Short Carbon Nanotube-Based Delivery of mRNA for HIV-1 Vaccines.

Developing a safe and effective preventive for HIV-1 remains the hope for controlling the global AIDS epidemic. Recently, mRNA vaccines have emerged as a promising alternative to conventional vaccine approaches, primarily due to their rapid development and potential for low-cost manufacture. Despite the advantages of mRNA vaccines, challenges remain, especially due to the adverse effects of the delivery vehicle and low delivery efficiency. As a result, Luna Labs is developing a short carbon nanotube-based delivery platform (NanoVac) that can co-deliver mRNA and HIV-1 glycoproteins to the immune system efficiently with negligible toxicity. Surface chemistries of NanoVac were optimized to guide antigen/mRNA loading density and presentation. Multiple formulations were engineered for compatibility with both intramuscular and intranasal administration. NanoVac candidates demonstrated immunogenicity in rabbits and generated human-derived humoral and cellular responses in humanized mice (HIS). Briefly, 33% of the HIV-1-infected HIS mice vaccinated with NanoVac-mRNA was cleared of virus infection by 8-weeks post-infection. Finally, NanoVac stabilized the loaded mRNA against degradation under refrigeration for at least three months, reducing the cold chain burden for vaccine deployment.

Mucosal Delivery of HIV-1 Glycoprotein Vaccine Candidate Enabled by Short Carbon Nanotubes.

The HIV-1 envelope glycoprotein spike is the target of antibodies, and therefore represents the main viral antigen for antibody-based vaccine design. One of the challenges in HIV-1 vaccine development is finding efficient ways for the immune system to recognize and respond to HIV-1 without establishing an infection. Since HIV-1 enters the body at mucosal surfaces, induction of immune response at these sites is a preferred preventive approach. Nasal administration is a very effective route for mucosal immunization since it can stimulate mucosal immune responses both locally and distantly. In this paper, Luna develops a safe, short carbon nanotube (CNT)-based, needle-free delivery platform known as "CNTVac". The size of short CNT was controlled to possess HIV-1 particle-like morphology (100-200 nm) capable of efficiently delivering a broad range of antigens intranasally. PEG-Lipid served as the antigen conformation protector and mucosal barrier penetration enhancer (Schematic Figure) was localized between V1V2 antigens, which caused highly enhanced local IgA and systemic antibody IgG responses in mice and rabbits. The short CNT incorporated with PEG-Lipid could not only serve as efficient delivery system but also reduce the amount of lipid usage in order to balance the vaccine dosage in order to eliminate the potential adverse effect. These data suggest a promising platform technology for vaccine delivery.

Layer-by-Layer Delivery of Multiple Antigens Using Trimethyl Chitosan Nanoparticles as a Malaria Vaccine Candidate.

Developing a safe and effective malaria vaccine is critical to reducing the spread and resurgence of this deadly disease, especially in children. In recent years, vaccine technology has seen expanded development of subunit protein, peptide, and nucleic acid vaccines. This is due to their inherent safety, the ability to tailor their immune response, simple storage requirements, easier production, and lower expense compared to using attenuated and inactivated organism-based approaches. However, these new vaccine technologies generally have low efficacy. Subunit vaccines, due to their weak immunogenicity, often necessitate advanced delivery vectors and/or the use of adjuvants. A new area of vaccine development involves design of synthetic micro- and nano-particles and adjuvants that can stimulate immune cells directly through their physical and chemical properties. Further, the unique and complex life cycle of the Plasmodium organism, with multiple stages and varying epitopes/antigens presented by the parasite, is another challenge for malaria vaccine development. Targeting multistage antigens simultaneously is therefore critical for an effective malaria vaccine. Here, we rationally design a layer-by-layer (LbL) antigen delivery platform (we called LbL NP) specifically engineered for malaria vaccines. A biocompatible modified chitosan nanoparticle (trimethyl chitosan, TMC) was synthesized and utilized for LbL loading and release of multiple malaria antigens from pre-erythrocytic and erythrocytic stages. LbL NP served as antigen/protein delivery vehicles and were demonstrated to induce the highest Plasmodium falciparum Circumsporozoite Protein (PfCSP) specific T-cell responses in mice studies as compared to multiple controls. From immunogenicity studies, it was concluded that two doses of intramuscular injection with a longer interval (4 weeks) than traditional malaria vaccine candidate dosing would be the vaccination potential for LbL NP vaccine candidates. Furthermore, in PfCSP/Py parasite challenge studies we demonstrated protective efficacy using LbL NP. These LbL NP provided a significant adjuvant effect since they may induce innate immune response that led to a potent adaptive immunity to mediate non-specific anti-malarial effect. Most importantly, the delivery of CSP full-length protein stimulated long-lasting protective immune responses even after the booster immunization 4 weeks later in mice.

LNA Thymidine Monomer Enables Differentiation of the Four Single-Nucleotide Variants by Melting Temperature.

High-resolution melting (HRM) analysis of DNA is a closed-tube single-nucleotide polymorphism (SNP) detection method that has shown many advantages in point-of-care diagnostics and personalized medicine. While recently developed melting probes have demonstrated significantly improved discrimination of mismatched (mutant) alleles from matched (wild-type) alleles, no effort has been made to design a simple melting probe that can reliably distinguish all four SNP alleles in a single experiment. Such a new probe could facilitate the discovery of rare genetic mutations at lower cost. Here we demonstrate that a melting probe embedded with a single locked thymidine monomer (tL) can reliably differentiate the four SNP alleles by four distinct melting temperatures (termed the "4Tm probe"). This enhanced discriminatory power comes from the decreased melting temperature of the tL·C mismatched hybrid as compared to that of the t·C mismatched hybrid, while the melting temperatures of the tL-A, tL·G and tL·T hybrids are increased or remain unchanged as compared to those of their canonical counterparts. This phenomenon is observed not only in the HRM experiments but also in the molecular dynamics simulations.

3D single-molecule tracking enables direct hybridization kinetics measurement in solution.

Single-molecule measurements of DNA hybridization kinetics are mostly performed on a surface or inside a trap. Here we demonstrate a time-resolved, 3D single-molecule tracking (3D-SMT) method that allows us to follow a freely diffusing ssDNA molecule in solution for hundreds of milliseconds or even seconds and observe multiple annealing and melting events taking place on the same molecule. This is achieved by combining confocal-feedback 3D-SMT with time-domain fluorescence lifetime measurement, where fluorescence lifetime serves as the indicator of hybridization. With sub-diffraction-limit spatial resolution in molecular tracking and 15 ms temporal resolution in monitoring the change of reporter's lifetime, we have demonstrated a full characterization of annealing rate (kon = 5.13 × 106 M-1 s-1), melting rate (koff = 9.55 s-1), and association constant (Ka = 0.54 µM-1) of an 8 bp duplex model system diffusing at 4.8 µm2 s-1. As our method completely eliminates the photobleaching artifacts and diffusion interference, our kon and koff results well represent the real kinetics in solution. Our binding kinetics measurement can be carried out in a low signal-to-noise ratio condition (SNR ≈ 1.4) where ∼130 recorded photons are sufficient for a lifetime estimation. Using a population-level analysis, we can characterize hybridization kinetics over a wide range (0.5-125 s-1), even beyond the reciprocals of the lifetime monitoring temporal resolution and the average track duration.

NanoCluster Beacons Enable Detection of a Single N6-Methyladenine.

While N(6)-methyladenine (m(6)A) is a common modification in prokaryotic and lower eukaryotic genomes and has many biological functions, there is no simple and cost-effective way to identify a single N(6)-methyladenine in a nucleic acid target. Here we introduce a robust, simple, enzyme-free and hybridization-based method using a new silver cluster probe, termed methyladenine-specific NanoCluster Beacon (maNCB), which can detect single m(6)A in DNA targets based on the fluorescence emission spectra of silver clusters. Not only can maNCB identify m(6)A at the single-base level but it also can quantify the extent of adenine methylation in heterogeneous samples. Our method is superior to high-resolution melting analysis as we can pinpoint the location of m(6)A in the target.

NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection.

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.

A complementary palette of NanoCluster Beacons.

NanoCluster Beacons (NCBs), which use few-atom DNA-templated silver clusters as reporters, are a type of activatable molecular probes that are low-cost and easy to prepare. While NCBs provide a high fluorescence enhancement ratio upon activation, their activation colors are currently limited. Here we report a simple method to design NCBs with complementary emission colors, creating a set of multicolor probes for homogeneous, separation-free detection. By systematically altering the position and the number of cytosines in the cluster-nucleation sequence, we have tuned the activation colors of NCBs to green (C8-8, 460 nm/555 nm); yellow (C5-5, 525 nm/585 nm); red (C3-4, 580 nm/635 nm); and near-infrared (C3-3, 645 nm/695 nm). At the same NCB concentration, the activated yellow NCB (C5-5) was found to be 1.3 times brighter than the traditional red NCB (C3-4). Three of the four colors (green, yellow, and red) were relatively spectrally pure. We also found that subtle changes in the linker sequence (down to the single-nucleotide level) could significantly alter the emission spectrum pattern of an NCB. When the length of linker sequences was increased, the emission peaks were found to migrate in a periodic fashion, suggesting short-range interactions between silver clusters and nucleobases. Size exclusion chromatography results indicated that the activated NCBs are more compact than their native duplex forms. Our findings demonstrate the unique photophysical properties and environmental sensitivities of few-atom DNA-templated silver clusters, which are not seen before in common organic dyes or luminescent crystals.

Fluorescent silver nanoclusters as DNA probes.

Fluorescent silver nanoclusters (few atoms, quantum sized) have attracted much attention as promising substitutes for conventional fluorophores. Due to their unique environmental sensitivities, new fluorescent probes have been developed based on silver nanoclusters for the sensitive and specific detection of DNA. In this review we present the recent discoveries of activatable and color-switchable properties of DNA-templated silver nanoclusters and discuss the strategies to use these new properties in DNA sensing.

DNA/RNA Detection Using DNA-Templated Few-Atom Silver Nanoclusters.

DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors.

Probing quenched dye fluorescence of Cy3-DNA-Au-nanoparticle hybrid conjugates using solution and array platforms.

Tuning the luminescence intensity of fluorophores using nanoparticles has shown great potential for the detection of inorganic metal ions, viruses, and proteins. The enhancement or quenching of a dye's fluorescence intensity is strongly dependent on the spatial separation of the dye from the nanoparticle surface. To extend luminescence probing from the solution platform to the solid-state platform, we explored and performed dye quenching assessment using an array format in this study. We report the distance-dependent fluorescence behavior of Au-DNA conjugates prepared by equilibrating phosphine-stabilized gold nanoparticles (AuNPs) of 10-nm size with the designed spacer ds-DNA consisting of thiol-modified target and Cy3-labeled complementary probe of different lengths (5-20 nm). The Cy3-labeled products were immobilized onto MPTMS (3-mercaptopropyltrimethoxysilane)-modified glass substrates and then excited with a 532-nm laser source. Quenching efficiency of AuNPs with increasing Au-to-dye distance was assessed using ligand exchange of the thiolated oligonucleotide by 2-mercaptoethanol (ME) to obtain free Cy3-DNA probe, thus eliminating nanoparticle effect on the dye's luminescence intensity. Effective exchange, revealed by UV-vis absorption and fluorescence profiles, was achieved in a few minutes. It was observed that fluorescence quenching of Au-DNA-Cy3 assessed using the array format was consistent with the result in solution phase for the conjugates with up to 10-nm Au-to-Cy3 separation distance.

Synthesis and optical properties of 1-alkyl-3-methylimidazolium lauryl sulfate ionic liquids.

We report the synthesis of a new series of imidazolium-based halogen-free ionic liquids 1-alkyl-3-methylimidazolium lauryl sulfates. By reacting 1-methylimidazole (MIM) with butyl, hexyl, octyl, and decyl bromides and exchanging bromide ion with lauryl sulfate anion, a series of ionic liquids [RMIM][C(12)H(25)OSO(3)] were produced. The high purity of these ionic liquids was verified with (1)H-NMR, (13)C-NMR, FT-IR and mass spectrometry (MS), demonstrating the effectiveness of this synthetic approach. Solubility test of these ionic liquids showed that they are soluble in most organic solvents except nonpolar solvents such as hexane and cyclohexane. The optical properties of [BMIM]Br and [BMIM][C(12)H(25)OSO(3)], where B refers to butyl, were examined. Both ionic liquids absorbed light in the UV region, yet essentially no absorption was recorded beyond 450 nm. Furthermore, both ionic liquids showed excitation wavelength-dependent fluorescence behavior. As an example, with an excitation wavelength of 360 nm, [BMIM][C(12)H(25)OSO(3)] showed an emission band maximum at 447 nm. Increasing the excitation wavelength to 440 nm, the emission band maximum was shifted to approximately 500 nm.