Current Pharmaceutical Biotechnology - Volume 14, Issue 2, 2013

Raman spectroscopy has long been used as a powerful tool for chemical composition analyses. Raman scattering light measurement has the advantage of being able to examine biomolecules in a nondestructive and non-labeling manner. However, molecular imaging of cells and tissues by using Raman microscopy had been hampered due to weak Raman signals and required long acquisition time. Recent advances in imaging devices, such as development of slitscanning Raman microscopy, have enabled us to acquire high-resolution Raman images of biomedical samples. Thus, Raman molecular imaging now has wide application potential for in-situ functional analysis of biomolecules in living bodies, such as studies of intracellular drug pharmacokinetics and oxygen saturation of blood capillaries. There is a possibility that Raman scattering light measurement can be applied for functional diagnostic imaging of human bodies, taking advantage of its noninvasive merits in the future. This review briefly highlights recent topics in spontaneous Raman molecular imaging of cells and tissues.

Raman spectroscopy is a promising technique for the identification and analysis of molecules in a sample without any labeling or modification. Since Raman scattering spectra provide information about intracellular molecular distributions, metabolism and chemical reactions, Raman microscopy has been widely utilized for bio-imaging and biofunctional analysis. By using metallic nanostructures, Raman scattering from molecules at the vicinity of a metal surface is significantly enhanced, which is called surface-enhanced Raman scattering (SERS). Due to localized field enhancement, the highly sensitive detection of molecules can be achieved and many attempts to apply it for biological and biomedical research have been made. In this article, we review the applications of metallic nanoparticles as SERS agents for molecular analysis and imaging of biological cells and tissues.

Vibrational microscopy (Raman microscopy and infrared microscopy), which observes molecular vibrations, gives us the information of molecular species without staining because the observed signals are originated from intrinsic molecules of a cell. However, infrared radiation is absorbed with water, and the long wavelength (3-10 μm) limits the spatial resolution to several micrometers. Spontaneous emission of Raman scattering is quite feeble, and the Raman scattering often overlaps with one-photon fluorescence from a specimen. Coherent anti-Stokes Raman scattering (CARS) microscopy, which is one of the nonlinear Raman microscopy, is a method to overcome those problems. In this review, present system of CARS microscopy, the methods of background rejection, and applications are introduced.

For many years, spatial resolution is the most critical problem in IR microspectroscopy. This is because the spatial resolution of a conventional infrared microscope is restricted by the diffraction limit, which is almost the same as the wavelength of IR light, ranging from 2.5 to 25 μm. In the recent years, we have developed two novel types of far-field IR super-resolution microscopes using 2-color laser spectroscopies, those are transient fluorescence detected IR (TFD-IR) spectroscopy and vibrational sum-frequency generation (VSFG) spectroscopy. In these ways, because both transient fluorescence and VSFG signal have a wavelength in the visible region, the image is observed at the resolution of visible light, which is about 10 times smaller than that of IR light (that is, IR super-resolution). By using these techniques, we can map the specific IR absorption band with sub-micrometer spatial resolution, visualization of the molecular structure and reaction dynamics in a non-uniform environment such as a cell becomes a possibility. In the present reviews, we introduce our novel IR super-resolution microspectroscopy and its application to single cells in detail.

Near-infrared spectroscopy (NIRS) is widely used in studies of brain activity. In this article I explain the principles and theoretical limits of NIRS. I also discuss the need for the development of portable NIRS.

Purpose: Tissue autofluorescence study is a promising means of endoscopic detection of colonic neoplasia, but the mechanism of autofluorescence eruption has still not been verified. The purpose of this study was to precisely analyze the autofluorescence characteristics of freshly prepared normal rat colon under UVA and violet light excitation. Methods: Excised rat colons were studied by using multichannel spectrophotometry, spectroscopic imaging, confocal microscopy, combined two-photon excited fluorescence and second-harmonic generation (SHG) microscopy, and fluorescence lifetime imaging microscopy. Results: Spectroscopic analysis of freshly prepared colon sections revealed that the mucosa and the submucosa showed strong autofluorescence under UVA and violet light excitation. The combined images of two-photon and SHG microscopy revealed that the mucosal epithelia are the important source of autofluorescence. Nicotinamide adenine dinucleotide seems to be one of the major substances involved in the autofluorescence of the mucosal layer on 365- nm light excitation. The autofluorescence spectra of the luminal surfaces were identical to those of the mucosa on crosssectional examinations with 365-nm excitation. The main origin of autofluorescence of the luminal surface with 365-nm excitation is the epithelial cells in the mucosa without overlay of submucosal fluorescence. Conclusions: The mucosal layer is the important source of the autofluorescence observed under excitation with UVA/violet light in multilayered colonic structures. Illumination of 365-nm wavelength light is a suitable means of analyzing the autofluorescence of mucosal epithelia.

The objective of this study was to investigate the use of iontophoresis and/or microneedles to enhance transdermal delivery of leuprolide acetate in vivo in hairless rats. Microporation was achieved using 500 μm long maltose microneedles and pore formation was confirmed using dye binding studies, histology studies, calcein imaging studies, pore permeability index calculation and trans-epidermal water loss measurement. Iontophoresis was performed using liquid reservoir patch with inbuilt silver wire electrode and a current density of 0.1 mA/cm2 was applied for 4 hours. Delivery studies were performed using microneedles and iontophoresis alone and in combination. Passive studies involving delivery through intact skin and injections of drug solution administered subcutaneously served as controls. Blood samples were collected at predetermined time points and plasma samples were analyzed for drug using ELISA. Significantly higher drug levels were detected at the end of 6 hours treatment by microneedles alone treatment (0.98 ± 0.08 ng/ml) as compared to passive (0.36 ± 0.22 ng/ml) delivery (p < 0.05). Further, three times more drug was found to be present systemically with iontophoresis alone (3.47 ± 0.03 ng/ml) or by combination (3.54 ± 0.08 ng/ml) treatments as compared to microneedles alone treatment (p < 0.05) at the end of treatment duration. When compared to iontophoresis alone treatment, combination treatment resulted in faster drug delivery due to propulsion of the drug through the preformed micropores. In conclusion, the use of microneedles and/or iontophoresis seems promising for the transdermal delivery of peptide like leuprolide acetate.

Mucopolysaccharidosis I (MPS I) is a genetic disorder caused by mutations on α-L-iduronidase (IDUA) gene, leading to low or null enzyme activity. As nonsense mutations are present in about two thirds of the patients, stop codon read through (SCRT) is a potential alternative to achieve enhanced enzyme activity. This mechanism suppresses premature stop codon mutations allowing the protein to be fully translated. Chloramphenicol is a peptidyl transferase inhibitor able to induce readthrough and is efficient in cross the blood brain barrier. In this work, fibroblasts from MPS I patients (p.W402X/p.W402X; p.R89W/p.W402X and p.Q70X/c.1739-1g>t) were treated with chloramphenicol, which resulted in 100-fold increase on IDUA activity on compound heterozygous fibroblasts. cDNA sequencing showed that only the alleles without the nonsense mutation were being amplified, even after treatment, leading us to suggest that the nonsense alleles were targeted to nonsense-mediated mRNA decay and that chloramphenicol acts through a mechanism other than SCRT.

Better understanding of protein higher order structures (HOS) is of major interest to researchers in the field of biotechnology and biopharmaceutics. Monitoring a protein's HOS is crucial towards understanding the impact of molecular conformation on the biotechnological application. In addition, maintaining the HOS is critical for achieving robust processes and developing stable formulations of therapeutic proteins. Loss of HOS contributes to increased aggregation, enhanced immunogenicity and loss of function. Selecting the proper biophysical methods to monitor the secondary and tertiary structures of therapeutic proteins remains the central question in this field. In this study, both Fourier Transform Infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy are employed to characterize the secondary structures of various proteins as a function of temperature and pH. Three proteins with different secondary structures were examined, human serum albumin (HSA), myoglobin, and the monoclonal antibody, ofatumumab. This work demonstrates that VCD is useful technique for monitoring subtle secondary structure changes of protein therapeutics that may occur during processing or handling.

Salmonella enterica is a Gram-negative intracellular bacterial pathogen which causes salmonellosis in humans and animals. During the past several decades, extensive studies have shown that the attenuated Salmonella vaccine vector is an optimal vehicle for delivering passenger antigens to mucosal sites to induce humoral, cellular, and mucosal immunity. This immunity leads to protection against challenges with the wild-type pathogens from which the passenger antigens were derived. A myriad of studies have demonstrated that using attenuated Salmonella vaccines for recombinant multivalent vaccine construction has multiple advantages. In this review, we summarize these advantages and further evaluate the Salmonella-based vaccines against five bacterial diseases. Four of these are Gram-negative pathogens— Escherichia coli, Helicobacter pylori, Shigella dysenteriae, and Yersinia pestis—and one is a mycobacterial pathogen, Mycobacterium tuberculosis. Apart from H. pylori, the Salmonella-based vaccines against the other four pathogens exhibit excellent performance in safety, immunogenicity, and protection. These properties qualify them to be as a new generation of vaccines for preventing infections from bacterial pathogens.

A major limitation of biological therapeutics is their propensity for degradation particularly in aqueous solutions hence resulting in their short shelf-life. In this study, the stability of trastuzumab (Herceptin®) intravenous (i.v.) solutions, an IgG1 monoclonal antibody (mAb), indicated for the treatment of HER2 positive breast cancer, stored under refrigerated conditions, was evaluated over 28 days. No change in visual appearance or average particle size was observed. The pH values of the trastuzumab i.v. solutions remained stable over time. Interestingly, no change in trastuzumab monomer concentration was observed throughout the 28-day study, as determined by SEC-HPLC. SDSPAGE showed only a monomer band corresponding to the molecular weight of trastuzumab. Circular dichroism spectra obtained following 28-day storage demonstrated integrity of the secondary structural conformation of trastuzumab. Results from this study show that trastuzumab i.v. solutions remain physically and structurally stable on storage at 2-8°C for 28 days. These findings suggest that trastuzumab in solution may not be as sensitive to degradation as expected for a mAb and therefore may have important implications in extending trastuzumab shelf life for clinical use and reducing associated healthcare cost.

There is continuous emergence of resistant strains which leads to urgent need to discover new antifungal agents. The investigation of adjunctive agents for antifungal activity might help to optimize the therapy for Invasive Aspergillosis (IA). The chelating agents Ethylenediamine Tetraacetic Acid (EDTA) & Disodium salt of EDTA (DiEDTA) as adjunct to antifungal drugs have been investigated against 8 pathogenic isolates of Aspergillus spp. The MIC (Minimum Inhibitory Concentration) found by DDA (Disc Diffusion Assay) is 7.50-15.0

Staphylococcus aureus is the most prevalent etiologic agent of sepsis. Statins, primarily prescribed for their cholesterol-lowering capabilities, may be beneficial for treating sepsis due to their anti-inflammatory properties. This study examined the effect of low dose, short term simvastatin pretreatment in conjunction with antibiotic treatment on host survival and demonstrated that pretreatment with simvastatin increased survival of C57BL/6 mice in response to S. aureus infection. In vitro studies revealed that short term simvastatin pretreatment did not reduce S. aureus-stimulated expression of surface proteins necessary for macrophage presentation of antigen to T cells, such as MHC Class II and costimulatory molecules CD80 and CD86, but did reduce both basal and S. aureus-stimulated levels of C5aR. Additionally, this work demonstrated the ability of simvastatin to dampen macrophage responses initiated not only by bacteria directly but by membrane vesicles shed in response to infection, revealing a new mechanism of immune modulation by statins. These data demonstrate the ability of short term simvastatin pretreatment to modulate immune responses and identify new insights into the underlying mechanisms of the anti-inflammatory properties of simvastatin that may decrease the pathophysiological effects leading to sepsis.

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.