A Self-Immolative DNA Nanogel Vaccine toward Cancer Immunotherapy.

The development of precisely engineered vehicles for intracellular delivery and the controlled release of payloads remains a challenge. DNA-based nanomaterials offer a promising solution based on the A-T-G-C alphabet-dictated predictable assembly and high programmability. Herein, we present a self-immolative DNA nanogel vaccine, which can be tracelessly released in the intracellular compartments and activate the immune response. Three building blocks with cytosine-rich overhang domains are designed to self-assemble into a DNA nanogel framework with a controlled size. Two oligo agonists and one antigen peptide are conjugated to the building blocks via an acid-labile chemical linker. Upon internalization into acidic endosomes, the formation of i-motif configurations leads to dissociation of the DNA nanogel vaccine. The acid-labile chemical linker is cleaved, releasing the agonists and antigen in their traceless original form to activate antigen-presenting cells and an immune response. This study presents a novel strategy for constructing delivery platforms for intracellularly stimuli-triggered traceless release of therapeutics.

Colorimetric Detection of Small Molecules in Complex Matrixes via Target-Mediated Growth of Aptamer-Functionalized Gold Nanoparticles.

A versatile and sensitive colorimetric assay that allows the rapid detection of small-molecule targets using the naked eye is demonstrated. The working principle of the assay integrates aptamer-target recognition and the aptamer-controlled growth of gold nanoparticles (Au NPs). Aptamer-target interactions modulate the amount of aptamer strands adsorbed on the surface of aptamer-functionalized Au NPs via desorption of the aptamer strands when target molecules bind with the aptamer. Depending on the resulting aptamer coverage, Au NPs grow into morphologically varied nanostructures, which give rise to different colored solutions. Au NPs with low aptamer coverage grow into spherical NPs, which produce red-colored solutions, whereas Au NPs with high aptamer coverage grow into branched NPs, which produce blue-colored solutions. We achieved visible colorimetric response and nanomolar detection limits for the detection of ochratoxin A (1 nM) in red wine samples, as well as cocaine (1 nM) and 17ß-estradiol (0.2 nM) in spiked synthetic urine and saliva, respectively. The detection limits were well within clinically and physiologically relevant ranges, and below the maximum food safety limits. The assay is highly sensitive, specific, and able to detect an array of analytes rapidly without requiring sophisticated equipment, making it relevant for many applications, such as high-throughput drug and clinical screening, food sampling, and diagnostics. Furthermore, the assay is easily adapted as a chip-based platform for rapid and portable target detection.

Enhancing Protein Adsorption for Improved Lateral Flow Assay on Cellulose Paper by Depleting Inert Additive Films Using Reactive Plasma.

Paper-based platforms are ideal for on-site surveillance of infectious diseases in low-resource settings due to their simplicity, self-containment, and low cost. The two most popular materials used in paper-based platforms are nitrocellulose and cellulose. The nitrocellulose membrane has a high protein binding affinity, but its high price is an issue. Cellulose paper is inexpensive and allows intricate fluidic control for more sophisticated biochemical reactions, but it has a low protein binding affinity. By examining the microstructure of cellulose paper, we discover that cellulose fibers in the paper matrix are covered by thin films, which possibly result from the additives used in the paper-making process. Our finding suggests that the thin films are inert to protein adsorption. By selectively depleting the inert films with reactive plasma, we were able to enhance the protein adsorption to the cellulose paper and improve the performance of lateral flow assays. The performance of certain lateral flow assays on the plasma-treated cellulose paper is equivalent to or better than that on the nitrocellulose membrane. This leads us to believe that cellulose paper with a microstructure exclusively designed for protein binding, either by refined paper manufacturing process or by post-manufacture modification such as the plasma treatment presented herein, can potentially replace nitrocellulose as a less expensive paper substrate for point-of-care rapid test kits.

Neural cell membrane-coated DNA nanogels as a potential target-specific drug delivery tool for the central nervous system.

A major bottleneck in drug/gene delivery to enhance tissue regeneration after injuries is to achieve targeted delivery to the cells of interest. Unfortunately, we have not been able to attain effective targeted drug delivery in tissues due to the lack of efficient delivery platforms. Since specific cell-cell interactions exist to impart the unique structure and functionality of tissues and organs, we hypothesize that such specific cellular interactions may also be harnessed for drug delivery applications in the form of cell membrane coatings. Here, we employed neural cell-derived membrane coating technique on DNA nanogels to improve target specificity. The efficacy of neural cell membrane-coated DNA nanogels (NCM-nanogels) was demonstrated by using four types of cell membranes derived from the central nervous system (CNS), namely, astrocytes, microglia, cortical neurons, and oligodendrocyte progenitor cells (OPCs). A successful coating of NCMs over DNA nanogels was confirmed by dynamic light scattering, zeta potential measurements and transmission electron microscopy. Subsequently, an overall improvement in cellular uptake of NCM-nanogels over uncoated DNA nanogels (p < 0.005) was seen. Additionally, we observed a selective uptake of OPC membrane-coated DNA nanogels (NCM-O mem) by oligodendrocytes over other cell types both in vitro and in vivo. Our quantitative polymerase chain reaction (qPCR) results also showed selective and effective gene knockdown capacity of NCM-O mem for OPC transfection. The findings in this work may be beneficial for future drug delivery applications targeted at the CNS.

Functionalization of inorganic nanoparticles for bioimaging applications.

Modern biomedical imaging technologies have led to significant advances in diagnosis and therapy. Because most disease processes occur at the molecular and cellular levels, researchers continue to face challenges in viewing and understanding these processes precisely and in real time. The ideal imaging resolution would be in nanometers, because most biological processes take place on this length scale. Therefore, the functionalization of nanoparticles (NPs) and their use in therapeutic and diagnostic applications are of great interest. Molecular and cellular imaging agents made from inorganic NPs have been developed to probe such biological events noninvasively. The conjugation of tiny NPs with specific biomolecules allows researchers to target the desired location, reduce overall toxicity, and boost the efficiency of the imaging probes. In this Account, we review recent research on the functionalization of NPs for bioimaging applications. Several types of NPs have been employed for bioimaging applications, including metal (Au, Ag), metal oxide (Fe(3)O(4)), and semiconductor nanocrystals (e.g. quantum dots (QDs) and magnetic quantum dots (MQDs)). The preparation of NPs for bioimaging applications can include a variety of steps: synthesis, coating, surface functionalization, and bioconjugation. The most common strategies of engineering NP surfaces involve physical adsorption or chemisorption of the desired ligands onto the surface. Chemisorption or covalent linkages are preferred, and the coated NPs should possess high colloidal stability, biocompatibility, water solubility, and functional groups for further bioconjugation. Many of the functionalization techniques that have been reported in the literature suffer from limitations such as complex synthesis steps, poor biocompatibility, low stability, and hydrophobic products. Coating strategies based on chemisorption and ligand exchange often provide a better way to tailor the surface properties of NPs. After conjugation with the appropriate targeting ligands, antibodies, or proteins, the NPs may exhibit highly selective binding, making them useful for fluorescence imaging, magnetic resonance imaging (MRI), positron emission tomography (PET) imaging, and multimodal imaging.

A self-contained all-in-one cartridge for sample preparation and real-time PCR in rapid influenza diagnosis.

Herein we present a fully automated system with pseudo-multiplexing capability for rapid infectious disease diagnosis. The all-in-one system was comprised of a polymer cartridge, a miniaturized thermal cycler, 1-color, 3-chamber fluorescence detectors for real-time reverse transcription polymerase chain reaction (RRT-PCR), and a pneumatic fluidic delivery unit consisting of two pinch-valve manifolds and two pneumatic pumps. The disposable, self-contained cartridge held all the necessary reagents for viral RNA purification and reverse transcription polymerase chain reaction (RT-PCR) detection, which took place all within the completely sealed cartridge. The operator only needed to pipette the patient's sample with lysis buffer into the cartridge, and the system would automatically perform the entire sample preparation and diagnosis within 2.5 h. We have successfully employed this system for seasonal influenza A H1N1 typing and sub-typing, obtaining comparable sensitivity as the experiments conducted using manual RNA extraction and commercial thermal cycler. A minimum detectable virus loading of 100 copies per µl has been determined by serial dilution experiments. This all-in-one desktop system would be suitable for decentralized disease diagnosis at immigration check points and outpatient clinics, and would not require highly skilled operators.

Cysteine-functionalized polyaspartic acid: a polymer for coating and bioconjugation of nanoparticles and quantum dots.

We have synthesized a biocompatible polyaspartic acid-based polymer (molecular weight approximately 15,000-25,000) with cysteine on its backbone for use as a capping ligand for functionalized Au, Ag, and CdSe@ZnS nanoparticles. Nearly monodisperse, hydrophobic Au and Ag nanoparticles and CdSe@ZnS quantum dots were first prepared in organic solvents via conventional synthesis and then ligand exchanged to derive polymer-coated water-soluble nanoparticles. Multiple thiol groups in the polymer backbone conferred excellent protection against aggregation of the nanoparticles, and the carboxylic acid groups in the polymer provided the possibility of covalent binding with antibodies. Compared to the conventional thiol-based ligands, this polymer coating led to superior colloidal stability under the experimental conditions involved in the bioconjugation and purification steps. Goat antihuman-IgG (anti-h-IgG) and antimouse epidermal growth factor receptor (anti-m-EGFR) antibodies were conjugated with the polymer-coated nanoparticles and successfully applied to protein detection. This polymer coating exhibited minimal nonspecific interaction with cells and could be broadly applied to cell labeling.

Magnetic PEDOT hollow capsules with single holes.

We demonstrate magnetic capsules with high uptake capacity by combining single-hole hydroxyl-functionalized PEDOT hollow spheres and Fe(3)O(4) nanoparticles; potential applications include pollutant removal and controlled drug delivery.

Interfacial properties and in vitro cytotoxic effects of surface-modified near infrared absorbing Au-Au(2)S nanoparticles.

Near infrared (NIR) absorbing Au-Au(2)S nanoparticles were modified with surfactants of different hydrocarbon chain lengths to allow loading of anticancer drug, cisplatin. The interfacial interactions and surfactant chain length effects on drug loading, optical properties and cytotoxicity were discussed in this work. Short-chain surfactants were oriented closer to the surface normal and were adsorbed at higher densities. Surface modification also changed the optical properties of the particles. Notably, particles modified with short-chain surfactants exhibited a red shift, whereas particles modified with long-chain surfactants showed a blue shift. The in vitro cytotoxicity of drug-loaded surface-modified particles was dependent on the surfactants' chain length. Significant cytotoxicity was observed for 1 mg/ml of drug-loaded particles using surfactants with the shortest chain length. After NIR triggered drug release, the released Pt compounds were observed to be cytotoxic, while remaining nanoparticles did not exhibit any cytotoxicity. Also, the released Pt compounds upon NIR irradiation of drug-loaded particles were observed to be more toxic and had a different molecular structure from cisplatin.

Sieve-through vertical flow platform for efficient liquid exchange in particle-based assays.

Particle-based assays are widely used in many biomedical applications. However, the performance of particle-based systems is often compromised by the carry-over contamination caused by the residual reagents during the liquid-exchange process. We have developed a sieve-through platform that utilizes a porous membrane to sieve out the particles, and an absorbent pad to remove the waste liquid by capillary force. The porous membrane is able to contain the liquid in the reaction chamber, and allows the waste liquid to flow through when it is brought into contact with the absorbent pad. The sieve-through platform is able to effectively remove the waste liquid, thereby achieving a more efficient liquid exchange as compared to the conventional process, and minimizing the carry-over contamination. In this study, we have determined the factors that affect the flow characteristics through the porous membrane on the sieve-through platform. We have shown that the sieve-through platform effectively reduces the carry-over contamination. In addition, we have shown particle-based ELISA on the sieve-through platform for the analysis of proteins and cells. We have further demonstrated the potential of the sieve-through platform for high-throughput analysis by presenting a sieve-array, which allows concurrent analysis of multiple samples in parallel. The sieve-through platform can significantly improve the performance of particle-based systems.

Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea-Based Micellar Nanocomplex beyond Tumor-Targeted Delivery.

Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumor-targeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier-drug synergies with the high-performance carrier of PEG-EGCG besides tumor-targeted delivery.

A general synthesis for PEDOT-coated nonconductive materials and PEDOT hollow particles by aqueous chemical polymerization.

A method for coating functionalized poly(3,4-ethylenedioxythiophene) (PEDOT) on nonconductive substrates in aqueous solution allows the deposition of PEDOT thin layers on various substrates, including silica and polystyrene (PS) nanoparticles, siliceous mesocellular foam, and chitosan-alginate fibers. The surface property is tuned by controlling the monomer composition in the aqueous solutions. Using appropriate organic solvents to remove the PS cores of PEDOT-coated PS nanoparticles, hollow PEDOT particles with single holes and PEDOT capsules can be formed.

Highly potent antimicrobial polyionenes with rapid killing kinetics, skin biocompatibility and in vivo bactericidal activity.

Effective antimicrobial agents are important arsenals in our perennial fight against communicable diseases, hospital-acquired and surgical site multidrug-resistant infections. In this study, we devise a strategy for the development of highly efficacious and skin compatible yet inexpensive water-soluble macromolecular antimicrobial polyionenes by employing a catalyst-free, polyaddition polymerization using commercially available monomers. A series of antimicrobial polyionenes are prepared through a simple polyaddition reaction with both polymer-forming reaction and charge installation occurring simultaneously. The compositions and structures of polymers are modulated to study their effects on antimicrobial activity against a broad spectrum of pathogenic microbes. Polymers with optimized compositions have potent antimicrobial activity with low minimum inhibitory concentrations of 1.95-7.8 µg/mL and high selectivity over mammalian cells. In particular, a killing efficiency of more than 99.9% within 2 min is obtained. Moreover, the polymers demonstrate high antimicrobial efficacy against various clinically-isolated multidrug-resistant microbes, yet exhibit vastly superior skin biocompatibility in mice as compared to other clinically used surgical scrubs (chlorhexidine and betadine). Microbicidal activity of the polymer is mediated via membrane lysis as demonstrated by confocal microscopy. Unlike small molecular antibiotics, repeated use of the polymer does not induce drug resistance. More importantly, the polymer shows excellent bactericidal activity in a P. aeruginosa-contaminated mouse skin model. Given their rapid and efficacious microbicidal activity and skin compatibility, these polymers have tremendous potential to be developed as surgical scrubs/hand sanitizers to prevent multidrug-resistant infections.

A microarray platform for detecting disease-specific circulating miRNA in human serum.

Circulating microRNAs (miRNAs) are emerging as potential blood-based biomarkers for cancer and other critical diseases. To profile the expression levels of these tiny molecules, especially in a point-of-care setting, it is imperative to quantify them directly in complex biological fluids. Herein, we report the development of a microarray platform with carboxyl-polyethylene glycol (PEG) as a functional layer and aminated hairpin nucleic acid molecules as target-specific capture probes (CPs). Due to the anti-fouling effect conferred by the carboxyl-PEG layer, we could directly detect as little as 10fM of miRNA targets in 20µl of unprocessed human serum. In contrast to the conventional miRNA microarrays, our platform does not require RNA extraction, labeling and target amplification, thus significantly reducing both the sample preparation steps as well as the total assay duration. The use of specially designed hairpin CPs entails reliable discrimination of miRNA sequences with high sequence homology. A nanoparticle-based detection technique, with the help of differential interference contrast (DIC) microscopy, offers excellent resolution down to a single molecule. With the capability of detecting disease-specific miRNA targets directly in human serum, our microarray platform has potential applications in rapid, minimally invasive clinical diagnostic assays.

Transparent nanostructured photochromic UV-blocking soft contact lenses.

AIM: We aim to develop transparent UV-blocking photochromic soft contact lenses via polymerization of a bicontinuous nanoemulsion. MATERIALS & METHODS: Transparent nanostructured polymers were prepared by incorporating a polymerizable surfactant and thermal initiator together with water, monomers, UV blockers and photochromic dyes. The polymers were characterized using oxygen permeometer, tensile tester, electron microscope, UV spectrophotometer, corneal cell culture and testing in rabbits. RESULTS: The polymers have good oxygen permeability, water content, stiffness, strength and UV-blocking ability comparable to commercial UV-blocking soft contact lenses. Their response to UV light is comparable to photochromic spectacle lenses, particularly in reverse transition from colored to colorless state. They are nontoxic and nonleaching. CONCLUSION: Our photochromic UV-blocking contact lenses provide a novel alternative to photochromic spectacles.

Long-Term Subconjunctival Delivery of Brimonidine Tartrate for Glaucoma Treatment Using a Microspheres/Carrier System.

Core-shell polymer microspheres with poly(d,l-lactic-co-glycolic acid) core and poly(l-lactic acid) (PLLA) shell are developed for the long-term subconjunctival release of brimonidine tartrate (BT) in order to reduce intraocular pressure (IOP) in the treatment of glaucoma. The PLLA-rich shell acts as a diffusion barrier, enabling linear release of BT over an extended period of 40 d. The microspheres are encased in a porous non-degradable methacrylate-based carrier for ease of subconjunctival implantation in a glaucoma-induced rabbit model. In vivo release of BT from the microspheres/carrier system has enabled a significant, immediate IOP reduction of 20 mmHg, which is sustained for 55 d. Long-term IOP reduction may be maintained by periodic replacement of the microspheres/carrier system.

Targeting Warburg Effect in Cancers with PEGylated Glucose.

In highly proliferative cancer cells, energy is predominantly produced by a high rate of glycolysis, followed by lactic acid fermentation, despite the availability of oxygen - an observation known as the Warburg effect. As a consequence, cells employing this glycolytic pathway require high uptake of glucose and increased metabolic rates to maintain their proliferation. It has been hypothesized that by blocking glucose uptake using modified glucose molecules, apoptosis in the cancer cells can be induced. In this study, it has been showed that several poly(ethylene glycol) (PEG)-modified glucose compounds could reduce cell proliferation in various cancer cell lines by a phenomenon that blocked the availability of the glucose transporters and reduced AKT1 (serine/threonine-specific protein kinase) activation. Xenograft cancer models that are intravenously administered with glucose-conjugated branched PEG (GBrP) daily for 14 d show little tumor development, as compared to the control group without GBrP treatment. The toxicological effects and the pharmacokinetics of the PEGylated glucose are studied in rodents. The PEGylated glucose exerts no systemic toxicity at 40 mg kg(-1) dosage. However, doses above 80 mg kg(-1) show dose-dependent toxicity in all the organs analyzed. The present results suggest PEGylated glucose as a promising "metabolic therapy" approach for the treatment of cancer.

A stacking flow immunoassay for the detection of dengue-specific immunoglobulins in salivary fluid.

Paper-based immunoassays, usually in the form of lateral flow tests, are currently the standard platform for home diagnostics. However, conventional lateral tests are often complicated by severe non-specific adsorption of detector particles when applied to test samples containing salivary fluid. It is believed that a high concentration of proteinaceous substances in salivary fluid causes particle aggregation and adhesion. In this study, we developed a stacking flow platform for single-step detection of a target antibody in salivary fluid. Stacking flow circumvents the need for separate sample pre-treatments, such as filtration or centrifugation, which are often required prior to testing saliva samples using paper-based immunoassays. This is achieved by guiding the samples and reagents to the test strip through different paths. By doing so, salivary substances that interfere with the particle-based sensing system are removed before they come into contact with the detection reagents, which greatly reduces the background. In addition, the stacking flow configuration enables uniform flow with a unique flow regulator, which leads to even test lines with good quantification capability, enabling the detection of ~20 ng mL(-1) α-fetoprotein in the serum. We have successfully applied the stacking flow device to detect dengue-specific immunoglobulins that are present in salivary fluid.

Mesoporous poly(melamine-formaldehyde) solid sorbent for carbon dioxide capture.

Feed the pore: A highly mesoporous melamine-formaldehyde resin is synthesized through a simple, one-step polycondensation reaction by using inexpensive and abundant common industrial chemicals. The material is demonstrated to have a high surface area and a well-defined pore structure. Its high density of CO2 binding pockets with low CO2 binding energy facilitates rapid and reversible CO2 sorption.