Greatly isolated heterogeneous circulating tumor cells using hybrid engineered cell membrane-camouflaged magnetic nanoparticles.

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.

Genetically engineered membrane-based nanoengagers for immunotherapy of pancreatic cancer.

Modulating macrophages presents a promising avenue in tumor immunotherapy. However, tumor cells have evolved mechanisms to evade macrophage activation and phagocytosis. Herein, we introduced a bispecific antibody-based nanoengager to facilitate the recognition and phagocytosis of tumor cells by macrophages. Specifically, we genetically engineered two single chain variable fragments (scFv) onto cell membrane: anti-CD40 scFv for engaging with macrophages and anti-Claudin18.2 (CLDN18.2) scFv for interacting with tumor cells. These nanoengagers were further constructed by coating scFv-anchored membrane into PLGA nanoparticle core. Our developed nanoengagers significantly boosted immune responses, including increased recognition and phagocytosis of tumor cells by macrophages, enhanced activation and antigen presentation, and elevated cytotoxic T lymphocyte activity. These combined benefits resulted in enhancing antitumor efficacy against highly aggressive "cold" pancreatic cancer. Overall, this study offers a versatile nanoengager design for immunotherapy, achieved through genetically engineering to incorporate antibody-anchored membrane.

Screening of Protein-Based Ultrasmall Nanozymes for Building Cell-Mimicking Catalytic Vesicles.

Enzymes are an important component for bottom-up building of synthetic/artificial cells. Nanozymes are nanomaterials with intrinsic enzyme-like properties, however, the construction of synthetic cells using nanozymes is difficult owing to their high surface energy or large size. Herein, the authors show a protein-based general platform that biomimetically integrates various ultrasmall metal nanozymes into protein shells. Specifically, eight metal-based ultrasmall nano-particles/clusters are in situ incorporated into ferritin nanocages that are self-assembled by 24 subunits of ferritin heavy chain. As a nanozyme generator, such a platform is suitable for screening the desired enzyme-like activities, including peroxidase (POD), oxidase (OXD), catalase (CAT) and superoxide dismutase (SOD). After screening, it is found that Ru intrinsically possesses the highest POD-like and CAT-like activities, while Mn and Pt show the highest OXD-like and SOD-like activities, respectively. Additionally, the inducers/inhibitors of various nanozymes are screened from more than 50 compounds to improve or inhibit their enzyme-like activities. Based on the screened nanozymes and their inhibitors, a proof-of-conceptually constructs cell-mimicking catalytic vesicles to mimic or modulate the events of redox homeostasis in living cells. This study offers a type of artificial metalloenzyme based on nanotechnology and shows a choice for bottom-up enzyme-based synthetic cell systems in a fully synthetic manner.

Combining Doxorubicin-Conjugated Polymeric Nanoparticles and 5-Aminolevulinic Acid for Enhancing Radiotherapy against Lung Cancer.

Radiation therapy (RT) concurrent with chemotherapy improves local lung cancer control but may cause systemic toxicity. There is an unmet clinical need of treatments that can selectively sensitize cancer cells to RT. Herein, we explored a radiosensitizing strategy that combines doxorubicin (DOX)-encapsulated polyaspartamide nanoparticles and 5-aminolevulinic acid (5-ALA). The DOX-polyaspartamide nanoparticles were coupled with NTSmut, a ligand specific to neurotensin receptor type 1 (NTSR1), for lung cancer targeting. DOX was coupled to the polymer backbone through a pH-sensitive hydrazone linker, which allows for controlled release of the drug in an acidic tumor micromovement. Meanwhile, 5-ALA accumulates in the cancer cell's mitochondria, forming protoporphyrin (PpIX) that amplifies RT-induced oxidative stress. When tested in vitro in H1299 cells, DOX-encapsulated nanoparticles in conjugation with 5-ALA enhanced cancer cell killing owing to the complementary radiosensitizing effects of DOX and 5-ALA. In vivo studies confirmed that the combination improved tumor suppression relative to RT alone without causing toxicity to normal tissues. Overall, our study suggests an effective and selective radiosensitizing approach.

A Novel Model System for Understanding Anticancer Activity of Hypoxia-Activated Prodrugs.

Reports on the comprehensive factors for design considerations of hypoxia-activated prodrugs (HAPs) are rare. We introduced a new model system composed of a series of highly water-soluble HAPs, providing a platform to comprehensively understand the interaction between HAPs and hypoxic biosystems. Specifically, four kinds of new HAPs were designed and synthesized, containing the same biologically active moiety but masked by different bioreductive groups. Our results demonstrated that the activity of the prodrugs was strongly dependent on not only the molecular structure but also the hypoxic tumor microenvironment. We found the presence of a direct linear relationship between cytotoxicity of the HAPs and the reduction potential of whole molecule/oxygen concentration/reductase expression. Moreover, limited blood vasculature in hypoxic regions was also a critical barrier for effective activation of the HAPs. This study offers a comprehensive insight into understanding the design factors required for HAPs.

Protein nanocages that penetrate airway mucus and tumor tissue.

Reports on drug delivery systems capable of overcoming multiple biological barriers are rare. We introduce a nanoparticle-based drug delivery technology capable of rapidly penetrating both lung tumor tissue and the mucus layer that protects airway tissues from nanoscale objects. Specifically, human ferritin heavy-chain nanocages (FTn) were functionalized with polyethylene glycol (PEG) in a unique manner that allows robust control over PEG location (nanoparticle surface only) and surface density. We varied PEG surface density and molecular weight to discover PEGylated FTn that rapidly penetrated both mucus barriers and tumor tissues in vitro and in vivo. Upon inhalation in mice, PEGylated FTn with optimized PEGylation rapidly penetrated the mucus gel layer and thus provided a uniform distribution throughout the airways. Subsequently, PEGylated FTn preferentially penetrated and distributed within orthotopic lung tumor tissue, and selectively entered cancer cells, in a transferrin receptor 1-dependent manner, which is up-regulated in most cancers. To test the potential therapeutic benefits, doxorubicin (DOX) was conjugated to PEGylated FTn via an acid-labile linker to facilitate intracellular release of DOX after cell entry. Inhalation of DOX-loaded PEGylated FTn led to 60% survival, compared with 10% survival in the group that inhaled DOX in solution at the maximally tolerated dose, in a murine model of malignant airway lung cancer. This approach may provide benefits as an adjuvant therapy combined with systemic chemo- or immunotherapy or as a stand-alone therapy for patients with tumors confined to the airways.

Mitochondrial disulfide relay mediates translocation of p53 and partitions its subcellular activity.

p53, a critical tumor suppressor, regulates mitochondrial respiration, but how a nuclear protein can orchestrate the function of an organelle encoded by two separate genomes, both of which require p53 for their integrity, remains unclear. Here we report that the mammalian homolog of the yeast mitochondrial disulfide relay protein Mia40 (CHCHD4) is necessary for the respiratory-dependent translocation of p53 into the mitochondria. In the setting of oxidative stress, increased CHCHD4 expression partitions p53 into the mitochondria and protects its genomic integrity while decreasing p53 nuclear localization and transcriptional activity. Conversely, decreased CHCHD4 expression prevents the mitochondrial translocation of p53 while augmenting its nuclear localization and activity. Thus, the mitochondrial disulfide relay system allows p53 to regulate two spatially segregated genomes depending on oxidative metabolic activity.

Self-illuminating 64Cu-doped CdSe/ZnS nanocrystals for in vivo tumor imaging.

Construction of self-illuminating semiconducting nanocrystals, also called quantum dots (QDs), has attracted much attention recently due to their potential as highly sensitive optical probes for biological imaging applications. Here we prepared a self-illuminating QD system by doping positron-emitting radionuclide (64)Cu into CdSe/ZnS core/shell QDs via a cation-exchange reaction. The (64)Cu-doped CdSe/ZnS QDs exhibit efficient Cerenkov resonance energy transfer (CRET). The signal of (64)Cu can accurately reflect the biodistribution of the QDs during circulation with no dissociation of (64)Cu from the nanoparticles. We also explored this system for in vivo tumor imaging. This nanoprobe showed high tumor-targeting ability in a U87MG glioblastoma xenograft model (12.7% ID/g at 17 h time point) and feasibility for in vivo luminescence imaging of tumor in the absence of excitation light. The availability of these self-illuminating integrated QDs provides an accurate and convenient tool for in vivo tumor imaging and detection.

Glucose oxidase-catalyzed growth of gold nanoparticles enables quantitative detection of attomolar cancer biomarkers.

Ultrasensitive and quantitative detection of cancer biomarkers is an unmet challenge because of their ultralow concentrations in clinical samples. Although gold nanoparticle (AuNP)-based immunoassays offer high sensitivity, they were unable to quantitatively detect targets of interest most likely due to their very narrow linear ranges. This article describes a quantitative colorimetric immunoassay based on glucose oxidase (GOx)-catalyzed growth of 5 nm AuNPs that can detect cancer biomarkers from attomolar to picomolar levels. In addition, the limit of detection (LOD) of prostate-specific antigen (PSA) of this approach (93 aM) exceeds that of commercial enzyme-linked immunosorbent assay (ELISA) (6.3 pM) by more than 4 orders of magnitude. The emergence of red or purple color based on enzyme-catalyzed growth of 5 nm AuNPs in the presence of target antigen is particularly suitable for point-of-care (POC) diagnostics in both resource-rich and resource-limited settings.

Machine-Learning-Assisted Nanozyme Design: Lessons from Materials and Engineered Enzymes.

Nanozymes are nanomaterials that exhibit enzyme-like biomimicry. In combination with intrinsic characteristics of nanomaterials, nanozymes have broad applicability in materials science, chemical engineering, bioengineering, biochemistry, and disease theranostics. Recently, the heterogeneity of published results has highlighted the complexity and diversity of nanozymes in terms of consistency of catalytic capacity. Machine learning (ML) shows promising potential for discovering new materials, yet it remains challenging for the design of new nanozymes based on ML approaches. Alternatively, ML is employed to promote optimization of intelligent design and application of catalytic materials and engineered enzymes. Incorporation of the successful ML algorithms used in the intelligent design of catalytic materials and engineered enzymes can concomitantly facilitate the guided development of next-generation nanozymes with desirable properties. Here, recent progress in ML, its utilization in the design of catalytic materials and enzymes, and how emergent ML applications serve as promising strategies to circumvent challenges associated with time-expensive and laborious testing in nanozyme research and development are summarized. The potential applications of successful examples of ML-aided catalytic materials and engineered enzymes in nanozyme design are also highlighted, with special focus on the unified aims in enhancing design and recapitulation of substrate selectivity and catalytic activity.

Self-Assembly of Heterogeneous Ferritin Nanocages for Tumor Uptake and Penetration.

Well-defined nanostructures are crucial for precisely understanding nano-bio interactions. However, nanoparticles (NPs) fabricated through conventional synthesis approaches often lack poor controllability and reproducibility. Herein, a synthetic biology-based strategy is introduced to fabricate uniformly reproducible protein-based NPs, achieving precise control over heterogeneous components of the NPs. Specifically, a ferritin assembly toolbox system is developed that enables intracellular assembly of ferritin subunits/variants in Escherichia coli. Using this strategy, a proof-of-concept study is provided to explore the interplay between ligand density of NPs and their tumor targets/penetration. Various ferritin hybrid nanocages (FHn) containing human ferritin heavy chains (FH) and light chains are accurately assembled, leveraging their intrinsic binding with tumor cells and prolonged circulation time in blood, respectively. Further studies reveal that tumor cell uptake is FH density-dependent through active binding with transferrin receptor 1, whereas in vivo tumor accumulation and tissue penetration are found to be correlated to heterogeneous assembly of FHn and vascular permeability of tumors. Densities of 3.7 FH/100 nm2 on the nanoparticle surface exhibit the highest degree of tumor accumulation and penetration, particularly in tumors with high permeability compared to those with low permeability. This study underscores the significance of nanoparticle heterogeneity in determining particle fate in biological systems.

Versatile Design of NO-Generating Proteolipid Nanovesicles for Alleviating Vascular Injury.

Vascular injury is central to the pathogenesis and progression of cardiovascular diseases, however, fostering alternative strategies to alleviate vascular injury remains a persisting challenge. Given the central role of cell-derived nitric oxide (NO) in modulating the endogenous repair of vascular injury, NO-generating proteolipid nanovesicles (PLV-NO) are designed that recapitulate the cell-mimicking functions for vascular repair and replacement. Specifically, the proteolipid nanovesicles (PLV) are versatilely fabricated using membrane proteins derived from different types of cells, followed by the incorporation of NO-generating nanozymes capable of catalyzing endogenous donors to produce NO. Taking two vascular injury models, two types of PLV-NO are tailored to meet the individual requirements of targeted diseases using platelet membrane proteins and endothelial membrane proteins, respectively. The platelet-based PLV-NO (pPLV-NO) demonstrates its efficacy in targeted repair of a vascular endothelium injury model through systemic delivery. On the other hand, the endothelial cell (EC)-based PLV-NO (ePLV-NO) exhibits suppression of thrombosis when modified onto a locally transplanted small-diameter vascular graft (SDVG). The versatile design of PLV-NO may enable a promising therapeutic option for various vascular injury-evoked cardiovascular diseases.

Data-Driven Design of Triple-Targeted Protein Nanoprobes for Multiplexed Imaging of Cancer Lymphatic Metastasis.

Targeted imaging of cancer lymphatic metastasis remains challenging due to its highly heterogeneous molecular and phenotypic diversity. Herein, triple-targeted protein nanoprobes capable of specifically binding to three targets for imaging cancer lymphatic metastasis, through a data-driven design approach combined with a synthetic biology-based assembly strategy, are introduced. Specifically, to address the diversity of metastatic lymph nodes (LNs), a combination of three targets, including C-X-C motif chemokine receptor 4 (CXCR4), transferrin receptor protein 1 (TfR1), and vascular endothelial growth factor receptor 3 (VEGFR3) is identified, leveraging machine leaning-based bioinformatics analysis and examination of LN tissues from patients with gastric cancer. Using this identified target combination, ferritin nanocage-based nanoprobes capable of specifically binding to all three targets are designed through the self-assembly of genetically engineered ferritin subunits using a synthetic biology approach. Using these nanoprobes, multiplexed imaging of heterogeneous metastatic LNs is successfully achieved in a polyclonal lymphatic metastasis animal model. In 19 freshly resected human gastric specimens, the signal from the triple-targeted nanoprobes significantly differentiates metastatic LNs from benign LNs. This study not only provides an effective nanoprobe for imaging highly heterogeneous lymphatic metastasis but also proposes a potential strategy for guiding the design of targeted nanomedicines for cancer lymphatic metastasis.

Self-assembly of amphiphilic plasmonic micelle-like nanoparticles in selective solvents.

Amphiphilic plasmonic micelle-like nanoparticles (APMNs) composed of gold nanoparticles (AuNPs) and amphiphilic block copolymers (BCPs) structurally resemble polymer micelles with well-defined architectures and chemistry. The APMNs can be potentially considered as a prototype for modeling a higher-level self-assembly of micelles. The understanding of such secondary self-assembly is of particular importance for the bottom-up design of new hierarchical nanostructures. This article describes the self-assembly, modeling, and applications of APMN assemblies in selective solvents. In a mixture of water/tetrahydrofuran, APMNs assembled into various superstructures, including unimolecular micelles, clusters with controlled number of APMNs, and vesicles, depending on the lengths of polymer tethers and the sizes of AuNP cores. The delicate interplay of entropy and enthalpy contributions to the overall free energy associated with the assembly process, which is strongly dependent on the spherical architecture of APMNs, yields an assembly diagram that is different from the assembly of linear BCPs. Our experimental and computational studies suggested that the morphologies of assemblies were largely determined by the deformability of the effective nanoparticles (that is, nanoparticles together with tethered chains as a whole). The assemblies of APMNs resulted in strong absorption in near-infrared range due to the remarkable plasmonic coupling of Au cores, thus facilitating their biomedical applications in bioimaging and photothermal therapy of cancer.

A facile, one-step nanocarbon functionalization for biomedical applications.

Despite their immense potential in biomedicine, carbon nanomaterials suffer from inefficient dispersion and biological activity in vivo. Here we utilize a single, yet multifunctional, hyaluronic acid-based biosurfactant to simultaneously disperse nanocarbons and target single-walled carbon nanotubes (SWCNTs) to CD44 receptor positive tumor cells with prompt uptake. Cellular uptake was monitored by intracellular enzyme-activated fluorescence, and localization of SWCNTs within cells was further confirmed by Raman mapping. In vivo photoacoustic, fluorescence, and positron emission tomography imaging of coated SWCNTs display high tumor targeting capability while providing long-term, fluorescence molecular imaging of targeted enzyme events. By utilizing a single biomaterial surfactant for SWCNT dispersion without additional bioconjugation, we designed a facile technique that brings nanocarbons closer to their biomedical potential.

Ferritin nanocages: a versatile platform for nanozyme design.

Nanozymes are a class of nanomaterials with enzyme-like activities and have attracted increasing attention due to their potential applications in biomedicine. However, nanozyme design incorporating the desired properties remains challenging. Natural or genetically engineered protein scaffolds, such as ferritin nanocages, have emerged as a promising platform for nanozyme design due to their unique protein structure, natural biomineralization capacity, self-assembly properties, and high biocompatibility. In this review, we highlight the intrinsic properties of ferritin nanocages, especially for nanozyme design. We also discuss the advantages of genetically engineered ferritin in the versatile design of nanozymes over natural ferritin. Additionally, we summarize the bioapplications of ferritin-based nanozymes based on their enzyme-mimicking activities. In this perspective, we mainly provide potential insights into the utilization of ferritin nanocages for nanozyme design.

Tumor Cell Nanovaccines Based on Genetically Engineered Antibody-Anchored Membrane.

Despite the promise in whole-tumor cell vaccines, a key challenge is to overcome the lack of costimulatory signals. Here, agonistic-antibody-boosted tumor cell nanovaccines are reported by genetically engineered antibody-anchored membrane (AAM) technology, capable of effectively activating costimulatory pathways. Specifically, the AAM can be stably constructed following genetic engineering of tumor cell membranes with anti-CD40 single chain variable fragment (scFv), an agonistic antibody to induce costimulatory signals. The nanovaccines are versatilely designed and obtained based on the anti-CD40 scFv-anchored membrane and nanotechnology. Following vaccination, the anti-CD40 scFv-anchored membrane nanovaccine (Nano-AAM/CD40) significantly facilitates dendritic cell maturation in CD40-humanized transgenic mice and subsequent adaptive immune responses. Compared to membrane-based nanovaccines alone, the enhanced antitumor efficacy in both "hot" and "cold" tumor models of the Nano-AAM/CD40 demonstrates the importance of agonistic antibodies in development of tumor-cell-based vaccines. To expand the design of nanovaccines, further incorporation of cell lysates into the Nano-AAM/CD40 to conceptually construct tumor cell-like nanovaccines results in boosted immune responses and improved antitumor efficacy against malignant tumors inoculated into CD40-humanized transgenic mice. Overall, this genetically engineered AAM technology provides a versatile design of nanovaccines by incorporation of tumor-cell-based components and agonistic antibodies of costimulatory immune checkpoints.

Machine-learning-assisted single-vessel analysis of nanoparticle permeability in tumour vasculatures.

The central dogma that nanoparticle delivery to tumours requires enhanced leakiness of vasculatures is a topic of debate. To address this, we propose a single-vessel quantitative analysis method by taking advantage of protein-based nanoprobes and image-segmentation-based machine learning (nano-ISML). Using nano-ISML, >67,000 individual blood vessels from 32 tumour models were quantified, revealing highly heterogenous vascular permeability of protein-based nanoparticles. There was a >13-fold difference in the percentage of high-permeability vessels in different tumours and >100-fold penetration ability in vessels with the highest permeability compared with vessels with the lowest permeability. Our data suggest passive extravasation and transendothelial transport were the dominant mechanisms for high- and low-permeability tumour vessels, respectively. To exemplify the nano-ISML-assisted rational design of nanomedicines, genetically tailored protein nanoparticles with improved transendothelial transport in low-permeability tumours were developed. Our study delineates the heterogeneity of tumour vascular permeability and defines a direction for the rational design of next-generation anticancer nanomedicines.

Synergistic enhancement of iron oxide nanoparticle and gadolinium for dual-contrast MRI.

PURPOSE: The use of MR contrast agents allows accurate diagnosis by exerting an influence on the longitudinal (T(1)) or transverse (T(2)) relaxation time of the surrounding tissue. In this study, we combined the use of iron oxide (IO) particles and nonspecific extracellular gadolinium chelate (Gd) in order to further improve the sensitivity and specificity of lesion detection. PROCEDURES: With a 7-Tesla scanner, pre-contrasted, IO-enhanced and dual contrast agent enhanced MRIs were performed in phantom, normal animals, and animal models of lymph node tumor metastases and orthotopic brain tumor. For the dual-contrast (DC) MRI, we focused on the evaluation of T(2) weighted DC MRI with IO administered first, then followed by the injection of a bolus of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). RESULTS: Based on the C/N ratios and MRI relaxometry, the synergistic effect of coordinated administration of Gd-DTPA and IO was observed and confirmed in phantom, normal liver and tumor models. At 30 min after administration of Feridex, Gd-DTPA further decreased T(2) relaxation in liver immediately after the injection. Additional administration of Gd-DTPA also immediately increased the signal contrast between tumor and brain parenchyma and maximized the C/N ratio to -4.12±0.71. Dual contrast MRI also enhanced the delineation of tumor borders and small lesions. CONCLUSIONS: DC-MRI will be helpful to improve diagnostic accuracy and decrease the threshold size for lesion detection.

Overcoming multidrug resistance with mesoporous silica nanorods as nanocarrier of doxorubicin.

Multidrug resistance (MDR) is a major obstacle to the effective chemotherapy in many human malignancies. Nanoparticulate drug delivery systems (NDDSs) have been reported to be able to bypass MDR, but the cancer therapeutic efficacy is still limited. In this study, we firstly designed the nonspherical mesoporous silica nanorods (MSNRs) with aspect ratio (AR) of 1.5 and 5 as drug delivery systems of doxorubicin to overcome multidrug resistance. For drug loading, the long-rod MSNRs (NLR, AR = 5) showed higher drug loading capacity of doxorubicin (DOX) than the short-rod MSNRs (NSR, AR = 1.5). NLR encapsulated DOX had increased intracellular DOX accumulation in drug-resistant Chinese hamster ovary (CHO) cells compared with free DOX by observablly increased cellular uptake and significantly prolonged intracellular drug retention. It further exhibited increased cytotoxicity compared with free DOX under different drug concentrations. These findings may provide a new perspective for designing high-performance nanoparticulate drug delivery systems for bypassing multidrug resistance of cancer therapy.