(Hyper)Thermophilic Enzymes: Production and Purification.

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

G-Protein-Coupled Receptor Expression and Purification.

G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs: (1) cell-free in vitro translation, (2) HEK cells, and (3) Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration, and can be analyzed with standard biophysical techniques and biochemical assays.

FDM-printed pH-responsive capsules for the oral delivery of a model macromolecular dye.

To this day, the oral delivery of biomacromolecules remains a major developmentally-oriented challenge. A combinatorial approach was followed at this study, to formulate an efficient carrier for the in vitro delivery of a model macromolecule, fluorescein isothiocyanate-dextran 4 kDa (FD4). The model macromolecule was formulated in a self-assembling peptide hydrogel (ac-(RADA)4-CONH2), prior to deposition in a hydroxypropyl methylcellulose-phthalate (HPMCP)-based 3D-printed capsule. Loading of FD4 was investigated for potential alterations on the structural (AFM) and gelling properties of the peptide carrier. Thermal analysis and morphological properties of the 3D-printed capsules were assessed by TGA, DSC and microscopy studies. For the peptide hydrogel, similar release profiles of FD4 were recorded in simulated gastric fluid pH 1.2 and phosphate buffer saline pH 7.4, indicating the need for a structural barrier, to protect the peptide carrier from the acidic environment of the stomach. The pH responsive character of the HPMCP-based capsule was evidenced in the release profiles of FD4 in a sequence of release media, i.e. simulated gastric fluid pH 1.2, simulated intestinal fluid pH 6.8 and phosphate buffer saline pH 7.4. The results supported the combinatorial formulation approach as a promising system for the efficient oral delivery of biomacromolecules.

Controlled Drug Delivery Systems for Oral Cancer Treatment-Current Status and Future Perspectives.

Oral squamous cell carcinoma (OSCC), which encompasses the oral cavity-derived malignancies, is a devastating disease causing substantial morbidity and mortality in both men and women. It is the most common subtype of the head and neck squamous cell carcinoma (HNSCC), which is ranked the sixth most common malignancy worldwide. Despite promising advancements in the conventional therapeutic approaches currently available for patients with oral cancer, many drawbacks are still to be addressed; surgical resection leads to permanent disfigurement, altered sense of self and debilitating physiological consequences, while chemo- and radio-therapies result in significant toxicities, all affecting patient wellbeing and quality of life. Thus, the development of novel therapeutic approaches or modifications of current strategies is paramount to improve individual health outcomes and survival, while early tumour detection remains a priority and significant challenge. In recent years, drug delivery systems and chronotherapy have been developed as alternative methods aiming to enhance the benefits of the current anticancer therapies, while minimizing their undesirable toxic effects on the healthy non-cancerous cells. Targeted drug delivery systems have the potential to increase drug bioavailability and bio-distribution at the site of the primary tumour. This review confers current knowledge on the diverse drug delivery methods, potential carriers (e.g., polymeric, inorganic, and combinational nanoparticles; nanolipids; hydrogels; exosomes) and anticancer targeted approaches for oral squamous cell carcinoma treatment, with an emphasis on their clinical relevance in the era of precision medicine, circadian chronobiology and patient-centred health care.

Synergistic Antitumor Potency of a Self-Assembling Peptide Hydrogel for the Local Co-delivery of Doxorubicin and Curcumin in the Treatment of Head and Neck Cancer.

Combination therapy has been conferred with manifold assets leveraging the synergy of different agents to achieve a sufficient therapeutic outcome with lower administered drug doses and reduced side effects. The therapeutic potency of a self-assembling peptide hydrogel for the co-delivery of doxorubicin and curcumin was assessed against head and neck cancer cells. The dual loaded peptide hydrogel enabled control over the rate of drug release based on drug's aqueous solubility. A significantly enhanced cell growth inhibitory effect was observed after treatment with the combination drug-loaded hydrogel formulations compared to the respective combination drug solution. The synergistic pharmacological effect of selected hydrogel formulations was further confirmed with enhanced apoptotic cell response, interference in cell cycle progression, and significantly altered apoptotic/anti-apoptotic gene expression profiles obtained in dose levels well below the half-maximal inhibitory concentrations of both drugs. The in vivo antitumor efficacy of the drug-loaded peptide hydrogel formulation was confirmed in HSC-3 cell-xenografted severe combined immunodeficient mice and visualized with µCT imaging. Histological and terminal deoxynucleotidyl transferase dUTP nick end labeling assay analyses of major organs were implemented to assess the safety of the topically administered hydrogel formulation. Overall, results demonstrated the therapeutic utility of the dual drug-loaded peptide hydrogel as a pertinent approach for the local treatment of head and neck cancer.

Chemotherapeutic Delivery from a Self-Assembling Peptide Nanofiber Hydrogel for the Management of Glioblastoma.

PURPOSE: Localized chemotherapy has gained significant impetus for the management of malignant brain tumors. In the present study, we appraised the versatility of an in-situ gel forming self-assembling peptide, ac-(RADA)4-CONH2, as a biocompatible delivery depot of the chemotherapeutic drug doxorubicin (DOX) and the anticancer agent curcumin (CUR), respectively. METHODS: The morphology and mechanical properties of ac-(RADA)4-CONH2 were assessed with scanning electron microscopy (SEM) and rheological studies. The in vitro drug release from ac-(RADA)4-CONH2 was monitored in phosphate-buffered saline pH 7.4. Distribution of the fluorescent actives within the peptide matrix was visualized with confocal laser scanning microscopy (CLSM). The in vitro biological performance of the ac-(RADA)4-CONH2-DOX and ac-(RADA)4-CONH2-CUR was evaluated on the human glioblastoma U-87 MG cell line. RESULTS: SEM studies revealed that the ac-(RADA)4-CONH2 hydrogel contains an entangled nanofiber network. Rheology studies showed that the more hydrophobic CUR resulted in a stiffer hydrogel compared with ac-(RADA)4-CONH2 and ac-(RADA)4-CONH2-DOX, due to the interaction of CUR with the hydrophobic domains of the peptide nanofibers as confirmed by CLSM. In vitro release studies showed a complete DOX release from ac-(RADA)4-CONH2 within 4 days and a prolonged release for ac-(RADA)4-CONH2-CUR over 20 days. An increased cellular uptake and a higher cytotoxic effect were observed for ac-(RADA)4-CONH2-DOX, compared with DOX solution. Higher levels of early apoptosis were observed for the cells treated with the ac-(RADA)4-CONH2-CUR, compared to CUR solution. CONCLUSIONS: The current findings highlight the potential utility of the in-situ depot forming ac-(RADA)4-CONH2 hydrogel for the local delivery of both water soluble and insoluble chemotherapeutic drugs.

Self-Assembling Peptide Nanofiber Hydrogels for Controlled Ocular Delivery of Timolol Maleate.

The self-assembling peptides Ac-(RADA)4-CONH2 and Ac-(IEIK)3I-CONH2, which form hydrogels in physiological conditions, were evaluated as carriers for ocular delivery of the ß-blocker timolol maleate. Electron microscopy studies revealed that hydrogels contain nanofibers, whereas rheological studies showed that the Ac-(IEIK)3I-CONH2 self-assembles in a stiffer hydrogel compared with the Ac-(RADA)4-CONH2 peptide. The in vitro release and ex vivo permeation studies demonstrated controlled release and transport of the drug through the cornea, which depended on the self-assembling peptide sequence. In vivo studies in rabbits showed significant increase in the area under the concentration-time curve (AUC) after administration of the drug through the Ac-(RADA)4-CONH2 hydrogel compared to drug solution, whereas a sustained reduction of intraocular pressure for up to 24 h after instillation was achieved for both drug-loaded hydrogels. Histological studies revealed good ocular tolerability upon application of the formulations, suggesting that self-assembling peptide hydrogels are promising systems for sustained ocular drug delivery.

Self-assembling peptide nanofiber hydrogels in tissue engineering and regenerative medicine: Progress, design guidelines, and applications.

Until the mid-1980s, mainly biologists were conducting peptide research. This changed with discoveries that opened new paths of research involving the use of peptides in bioengineering, biotechnology, biomedicine, nanotechnology, and bioelectronics. Peptide engineering and rational design of novel peptide sequences with unique and tailor-made properties further expanded the field. The discovery of short self-assembling peptides, which upon association form well-defined supramolecular architectures, created new and exciting areas of research. Depending on the amino acid sequence, the pH, and the type of the electrolyte in the medium, peptide self-assembly leads to the formation of nanofibers, which are further organized to form a hydrogel. In this review, the application of ionic complementary peptides which self-assemble to form nanofiber hydrogels for tissue engineering and regenerative medicine will be discussed through a selective presentation of the most important work performed during the last 25 years.

Bioactive self-assembling lipid-like peptides as permeation enhancers for oral drug delivery.

Amphiphilic, lipid-like, self-assembling peptides are functional biomaterials with surfactant properties. In this work, lipid-like peptides were designed to have a hydrophilic head composed of aspartic acid or lysine and a six alanine residue hydrophobic domain and have a length similar to that of biological lipids. The aim of this work was to examine the potential of using ac-A6 K-CONH2 , KA6 -CONH2 , ac-A6 D-COOH, and DA6 -COOH lipid-like peptides as permeability enhancers to facilitate transport through the intestinal barrier. In vitro transport studies of the macromolecular fluorescent marker fluorescein isothiocyanate (FITC)-dextran (4.4 kDa) through Caco-2 cell monolayers show the permeation enhancement ability of the lipid-like peptides. We observed increased FITC-dextran transport across the epithelial monolayer up to 7.6-fold in the presence of lipid-like peptides. Furthermore, we monitored the transepithelial resistance and performed immunofluorescence studies of the cell tight junctions. Ex vivo studies showed increased mucosal to serosal absorption of FITC-dextran in rat jejunum in the presence of the ac-A6 D-COOH peptide. Furthermore, a small increase in the serosal transport of bovine serum albumin was observed upon addition of ac-A6 D-COOH. Lipid-like peptides are biocompatible and they do not affect epithelial cell viability and epithelial monolayer integrity. Our results suggest that short, lipid-like peptides may be used as permeation enhancers to facilitate oral delivery of diagnostic and therapeutic molecules.

Lipid-like self-assembling peptide nanovesicles for drug delivery.

Amphiphilic self-assembling peptides are functional materials, which, depending on the amino acid sequence, the peptide length, and the physicochemical conditions, form a variety of nanostructures including nanovesicles, nanotubes, and nanovalves. We designed lipid-like peptides with an aspartic acid or lysine hydrophilic head and a hydrophobic tail composed of six alanines (i.e., ac-A6K-CONH2, KA6-CONH2, ac-A6D-COOH, and DA6-COOH). The resulting novel peptides have a length similar to biological lipids and form nanovesicles at physiological conditions. AFM microscopy and light scattering analyses of the positively charged lipid-like ac-A6K-CONH2, KA6-CONH2 peptide formulations showed individual nanovesicles. The negatively charged ac-A6D-COOH and DA6-COOH peptides self-assembled into nanovesicles that formed clusters that upon drying were organized into necklace-like formations of nanovesicles. Encapsulation of probe molecules and release studies through the peptide bilayer suggest that peptide nanovesicles may be good candidates for sustained release of pharmaceutically active hydrophilic and hydrophobic compounds. Lipid-like peptide nanovesicles represent a paradigm shifting system that may complement liposomes for the delivery of diagnostic and therapeutic agents.

G-protein-coupled receptor expression and purification.

G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the overexpression host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs by cell-free in vitro translation systems, HEK cells, and Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration and can be analyzed with standard biophysical techniques and biochemical assays.

(Hyper)thermophilic enzymes: production and purification.

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I.

Designer peptides with self-assembling properties form nanofibers which are further organized to form a hydrogel consisting of up to 99.5% water. We present here the encapsulation of neural stem cells into peptide nanofiber hydrogel scaffolds. This results in three-dimensional (3-D) neural tissue cultures in which neural stem cells differentiate into progenitor neural cells, neurons, astrocytes and oligodendrocytes when cultured in serum-free medium. Cell survival studies showed that neural cells in peptide hydrogels thrive for at least 5 months. In contrast, neural stem cells encapsulated in Collagen I were poorly differentiated and did not migrate significantly, thus forming clusters. We show that for culture periods of 1-2 weeks, neural stem cells proliferate and differentiate better in Matrigel. However, in long-term studies, the population of cells in Matrigel decreases whereas better cell survival rates are observed in neural tissue cultures in peptide hydrogels. Peptide functionalization with cell adhesion and cell differentiation motifs show superior cell survival and differentiation properties compared to those observed upon culturing neural cells in non-modified peptide hydrogels. These designed 3-D engineered tissue culturing systems have a potential use as tissue surrogates for tissue regeneration. The well-defined chemical and physical properties of the peptide nanofiber hydrogels and the use of serum-free medium allow for more realistic biological studies of neural cells in a biomimetic 3-D environment.

Molecular fabrications of smart nanobiomaterials and applications in personalized medicine.

Recent advances in nanotechnology adequately address many of the current challenges in biomedicine. However, to advance medicine we need personalized treatments which require the combination of nanotechnological progress with genetics, molecular biology, gene sequencing, and computational design. This paper reviews the literature of nanoscale biomaterials described to be totally biocompatible, non-toxic, non-immunogenic, and biodegradable and furthermore, have been used or have the potential to be used in personalized biomedical applications such as drug delivery, tissue regeneration, and diagnostics. The nanobiomaterial architecture is discussed as basis for fabrication of novel integrated systems involving cells, growth factors, proteins, cytokines, drug molecules, and other biomolecules with the purpose of creating a universal, all purpose nanobiomedical device for personalized therapies. Nanofabrication strategies toward the development of a platform for the implementation of nanotechnology in personalized medicine are also presented. In addition, there is a discussion on the challenges faced for designing versatile, smart nanobiomaterials and the requirements for choosing a material with tailor made specifications to address the needs of a specific patient.

Thermal stabilization of an endoglucanase by cyclization.

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-ß-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.

Two-layered injectable self-assembling peptide scaffold hydrogels for long-term sustained release of human antibodies.

The release kinetics for human immunoglobulin (IgG) through the permeable structure of nanofiber scaffold hydrogels consisting of the ac-(RADA)(4)-CONH(2) and ac-(KLDL)(3)-CONH(2) self-assembling peptides were studied during a period of 3 months. Self assembling peptides are a class of stimuli-responsive materials which undergo sol-gel transition in the presence of an electrolyte solution such as biological fluids and salts. IgG diffusivities decreased with increasing hydrogel nanofiber density providing a means to control the release kinetics. Two-layered hydrogel structures were created consisting of concentric spheres of ac-(RADA)(4)-CONH(2) core and ac-(KLDL)(3)-CONH(2) shell and the antibody diffusion profile was determined through the 'onion-like' architecture. Secondary and tertiary structure analyses as well as biological assays using single molecule analyses and quartz crystal microbalance of the released IgG showed that encapsulation and release did not affect the conformation of the antibody and the biological activity even after 3 months inside the hydrogel. The functionality of polyclonal human IgG to the phosphocholine antigen was determined and showed that IgG encapsulation and release did not affect the antibody binding efficacy to the antigen. Our experimental protocol allows for 100% IgG loading efficiency inside the hydrogel while the maximum amount of antibody loading depends solely on the solubility of the antibody in water because the peptide hydrogel consists of water up to 99.5%. Our results show that this fully biocompatible and injectable peptide hydrogel system may be used for controlled release applications as a carrier for therapeutic antibodies.

Designer peptide surfactants stabilize diverse functional membrane proteins.

Multi-spanning integral membrane proteins, including G-protein coupled receptors (GPCR), ion channels, and ion transporters, comprise a major class of drug targets. However, despite their vital importance, most molecular structures of membrane proteins remain elusive. This is largely due to lack of effective materials and methods to stabilize their functional conformation for sufficient time. Thus finding optimal surfactants and developing new approaches to study fundamental properties of unstable membrane proteins is urgently needed. In this tutorial review we summarize designer peptides with surfactant properties and their usefulness to stabilize membrane proteins. These peptide surfactants present new opportunities for the stabilization and characterization of diverse membrane proteins. Previous studies on the interaction between surfactant peptides and membrane proteins revealed strategies to design new peptides tailor-made for the stabilization of specific proteins. We review examples of solubilization, purification, long-term stabilization of membrane proteins, and the design principles of peptide sequences. We discuss future trends for exploiting spatial features, thermodynamic parameters, and self-assembling properties to create peptide surfactant structures to facilitate the characterization of diverse membrane proteins.

Enhanced electron transfer activity of photosystem I by polycations in aqueous solution.

The use of proteins in advanced nanotechnological applications requires extended stabilization of the functional protein conformation and enhanced activity. Here we report that simple cationic poly(amino acid)s can significantly increase the activity of the multidomain protein supercomplex Photosystem-I (PS-I) in solution better than other commonly used chemical detergents and anionic poly(amino acid)s. We carried out a systematic analysis using a series of poly(amino acid)s (i.e., poly-l-tyrosine, poly-l-histidine, poly-l-aspartic and poly-l-glutamic acid, poly-l-arginine, and poly-l-lysine). Our results show that the polycations poly-l-lysine and poly-l-arginine significantly enhance the photochemical activity of PS-I, whereas negatively charged and hydrophobic poly(amino acid)s did not increase the PS-I functionality in solution. Furthermore, we show that poly-l-lysine can stabilize highly active PS-I in the dry state, resulting in 84% activity recovery. These simple and inexpensive poly(amino acid)s will likely make significant contributions toward a highly active form of the PS-I membrane protein with important applications in nanotechnology and biotechnology.

NMR characterization of a 264-residue hyperthermostable endo-beta-1,3-glucanase.

Insight into the hyperthermostable endo-beta-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 degrees C and recently the X-ray structure of pfLamA has been obtained at 20 degrees C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 degrees C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-beta-1,3-glucanases at extreme temperatures.

Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold.

The release kinetics for a variety of proteins of a wide range of molecular mass, hydrodynamic radii, and isoelectric points through a nanofiber hydrogel scaffold consisting of designer self-assembling peptides were studied by using single-molecule fluorescence correlation spectroscopy (FCS). In contrast to classical diffusion experiments, the single-molecule approach allowed for the direct determination of diffusion coefficients for lysozyme, trypsin inhibitor, BSA, and IgG both inside the hydrogel and after being released into the solution. The results of the FCS analyses and the calculated pristine in-gel diffusion coefficients were compared with the values obtained from the Stokes-Einstein equation, Fickian diffusion models, and the literature. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein. Protein diffusivities decreased, with increasing hydrogel nanofiber density providing a means of controlling the release kinetics. Secondary and tertiary structure analyses and biological assays of the released proteins showed that encapsulation and release did not affect the protein conformation and functionality. Our results show that this biocompatible and injectable designer self-assembling peptide hydrogel system may be useful as a carrier for therapeutic proteins for sustained release applications.