Stimuli Responsive Molecular Exchange of Structure Directing Agents on Gold Nanobipyramids: Cancer Cell Detection and Synergistic Therapeutics.

Surface-engineered gold nanoparticles have been considered as versatile systems for theranostics applications. Moreover, surface covering or stabilizing agents on gold nanoparticles especially gold nanobipyramids (AuNBPs) provides an extra space for cargo molecules entrapment. However, it is not well studied yet and also the preparation of AuNBPs still remains dependent largely on cetyltrimethylammonium bromide (CTAB), a cytotoxic surfactant. Therefore, the direct use of CTAB stabilized nanoparticles is not recommended for cancer theranostics applications. Herein, we address an approach of dodecyl ethyl dimethylammonium bromide (DMAB) as biocompatible structure directing agent for AuNBPs, which also accommodate anticancer drug doxorubicin (45%), an additional chemotherapeutics agent. Upon near-infrared light (NIR, 808 nm) exposure, engineered AuNBPs exhibit (i) better phototransduction (51 °C) due to NIR absorption ability (650-900 nm), (ii) photo triggered drug release (more than 80%), and (iii) synergistic chemophototherapy for breast cancer cells. Drug release response has been evaluated in tumor microenvironment conditions (84% in acidic pH and 80% at high GSH) due to protonation and high affinity of thiol binding with AuNBPs followed by DMAB replacement. Intracellular glutathione (GSH, 5-7.5 mM) replaces DMAB from AuNBPs, which cause easy aggregation of nanoparticles as corroborated by colorimetric shifts, suggesting their utilization as a molecular sensing probe of early stage cancer biomarkers. Our optimized recipe yield is monodisperse DMAB-AuNBPs with ∼90% purity even at large scales (500 mL volume per batch). DMAB-AuNBPs show better cell viability (more than 90%) across all concentrations (5-500 ug/mL) when directly compared to CTAB-AuNBPs (less than 10%). Our findings show the potential of DMAB-AuNBPs for early stage cancer detection and theranostics applications.

Nucleic acid aptamers as stabilizers of proteins: the stability of tetanus toxoid.

PURPOSE: Exposure of tetanus toxoid to moisture leads to its aggregation and reduction of potency. The aim of this work was to use SELEX (systematic evolution of ligands by exponential enrichment) protocol and select aptamers which recognize tetanus toxoid (Mr ~150 kDa) with high affinity. METHODS: Colyophilized preparations of tetanus toxoid and specific aptamers were encapsulated in PLGA microspheres and sustained release of the antigen was observed up to 55 days using different techniques. RESULTS: The total protein released was between 40-55% (24-45% residual antigenicity) in the presence of the aptamers as compared to 25% (11% residual antigenicity) for the antigen alone. We show that instead of inhibiting absorption of moisture, the aptamers blocked the protein unfolding upon absorption of moisture, inhibiting the initiation of aggregation. When exposed to accelerated storage conditions, some of the RNA sequences were able to inhibit moisture-induced aggregation in vitro and retain antigenicity of tetanus toxoid. CONCLUSIONS: Nucleic acid aptamers represent a novel class of protein stabilizers which stabilize the protein by interacting directly with it. This mechanism is unlike that of small molecules which alter the medium properties and hence depend on the stress condition a protein is exposed to.

Accelerated stability studies for moisture-induced aggregation of tetanus toxoid.

PURPOSE: The study was carried out to evaluate the effect of exposing solid tetanus toxoid to moisture in two different ways on the structure and function of the toxoid. METHODS: Tetanus toxoid was exposed to moisture by (i) the addition of an optimized amount of buffer and (ii) incubation under an environment provided by a saturated solution of K(2)CrO(4.) The changes in the conformational, structural and antigenic properties of tetanus toxoid were measured and compared. RESULTS: Results show that even at a similar level of moisture-induced aggregation, the amounts of water absorbed by the two preparations of tetanus toxoid are different. Differences in antigenicity and changes in structure of the toxoid at primary, secondary and tertiary structure levels were seen. CONCLUSION: Although both conditions are used to mimic accelerated stability conditions in the laboratory, the final products are different in the two cases. Thus, conditions for 'accelerated stability studies' for therapeutic proteins need to be selected with care so that they resemble the fate of the actual product.

Synthesis and characterization of an injectable microparticles integrated hydrogel composite biomaterial: In-vivo biocompatibility and inflammatory arthritis treatment.

Polymeric hydrogels and microparticles have been widely used for localized drug delivery applications for the treatment of arthritis. Nonetheless, owing to initial burst drug release, non-specific biodistribution and low retention time at the target site in body, these polymeric drug delivery systems have been found with low in-vivo performance. Hence, the above limitations need to be resolved by designing a smart novel drug delivery system which is the current need in biomedicine. Herein, a novel localized injectable thermoresponsive microparticles embedded hydrogel composite drug delivery system has been developed for the treatment of inflammatory arthritis. In the current study, methotrexate (MTX) loaded alginate microparticles (MTX-Microparticles) are embedded into thermoreversible hydrogel matrix (MTX-MPs-H) prepared by physical blending of sodium hyaluronate and methylcellulose (SHMC). Microparticles-hydrogel composite system exhibited appropriate in-vitro thermoreversibility (sol at 4 °C and gel at 37 °C), biocompatibility (>80 %), hemocompatibility, and controlled drug release profile. The in-vivo biocompatibility studies for 10 days revealed that composite system is non-toxic in nature. The developed MTX-MPs-H composite drug delivery system effectively decreased the swelling/ inflammation of the arthritis affected paw in wistar rats in comparison to only alginate microparticles and pure MTX up to 30 days.

Characteristics of Molecularly Engineered Anticancer Drug Conjugated Organic Nanomicelles for Site-Selective Cancer Cell Rupture and Growth Inhibition of Tumor Spheroids.

Site-selective uptake and specific biodistribution of chemotherapeutic drugs are essential prerequisites for targeted cancer therapy. Especially, antibody and peptide conjugated drugs have been attempted as localized therapeutic agents. However, the characteristics of drug conjugated nanosystems are less explored, which are limited with their toxicity, low therapeutic efficacy, complicated synthesis, and high costs. Herein, we report a biocompatible (about 95%) molecularly engineered anticancer drug conjugated nanomicelles (∼200 nm in size) for site-selective CD44 overexpressed cancer cell rupture and tumor growth inhibition. Microscopic analysis demonstrates the distinct visualization of organic-organic interfaces (∼5 nm), which are corroborated with spectroscopic measurements confirmed the conjugation of niclosamide drug with hyaluronic acid (NIC-HA). Uniformly distributed hemocompatible (about 99%) organic nanomicelles exhibit the cellular membrane and cytoplasmic targeting with significant cellular rupture (IC50 of 4 µM for MDA MB 231 cells) indicating their inherent targeting ability for cancer cells and cancer stem cells. An inclusive in vitro and in vivo analysis for targeted antitumor activity (HT1080 tumor xenograft model) of NIC-HA nanoconjugates (∼24.6% loading) exhibited promising cancer cell death and tumor growth inhibition (60%, p < 0.05) due to STAT-3 signaling pathway inhibition and induction of apoptosis in CD44-positive triple negative breast cancer cells.

Niclosamide encapsulated polymeric nanocarriers for targeted cancer therapy.

Localized cancer rates are on an upsurge, severely affecting mankind across the globe. Timely diagnosis and adopting appropriate treatment strategies could improve the quality of life significantly reducing the mortality and morbidity rates. Recently, nanotherapeutics has precipitously shown increased efficacy for controlling abnormal tissue growth in certain sites in the body, among which ligand functionalized nanoparticles (NP) have caught much attention for improved survival statistics via active targeting. Our focus was to repurpose the antihelminthic drug, niclosamide (NIC), which could aid in inhibiting the abnormal growth of cells restricted to a specific region. The work here presents a one-pot synthesis of niclosamide encapsulated, hyaluronic acid functionalized core-shell nanocarriers [(NIC-PLGA NP)HA] for active targeting of localized cancer. The synthesized nanocarriers were found to possess spherical morphology with mean size of 150.8 ± 9 nm and zeta potential of -24.9 ± 7.21 mV. The encapsulation efficiency was found to be 79.19 ± 0.16% with a loading efficiency of 7.19 ± 0.01%. The nanohybrids exhibited extreme cytocompatibility upon testing with MDA-MB-231 and L929 cell lines. The rate of cancer cell elimination was approximately 85% with targeted cell imaging results being highly convincing. [(NIC-PLGA NP)HA] demonstrates increased cellular uptake leading to a hike in reactive oxygen species (ROS) generation, combating tumour cells aiding in the localized treatment of cancer and associated therapy.

Nucleic acid aptamers stabilize proteins against different types of stress conditions.

It has been observed that the same osmolyte cannot provide protection to a protein exposed to more than one stress condition. We wanted to study the effect of nucleic acid aptamers on the stabilization of proteins against a variety of stress conditions. Adjuvanted tetanus toxoid was exposed to thermal, freeze-thawing, and agitation stress. The stability and antigenicity of the toxoid were measured. Using nucleic acid aptamers selected against tetanus toxoid, we show that these specific RNA sequences were able to stabilize alumina-adsorbed tetanus toxoid against thermal-, agitation-, and freeze-thawing-induced stress. Binding affinity of the aptamer-protein complex did not show any significant change at elevated temperature as compared with that at room temperature, indicating that the aptamer protected the protein by remaining bound to it under stress conditions and did not allow either the protein to unfold or to promote protein-protein interaction. Thus, we show that by changing the stabilization strategy from a solvent-centric to a protein-centric approach, the same molecule can be employed as a stabilizer against more than one stress condition and thus probably reduce the cost of the product during its formulation.

Linking the solution viscosity of an IgG2 monoclonal antibody to its structure as a function of pH and temperature.

Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0-7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.

Biophysical characterization and immunization studies of dominant negative inhibitor (DNI), a candidate anthrax toxin subunit vaccine.

Dominant Negative Inhibitor (DNI) is a translocation-deficient homolog of recombinant protective antigen of Bacillus anthracis that is a candidate for a next generation anthrax vaccine. This study demonstrates that the biophysical characteristics of the DNI protein stored in lyophilized form at 4°C for 8 y were similar to recombinant Protective Antigen (rPA). To provide information on the accelerated stability of DNI, samples in the lyophilized form were subjected to thermal stress (40°C and 70°C for up to 4 weeks) and thoroughly evaluated using various biophysical and chemical characterization techniques. Results demonstrate preserved structural stability of the DNI protein under extreme conditions, suggesting long-term stability can be achieved for a vaccine that employs DNI, as desired for a biodefense countermeasure. Furthermore, the biological activity of the stressed DNI bound to the adjuvant Alhydrogel (®) was evaluated in mice and it was found that the immunogenicity DNI was not affected by thermal stress.

Trehalose and protein stability.

The role of osmolytes, and especially trehalose, in stabilizing proteins under stress conditions is now a widely accepted fact. The physical and chemical properties of trehalose, i.e., low chemical reactivity, nonreducing nature, high glass transition temperature, high affinity for water molecules, existence of a number of polymorphs, etc., make it uniquely suitable for stabilizing partially unfolded protein molecules and inhibiting protein aggregation. This article discusses the various adverse situations that protein molecules face, both within the cell and outside, leading to their aggregation and inactivation. The use of trehalose in stabilizing protein molecules and helping them retain their functionally active forms under such conditions is examined. The various theories and mechanisms used to explain the protective action of trehalose are briefly presented. The experimental tools that can be used to decipher the mechanism of aggregation and the role of trehalose are also discussed.

Effect of trehalose on protein structure.

Trehalose is a ubiquitous molecule that occurs in lower and higher life forms but not in mammals. Till about 40 years ago, trehalose was visualized as a storage molecule, aiding the release of glucose for carrying out cellular functions. This perception has now changed dramatically. The role of trehalose has expanded, and this molecule has now been implicated in a variety of situations. Trehalose is synthesized as a stress-responsive factor when cells are exposed to environmental stresses like heat, cold, oxidation, desiccation, and so forth. When unicellular organisms are exposed to stress, they adapt by synthesizing huge amounts of trehalose, which helps them in retaining cellular integrity. This is thought to occur by prevention of denaturation of proteins by trehalose, which would otherwise degrade under stress. This explanation may be rational, since recently, trehalose has been shown to slow down the rate of polyglutamine-mediated protein aggregation and the resultant pathogenesis by stabilizing an aggregation-prone model protein. In recent years, trehalose has also proved useful in the cryopreservation of sperm and stem cells and in the development of a highly reliable organ preservation solution. This review aims to highlight the changing perception of the role of trehalose over the last 10 years and to propose common mechanisms that may be involved in all the myriad ways in which trehalose stabilizes protein structures. These will take into account the structure of trehalose molecule and its interactions with its environment, and the explanations will focus on the role of trehalose in preventing protein denaturation.