Great Potential For Pharmaceutical Applications Research Articles

3D printing: Innovative solutions for patients and pharmaceutical industry

Three-dimensional (3D) printing is an emerging technology with great potential in pharmaceutical applications, providing innovative solutions for both patients and pharmaceutical industry. This technology offers precise construction of the structure of dosage forms and can benefit drug product design by providing versatile release modes to meet clinical needs and facilitating patient-centric treatment, such as personalized dosing, accommodate treatment of specific disease states or patient populations. Utilization of 3D printing also facilitates digital drug product development and manufacturing. Development of 3D printing at early clinical stages and commercial scale pharmaceutical manufacturing has substantially advanced in recent years. In this review, we discuss how 3D printing accelerates early-stage drug development, including pre-clinical research and early phase human studies, and facilitates late-stage product manufacturing as well as how the technology can benefit patients. The advantages, current status, and challenges of employing 3D printing in large scale manufacturing and personalized dosing are introduced respectively. The considerations and efforts of regulatory agencies to address 3D printing technology are also discussed.

Synthesis and characterization of surface-modified ultrafine titanium dioxide nanoparticles with an antioxidant functionalized biopolymer as a therapeutic agent: Anticancer and antimicrobial evaluation

Surface modified biodegradable protein-based nanoparticles have great potential in pharmaceutical applications. In the present study, we discuss the surface engineering of titanium dioxide nanoparticles (TiO2 NPs) with alpha-lipoic acid (ALA) functionalized bovine serum albumin (BSA) conjugate as a biocompatible therapeutic system. Sol-gel method is used to synthesize TiO2 NPs, which is characterized using ultraviolet (UV)/visible, Fourier transform infrared (FT-IR), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). BSA was functionalized with antioxidant ALA by Steglich esterification, and TiO2 NPs were loaded into this synthesized BSA-ALA conjugate. The finally developed product was studied using various characterization methods like UV-Visible, FT-IR, XRD, SEM-EDX, TEM, TG-DTG, DSC and fluorescence emission analysis. In vitro cytotoxicity of this system was examined by applying the trypan blue exclusion method, and antimicrobial analysis was implemented by well diffusion method against antibiotic-resistant bacterial and fungal strains.

Cyclodextrin-based low melting mixtures as a solubilizing vehicle: Application to non-steroidal anti-inflammatory drugs

Poor water solubility represents a serious issue for drug formulation and delivery. Cyclodextrins (CDs) and cosolvents are valuable approaches to overcome this issue. Currently, there is a growing interest in deep eutectic solvents (DESs) with high solubilizing ability and low toxicity. Low melting mixtures (LMMs) based on CDs have recently emerged, combining the solubilizing properties of solvents and drug carriers. In this study, we evaluated the solubility of ketoprofen, flurbiprofen and naproxen in two mixtures based on levulinic acid (Lev). The effect of water on the solubilizing potential and viscosity of these mixtures was assessed. Also, the stability of the studied drugs was monitored up to one year. The LMM based on randomly methylated-β-cyclodextrin (RAMEB) showed the highest solubilizing potential (up to 5012-fold the aqueous solubility) compared to the DES based on choline chloride. Formation of RAMEB/drug inclusion complex in the LMM was evidenced by nuclear magnetic resonance spectroscopy (NMR). Adding water (10 wt%) sharply reduced the viscosity of both mixtures while maintaining their high solubilizing ability. Moreover, the studied drugs were stable up to 90% in both mixtures after a year at 25 °C. Such low-viscous mixtures endowed with high solubilizing and stabilizing properties have great potential for pharmaceutical applications.

How does the hydrophobic content of methacrylate ABA triblock copolymers affect polymersome formation?

AbstractPolymersomes are exciting self‐assembled structures with great potential in pharmaceutical applications. A systematic investigation of a novel series of methacrylate‐based polymersomes is reported in this study. Five well‐defined ABA triblock copolymers with A being based on tri(ethylene glycol) methyl methacrylate and B being based on 2‐(diethylamino)ethyl methacrylate (DMAEMA) were synthesized using a living polymerization method. The effect of the composition of the ABA triblock copolymers on the thickness of the hydrophobic membrane of the polymersomes and the release of a model drug is demonstrated.

Alteration of resveratrol-dependent glycosyltransferase activity by elicitation in DJ-526 rice

ABSTRACT Since the successful creation of DJ-526, a resveratrol-enriched rice cultivar, research has focused on resveratrol production because of its great potential in pharmaceutical applications. However, the utilization of resveratrol in DJ-526 is limited by glycosylation, which converts resveratrol to its glucoside (piceid), in a process driven by glycosyltransferase. The verification of resveratrol-dependent glycosyltransferase activity is an essential strategy for improving resveratrol production in DJ-526 rice. In this study, 27 candidate glycosyltransferases were evaluated in germinated seeds. Among the candidates, only R12 exhibited upregulation related to increased resveratrol and piceid content during seed germination, whereas various effects on the activity of glycosyltransferase were observed by the presence of a bio-elicitor. Yeast extract tended to enhance glycosyltransferase activity by seven candidates, and a specific peak for an unknown compound production was identified. Conversely, chitosan acted as a glycosyltransferase inhibitor. Our results suggested that R12 and R19 are the most relevant candidate resveratrol-dependent glycosyltransferases in DJ-526 seeds during germination and elicitation. Future research should assess the possibility of silencing these candidate genes in an effort to improve resveratrol levels in DJ-526 rice.

Immune-enhancing effects of anionic macromolecules extracted from Codium fragile coupled with arachidonic acid in RAW264.7 cells.

Arachidonic acid (ARA) is an integral constituent of the biological cell membrane, conferring it with fluidity and flexibility, which are necessary for the function of all cells, especially nervous system, skeletal muscle, and immune system. Codium species biosynthesize sulfated polysaccharides with very distinct structural features. Some of them have different biological activities with great potential in pharmaceutical applications. In this study, anionic macromolecules extracted from Codium fragile were investigated for their cooperative immune-enhancing activities with ARA. The cooperation between ARA and Codium resulted in increased, dose-dependent nitric oxide production and iNOS gene expression. In addition, co-treatment of ARA and Codium effectively increased pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), compared with Codium alone. We also demonstrated that the expression of COX-2 mRNA was also increased, which is responsible for the production of inflammatory mediator prostaglandins and their metabolites. Compared to the Codium group, the co-treatment of Codium with ARA enhanced the phosphorylation of nuclear factor-κB p-65, p38, and extracellular signal-related kinase 1/2, indicating that this combination stimulated immune response through nuclear factor-κB and mitogen-activated protein kinase pathways. These results indicated that the coordination of arachidonic acid with polysaccharide extracted from seaweed may be a potential source of immunomodulatory molecules.

Supercritical antisolvent process-assisted fabrication of chrysin-polyvinylpyrrolidone sub-microparticles for improved anticancer efficiency

The practical application of chrysin (CHS) is significantly hindered due to its poor aqueous solubility. To address this issue, a delivery platform based on CHS-polyvinylpyrrolidone (CHS-PVP) sub-microparticles was fabricated using the supercritical antisolvent (SAS) process. Initially, the effects of different operating parameters, including temperature, CHS concentration, and acetone/ethanol ratio on the particle size of CHS-PVP sub-microparticles were validated. This eco-friendly approach resulted in spherical-shaped particles with an average particle size of 273.7 nm ± 38.9 nm at the optimal conditions: acetone/ethanol ratio of 3:7; CHS concentration of 3 mg/mL; and temperature of 318.15 K. Further, the molecular interactions, crystallinity, drug release, and anticancer efficiency in vitro of designed particles were systematically investigated. The CHS-PVP sub-microparticles exhibited higher antiproliferative effects over pure CHS in MDA-MB-231 cells, attributing to the enhanced solubility of CHS. These findings state that the SAS process-based CHS-PVP sub-microparticles have great potential in pharmaceutical applications.

Characterization of rotenoid stemonal by semiempirical methods and molecular docking

Clitoria fairchildiana is a common plant in Amazon rain forest. Popularly known as sombreiro, this plant has a great potential to use its phytochemical content for biological and medical applications. Stemonal, one of many phytochemical products extracted from “sobreiro”, presents cytotoxic and anti-inflamatory activities, which means that has great potential for pharmaceutical applications. In order to collaborate with stemonal research, this paper studied the molecular properties of this molecule. The molecular structure of stemonal was optimized using semi-empiric methods, such as PM3, using ArgusLab® software. Molecular parameters, such as frontier orbitals (HOMO and LUMO), MESP and formation enthalpy were obtained. Also, molecular docking experiments were conducted in order to evaluate the applicability over AChE, and the coupling of stemonal in this enzyme shows that the phytochemical binds into the catalytic site of the enzyme, which means that the molecule inhibits the hydrolysis of acethylcoline, the neurotransmitter evolved into skeletal muscules. Therefore, stemonal molecule has great potential to be applied into pharmaceutical formulations to achieve a better treatment for Alzheimer’s diesease.

Recent Advances in Pharmaceutical Potential of Brown Algal Polysaccharides and their Derivatives.

Marine plants, animals and microorganisms display steady growth in the ocean and are abundant carbohydrate resources. Specifically, natural polysaccharides obtained from brown algae have been drawing increasing attention owing to their great potential in pharmaceutical applications. This review describes the structural and biological features of brown algal polysaccharides, including alginates, fucoidans, and laminarins, and it highlights recently developed approaches used to obtain the oligo- and polysaccharides with defined structures. Functional modification of these polysaccharides promotes their advanced applications in biomedical materials for controlled release and targeted drug delivery, etc. Moreover, brown algal polysaccharides and their derivatives possess numerous biological activities with anticancer, anticoagulant, wound healing, and antiviral properties. In addition, we also discuss carbohydrate- based substrates from brown algae, which are currently in clinical and preclinical studies, as well as the marine drugs that are already on the market. The present review summarizes the recent development in carbohydratebased products from brown algae, with promising findings that could rapidly facilitate the future discovery of novel marine drugs.

In vitro construction of vascularized iPSC-derived 3D-cardiac myoblast tissues for drug assessment

Event Abstract Back to Event In vitro construction of vascularized iPSC-derived 3D-cardiac myoblast tissues for drug assessment Michiya Matsusaki1, Yuto Amano1, Manabu Seo2*, Takashi Yamaguchi2, Shigeru Miyagawa3, Yoshiki Sawa3* and Mitsuru Akashi1 1 Osaka University, Department of Applied Chemistry, Graduate School of Engineering, Japan 2 Ricoh Company, Ltd., Advanced Technology Research & Development Center, Ricoh Institute of Technology, Japan 3 Osaka University Graduate School of Medicine, Department of Cardiovascular Surgery, Japan Introduction: Recent drug discovery systems are time-consuming and costly because the pharmaceutical assays using cell monolayer and animal models have many issues due to different drug responses as compared to human body. Therefore, the use of induced pluripotent stem cell (iPSC), which can provide many types of normal and diseased human cell sources, has enormous potential for pharmaceutical assays. However, since nearly all tissues are integrated three-dimensional (3D) structures of multiple types of cells and extracellular matrices (ECMs), and since intercellular signaling is important for tissue functions, it is difficult to evaluate actual tissue functions by 2D-culture method. In this study, we aim to develop 3D-cardiac myoblast tissues (3D-iPSC-CM) composed of iPS-CMs, cardiac fibroblast, and cardiac endothelial cells by the cell-accumulation technique (Figure 1). We reported a bottom-up approach, termed “accumulation technique”[1] which was improved method of our previous technique, hierarchical cell manipulation[2], to develop multilayered thick tissues (>100 µm) by cell surface coating with nanometer-sized ECM-films[3]. Less than 10 nm sized ECM-films, such as fibronectin (FN)-gelatin (G), induced cell-cell interaction in three dimensions. By this method, iPSC-derived 3D-cardiac muscle tissues were successfully fabricated. The obtained tissues showed the synchronized contractions, and the tolerance to the cytotoxicity of anticancer drugs[4]. The iPSC-derived 3D-cardiac muscle tissues have a great potential for pharmaceutical applications. Figure 1. Schematic illustration of the vascularized 3D-iPSC-CM for drug assessments. Methods: iPSC-CM with 50~60% differentiation was used in this study. After repeating the nine steps of layer-by-layer (LbL) deposition, the (FN/G)4 FN films with about 10 nm thickness were prepared on the cell surface by cell accumulation technique. Normal human cardiac fibroblasts (NHCF) and normal human cardiac endothelial cells (NHCMEC) were also coated as the same way. The coated cells were seeded into a 24 micro well cell culture insert (1 x 105 cells/layers) and cultured to construct multilayered tissues. The ratio of NHCF was varied to optimize for the highest beating number, synchronization, and vascularization. 10% NHCMEC was added for blood capillary formation. Results: The ratio of NHCF was varied to 0, 25, 50, 75, and 100% to optimize beating number and synchronization of the tissues. The 3D-iPSC-CM tissues with 25~50% of NHCF clearly showed the highest beating number and synchronization. In the case of blood capillary formation, the network clearly found at over 25% NHCF condition and the network area increased with increasing NHCF ratio. Accordingly, 50% NHCF was used for the drug assessment. Interestingly, the vascularized 3D-tissues showed higher tolerance to the drug as compared to the monolayer culture (2D). We also found that the capillary was damaged by the treatment of doxorubicin in dose dependent manner. These results indicated that the vascularized 3D-iPSC-CM tissues might be useful for drug assessment. Figure 2. (a) Beating number of 3D-iPSC-CM tissues (NHCF50) and 2D monolayr of iPSC-CMs (NHCF50) after 3 days of incubation with 1, 50, or 1000 nM doxorubicin. *indicates 0. (b) CLSM images of the 3D-tissues and 2D-monolayer after incubation with doxorubicin by immunostaining with troponin T antibody. (c) CLSM images of blood capillary networks in 3D-iPSC-CM tissues after treatment with doxorubicin. The networks were immunostained with anti CD31 antibody. (d) Quantified capillary area in images of (c). ††p < 0.01 statistically significant difference as compared to 2D. Conclusions: We successfully constructed vascularized iPSC-CM tissues by coating of FN-G nanofilms. Drug responses and tolerance mechanism of the 3D-tissues will report in the conference. NEXT Program (LR026); Grant-in-Aid for Scientific Research (S) (A232250040); Grant-in-Aid for Scientific Research (B) (26282138); Grant-in-Aid for Scientific Research on Innovative Areas (26106717); SENTAN-JST Program (13A1204); Health Labour Sciences Research Grant from the Japanese Ministry of Health, Labour and Welfare, and Grand-in-Aid for JSPS Fellows (24・622)

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

Boesenbergia rotunda, a herb in the ginger family, contains numerous beneficial compounds, such as flavonoids, flavones and cyclohexenyl chalcone derivatives, that have great potential for pharmaceutical applications. However, the low concentration of the bioactive compounds limits their commercial application. In this study, a simple and reliable Agrobacterium-mediated transformation protocol for B. rotunda cell suspension culture was successfully developed. The minimal inhibitory concentration and natural tolerance of the selective agent, hygromycin, against the cells were 20 mg l−l and 30 mg l−l in liquid media and solid media, respectively. The highest number of transformed regenerants (18 ± 0.00 per ml settled cell volume) was recorded when cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA1304 for 10 min and co-cultivated for 2 days. Prolonged infection time (> 10 min) and co-cultivation period (> 2 days), however, did not increase the transformation efficiencies. The results clearly show that infection and co-cultivation periods strongly influenced the transformation efficiency in ginger. The transformed cells were recovered and showed green fluorescent signals under ultraviolet excitation. An intense blue colour was observed in the transformed cells after β-glucuronidase (GUS) histochemical staining, further confirming the functionality of the GUS enzymes in the regenerants. Polymerase chain reaction analysis of 3-, 6-, 9- and 12-month-old transformed cells confirmed that the protocol enabled stable integration of the mgfp5 gene. Moreover, the comparatively high number of transformed regenerants in this study made it possible to generate a large number of transgenic cells in a short period, which would be useful for high-throughput functional screening of enzymes involved in the biosynthetic pathways of bioactive compounds.

Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain

BackgroundThe phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms.ResultsWe develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation.ConclusionsWe achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

A novel method to chemically bind thiazolidine-2,4-dione through cross-linked chitosan nanoparticles using malanodialdehyde as a cross-linker

Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications because of its biocompatibility, high charge density, nontoxicity and mucoadhesion. It has been shown that it not only improves the dissolution of poorly soluble drugs but also exerts a significant effect on fat metabolism in the body. Gel formation can be obtained by interactions of chitosans with low molecular counterions such as polyphosphates, sulfates, and cross-linkers. This gelling property of chitosan allows a wide range of applications such as the coating of pharmaceuticals and food products, gel entrapment of biochemicals, plant embryos, whole cells, microorganisms, and algae. This manuscript is an insight into the exploitation of its properties for microencapsulating drugs. Herein, we report a novel method to chemically bind thiazolidine-2,4-dione through cross-linked chitosan nanoparticles using malanodialdehyde as a new cross-linker and well characterized by FT-IR, NMR, TGA, DSC, powder X-ray diffraction, TEM, and cyclic voltametry.

Vitamin A and vitamin E interaction behavior on chitosan microspheres

Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications due to its biocompatibility, high charge density, and non-toxicity. In this study, chitosan microspheres were successfully prepared by an adapted method of coagulation/dispersion. The degree of deacetylation of chitosan powder was obtained by NMR 1H and FTIR techniques. Chitosan powder and chitosan microspheres were characterized by BET surface area and scanning electron microscopy (SEM). The interactions among the chitosan microspheres and the vitamins A and E were characterized by FTIR. In order to evaluate the ability of interaction of vitamin A and vitamin E with the chitosan microspheres, the thermodynamic parameters were followed by calorimetric titration. Different experimental approaches were applied, such as adsorption isotherms, kinetics and thermodynamics studies. The obtained results showed that the interactions of chitosan microspheres with the vitamins were spontaneous, enthalpically and entropically favorable, indicating that the chitosan microspheres can be used with success in the controlled release of these vitamins.

One‐Step Preparation of Multifunctional Chitosan Microspheres by a Simple Sonochemical Method

Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications due to its biocompatibility, high charge density, nontoxicity, and mucoadhesion properties. Processing techniques for the preparation of chitosan microspheres have been extensively developed since the 1980s. The present paper describes for the first time a fast and one-step process for the preparation of stable chitosan microspheres by a simple sonochemical method. The microspheres were characterized by their particle size, surface morphology, stability, and drug-entrapment efficiency. The average size of the microspheres was found to be around 1 mum with a narrow size distribution, which enabled them to be used for in vivo applications. The encapsulation of different dyes into these microspheres was readily achieved with more than 75 % efficacy by dissolving them into the organic phase before sonication. The chitosan microspheres demonstrated excellent stability toward acidic and basic conditions ranging from pH 4 to 9, thereby indicating their implementation as possible therapeutic and diagnostic agents. The stability of these microspheres appears to be contributed from intermolecular imine cross-linking in addition to other noncovalent interactions. The ability of the surface-exposed amino groups of chitosan microspheres to undergo chemical conjugation with potential drugs and/or targeting vectors was determined by their reaction with fluorescein isothiocyanate (FITC) and fluorescamine followed by confocal microscopy.

Tumor targeting of humanized fragment antibody secreted from transgenic rice cell suspension culture

The tumor-associated glycoprotein 72 (TAG 72) has been shown to be expressed in the majority of human adenocarcinomas. In an effort to develop a technique for the safe and inexpensive production of large quantities of anti-TAG 72 humanized antibody fragments (hzAb) as a future source of clinical-grade proteins, we developed a transgenic rice cell suspension culture system. The in vivo assembly and secretion of hzAb were achieved in a transgenic rice cell culture under the control of the rice alpha amylase 3D (RAmy 3D) expression system, and the biological activities of plant-derived hzAb were determined to be quite similar to those of animal-derived antibody. Purified hzAb was shown to bind to the recombinant antigen, TAG 72, and to bind specifically to human LS 174T colon adenocarcinoma cells expressing the TAG 72 antigen, and this binding occurred to the same extent as was seen with animal-derived antibody. Plant-derived hzAb proved as effective as animal-derived antibody in targeting tumors of xenotransplanted LS 174T cells in nude mice. The results of this study indicate that the hzAb derived from plant cell suspension cultures may have great potential for pharmaceutical applications in the development of future cancer therapeutic and diagnostic protocols.