Nítrico , Técnicas Fotoacústicas , Técnicas Fotoacústicas/métodos , Xantenos , Diagnóstico por Imagem/métodos , Corantes Fluorescentes , Imagem Óptica
5.
J Clin Microbiol ; 61(2): e0149822, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36688643

RESUMO

Testing of cellular therapy products for Mycoplasma is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of Mycoplasma contamination in cell culture is high. Gold standard testing follows USP 63 which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ Mycoplasma detection kit (Life Technologies), the MycoTOOL Mycoplasma real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal Mycoplasma detection kit, and the Biofire Mycoplasma assay (bioMérieux Industry) using 10 cultured Mollicutes spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire Mycoplasma assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.


Assuntos
Mycoplasma , Humanos , Limite de Detecção , Técnicas de Cultura de Células , Padrões de Referência , Terapia Baseada em Transplante de Células e Tecidos
6.
Org Lett ; 24(46): 8509-8513, 2022 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-36374323

RESUMO

SWIR dyes offer many advantages over their more common NIR congeners; however, the available options are limited. New SWIR imaging agents can be accessed by remodeling existing NIR molecules (i.e., hemicyanines (HDs)). In this study, we synthesized SWIR-HD, a modified HD featuring dimethylsilicon and benzo[cd]indolium groups that are designed to red-shift the absorbance and emission to 988 and 1126 nm, respectively. SWIR-HD was employed to visualize the liver and tumors via multimodal SWIR imaging.


Assuntos
Silício
7.
Methods Enzymol ; 657: 157-180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34353486

RESUMO

In this chapter, we introduce a two-phase tuning approach for developing highly sensitive photoacoustic probes for imaging nitric oxide (NO) in the near-infrared (NIR)-II window. Due to the synthetically challenging nature of current NIR-II dye platforms, our two-phase tuning approach circumvents this issue by first allowing one to tune the reactivity using a synthetically accessible dye. We have used a physical organic workflow to understand the reaction kinetics and identify the most reactive sensing component. The selected reactive trigger is then introduced to phase two where it is appended to a range of well-established NIR-II dyes. This strategy is used to select the ideal photoacoustic probe for NIR-II imaging in vivo. Here, we have detailed procedures for synthesis, in vitro studies, and in vivo imaging.


Assuntos
Técnicas Fotoacústicas , Corantes , Óxido Nítrico , Análise Espectral
8.
J Am Chem Soc ; 143(18): 7196-7202, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33905646

RESUMO

Photoacoustic (PA) imaging has emerged as a reliable in vivo technique for diverse biomedical applications ranging from disease screening to analyte sensing. Most contemporary PA imaging agents employ NIR-I light (650-900 nm) to generate an ultrasound signal; however, there is significant interference from endogenous biomolecules such as hemoglobin that are PA active in this window. Transitioning to longer excitation wavelengths (i.e., NIR-II) reduces the background and facilitates the detection of low abundance targets (e.g., nitric oxide, NO). In this study, we employed a two-phase tuning approach to develop APNO-1080, a NIR-II NO-responsive probe for deep-tissue PA imaging. First, we performed Hammett and Brønsted analyses to identify a highly reactive and selective aniline-based trigger that reacts with NO via N-nitrosation chemistry. Next, we screened a panel of NIR-II platforms to identify chemical structures that have a low propensity to aggregate since this can diminish the PA signal. In a head-to-head comparison with a NIR-I analogue, APNO-1080 was 17.7-fold more sensitive in an in vitro tissue phantom assay. To evaluate the deep-tissue imaging capabilities of APNO-1080 in vivo, we performed PA imaging in an orthotopic breast cancer model and a heterotopic lung cancer model. Relative to control mice not bearing tumors, the normalized turn-on response was 1.3 ± 0.12 and 1.65 ± 0.07, respectively.


Assuntos
Desenvolvimento de Medicamentos , Corantes Fluorescentes/química , Óxido Nítrico/análise , Imagem Óptica , Técnicas Fotoacústicas , Células A549 , Animais , Corantes Fluorescentes/síntese química , Humanos , Raios Infravermelhos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem
9.
Diagn Microbiol Infect Dis ; 99(1): 115209, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33080426

RESUMO

Nasopharyngeal flocked swabs placed in viral transport media (VTM) are the preferred collection methodology for respiratory virus testing. Due to the rapid depletion of available reagents and swabs, we have validated an alternative swab placed in phosphate-buffered saline (PBS) for use in respiratory virus testing in a SARS-CoV-2 real-time polymerase chain reaction assay and a multiplexed respiratory virus panel. We collected nasopharyngeal (NP) swabs and oropharyngeal (OP) swabs from 10 healthy volunteers. Flocked swabs were placed in VTM and alternative swabs in PBS. In this feasibility study, we show that NP collection is better for detection of human material than OP collection, as measured by significantly lower RNase P gene cycle threshold values, and that a Dacron polyester swab in PBS shows equivalent detection of SARS-CoV-2 and RSV to a flocked swab in VTM in contrived specimens. Diluted SARS-CoV-2-positive patient specimens are detectable for up to 72 h at 4 °C.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Teste de Ácido Nucleico para COVID-19 , Meios de Cultura , Estudos de Viabilidade , Humanos , Nasofaringe/virologia , Orofaringe/virologia , Polietilenotereftalatos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , SARS-CoV-2/genética
10.
Chem Sci ; 12(10): 3393-3405, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34163614

RESUMO

In vivo imaging is a powerful approach to study biological processes. Beyond cellular methods, in vivo studies allow for biological stimuli (small molecules or proteins) to be studied in their native environment. This has the potential to aid in the discovery of new biology and guide the development of diagnostics and therapies for diseases. To ensure selectivity and an observable readout, the probe development field is shifting towards activity-based sensing (ABS) approaches and near-infrared (NIR) imaging modalities. This perspective will highlight recent in vivo ABS applications that utilize NIR imaging platforms.

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