RESUMO
Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2 times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cells in nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.
Assuntos
Neoplasias da Mama , Células Endoteliais , Nanomedicina , Nanopartículas , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , Nanopartículas/química , Nanomedicina/métodos , FemininoRESUMO
Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope. By exploiting the nanoparticles' light scattering properties, we demonstrated quantitative label-free imaging of intracellular nanoparticles with ultrastructural context. We confirmed the compatibility of two expansion microscopy protocols, protein retention and pan-expansion microscopy, with nanoparticle uptake studies. We validated relative differences between nanoparticle cellular accumulation for various surface modifications using mass spectrometry and determined the intracellular nanoparticle spatial distribution in 3D for entire single cells. This super-resolution imaging platform technology may be broadly used to understand the nanoparticle intracellular fate in fundamental and applied studies to potentially inform the engineering of safer and more effective nanomedicines.
Assuntos
Nanopartículas Metálicas , Animais , Nanopartículas Metálicas/química , Microscopia Eletrônica , Nanomedicina , Espectrometria de Massas , Imageamento Tridimensional , MamíferosRESUMO
We used heparosan (HEP) polysaccharides for controlling nanoparticle delivery to innate immune cells. Our results show that HEP-coated nanoparticles were endocytosed in a time-dependent manner by innate immune cells via both clathrin-mediated and macropinocytosis pathways. Upon endocytosis, we observed HEP-coated nanoparticles in intracellular vesicles and the cytoplasm, demonstrating the potential for nanoparticle escape from intracellular vesicles. Competition with other glycosaminoglycan types inhibited the endocytosis of HEP-coated nanoparticles only partially. We further found that nanoparticle uptake into innate immune cells can be controlled by more than 3 orders of magnitude via systematically varying the HEP surface density. Our results suggest a substantial potential for HEP-coated nanoparticles to target innate immune cells for efficient intracellular delivery, including into the cytoplasm. This HEP nanoparticle surface engineering technology may be broadly used to develop efficient nanoscale devices for drug and gene delivery as well as possibly for gene editing and immuno-engineering applications.
Assuntos
Nanopartículas , Clatrina/metabolismo , Dissacarídeos , Endocitose , Imunidade Inata , PolissacarídeosRESUMO
Activation of the innate immune STimulator of INterferon Genes (STING) pathway potentiates antitumour immunity, but systemic delivery of STING agonists to tumours is challenging. We conjugated STING-activating cyclic dinucleotides (CDNs) to PEGylated lipids (CDN-PEG-lipids; PEG, polyethylene glycol) via a cleavable linker and incorporated them into lipid nanodiscs (LNDs), which are discoid nanoparticles formed by self-assembly. Compared to state-of-the-art liposomes, intravenously administered LNDs carrying CDN-PEG-lipid (LND-CDNs) exhibited more efficient penetration of tumours, exposing the majority of tumour cells to STING agonist. A single dose of LND-CDNs induced rejection of established tumours, coincident with immune memory against tumour rechallenge. Although CDNs were not directly tumoricidal, LND-CDN uptake by cancer cells correlated with robust T-cell activation by promoting CDN and tumour antigen co-localization in dendritic cells. LNDs thus appear promising as a vehicle for robust delivery of compounds throughout solid tumours, which can be exploited for enhanced immunotherapy.
Assuntos
Nanopartículas , Neoplasias , Humanos , Imunoterapia , Lipídeos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
We report on the absolute quantification of nanoparticle interactions with individual human B cells using quadrupole-based inductively coupled plasma mass spectrometry (ICP-MS). This method enables the quantification of nanoparticle-cell interactions at single nanoparticle and single cell levels. We demonstrate the efficient and accurate detection of individually suspended B cells and found an â¼100-fold higher association of colloidally stable positively charged nanoparticles with single B cells than neutrally charged nanoparticles. We confirmed that these nanoparticles were internalized by individual B cells and determined that the internalization occurred via energy-dependent pathways consistent with endocytosis. Using dual analyte ICP-MS, we determined that >80% of single B cells were positive for nanoparticles. Our study demonstrates an ICP-MS workflow for the absolute quantification of nanoparticle-cell interactions with single cell and single nanoparticle resolution. This unique workflow could inform the rational design of various nanomaterials for controlling cellular interactions, including immune cell-nanoparticle interactions.
Assuntos
Nanopartículas , Humanos , Espectrometria de Massas/métodos , Análise EspectralRESUMO
Nanoparticle modification with poly(ethylene glycol) (PEG) is a widely used surface engineering strategy in nanomedicine. However, since the artificial PEG polymer may adversely impact nanomedicine safety and efficacy, alternative surface modifications are needed. Here, we explored the "self" polysaccharide heparosan (HEP) to prepare colloidally stable HEP-coated nanoparticles, including gold and silver nanoparticles and liposomes. We found that the HEP-coating reduced the nanoparticle protein corona formation as efficiently as PEG coatings upon serum incubation. Liquid chromatography-mass spectrometry revealed the protein corona profiles. Heparosan-coated nanoparticles exhibited up to 230-fold higher uptake in certain innate immune cells, but not in other tested cell types, than PEGylated nanoparticles. No noticeable cytotoxicity was observed. Serum proteins did not mediate the high cell uptake of HEP-coated nanoparticles. Our work suggests that HEP polymers may be an effective surface modification technology for nanomedicines to safely and efficiently target certain innate immune cells.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Coroa de Proteína , Adsorção , Proteínas Sanguíneas , Dissacarídeos , Nanopartículas/química , Polietilenoglicóis/química , Polímeros , Polissacarídeos , PrataRESUMO
To control a nanoparticle's chemical composition and thus function, researchers require readily accessible and economical characterization methods that provide quantitative in situ analysis of individual nanoparticles with high throughput. Here, we established dual analyte single-particle inductively coupled plasma quadrupole mass spectrometry to quantify the chemical composition and reaction kinetics of individual colloidal nanoparticles. We determined the individual bimetallic nanoparticle mass and chemical composition changes during two different chemical reactions: (i) nanoparticle etching and (ii) element deposition on nanoparticles at a rate of 300+ nanoparticles/min. Our results revealed the heterogeneity of chemical reactions at the single nanoparticle level. This proof-of-concept study serves as a framework to quantitatively understand the dynamic changes of physicochemical properties that individual nanoparticles undergo during chemical reactions using a commonly available mass spectrometer. Such methods will broadly empower and inform the synthesis and development of safer, more effective, and more efficient nanotechnologies that use nanoparticles with defined functions.
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Nanopartículas , Cinética , Espectrometria de Massas/métodos , Análise EspectralRESUMO
Combination immunotherapy treatments that recruit both innate and adaptive immunity have the potential to increase cancer response rates by engaging a more complete repertoire of effector mechanisms. Here, we combined intratumoral STimulator of INterferon Genes (STING) agonist therapy with systemically injected extended half-life IL2 and anti-PD-1 checkpoint blockade (hereafter CIP therapy) to drive innate and adaptive antitumor immunity in models of triple-negative breast cancer. Unlike treatment with the individual components, this trivalent immunotherapy halted primary tumor progression and led to long-term remission for a majority of animals in two spontaneously metastasizing orthotopic breast tumor models, though only as a neoadjuvant therapy but not adjuvant therapy. CIP therapy induced antitumor T-cell responses, but protection from metastatic relapse depended on natural killer (NK) cells. The combination of STING agonists with IL2/anti-PD-1 synergized to stimulate sustained granzyme and cytokine expression by lung-infiltrating NK cells. Type I IFNs generated as a result of STING agonism, combined with IL2, acted in a positive-feedback loop by enhancing the expression of IFNAR-1 and CD25 on lung NK cells. These results suggest that NK cells can be therapeutically targeted to effectively eliminate tumor metastases.See related Spotlight by Demaria, p. 3.
Assuntos
Neoplasias da Mama/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Terapia Neoadjuvante , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/secundário , Linhagem Celular Tumoral , Meia-Vida , Imunoterapia , Interferon Tipo I/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Metástase NeoplásicaRESUMO
There is growing evidence indicating the need to combine the rehabilitation and regenerative medicine fields to maximize functional recovery after spinal cord injury (SCI), but there are limited methods to synergistically combine the fields. Conductive biomaterials may enable synergistic combination of biomaterials with electric stimulation (ES), which may enable direct ES of neurons to enhance axon regeneration and reorganization for better functional recovery; however, there are three major challenges in developing conductive biomaterials: (1) low conductivity of conductive composites, (2) many conductive components are cytotoxic, and (3) many conductive biomaterials are pre-formed scaffolds and are not injectable. Pre-formed, noninjectable scaffolds may hinder clinical translation in a surgical context for the most common contusion-type of SCI. Alternatively, an injectable biomaterial, inspired by lessons from bioinks in the bioprinting field, may be more translational for contusion SCIs. Therefore, in the current study, a conductive hydrogel was developed by incorporating high aspect ratio citrate-gold nanorods (GNRs) into a hyaluronic acid and gelatin hydrogel. To fabricate nontoxic citrate-GNRs, a robust synthesis for high aspect ratio GNRs was combined with an indirect ligand exchange to exchange a cytotoxic surfactant for nontoxic citrate. For enhanced surgical placement, the hydrogel precursor solution (i.e., before crosslinking) was paste-like, injectable/bioprintable, and fast-crosslinking (i.e., 4 min). Finally, the crosslinked hydrogel supported the adhesion/viability of seeded rat neural stem cells in vitro. The current study developed and characterized a GNR conductive hydrogel/bioink that provided a refinable and translational platform for future synergistic combination with ES to improve functional recovery after SCI.
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Bioimpressão , Nanotubos , Animais , Axônios , Bioimpressão/métodos , Gelatina , Ouro , Ácido Hialurônico , Hidrogéis , Regeneração Nervosa , Impressão Tridimensional , Ratos , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Tissue-resident memory T cells (TRMs) can profoundly enhance mucosal immunity, but parameters governing TRM induction by vaccination remain poorly understood. Here, we describe an approach exploiting natural albumin transport across the airway epithelium to enhance mucosal TRM generation by vaccination. Pulmonary immunization with albumin-binding amphiphile conjugates of peptide antigens and CpG adjuvant (amph-vaccines) increased vaccine accumulation in the lung and mediastinal lymph nodes (MLNs). Amph-vaccines prolonged antigen presentation in MLNs over 2 weeks, leading to 25-fold increased lung-resident T cell responses over traditional immunization and enhanced protection from viral or tumor challenge. Mimicking such prolonged exposure through repeated administration of soluble vaccine revealed that persistence of both antigen and adjuvant was critical for optimal TRM induction, mediated through T cell priming in MLNs after prime, and directly in the lung tissue after boost. Thus, vaccine persistence strongly promotes TRM induction, and amph-conjugates may provide a practical approach to achieve such kinetics in mucosal vaccines.
Assuntos
Adjuvantes Imunológicos , Albuminas/imunologia , Imunidade nas Mucosas , Memória Imunológica , Pulmão/imunologia , Células T de Memória/imunologia , Animais , Biomarcadores , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunização , Imunofenotipagem , Pulmão/metabolismo , Ativação Linfocitária/imunologia , Células T de Memória/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas/imunologiaRESUMO
Biological interactions, toxicity, and environmental fate of engineered nanoparticles are affected by colloidal stability and aggregation. To assess nanoparticle aggregation, analytical methods are needed that allow quantification of individual nanoparticle aggregates. However, most techniques used for nanoparticle aggregation analysis are limited to ensemble measurements or require harsh sample preparation that may introduce artifacts. An ideal method would analyze aggregate size in situ with single-nanoparticle resolution. Here, we established and validated single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) as an unbiased high-throughput analytical technique to quantify nanoparticle size distributions and aggregation in situ. We induced nanoparticle aggregation by exposure to physiologically relevant saline conditions and applied SP-ICP-MS to quantify aggregate size and aggregation kinetics at the individual aggregate level. In situ SP-ICP-MS analysis revealed rational surface engineering principles for the preparation of colloidally stable nanoparticles. Our quantitative SP-ICP-MS technique is a platform technology to evaluate aggregation characteristics of various types of surface-engineered nanoparticles under physiologically relevant conditions. Potential widespread applications of this method may include the study of nanoparticle aggregation in environmental samples and the preparation of colloidally stable nanoparticle formulations for bioanalytical assays and nanomedicine. Graphical abstract.
RESUMO
Nanoparticle-based therapeutics and diagnostics are commonly referred to as nanomedicine and may significantly impact the future of healthcare. However, the clinical translation of these technologies is challenging. One of these challenges is the efficient delivery of nanoparticles to specific cell populations and subcellular targets in the body to elicit desired biological and therapeutic responses. It is critical for researchers to understand the fundamental concepts of how nanoparticles interact with biological systems to predict and control in vivo nanoparticle transport for improved clinical benefit. In this overview article, we review and discuss cellular internalization pathways, summarize the field`s understanding of how nanoparticle physicochemical properties affect cellular interactions, and explore and discuss intracellular nanoparticle trafficking and kinetics. Our overview may provide a valuable resource for researchers and may inspire new studies to expand our current understanding of nanotechnology-biology interactions at cellular and subcellular levels with the goal to improve clinical translation of nanomedicines.
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Nanopartículas/metabolismo , Animais , Transporte Biológico , Endocitose , Humanos , Cinética , NanomedicinaRESUMO
A growing need for individuals with behavior analytic training at the undergraduate level has led to an increase in baccalaureate programs with a strong behavior analytic focus. Although research has been conducted examining essential and assigned readings at the graduate level, no research to date has focused on identifying suggested readings that should be a focal point of undergraduate training programs. The purpose of the present study was to identify what individuals from across the behavior analytic field believe are essential readings for undergraduate students as they prepare for employment in the field or admission into graduate programs. Respondents were asked to provide answers to a variety of questions about essential readings in the field and whether these would be critical to undergraduate training. This paper presents those texts that were deemed essential, as well as areas where opinions varied.