Proteomimetic Polymers Trigger Potent Antigen-Specific T Cell Responses to Limit Tumor Growth.

Elicitation of effective antitumor immunity following cancer vaccination requires the selective activation of distinct effector cell populations and pathways. Here we report a therapeutic approach for generating potent T cell responses using a modular vaccination platform technology capable of inducing directed immune activation, termed the Protein-like Polymer (PLP). PLPs demonstrate increased proteolytic resistance, high uptake by antigen-presenting cells (APCs), and enhanced payload-specific T cell responses. Key design parameters, namely payload linkage chemistry, degree of polymerization, and side chain composition, were varied to optimize vaccine formulations. Linking antigens to the polymer backbone using an intracellularly cleaved disulfide bond copolymerized with a diluent amount of oligo(ethylene glycol) (OEG) resulted in the highest payload-specific potentiation of antigen immunogenicity, enhancing dendritic cell (DC) activation and antigen-specific T cell responses. Vaccination with PLPs carrying either gp100, E7, or adpgk peptides significantly increased the survival of mice inoculated with B16F10, TC-1, or MC38 tumors, respectively, without the need for adjuvants. B16F10-bearing mice immunized with gp100-carrying PLPs showed increased antitumor CD8+ T cell immunity, suppressed tumor growth, and treatment synergy when paired with two distinct stimulator of interferon gene (STING) agonists. In a human papillomavirus-associated TC-1 model, combination therapy with PLP and 2'3'-cGAMP resulted in 40% of mice completely eliminating implanted tumors while also displaying curative protection from rechallenge, consistent with conferment of lasting immunological memory. Finally, PLPs can be stored long-term in a lyophilized state and are highly tunable, underscoring the unique properties of the platform for use as generalizable cancer vaccines.

Programming "Atomic Substitution" in Alloy Colloidal Crystals Using DNA.

Although examples of colloidal crystal analogues to metal alloys have been reported, general routes for preparing 3D analogues to random substitutional alloys do not exist. Here, we use the programmability of DNA (length and sequence) to match nanoparticle component sizes, define parent lattice symmetry and substitutional order, and achieve faceted crystal habits. We synthesized substitutional alloy colloidal crystals with either ordered or random arrangements of two components (Au and Fe3O4 nanoparticles) within an otherwise identical parent lattice and crystal habit, confirmed via scanning electron microscopy and small-angle X-ray scattering. Energy dispersive X-ray spectroscopy reveals information regarding composition and local order, while the magnetic properties of Fe3O4 nanoparticles can direct different structural outcomes for different alloys in an applied magnetic field. This work constitutes a platform for independently defining substitution within multicomponent colloidal crystals, a capability that will expand the scope of functional materials that can be realized through programmable assembly.

Regioselective Deposition of Metals on Seeds within a Polymer Matrix.

We use scanning probe block copolymer lithography in a two-step sequential manner to explore the deposition of secondary metals on nanoparticle seeds. When single element nanoparticles (Au, Ag, Cu, Co, or Ni) were used as seeds, both heterogeneous and homogeneous growth occurred, as rationalized using the thermodynamic concepts of bond strength and lattice mismatch. Specifically, heterogeneous growth occurs when the heterobond strength between the seed and growth atoms is stronger than the homobond strength between the growth atoms. Moreover, the resulting nanoparticle structure depends on the degree of lattice mismatch between the seed and growth metals. Specifically, a large lattice mismatch (e.g., 13.82% for Au and Ni) typically resulted in heterodimers, whereas a small lattice mismatch (e.g., 0.19% for Au and Ag) resulted in core-shell structures. Interestingly, when heterodimer nanoparticles were used as seeds, the secondary metals deposited asymmetrically on one side of the seed. By programming the deposition conditions of Ag and Cu on AuNi heterodimer seeds, two distinct nanostructures were synthesized with (1) Ag and Cu on the Au domain and (2) Ag on the Au domain and Cu on the Ni domain, illustrating how this technique can be used to predictively synthesize structurally complex, multimetallic nanostructures.

Photopolymerized Features via Beam Pen Lithography as a Novel Tool for the Generation of Large Area Protein Micropatterns.

A cantilever-free scanning probe lithography (CF-SPL)-based method for the rapid polymerization of nanoscale features on a surface via crosslinking and thiol-acrylate photoreactions is described, wherein the nanoscale position, height, and diameter of each feature can be finely and independently tuned. With precise spatiotemporal control over the illumination pattern, beam pen lithography (BPL) allows for the photo-crosslinking of polymers into ultrahigh resolution features over centimeter-scale areas using massively parallel >160 000 pen arrays of individually addressable pens that guide and focus light onto the surface with sub-diffraction resolution. The photoinduced crosslinking reaction of the ink material, which is composed of photoinitiator, diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide, poly(ethylene glycol) diacrylate, and thiol-modified functional binding molecules (i.e., thiol-PEG-biotin or 16-mercaptohexanoic acid), proceeds to ≈80% conversion with UV exposure (72 mW cm-2 ) for short time periods (0.5 s). Such polymer patterns are further reacted with proteins (streptavidin and fibronectin) to yield protein arrays with feature arrangements at high resolution and densities controlled by local UV exposure. This platform, which combines polymer photochemistry and massive arrays of scanning probes, constitutes a new approach to making biomolecular microarrays in a high-throughput and high-yielding manner, opening new routes for biochip synthesis, bioscreening, and cell biology research.

Evolution of Dip-Pen Nanolithography (DPN): From Molecular Patterning to Materials Discovery.

Dip-pen nanolithography (DPN) is a nanofabrication technique that can be used to directly write molecular patterns on substrates with high resolution and registration. Over the past two decades, DPN has evolved in its ability to transport molecular and material "inks" (e.g., alkanethiols, biological molecules like DNA, viruses, and proteins, polymers, and nanoparticles) to many surfaces in a high-throughput fashion, enabling the synthesis and study of complex chemical and biological structures. In addition, DPN has laid the foundation for a series of related scanning probe methodologies, for example, polymer pen lithography (PPL), scanning probe block copolymer lithography (SPBCL), and beam-pen lithography (BPL), which do not rely on cantilever tips. Structures prepared with these methodologies have been used to understand the consequences of miniaturization and open a door to new capabilities in catalysis, optics, biomedicine, and chemical synthesis, where, in sum, a process originally intended to compete with tools used by the semiconductor industry for rapid prototyping has transcended that application to advanced materials discovery. This review outlines the major DPN advances, the subsequent methods based on the technique, and the opportunities for future fundamental and technological exploration. Most importantly, it commemorates the 20th anniversary of the discovery of DPN.

Catalyst discovery through megalibraries of nanomaterials.

The nanomaterial landscape is so vast that a high-throughput combinatorial approach is required to understand structure-function relationships. To address this challenge, an approach for the synthesis and screening of megalibraries of unique nanoscale features (>10,000,000) with tailorable location, size, and composition has been developed. Polymer pen lithography, a parallel lithographic technique, is combined with an ink spray-coating method to create pen arrays, where each pen has a different but deliberately chosen quantity and composition of ink. With this technique, gradients of Au-Cu bimetallic nanoparticles have been synthesized and then screened for activity by in situ Raman spectroscopy with respect to single-walled carbon nanotube (SWNT) growth. Au3Cu, a composition not previously known to catalyze SWNT growth, has been identified as the most active composition.

Electrochemical Polymer Pen Lithography.

The development of a massively parallel lithographic technique called electrochemical polymer pen lithography is reported. Pyramidal pen arrays, consisting of more than 10 000 hydrogel pens loaded with metal salts, are integrated into a three-electrode cell and used to locally reduce ions at each pen tip. This system enables high-throughput patterning of a variety of metallic inks (e.g., Ni2+ , Pt2+ , Ag+ ) on the nanometer to micrometer length scale. By incorporating a z-direction piezo actuator, the extension length and dwell time can be used to precisely define feature dimensions (210 to 10 µm in width, and up to 900 nm in height, thus far). Furthermore, by controlling the potential and precursor concentrations, more than one element can be simultaneously deposited, creating a new tool for the synthesis of alloy features, such as NiCo, which are relevant for catalysis. Importantly, this methodology enables fine control over feature size and composition in a single pattern, which may make it ultimately useful for rapid, high-throughput combinatorial screening of metallic features.

Multi-State Dynamic Coordination Complexes Interconverted through Counterion-Controlled Phase Transfer.

We studied a series of dynamic weak-link approach (WLA) complexes that can be shuttled between two immiscible solvents and switched between two structural states via ion exchange. Here, we established that hydrophobic anions transfer cationic, amphiphilic complexes from the aqueous phase to the organic phase, while a chloride source reverses the process. As a result of the dynamic metal coordination properties of WLA complexes, the denticity of these complexes (mono- to bi-) can be modulated as they partition into different phases. In addition, we discovered that heteroligated complexes bearing ligands of different donor strengths preferentially rearrange into two homoligated complexes that are phase-partitioned to maximize the number of stronger coordination bonds. This behavior is not observed in systems with one solvent, highlighting the dynamic and stimuli-responsive nature of hemilabile ligands in a multiphasic solvent environment. Taken together, this work shows that the highly reconfigurable WLA modality can enable the design of biphasic reaction networks or chemical separations driven by straightforward salt metathesis reactions.

Dual Toll-Like Receptor Targeting Liposomal Spherical Nucleic Acids.

Liposomal spherical nucleic acids (LSNAs) are a class of nanomaterial used broadly for biomedical applications. Their intrinsic capacity to rapidly enter cells and engage cell surface and intracellular ligands stems from their unique three-dimensional architecture, which consists of densely packed and uniformly oriented oligonucleotides on the surface of a liposomal core. Such structures are promising for therapeutics because they can carry chemical cargo within the lipid core in addition to the nucleic acids that define them, in principle enabling delivery of multiple signals to a single cell. On the basis of these traits, we have designed novel dual-targeting LSNAs that deliver a nucleic acid specific for TLR9 inhibition and a small molecule (TAK-242) that inhibits TLR4. Toll-like receptors (TLRs) play a large role in pathogen recognition and disease initiation, and TLR subtypes are differentially located within the lipid membranes of the cell surface and within intracellular endosomes. Oftentimes, in acute or chronic inflammatory conditions, multiple TLRs are activated, leading to stimulation of distinct, and sometimes overlapping, downstream pathways. As such, these inflammatory conditions may respond to attenuation of more than one initiating receptor. We show that dual targeting LSNAs, comprised of unilamellar liposomal cores, the INH-18 oligonucleotide sequence, and TAK-242 robustly inhibit TLR-9 and TLR-4 respectively, in engineered TLR reporter cells and primary mouse peritoneal macrophages. Importantly, the LSNAs exhibit up to a 10- and a 1000-fold increase, respectively, in TLR inhibition compared to the linear sequence and TAK-242 alone. Moreover, the timing of delivery is shown to be a critical factor in effecting TLR-inhibition, with near-complete TLR-4 inhibition occurring when cells were pretreated with SNAs for 4 h prior to stimulation. The most pronounced effect observed from this approach is the benefit of delivering the small molecule within the SNA via the receptor-mediated internalization pathway common to SNAs.

Windowless Observation of Evaporation-Induced Coarsening of Au-Pt Nanoparticles in Polymer Nanoreactors.

The interactions between nanoparticles and solvents play a critical role in the formation of complex, metastable nanostructures. However, direct observation of such interactions with high spatial and temporal resolution is challenging with conventional liquid-cell transmission electron microscopy (TEM) experiments. Here, a windowless system consisting of polymer nanoreactors deposited via scanning probe block copolymer lithography (SPBCL) on an amorphous carbon film is used to investigate the coarsening of ultrafine (1-3 nm) Au-Pt bimetallic nanoparticles as a function of solvent evaporation. In such reactors, homogeneous Au-Pt nanoparticles are synthesized from metal-ion precursors in situ under electron irradiation. The nonuniform evaporation of the thin polymer film not only concentrates the nanoparticles but also accelerates the coalescence kinetics at the receding polymer edges. Qualitative analysis of the particle forces influencing coalescence suggests that capillary dragging by the polymer edges plays a significant role in accelerating this process. Taken together, this work (1) provides fundamental insight into the role of solvents in the chemistry and coarsening behavior of nanoparticles during the synthesis of polyelemental nanostructures, (2) provides insight into how particles form via the SPBCL process, and (3) shows how SPBCL-generated domes, instead of liquid cells, can be used to study nanoparticle formation. More generally, it shows why conventional models of particle coarsening, which do not take into account solvent evaporation, cannot be used to describe what is occurring in thin film, liquid-based syntheses of nanostructures.

Enhancing the Stability and Immunomodulatory Activity of Liposomal Spherical Nucleic Acids through Lipid-Tail DNA Modifications.

Liposomal spherical nucleic acids (LSNAs) are an attractive therapeutic platform for gene regulation and immunomodulation due to their biocompatibility, chemically tunable structures, and ability to enter cells rapidly without the need for ancillary transfection agents. Such structures consist of small (<100 nm) liposomal cores functionalized with a dense, highly oriented nucleic acid shell, both of which are key components in facilitating their biological activity. Here, the properties of LSNAs synthesized using conventional methods, anchoring cholesterol terminated oligonucleotides into a liposomal core, are compared to LSNAs made by directly modifying the surface of a liposomal core containing azide-functionalized lipids with dibenzocyclooctyl-terminated oligonucleotides. The surface densities of the oligonucleotides are measured for both types of LSNAs, with the lipid-modified structures having approximately twice the oligonucleotide surface coverage. The stabilities and cellular uptake properties of these structures are also evaluated. The higher density, lipid-functionalized structures are markedly more stable than conventional cholesterol-based structures in the presence of other unmodified liposomes and serum proteins as evidenced by fluorescence assays. Significantly, this new form of LSNA exhibits more rapid cellular uptake and increased sequence-specific toll-like receptor activation in immune reporter cell lines, making it a promising candidate for immunotherapy.

RNA-Based Immunostimulatory Liposomal Spherical Nucleic Acids as Potent TLR7/8 Modulators.

Immunostimulatory spherical nucleic acids (IS-LSNAs) comprised of RNA selective for toll-like receptors (TLRs) 7/8 are synthesized and characterized. These structures consist of liposomal cores functionalized with a dense shell of RNA inserted into the wall of the lipid core via hydrophobic cholesterol moieties. IS-LSNAs potently activate TLR7/8 via NF-κΒ signaling in reporter cell lines and in primary immune cells as evidenced by cytokine production and the upregulation of costimulatory receptors. Importantly, they are preferentially taken up by plasmacytoid dendritic cells, an observation that makes them potentially useful for immunotherapy. In addition, these structures contain a core that can be loaded with antigens and used to prime T cells. In this regard, it is shown that dendritic cells treated with IS-SNAs loaded with ovalbumin peptide can prime ova specific CD8+ T cells. In addition to introducing the first IS-LSNAs consisting of RNA, these experiments show that one can facilitate an antigen-specific T cell response greater than that of free or cationic lipid-transfected RNA of the same sequence selective for TLR7/8. This work points toward the promise of using IS-LSNAs comprised of RNA as potent and highly tunable TLR-specific agents for the development of vaccines and other pharmaceuticals that require selective immunomodulation.

Design Rules for Template-Confined DNA-Mediated Nanoparticle Assembly.

Template-based strategies are becoming increasingly important for controlling the position of nanoparticle-based (NP-based) structures on surfaces for a wide variety of encoding and device fabrication strategies. Thus, there is an increasing need to understand the behavior of NPs in confined spaces. Herein, a systematic investigation of the diffusion and adsorption properties of DNA-modified NPs is presented in lithographically defined, high-aspect-ratio pores using a template-confined, DNA-mediated assembly. Leveraging the sequence-specific binding affinity of DNA, it is discovered that although NP adsorption in deep polymer pores follows a traditional Langmuir adsorption model when under thermodynamic control, such NPs kinetically follow Fick's classical law of diffusion. Importantly, these observations allow one to establish design rules for template-confined, DNA-mediated NP assembly on substrates based on pore dimensions, NP size and shape, NP concentration, temperature, and time. As a proof-of-concept example, these design rules are used to engineer a vertical, four-layer assembly consisting of individual octahedral NPs stacked on top of one another, with in-plane positioning defined by pores generated by e-beam lithography.

Structural Evolution of Three-Component Nanoparticles in Polymer Nanoreactors.

Recent developments in scanning probe block copolymer lithography (SPBCL) enable the confinement of multiple metal precursors in a polymer nanoreactor and their subsequent transformation into a single multimetallic heterostructured nanoparticle through thermal annealing. However, the process by which multimetallic nanoparticles form in SPBCL-patterned nanoreactors remains unclear. Here, we utilize the combination of PEO-b-P2VP and Au, Ag, and Cu salts as a model three-component system to investigate this process. The data suggest that the formation of single-component Au, Ag, or Cu nanoparticles within polymer nanoreactors consists of two stages: (I) nucleation, growth, and coarsening of the particles to yield a single particle in each reactor; (II) continued particle growth by depletion of the remaining precursor in the reactor until the particle reaches a stable size. Also, different aggregation rates are observed for single-component particle formation (Au > Ag > Cu). This behavior is also observed for two-component systems, where nucleation sites have greater Au content than the other metals. This information can be used to trap nanoparticles with kinetic structures. High-temperature treatment ultimately facilitates the structural evolution of the kinetic particle into a particle with a fixed structure. Therefore, with multicomponent systems, a third stage that involves elemental redistribution within the particle must be part of the description of the synthetic process. This work not only provides a glimpse at the mechanism underlying multicomponent nanoparticle formation in SPBCL-generated nanoreactors but also illustrates, for the first time, the utility of SPBCL as a platform for controlling the architectural evolution of multimetallic nanoparticles in general.

Liposomal Spherical Nucleic Acids for Regulating Long Noncoding RNAs in the Nucleus.

Emerging evidence indicates that long noncoding RNAs (lncRNAs) are actively involved in a number of developmental and tumorigenic processes. Here, the authors describe the first successful use of spherical nucleic acids as an effective nanoparticle platform for regulating lncRNAs in cells; specifically, for the targeted knockdown of the nuclear-retained metastasis associated lung adenocarcinoma transcript 1 (Malat1), a key oncogenic lncRNA involved in metastasis of several cancers. Utilizing the liposomal spherical nucleic acid (LSNA) constructs, the authors first explored the delivery of antisense oligonucleotides to the nucleus. A dose-dependent inhibition of Malat1 upon LSNA treatment as well as the consequent up-regulation of tumor suppressor messenger RNA associated with Malat1 knockdown are shown. These findings reveal the biologic and therapeutic potential of a LSNA-based antisense strategy in targeting disease-associated, nuclear-retained lncRNAs.

Universal Biotin-PEG-Linked Gold Nanoparticle Probes for the Simultaneous Detection of Nucleic Acids and Proteins.

Novel biotin-polyethylene glycol (biotin-PEG) gold nanoparticle probes have been synthesized and used as universal constructs for the detection of protein (prostate-specific antigen, PSA) and nucleic acid targets (microRNAs) from a single sample. Microarray assays based upon these probes enabled sensitive detection of biomarker targets (50 fM for nucleic acid targets and 1 pg/µL for the PSA target). Ways of detecting biomarkers, including nucleic acids and proteins, are necessary for the clinical diagnosis of many diseases, but currently available diagnostic platforms rely primarily on the independent detection of proteins or nucleic acids. In addition to the economic benefits associated with the use of a single platform to detect both classes of analytes, studies have shown that the simultaneous identification of multiple classes of biomarkers in the same sample could be useful for the detection and management of early stage diseases, especially when sample amounts are limited. Therefore, these new probes and the assays based upon them open the door for high-sensitivity combination-target assays for studying and tracking biological pathways and diseases.

Drug-Loaded Polymeric Spherical Nucleic Acids: Enhancing Colloidal Stability and Cellular Uptake of Polymeric Nanoparticles through DNA Surface-Functionalization.

Small-sized (∼65 nm) doxorubicin (Dox)-loaded polymeric nanoparticles (PNPs) were modified with oligonucleotides to form colloidally stable Dox-loaded polymeric spherical nucleic acid (Dox-PSNA) nanostructures in biological media. The nucleic acid shell facilitates the cellular uptake of Dox-PSNA, which results in in vitro cytotoxicity against SKOV3 cancer cells.

The Structural Fate of Individual Multicomponent Metal-Oxide Nanoparticles in Polymer Nanoreactors.

Multicomponent nanoparticles can be synthesized with either homogeneous or phase-segregated architectures depending on the synthesis conditions and elements incorporated. To understand the parameters that determine their structural fate, multicomponent metal-oxide nanoparticles consisting of combinations of Co, Ni, and Cu were synthesized by using scanning probe block copolymer lithography and characterized using correlated electron microscopy. These studies revealed that the miscibility, ratio of the metallic components, and the synthesis temperature determine the crystal structure and architecture of the nanoparticles. A Co-Ni-O system forms a rock salt structure largely owing to the miscibility of CoO and NiO, while Cu-Ni-O, which has large miscibility gaps, forms either homogeneous oxides, heterojunctions, or alloys depending on the annealing temperature and composition. Moreover, a higher-ordered structure, Co-Ni-Cu-O, was found to follow the behavior of lower ordered systems.

Biomimicking Nano-Micro Binary Polymer Brushes for Smart Cell Orientation and Adhesion Control.

A new biomimetic surface named nano-micro binary polymer brushes is fabricated by large-area bench-top dip-pen nanodisplacement lithography technique. It is composed of gelatin-modified poly(glycidyl methacrylate) nanolines which are spaced by microstripes of poly(N-isopropylacrylamide). Cells are not only adhered and oriented well on the re-used surface, but also detachable from the surface with well-preserved extracellular matrix and aligned morphology.

Relationships between Poly(ethylene glycol) Modifications on RNA-Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time.

Two synthetic approaches that allow one to control PEG content within spherical nucleic acids (SNAs) have been developed. One approach begins with RNA-modified gold nanoparticles followed by a backfill of PEG 2K alkanethiols, and the other involves co-adsorption of the two entities on a gold nanoparticle template. These two methods have been used to explore the role of PEG density on the chemical and biological properties of RNA-SNAs. Such studies show that while increasing the extent of PEGylation within RNA-SNAs extends their blood circulation half-life in mice, it also results in decreased cellular uptake. Modified ELISA assays show that constructs, depending upon RNA and PEG content, have markedly different affinities for class A scavenger receptors, the entities responsible, in part, for cellular internalization of SNAs. In designing SNAs for therapeutic purposes, these competing factors must be considered and appropriately adjusted depending upon the desired use.