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Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents, which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1âS0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1âS0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches with cyanine dyes to give ultrafast relaxation (<10â ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (>3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.
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Compostos Azo , Carbocianinas , Corantes Fluorescentes , Técnicas Fotoacústicas , Compostos Azo/química , Técnicas Fotoacústicas/métodos , Corantes Fluorescentes/química , Carbocianinas/química , Humanos , Raios Infravermelhos , Estrutura Molecular , Imagem ÓpticaRESUMO
Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 °C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 °C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR.
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Despite the appreciable success of synthetic nanomaterials for targeted cancer therapy in preclinical studies, technical challenges involving their large-scale, cost-effective production and intrinsic toxicity associated with the materials, as well as their inability to penetrate tumor tissues deeply, limit their clinical translation. Here, we describe biologically derived nanocarriers developed from a bioengineered yeast strain that may overcome such impediments. The budding yeast Saccharomyces cerevisiae was genetically engineered to produce nanosized vacuoles displaying human epidermal growth factor receptor 2 (HER2)-specific affibody for active targeting. These nanosized vacuoles efficiently loaded the anticancer drug doxorubicin (Dox) and were effectively endocytosed by cultured cancer cells. Their cancer-targeting ability, along with their unique endomembrane compositions, significantly enhanced drug penetration in multicellular cultures and improved drug distribution in a tumor xenograft. Furthermore, Dox-loaded vacuoles successfully prevented tumor growth without eliciting any prolonged immune responses. The current study provides a platform technology for generating cancer-specific, tissue-penetrating, safe, and scalable biological nanoparticles for targeted cancer therapy.
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Bioengenharia , Terapia de Alvo Molecular , Especificidade de Órgãos , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Doxorrubicina/sangue , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Células RAW 264.7 , Receptor ErbB-2/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Self-assembled nanoparticles (NPs) have been intensively utilized as cancer drug delivery carriers because hydrophobic anticancer drugs may be efficiently loaded into the particle cores. In this study, we synthesized and evaluated the therapeutic index of self-assembled NPs chemically conjugated to a fibronectin extra domain B-specific peptide (APTEDB) and an anticancer agent SN38. The APTEDB-SN38 formed self-assembled structures with a diameter of 58 ± 3 nm in an aqueous solution and displayed excellent drug loading, solubility, and stability properties. A pharmacokinetic study revealed that the blood circulation half-life of SN38 following injection of the APTEDB-SN38 NPs was markedly higher than that of the small molecule CPT-11. The APTEDB-SN38 NPs delivered SN38 to tumor sites by both passive and active targeting. Finally, the APTEDB-SN38 NPs exhibited potent antitumor activities and low toxicities against EDB-expressing tumors (LLC, U87MG) in mice. This system merits further preclinical and clinical investigations for SN38 delivery.
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Nanopartículas , Animais , Antineoplásicos , Linhagem Celular Tumoral , Portadores de Fármacos , Camundongos , Camundongos Endogâmicos BALB C , NeoplasiasRESUMO
Single-cell chemical and metabolic imaging technologies provide unprecedented insights into individual cell dynamics, advancing our understanding of cellular processes, molecular interactions, and metabolic activities. Advances in fluorescence, Raman, optoacoustic (photoacoustic), or mass spectrometry methods have paved the way to characterize metabolites, signaling molecules, and other moieties within individual cells. These modalities can also lead to single-cell imaging capabilities by targeting endogenous cell contrast or by employing exogenous contrast generation techniques, including contrast agents that target specific cell structure or function. In this review, we present key developments, summarize recent applications in single-cell interrogation and imaging, and illustrate their advantages, limitations, and outlook.
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Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Imagem Molecular/métodos , Animais , Imagem Multimodal/métodosRESUMO
Cyanine derivatives are organic dyes widely used for optical imaging. However, their potential in longitudinal optoacoustic imaging and photothermal therapy remains limited due to challenges such as poor chemical stability, poor photostability, and low photothermal conversion. In this study, we present a new structural modification for cyanine dyes by introducing a strongly electron-withdrawing group (barbiturate), resulting in a new series of barbiturate-cyanine dyes (BC810, BC885, and BC1010) with suppressed fluorescence and enhanced stability. Furthermore, the introduction of BC1010 into block copolymers (PEG114-b-PCL60) induces aggregation-caused quenching, further boosting the photothermal performance. The photophysical properties of nanoparticles (BC1010-NPs) include their remarkably broad absorption range from 900 to 1200 nm for optoacoustic imaging, allowing imaging applications in NIR-I and NIR-II windows. The combined effect of these strategies, including improved photostability, enhanced nonradiative relaxation, and aggregation-caused quenching, enables the detection of optoacoustic signals with high sensitivity and effective photothermal treatment of in vivo tumor models when BC1010-NPs are administered before irradiation with a 1064 nm laser. This research introduces a barbiturate-functionalized cyanine derivative with optimal properties for efficient optoacoustics-guided theranostic applications. This new compound holds significant potential for biomedical use, facilitating advancements in optoacoustic-guided diagnostic and therapeutic approaches.
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Barbitúricos , Carbocianinas , Nanopartículas , Técnicas Fotoacústicas , Fototerapia , Animais , Técnicas Fotoacústicas/métodos , Carbocianinas/química , Carbocianinas/administração & dosagem , Nanopartículas/química , Barbitúricos/química , Barbitúricos/administração & dosagem , Fototerapia/métodos , Humanos , Camundongos Endogâmicos BALB C , Feminino , Camundongos Nus , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Corantes Fluorescentes/administração & dosagem , Camundongos , Terapia Fototérmica/métodos , Neoplasias/terapiaRESUMO
SIGNIFICANCE: Quantitative optoacoustic imaging (QOAI) continues to be a challenge due to the influence of nonlinear optical fluence distribution, which distorts the optoacoustic image representation. Nonlinear optical fluence correction in OA imaging is highly ill-posed, leading to the inaccurate recovery of optical absorption maps. This work aims to recover the optical absorption maps using deep learning (DL) approach by correcting for the fluence effect. AIM: Different DL models were compared and investigated to enable optical absorption coefficient recovery at a particular wavelength in a nonhomogeneous foreground and background medium. APPROACH: Data-driven models were trained with two-dimensional (2D) Blood vessel and three-dimensional (3D) numerical breast phantom with highly heterogeneous/realistic structures to correct for the nonlinear optical fluence distribution. The trained DL models such as U-Net, Fully Dense (FD) U-Net, Y-Net, FD Y-Net, Deep residual U-Net (Deep ResU-Net), and generative adversarial network (GAN) were tested to evaluate the performance of optical absorption coefficient recovery (or fluence compensation) with in-silico and in-vivo datasets. RESULTS: The results indicated that FD U-Net-based deconvolution improves by about 10% over reconstructed optoacoustic images in terms of peak-signal-to-noise ratio. Further, it was observed that DL models can indeed highlight deep-seated structures with higher contrast due to fluence compensation. Importantly, the DL models were found to be about 17 times faster than solving diffusion equation for fluence correction. CONCLUSIONS: The DL methods were able to compensate for nonlinear optical fluence distribution more effectively and improve the optoacoustic image quality.
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Aprendizado Profundo , Técnicas Fotoacústicas , Processamento de Imagem Assistida por Computador/métodos , Imagens de Fantasmas , Técnicas Fotoacústicas/métodos , Razão Sinal-RuídoRESUMO
Optoacoustic (photoacoustic) imaging offers unique opportunities for visualizing biological function in vivo by achieving high-resolution images of optical contrast much deeper than any other optical technique. The method detects ultrasound waves that are generated inside tissue by thermo-elastic expansion, i.e., the conversion of light absorption by tissue structures to ultrasound when the tissue is illuminated by the light of varying intensity. Listening instead of looking to light offers the major advantage of image formation with a resolution that obeys ultrasonic diffraction and not photon diffusion laws. While the technique has been widely used to explore contrast from endogenous photo-absorbing molecules, such as hemoglobin or melanin, the use of exogenous agents can extend applications to a larger range of biological and possible clinical applications, such as image-guided surgery, disease monitoring, and the evaluation of drug delivery, biodistribution, and kinetics. This review summarizes recent developments in optoacoustic agents, and highlights new functions visualized and potent pharmacology applications enabled with the use of external contrast agents.
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Técnicas Fotoacústicas , Meios de Contraste , Diagnóstico por Imagem , Humanos , Melaninas , Técnicas Fotoacústicas/métodos , Distribuição TecidualRESUMO
Genetically engineered bacterial outer membrane vesicles (OMVs) offer promising applications for gene therapy, immunotherapy, and vaccine delivery. Importantly, OMVs are biocompatible, biodegradable, and easy to engineer and produce on a large scale. In this chapter, we discuss the development and application of bioengineered OMVs for optoacoustics-guided phototherapy applications (theranostics). We provide detailed protocols for OMVs preparation, characterization, and in vitro and in vivo validation. The engineered OMVs carry the biopolymer melanin, which generates a strong optoacoustic (OA) signal and intense heat upon absorption of near-infrared (NIR) light, enabling optoacoustics-guided cancer diagnosis and photothermal therapy in vivo.
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Vesículas Extracelulares , Fototerapia , Biopolímeros , MelaninasRESUMO
Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.
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Neoplasias do Colo/terapia , Escherichia coli/metabolismo , Monofenol Mono-Oxigenase/uso terapêutico , Técnicas Fotoacústicas/métodos , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Near-infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material-associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine-based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the αv ß3 integrin ligand c(RGDyC), resulting in self-assembled nanoparticles (NPs) with cancer-targeting capability. Such CR760RGD-NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor-targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD-NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD-NPs as a promising nanoagent for NIR-based cancer phototheranostics.
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Nanopartículas , Neoplasias , Técnicas Fotoacústicas , Animais , Camundongos , Nanopartículas/uso terapêutico , Neoplasias/terapia , Fototerapia , Terapia Fototérmica , Nanomedicina TeranósticaRESUMO
Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.
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The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 µm throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations.
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We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.
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Técnicas Fotoacústicas , Animais , Camundongos , Técnicas Fotoacústicas/métodos , Proteínas , SoftwareRESUMO
Advances in genetic engineering have enabled the use of bacterial outer membrane vesicles (OMVs) to deliver vaccines, drugs and immunotherapy agents, as a strategy to circumvent biocompatibility and large-scale production issues associated with synthetic nanomaterials. We investigate bioengineered OMVs for contrast enhancement in optoacoustic (photoacoustic) imaging. We produce OMVs encapsulating biopolymer-melanin (OMVMel) using a bacterial strain expressing a tyrosinase transgene. Our results show that upon near-infrared light irradiation, OMVMel generates strong optoacoustic signals appropriate for imaging applications. In addition, we show that OMVMel builds up intense heat from the absorbed laser energy and mediates photothermal effects both in vitro and in vivo. Using multispectral optoacoustic tomography, we noninvasively monitor the spatio-temporal, tumour-associated OMVMel distribution in vivo. This work points to the use of bioengineered vesicles as potent alternatives to synthetic particles more commonly employed for optoacoustic imaging, with the potential to enable both image enhancement and photothermal applications.
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Nanopartículas , Técnicas Fotoacústicas/métodos , Animais , Proteínas da Membrana Bacteriana Externa/química , Bioengenharia , Biopolímeros/química , Feminino , Temperatura Alta/uso terapêutico , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/terapia , Melaninas/química , Camundongos , Camundongos Nus , Nanopartículas/química , Nanotecnologia , Nanomedicina TeranósticaRESUMO
Intravenously administered liposomes and other nano-sized particles are known to passively accumulate in solid tumors via the enhanced permeability and retention (EPR) effect, which is extensively explored toward the improvement of diagnosis and drug delivery in oncology. Agent extravasation into tumors is often hampered by the mononuclear phagocytic and renal systems, which sequester and/or eliminate most of the nanoparticles from the body. Dynamic imaging of the tumor microcirculation and bolus perfusion can thus facilitate optimization of the nanoparticle delivery. When it comes to non-invasive visualization of rapid biological dynamics in whole tumors, the currently available pre-clinical imaging modalities are commonly limited by shallow penetration, lack of suitable contrast or otherwise insufficient spatial or temporal resolution. Herein, we demonstrate the unique capabilities of a combined epi-fluorescence and optoacoustic tomography (FLOT) system for characterizing contrast agent dynamics in orthotopic breast tumors in mice. A liposomal indocyanine green (Lipo-ICG) preparation was administered intravenously with the time-lapse data continuously acquired during and after the injection procedure. In addition to the highly sensitive detection of the fluorescence agent by the epi-fluorescence modality, the volumetric multi-spectral optoacoustic tomography readings further enabled resolving deep-seated vascular structures with high spatial resolution and hence provided accurate readings of the dynamic bio-distribution of nanoparticles in the entire tumor in 3D. The synergetic combination of the two modalities can become a powerful tool in cancer research and potentially aid the diagnosis, staging and treatment guidance of certain types of cancer in the clinical setting.
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Multi-Spectral Optoacoustic Tomography (MSOT) merges the power of high-resolution imaging at tissue depths of several millimeters to centimeters with the advantages of optical imaging, in large part by exploiting spectral detection of endogenous molecules in tissue or exogenous photoabsorbing probes. Current advances in fast-tuning laser technology, image reconstruction and spectral detection schemes have yielded real-time optoacoustic (photoacoustic) imaging spanning applications from microscopy to human imaging. This progress has generated an unprecedented need for imaging probes and reporter gene approaches optimized for optoacoustic signal generation. New classes of probes are emerging and create new opportunities for visualizing morphological and pathophysiological features in vivo, in a non-invasive manner. Here we review recent progress in optoacoustic probes and discuss applications and challenges for biological imaging as well as prospects for clinical translation.
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Cell-penetrating peptides (CPPs) have been widely used to deliver nucleic acid molecules. Generally, CPPs consisting of short amino acid sequences have a linear structure, resulting in a weak complexation and low transfection efficacy. To overcome these drawbacks, a novel type of CPP is required to enhance the delivery efficacy while maintaining its safe use at the same time. Herein, we report that a bioreducible branched poly-CPP structure capable of responding to reducing conditions attained both outstanding delivery effectiveness and selective gene release in carcinoma cells. Branched structures provide unusually strong electrostatic attraction between DNA and siRNA molecules, thereby improving the transfection capability through a tightly condensed form. We designed a modified type of nona-arginine (mR9) and synthesized a branched-mR9 (B-mR9) using disulfide bonds. A novel B-mR9/pDNA polyplex exhibited redox-cleavability and high transfection efficacy compared to conventional CPPs, with higher cell viability as well. B-mR9/VEGF siRNA polyplex exhibited significant serum stability and high gene-silencing effects in vitro. Furthermore, the B-mR9 polyplex showed outstanding tumor accumulation and inhibition ability in vivo. The results suggest that the bioreducible branched poly CPP has great potential as a gene delivery platform.
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Arginina/análogos & derivados , Peptídeos Penetradores de Células/química , DNA/administração & dosagem , Técnicas de Transferência de Genes , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Sobrevivência Celular , DNA/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Although it has been shown that the size of nanoparticle-based vaccines is a key determining factor for the induction of immune responses, few studies have provided detailed analyses of thresholds or critical sizes of nanoparticle vaccines. Here we report effects of the size of gold nanoparticle (GNP)-based vaccines on their efficiency of delivery to lymph nodes (LNs) and induction of CD8+ T-cell responses. We further propose a threshold size of GNPs for use as an effective vaccine. To examine the effects of GNP size, we synthesized GNPs with diameters of 7, 14 and 28nm, and then conjugated them with recombinant ovalbumin (OVA) as a model antigen. The resulting OVA-GNPs had hydrodynamic diameter (HD) of ~10, 22, and 33nm for 7, 14 and 28nm GNPs, respectively and exhibited a size-dependent increase in cellular uptake by dendritic cells (DCs) and subsequent T-cell cross-priming and activation. Upon injection into a mouse footpad, both 22- and 33-nm OVA-GNPs showed much higher delivery efficiency to draining LNs than did 10-nm OVA-GNPs. An ex vivo restimulation assay using OVA as an antigen revealed that frequencies of OVA-specific CD8+ T cells were higher in mice immunized with 22- and 33-nm OVA-GNPs than in those immunized with 10-nm OVA-GNPs; moreover, these cells were shown to be poly-functional. In a tumor-prevention study, 22-nm OVA-GNPs showed greater antitumor efficacy, and higher infiltration of CD8+ T-cells and greater tumor cell apoptosis and cell death than 10-nm OVA-GNPs. Taken together, our results suggest that the size threshold for induction of potent cellular responses and T-cell poly-functionality by GNPs lies between 10nm and 22nm, and highlight the importance of nanoparticle size as a critical parameter in designing and developing nanoparticle-based vaccines.
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Antígenos/administração & dosagem , Ouro/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Ovalbumina/administração & dosagem , Vacinas/administração & dosagem , Animais , Antígenos/química , Antígenos/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Escherichia coli/genética , Feminino , Ouro/química , Linfonodos/metabolismo , Nanopartículas Metálicas/química , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ovalbumina/química , Ovalbumina/genética , Tamanho da Partícula , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Carga Tumoral/efeitos dos fármacos , Vacinas/químicaRESUMO
There is considerable interest in developing a common, universal platform for delivering biomacromolecules such as proteins and RNAs into diverse cells with high efficiency. Here, it is shown that carbon nanosyringe arrays (CNSAs) under an applied centrifugal g-force (cf-CNSAs) can deliver diverse bioactive cargos directly into the cytosol of hard-to-transfect cells with relatively high efficiency and reproducibility. The cf-CNSA platform, an optimized version of a previous CNSA-mediated intracellular delivery platform that adds a g-force feature, exhibits more rapid and superior delivery of cargos to various hard-to-transfect cells than is the case in the absence of g-force. Active species, including small interfering RNAs, plasmids, and proteins are successfully transported across plasma membrane barriers into various cells. By overcoming the limitations of currently available transfection methods, the cf-CNSA platform paves the way to universal delivery of a variety of cargos, facilitating the analysis of cellular responses in diverse cell types.