Stoichiometric control of DNA-grafted colloid self-assembly.

There has been considerable interest in understanding the self-assembly of DNA-grafted nanoparticles into different crystal structures, e.g., CsCl, AlB2, and Cr3Si. Although there are important exceptions, a generally accepted view is that the right stoichiometry of the two building block colloids needs to be mixed to form the desired crystal structure. To incisively probe this issue, we combine experiments and theory on a series of DNA-grafted nanoparticles at varying stoichiometries, including noninteger values. We show that stoichiometry can couple with the geometries of the building blocks to tune the resulting equilibrium crystal morphology. As a concrete example, a stoichiometric ratio of 3:1 typically results in the Cr3Si structure. However, AlB2 can form when appropriate building blocks are used so that the AlB2 standard-state free energy is low enough to overcome the entropic preference for Cr3Si. These situations can also lead to an undesirable phase coexistence between crystal polymorphs. Thus, whereas stoichiometry can be a powerful handle for direct control of lattice formation, care must be taken in its design and selection to avoid polymorph coexistence.

MUC1 Aptamer Targeted SERS Nanoprobes.

Recently, surface-enhanced Raman scattering (SERS) nanoprobes (NPs) have shown promise in the field of cancer imaging due to their unparalleled signal specificity and high sensitivity. Here we report the development of a DNA aptamer targeted SERS NP. Recently, aptamers are being investigated as a viable alternative to more traditional antibody targeting due to their low immunogenicity and low cost of production. We developed a strategy to functionalize SERS NPs with DNA aptamers, which target Mucin1 (MUC1) in human breast cancer (BC). Thorough in vitro characterization studies demonstrated excellent serum stability and specific binding of the targeted NPs to MUC1. In order to test their in vivo targeting capability, we co-injected MUC1-targeted SERS NPs, and as controls non-targeted and blocked MUC1-targeted SERS NPs in BC xenograft mouse models. A two-tumor mouse model with differential expression of MUC1 (MDA-MB-468 and MDA-MB-453) was used to control for active versus passive targeting in the same animals. The results showed that the targeted SERS NPs home to the tumors via active targeting of MUC1, with low levels of passive targeting. We expect this strategy to be an advantageous alternative to antibody-based targeting and useful for targeted imaging of tumor extent, progression, and therapeutic response.

Selective transformations between nanoparticle superlattices via the reprogramming of DNA-mediated interactions.

The rapid development of self-assembly approaches has enabled the creation of materials with desired organization of nanoscale components. However, achieving dynamic control, wherein the system can be transformed on demand into multiple entirely different states, is typically absent in atomic and molecular systems and has remained elusive in designed nanoparticle systems. Here, we demonstrate with in situ small-angle X-ray scattering that, by using DNA strands as inputs, the structure of a three-dimensional lattice of DNA-coated nanoparticles can be switched from an initial 'mother' phase into one of multiple 'daughter' phases. The introduction of different types of reprogramming DNA strands modifies the DNA shells of the nanoparticles within the superlattice, thereby shifting interparticle interactions to drive the transformation into a particular daughter phase. Moreover, we mapped quantitatively with free-energy calculations the selective reprogramming of interactions onto the observed daughter phases.

Dynamic tuning of DNA-nanoparticle superlattices by molecular intercalation of double helix.

Nanoparticle (NP) assembly using DNA recognition has emerged as a powerful tool for the fabrication of 3D superlattices. In addition to the vast structural diversity, this approach provides an avenue for dynamic 3D NP assembly, which is promising for the modulation of interparticle distances and, hence, for example, for in situ tuning of optical properties. While several approaches have been explored for changing NP separations in the lattices using responsiveness of single-stranded DNA (ss-DNA), far less work has been done for the manipulation of most abundant double-stranded DNA (ds-DNA) motifs. Here, we present a novel strategy for modulation of interparticle distances in DNA linked 3D self-assembled NP lattices by molecular intercalator. We utilize ethidium bromide (EtBr) as a model intercalator to demonstrate selective and isotropic lattice expansion for three superlattice types (bcc, fcc, and AlB2) due to the intercalation of ds-DNA linking NPs. We further show the reversibility of the lattice parameter using n-butanol as a retrieving agent as well as an increased lattice thermal stability by 12-14 °C due to the inclusion of EtBr. The proposed intercalator-based strategy permits the creation of reconfigurable and thermally stable superlattices, which could lead to tunable and functionally responsive materials.

Extracellular Vesicles for Drug Delivery and Theranostics In Vivo.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanopouches generated by all cells and are abundant in various body fluids. Depending on the parent and recipient cells, EVs exchange diverse constituents including nucleic acids, proteins, carbohydrates, and metabolites. Morphologically, EVs suffer from low zeta potentials and short circulation times, but they also offer low intrinsic immunogenicity and inherent stability. Some crucial factors for the effective clinical application of EVs include controlling immune system clearance, achieving the large-scale production of EVs with efficient quality control, and determining the dominant mechanism of the in vivo action of EVs. In this Perspective, we shed light on how these intriguing nano-objects are utilized in cellular imaging and drug delivery for disease therapeutics. We also discuss potential strategies for overcoming the associated limitations.

Herb Extracellular Vesicle-Chitosan-PEGylated Graphene Oxide Conjugate Delivers Estrogen Receptor α Targeting siRNA to Breast Cancer Cells.

Herb-based extracellular vesicles (EV), inherently replete with bioactive proteins, RNA, lipids, and other medicinal compounds, are noncytotoxic and uniquely capable of cellular delivery to meet the ever-stringent challenges of ongoing clinical applications. EVs are abundant in nature, affordable, and scalable, but they are also incredibly fragile and stuffed with many biomolecules. To address the low drug binding abilities and poor stability of EVs, we demonstrated herb-based EVs (isolated from neem, mint, and curry leaves) conjugated with chitosan (CS) and PEGylated graphene oxide (GP) that led to their transformation into robust and efficient vectors. The designed conjugates successfully delivered estrogen receptor α (ERα1)-targeting siRNA to breast cancer MCF7 cells. Our data revealed that neem-based EV-CS-GP conjugates were most efficient in cellular siRNA delivery, which could be attributed to hyaluronic acid-mediated recognition of neem EVs by MCF7 cells via CD44 receptors. Our approach shows a futuristic direction in designing clinically viable, sustainable, nontoxic EV-based vehicles that can deliver a variety of functional siRNA cargos.

In Situ-Forming Protein-Polymer Hydrogel for Glucose-Responsive Insulin Release.

Phenylboronic acid (PBA)-containing hydrogels (HGs), capable of glucose-responsive insulin release, have shown promise in diabetes management in preclinical studies. However, sustainable material usage and attaining an optimum insulin release profile pose a significant challenge in such HG design. Herein, we present the development of a straightforward fabrication strategy for glucose-responsive protein-polymer hybrid HGs (PPHGs). We prepare PPHGs by crosslinking polyvinyl alcohol (PVA) with various nature-abundant proteins, such as bovine serum albumin (BSA), egg albumin, casein, whey protein, and so forth, using formylphenylboronic acid (FPBA)-based crosslinkers. We showcase PPHGs with diverse bulk rheological properties that are appropriately modulated by the positions of aldehyde, boronic acid, and fluorine substitutions in the FPBA-crosslinker. The orthogonal imine and boronate ester bonds formed by FPBAs are susceptible to the acidic pH environment and glucose concentrations, leading to the glucose-responsive dissolution of the PPHGs. We further demonstrate that by an appropriate selection of FPBAs, glucose-responsive insulin release profiles of the PPHGs can be precisely engineered at the molecular level. Importantly, PPHGs are injectable, incur no cytotoxicity, and, therefore, hold great potential as smart insulin for in vivo applications in the near future.

Probing Aberrantly Glycosylated Mucin 1 in Breast Cancer Extracellular Vesicles.

Aberrantly glycosylated mucin 1 is a critical prognostic biomarker in breast epithelial cancers. Hypoglycosylated mucin 1 coats the surface of the cancer cells, where O-glycans are predominantly linked via an N-acetylgalactosamine moiety (GalNAc). Cancer cell-derived extracellular vesicles (EVs) carry biomarkers from parent cancer cells to the recipient cells and, therefore, could potentially be leveraged for diagnostics and noninvasive disease monitoring. We devised a label-free approach for identifying glycoprotein mucin 1 overexpression on breast cancer EVs. While exploring a plethora of biochemical (enzyme-linked immunosorbent assay, flow cytometry, and SDS-PAGE) and label-free biophysical techniques (circular dichroism and infrared spectroscopy (IR)) along with multivariate analysis, we discovered that mucin 1 is significantly overexpressed in breast cancer EVs and aberrant glycosylation in mucin 1 could be critically addressed using IR and multivariate analysis targeting the GalNAc sugar. This approach emerges as a convenient and comprehensive method of distinguishing cancer EVs from normal samples and holds potential for nonintrusive breast cancer liquid biopsy screening.

Glucose-Responsive Chitosan Nanoparticle/Poly(vinyl alcohol) Hydrogels for Sustained Insulin Release In Vivo.

Stimuli-responsive hydrogels (HGs) with a controlled drug release profile are the current challenge for advanced therapeutic applications. Specifically, antidiabetic drug-loaded glucose-responsive HGs are being investigated for closed-loop insulin delivery in insulin-dependent diabetes patients. In this direction, new design principles must be exploited to create inexpensive, naturally occurring, biocompatible glucose-responsive HG materials for the future. In this work, we developed chitosan nanoparticle/poly(vinyl alcohol) (PVA) hybrid HGs (CPHGs) for controlled insulin delivery for diabetes management. In this design, PVA and chitosan nanoparticles (CNPs) are cross-linked with a glucose-responsive formylphenylboronic acid (FPBA)-based cross-linker in situ. Leveraging the structural diversity of FPBA and its pinacol ester-based cross-linkers, we fabricate six CPHGs (CPHG1-6) with more than 80% water content. Using dynamic rheological measurements, we demonstrate elastic solid-like properties of CPHG1-6, which are dramatically reduced under low-pH and high-glucose environments. An in vitro drug release assay reveals size-dependent glucose-responsive drug release from the CPHGs under physiological conditions. It is important to note that the CPHGs show appreciable self-healing and noncytotoxic properties. Promisingly, we observe a significantly slower insulin release profile from the CPHG matrix in the type-1 diabetes (T1D) rat model. We are actively pursuing scaling up of CPHGs and the in vivo safety studies for clinical trial in the near future.

Label-Free Physical-Analytical Techniques Reveal Epigenetic Modifications of Breast Cancer Chromosomes.

Epigenetic dysregulation including DNA methylation and histone modifications is being increasingly recognized as a promising biomarker for the diagnosis and prognosis of cancer. Herein, we devised a label-free analytical toolbox comprising IR, UV-vis, CD spectroscopy, and cyclic voltammetry, which is capable to differentiate significantly hyper-methylated breast cancer chromosomes from the normal breast epithelial counterparts.

Correction: Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Correction for 'Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation' by Chrysafis Andreou et al., Nanoscale Horiz., 2022, 7, 1540-1552, https://doi.org/10.1039/d2nh00331g.

Importance of Extracellular Vesicle Derived RNAs as Critical Colorectal Cancer Biomarkers.

Colorectal cancer typically begins from a nonmalignant polyp formation in the large intestine that, over time, develops into colorectal cancer. The growth of benign polyps can be checked if detected in the early stages of the disease. Doctors usually recommend colonoscopy to average and high-risk individuals for colorectal cancer screening. Elevated carcinoembryonic antigen (CEA) is a broadly used biomarker for colorectal cancer. The genetic and epigenetic alteration of genes such as p53, BRAF, APC, and PIK3CA is also correlated with colorectal cancer in various clinical studies. In general, tissue biopsy is most frequently used for colorectal cancer diagnosis, but the whole tumor heterogeneity cannot be accessed by this technique. Furthermore, such a highly invasive technique is not suitable for repeated testing. Recently, extracellular vesicles (EVs), lipid bilayer enclosed sacs secreted from colorectal cancer cells, are emerging as a diagnostic tool for colon cancer detection. The major advantages of using EVs for colon cancer diagnosis are (i) EVs can be isolated in a noninvasive manner from the body fluid and (ii) EV incorporated cargoes (mostly RNAs) reveal various aspects of colorectal cancer. EV-RNAs are also implicated in tumor invasion and influence the immune system for the further spread of tumors. However, due to the lack of standardized EV detection strategies, diagnostic applicability is limited. Herein, we review the recent literature on the pathobiological dependence of colorectal cancer on EV-RNAs. Further, we present the advantages of identification and characterization of EV-RNAs to explore the connection between differential expression of extracellular vesicle incorporated RNAs and colorectal cancer. How this approach may potentially translate into point of care colorectal cancer diagnostics is also discussed.

Repurposing pinacol esters of boronic acids for tuning viscoelastic properties of glucose-responsive polymer hydrogels: effects on insulin release kinetics.

In the era of the diabetes pandemic, injectable hydrogels (HGs) capable of releasing the desired amount of insulin under hyperglycemic conditions will significantly advance smart insulin development. Several smart boronic acid-based polymer HGs release insulin under high-glucose conditions. However, the correlation of insulin release characteristics with rheological properties is not well understood yet. Herein, we report a generalized and facile fabrication strategy of a new class of glucose-responsive hydrogels by crosslinking a biocompatible polymer, poly(vinyl alcohol) with pinacol esters of bisboronic acids via transesterification reactions. We show the versatility of the method by fabricating four hydrogels with diverse rheological properties. The HGs embody more than 70% water amenable for hosting insulin in the matrix. HG with high storage modulus, derived from 1,4-benzenediboronic acid bis(pinacol) ester releases ∼3 fold less insulin compared to softer HGs derived from acetylene-1,2-diyl bis(boronic acid pinacol ester) and bis[(pinacolato)boryl]methane under hyperglycemic conditions. We find that HG derived from the bis[(pinacolato)boryl]methane crosslinker exhibits superior insulin release properties due to the softness of the hydrogel matrix. We further show that the newly formulated gel is injectable without any structural change in the released insulin molecules and does not cause cytotoxicity. We believe that glucose-responsive hydrogels with tunable viscoelastic properties will pave the way for developing a variety of hydrogels with programmable insulin release properties.

A Mechanoelastic Glimpse on Hyaluronan-Coated Extracellular Vesicles.

Cancer cells secrete extracellular vesicles (EVs) covered with a carbohydrate polymer, hyaluronan (HA), linked to tumor malignancy. Herein, we have unravelled the contour lengths of HA on a single cancer cell-derived EV surface using single-molecule force spectroscopy (SMFS), which divulges the presence of low molecular weight HA (LMW-HA < 200 kDa). We also discovered that these LMW-HA-EVs are significantly more elastic than the normal cell-derived EVs. This intrinsic elasticity of cancer EVs could be directly allied to the LMW-HA abundance and associated labile water network on EV surface as revealed by correlative SMFS, hydration dynamics with fluorescence spectroscopy, and molecular dynamics simulations. This method emerges as a molecular biosensor of the cancer microenvironment.

Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Visualizing the presence and distribution of multiple specific molecular markers within a tumor can reveal the composition of its microenvironment, inform diagnosis, stratify patients, and guide treatment. Raman imaging with multiple molecularly-targeted surface enhanced Raman scattering (SERS) nanoprobes could help investigate emerging cancer treatments preclinically or enable personalized treatment assessment. Here, we report a comprehensive strategy for multiplexed imaging using SERS nanoprobes and machine learning (ML) to monitor the early effects of immune checkpoint blockade (ICB) in tumor-bearing mice. We used antibody-functionalized SERS nanoprobes to visualize 7 + 1 immunotherapy-related targets simultaneously. The multiplexed images were spectrally resolved and then spatially segmented into superpixels based on the unmixed signals. The superpixels were used to train ML models, leading to the successful classification of mice into treated and untreated groups, and identifying tumor regions with variable responses to treatment. This method may help predict treatment efficacy in tumors and identify areas of tumor variability and therapy resistance.

DNA directed self-assembly of anisotropic plasmonic nanostructures.

Programmable positioning of one-dimensional (1D) gold nanorods (AuNRs) was achieved by DNA directed self-assembly. AuNR dimer structures with various predetermined inter-rod angles and relative distances were constructed with high efficiency. These discrete anisotropic metallic nanostructures exhibit unique plasmonic properties, as measured experimentally and simulated by the discrete dipole approximation method.

Folate-Functionalized DNA Origami for Targeted Delivery of Doxorubicin to Triple-Negative Breast Cancer.

DNA origami has emerged as a versatile platform for diverse applications, namely, photonics, electronics, (bio) sensing, smart actuator, and drug delivery. In the last decade, DNA origami has been extensively pursued for efficient anticancer therapy. However, challenges remain to develop strategies that improve the targeting efficiency and drug delivery capability of the DNA origami nanostructures. In this direction, we developed folate-functionalized DNA origami that effectively targets and delivers doxorubicin (DOX), a well-known anticancer drug to the folate receptor alpha (FOLR1) expressing triple-negative breast cancer (TNBC) cells in vitro. We show that folate-functionalized DNA origami structure targets and kills FOLR1 overexpressing cells with better efficacy than nontargeted origami. We envision that this study will open up the possibility of target specific delivery of anticancer drug combinations using the versatile DNA origami nanostructures to the drug resistant cancer cells.

Structurally symmetric near-infrared fluorophore IRDye78-protein complex enables multimodal cancer imaging.

Rationale: Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current approved intraoperative contrast agents exemplified by indocyanine green (ICG) have a few drawbacks including the inability of pre-surgical localization. Alternative near-infrared (NIR) dyes including IRDye800cw are being explored in advanced clinical trials but often encounter low chemical yields and complex purifications owing to the asymmetric synthesis. A single contrast agent with ease of synthesis that works in multiple cancer types and simultaneously allows presurgical imaging, intraoperative deep-tissue three-dimensional visualization, and high-speed microscopic visualization of tumor margins via spatiotemporally complementary modalities would be beneficial. Methods: Due to the lack of commercial availability and the absence of detailed synthesis and characterization, we proposed a facile and scalable synthesis pathway for the symmetric NIR water-soluble heptamethine sulfoindocyanine IRDye78. The synthesis can be accomplished in four steps from commercially-available building blocks. Its symmetric resonant structure avoided asymmetric synthesis problems while still preserving the benefits of analogous IRDye800cw with commensurable optical properties. Next, we introduced a low-molecular-weight protein alpha-lactalbumin (α-LA) as the carrier that effectively modulates the hepatic clearance of IRDye78 into the preferred renal excretion pathway. We further implemented 89Zr radiolabeling onto the protein scaffold for positron emission tomography (PET). The multimodal imaging capability of the fluorophore-protein complex was validated in breast cancer and glioblastoma. Results: The scalable synthesis resulted in high chemical yields, typically 95% yield in the final step of the chloro dye. Chemical structures of intermediates and the final fluorophore were confirmed. Asymmetric IRDye78 exhibited comparable optical features as symmetric IRDye800cw. Its well-balanced quantum yield affords concurrent dual fluorescence and optoacoustic contrast without self-quenching nor concentration-dependent absorption. The NHS ester functionality modulates efficient covalent coupling to reactive side-chain amines to the protein carrier, along with desferrioxamine (DFO) for stable radiolabeling of 89Zr. The fluorophore-protein complex advantageously shifted the biodistribution and can be effectively cleared through the urinary pathway. The agent accumulates in tumors and enables triple-modal visualization in mouse xenograft models of both breast and brain cancers. Conclusion: This study described in detail a generalized strategic modulation of clearance routes towards the favorable renal clearance, via the introduction of α-LA. IRDye78 as a feasible alternative of IRDye800cw currently in clinical phases was proposed with a facile synthesis and fully characterized for the first time. This fluorophore-protein complex with stable radiolabeling should have great potential for clinical translation where it could enable an elegant workflow from preoperative planning to intraoperative deep tissue and high-resolution image-guided resection.

DNA Nanostructures and DNA-Functionalized Nanoparticles for Cancer Theranostics.

In the last two decades, DNA has attracted significant attention toward the development of materials at the nanoscale for emerging applications due to the unparalleled versatility and programmability of DNA building blocks. DNA-based artificial nanomaterials can be broadly classified into two categories: DNA nanostructures (DNA-NSs) and DNA-functionalized nanoparticles (DNA-NPs). More importantly, their use in nanotheranostics, a field that combines diagnostics with therapy via drug or gene delivery in an all-in-one platform, has been applied extensively in recent years to provide personalized cancer treatments. Conveniently, the ease of attachment of both imaging and therapeutic moieties to DNA-NSs or DNA-NPs enables high biostability, biocompatibility, and drug loading capabilities, and as a consequence, has markedly catalyzed the rapid growth of this field. This review aims to provide an overview of the recent progress of DNA-NSs and DNA-NPs as theranostic agents, the use of DNA-NSs and DNA-NPs as gene and drug delivery platforms, and a perspective on their clinical translation in the realm of oncology.

Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer.

Theranostic agents should ideally be renally cleared and biodegradable. Here, we report the synthesis, characterization and theranostic applications of fluorescent ultrasmall gold quantum clusters that are stabilized by the milk metalloprotein alpha-lactalbumin. We synthesized three types of these nanoprobes that together display fluorescence across the visible and near-infrared spectra when excited at a single wavelength through optical colour coding. In live tumour-bearing mice, the near-infrared nanoprobe generates contrast for fluorescence, X-ray computed tomography and magnetic resonance imaging, and exhibits long circulation times, low accumulation in the reticuloendothelial system, sustained tumour retention, insignificant toxicity and renal clearance. An intravenously administrated near-infrared nanoprobe with a large Stokes shift facilitated the detection and image-guided resection of breast tumours in vivo using a smartphone with modified optics. Moreover, the partially unfolded structure of alpha-lactalbumin in the nanoprobe helps with the formation of an anti-cancer lipoprotein complex with oleic acid that triggers the inhibition of the MAPK and PI3K-AKT pathways, immunogenic cell death and the recruitment of infiltrating macrophages. The biodegradability and safety profile of the nanoprobes make them suitable for the systemic detection and localized treatment of cancer.