Mesobiliverdin IXα ameliorates osteoporosis via promoting osteogenic differentiation of mesenchymal stem cells.

Heme oxygenase-1 (HO-1) expression promotes osteogenesis, but the mechanisms remain unclear and therapeutic strategies using it to target bone disorders such as osteoporosis have not progressed. Mesobiliverdin IXα is a naturally occurring bilin analog of HO-1 catalytic product biliverdin IXα. Inclusion of mesobiliverdin IXα in the feed diet of ovariectomized osteoporotic mice was observed to increase femur bone volume, trabecular thickness and osteogenesis serum markers osteoprotegrin and osteocalcin and to decrease bone resorption serum markers cross-linked N-teleopeptide and tartrate-resistant acid phosphatase 5b. Moreover, in vitro exposure of human bone marrow mesenchymal stem cells to mesobiliverdin IXα enhanced osteogenic differentiation efficiency by two-fold over non-exposed controls. Our results imply that mesobiliverdin IXα promotes osteogenesis in ways that reflect the potential therapeutic effects of induced HO-1 expression in alleviating osteoporosis.

Polymeric Nanovectors Incorporated with Ganciclovir and HSV-tk Encoding Plasmid for Gene-Directed Enzyme Prodrug Therapy.

In the area of gene-directed enzyme prodrug therapy (GDEPT), using herpes simplex virus thymidine kinase (HSV-tk) paired with prodrug ganciclovir (GCV) for cancer treatment has been extensively studied. It is a process involved with two steps whereby the gene (HSV-tk) is first delivered to malignant cells. Afterward, non-toxic GCV is administered to that site and activated to cytotoxic ganciclovir triphosphate by HSV-tk enzyme expressed exogenously. In this study, we presented a one-step approach that both gene and prodrug were delivered at the same time by incorporating them with polymeric micellar nanovectors. GCV was employed as an initiator in the ring-opening polymerization of ε-caprolactone (ε-CL) to synthesize hydrophobic GCV-poly(caprolactone) (GCV-PCL), which was furthered grafted with hydrophilic chitosan to obtain amphiphilic polymer (GCV-PCL-chitosan) for the fabrication of self-assembled micellar nanoparticles. The synthesized amphiphilic polymer was characterized using Fourier transform infrared spectroscopy and proton nuclear magnetic resonance. Micellar prodrug nanoparticles were analyzed by dynamic light scattering, zeta potential, critical micelle concentration, and transmission electron microscopy. Polymeric prodrug micelles with optimal features incorporated with HSV-tk encoding plasmids were cultivated with HT29 colorectal cancer cells and anticancer effectiveness was determined. Our results showed that prodrug GCV and HSV-tk cDNA encoded plasmid incorporated in GCV-PCL-chitosan polymeric nanocarriers could be delivered in a one-step manner to HT-29 cells and triggered high cytotoxicity.

Ring-opening polymerization of ε-caprolactone initiated by ganciclovir (GCV) for the preparation of GCV-tagged polymeric micelles.

Ganciclovir (GCV) is a nucleoside analogue with antiviral activity against herpes viral infections, and the most widely used antiviral to treat cytomegalovirus infections. However, the low bioavailability and short half-life of GCV necessitate the development of a carrier for sustained delivery. In this study, guanosine-based GCV was used as the initiator directly in ring-opening polymerization of ε-caprolactone (ε-CL) to form hydrophobic GCV-poly(caprolactone) (GCV-PCL) which was then grafted with hydrophilic chitosan to form amphiphilic copolymers for the preparation of stable micellar nanoparticles. Successful synthesis of GCV-PCL and GCV-PCL-chitosan were verified by 1H-NMR analysis. Self-assembled micellar nanoparticles were characterized by dynamic light scattering and zetasizer with an average size of 117 nm and a positive charge of 24.2 mV. The drug release kinetics of GCV was investigated and cytotoxicity assay demonstrated that GCV-tagged polymeric micelles were non-toxic. Our results showed that GCV could be used directly in the initiation of ring-opening polymerization of ε-CL and non-toxic polymeric micelles for GCV delivery can be formed.

Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes.

Plant-derived extracellular vesicles (EVs), containing a myriad of bioactive proteins, microRNAs, lipids, and secondary metabolites, have recently become the focus of rising interest due to their important roles in various applications. The widely accepted method for isolating plant EVs is differential ultracentrifugation plus density gradient centrifugation. However, the combination of differential ultracentrifugation and density gradient centrifugation for the isolation of plant EVs is time-consuming and labor-intensive. Hence, there is a need for more efficient methods to perform the separation of plant EVs. In this study, EVs were separated from Arabidopsis thaliana leaves by a cost-effective polyethylene glycol (PEG)-based precipitation approach. The mean size of purified Arabidopsis thaliana EVs determined by dynamic light scattering was 266 nm, which is consistent with nanoparticle tracking analysis. The size was also confirmed via transmission electron microscopy with morphology of a cup-shaped appearance which is the typical mammalian exosome's morphology. Additionally, Western blotting of the purified Arabidopsis thaliana EVs, using commercially available mammalian exosomal kits, displayed surface marker tetraspanin proteins (CD9, CD63, and CD81), and endosomal sorting complexes required for transport (ESCRT)-associated proteins (TSG101 and ALIX). This demonstrates that the purified Arabidopsis thaliana EVs reveal the typical proteins reported in mammalian exosomes.

Inhibition of SARS-CoV-2 Spike Protein Pseudotyped Virus Infection Using ACE2-Tethered Micro/Nanoparticles.

Coronavirus disease 2019 (COVID-19) has caused a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral infection is reliant upon the binding between angiotensin-converting enzyme 2 receptor (ACE2) and spike protein (S). Therefore, ACE2 is a key receptor for SARS-CoV-2 to infect the host. Nonetheless, as SARS-CoV-2 is constantly mutating into new variants that cause high infection rates, the development of prophylactic and therapeutic approaches remains a necessity to continue fighting mutated SARS-CoV-2 variants. In this study, ACE2-streptavidin fusion proteins expressed by recombinant DNA technology were anchored on biotinylated fluorescent polystyrene particles of various sizes ranging from 0.15 to 5 µm. The ACE2-tethered micro/nanoparticles were shown to prevent spike protein pseudotyped lentivirus entry into ACE2-expressing HEK293T cells. Compared to ACE2 in soluble form, micro-sized particles (2 and 5 µm) immobilized with ACE2 interfered more efficiently with viral attachment, entry, and the ensuing infection. Our results showed that particles functionalized with ACE2 could be used as efficient decoys to block the infection of SARS-CoV-2 strains.

Eradication of Myrosinase-Tethered Cancer Cells by Allyl Isothiocyanate Derived from Enzymatic Hydrolysis of Sinigrin.

Sinigrin is present in significant amounts in cruciferous vegetables. Epidemiological studies suggest that the consumption of such vegetables decreases the risk of cancer, and the effect is attributed mainly to allyl isothiocyanate (AITC), a hydrolysis product of sinigrin catalyzed by myrosinase. Anticancer activity of AITC has been previously investigated for several cancer models, but less attention was paid to delivering AITC on the target site. In this study, the gene sequences of core streptavidin (coreSA) and myrosinase (MYR) were cloned in a pET-30a(+) plasmid and transformed into BL21(DE3) E. coli competent cells. The MYR-coreSA chimeric protein was expressed and purified using immobilized metal affinity chromatography and further characterized by gel electrophoresis, Western blot, and enzyme activity assay. The purified MYR-coreSA chimeric protein was tethered on the outer membrane of biotinylated adenocarcinoma A549 cells and then treated with various concentrations of sinigrin. Our results showed that 20 µM of sinigrin inhibited the growth of A549 cells tethered with myrosinase by ~60% in 48 h. Furthermore, the levels of treated cells undertaken apoptosis were determined by Caspase-3/7 activation and Annexin-V. In summary, sinigrin harnessed like a prodrug catalyzed by myrosinase to the production of AITC, which induced cell apoptosis and arrested the growth of lung cancer cells.

Inactivation of SARS-CoV-2 Spike Protein Pseudotyped Virus Infection Using ACE2-Tethered Gold Nanorods under Near-Infrared Laser Irradiation.

Since the angiotensin-converting enzyme 2 (ACE2) protein is abundant on the surface of respiratory cells in the lungs, it has been confirmed to be the entry-point receptor for the spike glycoprotein of SARS-CoV-2. As such, gold nanorods (AuNRs) functionalized with ACE2 ectodomain (ACE2ED) act not only as decoys for these viruses to keep them from binding with the ACE2-expressing cells but also as agents to ablate infectious virions through heat generated from AuNRs under near-infrared (NIR) laser irradiation. Using plasmid containing the SARS-CoV-2 spike protein gene (with a D614G mutation), spike protein pseudotyped viral particles with a lentiviral core and green fluorescent protein reporter were constructed and used for transfecting ACE2-expressing HEK293T cells. Since these viral particles behave like their coronavirus counterparts, they are the ideal surrogates of native virions for studying viral entry into host cells. Our results showed that, once the surrogate pseudoviruses with spike protein encounter ACE2ED-tethered AuNRs, these virions are entrapped, resulting in decreased viral infection to ACE2-expressing HEK293T cells. Moreover, the effect of photothermolysis created by ACE2ED-tagged AuNRs under 808-nm NIR laser irradiation for 5 min led to viral breakdown. In summary, ACE2ED-tethered AuNRs with dual functions (virus decoy and destruction) could have an intriguing advantage in the treatment of diseases involving rapidly mutating viral species such as SARS-CoV-2.

Anticancer Activity, Mechanism, and Delivery of Allyl Isothiocyanate.

Allyl isothiocyanate (AITC) is a phytochemical that is abundantly present in cruciferous vegetables of the Brassicaceae family, such as cabbage, broccoli, mustard, wasabi, and cauliflower. The pungent taste of these vegetables is mainly due to the content of AITC present in these vegetables. AITC is stored stably in the plant as its precursor sinigrin (a type of glucosinolate), which is physically separated from myrosin cells containing myrosinase. Upon tissue disruption, myrosinase gets released and hydrolyzes the sinigrin to produce AITC and by-products. AITC is an organosulfur compound, both an irritant and toxic, but it carries pharmacological properties, including anticancer, antibacterial, antifungal, and anti-inflammatory activities. Despite the promising anticancer effectiveness of AITC, its clinical application still possesses challenges due to several factors, i.e., low aqueous solubility, instability, and low bioavailability. In this review, the anticancer activity of AITC against several cancer models is summarized from the literature. Although the mechanism of action is still not fully understood, several pathways have been identified; these are discussed in this review. Not much attention has been given to the delivery of AITC, which hinders its clinical application. However, the few studies that have demonstrated the use of nanotechnology to facilitate the delivery of AITC are addressed.

Photothermolysis of glioblastoma stem-like cells targeted by carbon nanotubes conjugated with CD133 monoclonal antibody.

CD133(+) cells in glioblastoma (GBM) display cancer stem cell-like properties and have been considered as the culprit of tumor recurrence, justifying exploration of potential therapeutic modalities targeting CD133(+) cancer stem-like cells (CSCs). For photothermolysis studies, GBM-CD133(+) and GBM-CD133(-) cells mixed with various ratios were challenged with single-walled carbon nanotubes (SWNTs) conjugated with CD133 monoclonal antibody (anti-CD133) and then irradiated with near-infrared laser light. Results show that GBM-CD133(+) cells were selectively targeted and eradicated, whereas GBM-CD133(-) cells remained viable. In addition, in vitro tumorigenic and self-renewal capability of GBM-CD133(+) treated with localized hyperthermia was significantly blocked. Furthermore, GBM-CD133(+) cells pretreated with anti-CD133-SWNTs and irradiated by near-infrared laser 2 days after xenotransplantation in nude mice did not exhibit sustainability of CSC features for tumor growth. Taken altogether, our studies demonstrated that anti-CD133-SWNTs have the potential to be utilized as a thermal-coupling agent to effectively target and destroy GBM CSCs in vitro and in vivo. FROM THE CLINICAL EDITOR: Glioblastoma remains one of the most notorious cancer from the standpoint of recurrence and overall resistance to therapy. CD133+ stem cells occur among GBM cells, and may be responsible for the huge recurrence risk. This paper discusses a targeted elimination method of these cells, which may enable more efficient therapy in an effort to minimize or prevent recurrence.

Blockade of macrophage adhesion to CD200-treated polystyrene culture surface.

CD200 is an anti-inflammatory transmembrane glycoprotein in the immunoglobulin superfamily. The interaction of CD200 and its receptor CD200R has shown to inhibit inflammatory response of myeloid cells to foreign materials. The purpose of this study is to create a CD200 immobilized biomaterial surface through polydopamine coating to suppress macrophage cell adhesion and reduce inflammatory cytokine secretion accordingly by macrophages. In this study, tissue-culture treated polystyrene (TCPS) surface was modified with biotin through polydopamine coating. Purified CD200-streptavidin fusion protein was then immobilized onto the biotinylated TCPS surface through the high affinity between biotin and streptavidin. Mouse J774A.1 macrophages were seeded on CD200-immobilized TCPS surface to evaluate the effect of CD200 on preventing macrophage attachment. The effects of CD200-immobilized TCPS surface on pro-inflammatory cytokine secretion from J774A.1 macrophages were measured by enzyme-linked immunosorbent assay. As a result, CD200-immobilized TCPS surface suppressed macrophage attachment for up to 9 hr. The level of IL-6 and TNF-α secreted from J774A.1 macrophages interacted with CD200-coated TCPS surface was reduced by 36.3% and 32.4%, respectively.

Less phagocytosis of viral vectors by tethering with CD47 ectodomain.

Many viral vectors, which are effective when administrated in situ, lack efficacy when delivered intravenously. The key reason for this is the rapid clearance of the viruses from the blood circulation via the immune system before they reach target sites. Therefore, avoiding their clearance by the immune system is essential. In this study, lentiviral vectors were tethered with the ectodomain of self-marker protein CD47 to suppress phagocytosis via interacting with SIRPα on the outer membrane of macrophage cells. CD47 ectodomain and core-streptavidin fusion gene (CD47ED-coreSA) was constructed into pET-30a(+) plasmid and transformed into Lemo21 (DE3) competent E. coli cells. The expressed CD47ED-coreSA chimeric protein was purified by cobalt-nitrilotriacetate affinity column and characterized by SDS-PAGE and western blot. The purified chimeric protein was anchored on biotinylated lentivirus via biotin-streptavidin binding. The CD47ED-capped lentiviruses encoding GFP were used to infect J774A.1 macrophage cells to assess the impact on phagocytosis. Our results showed that the overexpressed CD47ED-coreSA chimeric protein was purified and bound on the surface of biotinylated lentivirus which was confirmed via immunoblotting assay. The process to produce biotinylated lentivirus did not affect native viral infectivity. It was shown that the level of GFP expression in J774A.1 macrophages transduced with CD47ED-lentiviruses was threefold lower in comparison to control lentiviruses, indicating an antiphagocytic effect triggered by the interaction of CD47ED and SIRPα. Through the test of blocking antibodies against CD47ED and/or SIRPα, it was confirmed that the phagocytosis inhibition was mediated through the CD47ED-SIRPα axis signaling. In conclusion, surface immobilization of CD47ED on lentiviral vectors inhibits their phagocytosis by macrophages. The chimeric protein of CD47 ectodomain and core-streptavidin is effective in mediating the surface binding and endowing the lentiviral nanoparticles with the antiphagocytic property.

Mesenchymal stem cells anchored with thymidine phosphorylase for doxifluridine-mediated cancer therapy.

Many tumors express thymidine phosphorylase (TYMP) with various levels, however due to tumor heterogeneity, the amount of TYMP is usually not enough to convert prodrug doxifluridine (5'-DFUR) to toxic drug 5-fluorouracil (5-FU). Since human mesenchymal stem cells (hMSCs) have unique features of tumor-tropism and low immunogenicity, the purpose of this study is to use mesenchymal stem cells as carriers to deliver TYMP to cancer cells and then trigger their death by administrating doxifluridine. First, the TYMP gene sequence and core streptavidin (core SA) were constructed into pET-30a(+) plasmid. After bacterial transformation and colony screening, TYMP-SA fusion protein was expressed by IPTG induction and purified by immobilized metal affinity chromatography and characterized by SDS-PAGE and western blot with a clear band at 75 kDa. The characterized TYMP-SA was further anchored on the cell membrane of biotinylated hMSCs via biotin-streptavidin binding. hMSCs anchored with TYMP-SA were then co-cultured with adenocarcinoma A549 cells (with different ratios) and treated with 100 µM prodrug doxifluridine over the course of four days. Our results showed that a 2 : 1 ratio led to the eradication of A549 cells at the end of the experiment with less than 5% confluency, in comparison with the 1 : 1 and 1 : 2 ratios which still had about 13% and 20% confluency respectively. In conclusion, harnessing hMSCs as cell carriers for the delivery of TYMP enzyme to cancer cells could lead to significant cell death post-treatment of the prodrug doxifluridine.

Diminution of Phagocytosed Micro/Nanoparticles by Tethering with Immunoregulatory CD200 Protein.

CD200 is known as an anti-inflammatory transmembrane glycoprotein in the immunoglobulin superfamily. CD200 interacts with its receptor CD200R which is highly expressed on myeloid cells such as macrophages and neutrophils. CD200-CD200R interaction has known to reduce macrophage activation and chronic inflammation. To harness the immunomodulatory property of CD200 for surface modification, CD200-streptavidin fusion protein was expressed from bacteria transformed with pET20b plasmid encoded with CD200 extracellular domain and core streptavidin. The purified CD200-SA protein was bound to biotin-coated fluorescent polystyrene particles of various sizes ranging from 0.15 to 2 µm. THP-1 macrophages were cultivated with CD200-modified micro/nanoparticles in comparison with controls. Our results showed that both nano- and micro-sized particles decorated with CD200 decreased phagocytosis activities of THP-1 macrophages. Such diminution of phagocytosis was examined to be associated with downregulation of Toll-like receptor 4 (TLR4) expression on the surface of macrophages. Moreover, THP-1 macrophages treated with CD200-coated particles decreased the secretion of tumor necrosis factor-α (TNF-α).

Efficient Expression of Soluble Recombinant Protein Fused with Core-Streptavidin in Bacterial Strain with T7 Expression System.

The limited amount of fusion protein transported into cytosol milieu has made it challenging to obtain a sufficient amount for further applications. To avoid the laborious and expensive task, T7 promoter-driving pET-30a(+) coding for chimeric gene of thymidine phosphorylase and core streptavidin as a model system was constructed and transformed into a variety of E. coli strains with T7 expression system. Our results demonstrated that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system is able to provide efficient expression of soluble TP-coreSA fusion protein for purification. Moreover, the eluted TP-coreSA fusion protein tethered on biotinylated A549 carcinoma cells could effectively eliminate these malignant cells after administrating prodrug 5'-DFUR.

Effect of hypotonic stress on retroviral transduction.

Short half-life has long been known as a main barrier for retroviral gene delivery due to quick degradation that seriously limited application of retrovirus-mediated methodology in the clinical use. To circumvent this challenge, many physical and chemical approaches have been developed to maximize contact opportunity of retroviruses and cells before viral vectors decay. However, most of methods are not easy to be followed due to complicated equipment settings and/or long procedures. In this study, we introduced an easy, cost-effective, efficient, and scalable strategy to enhance retroviral transduction by hypo-osmotic stress. It has been demonstrated that under hypotonic exposure, cell membrane is permeabilized to allow numerous exterior molecules accessing to cytoplasm through an intensive endocytosis, yielding high efficiency of cellular uptake. We hypothesized this hypotonic stress-induced internalization may provide a unique opportunity of cell entry for retroviruses without the need of receptor binding, and thus overcome the insufficient transduction rate due to loss of envelope protein. Indeed, our results showed that with assistance of hypotonic stress, retroviral transduction rates dramatically increased about 5.6- and 17.7-fold using fresh and decayed retroviruses, respectively, in comparison with corresponding groups without hypotonic stress. In summary, hypotonic stress was shown as a promising tool for enhancement of retroviral transduction efficiency without limitation of short half-life.

In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody.

Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm(-2) for 10 min which was then maintained at 6 W cm(-2) for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

Migration of mesenchymal stem cells tethered with carbon nanotubes under a chemotactic gradient.

Carbon nanotubes (CNTs) have been extensively studied for photothermal ablation of malignant cells due to their ability to absorb near-infrared (NIR) laser light and convert it to thermal energy for the lysis of tumor cells. Functionalizing CNTs with tumor-targeting moieties can facilitate the delivery to tumor sites. Instead of using targeting moieties, mesenchymal stem cells (MSCs) have been considered as vehicles to deliver therapeutic agents to cancer cells. In this study, the effects of attaching CNTs to MSCs on cell migration in response to a chemotactic gradient were investigated. Multiwalled carbon nanotubes (MWCNTs) were functionalized with streptavidin-fluorescein isothiocyanate (SA-FITC). The surface of human MSCs was biotinylated by culturing MSCs with biotin-lipid containing medium. CNTs were then attached on the outer cell membrane of biotinylated MSCs through SA-biotin binding. Fluorescence microscopy confirmed CNTs were located on the surface of MSCs. Various amounts of CNTs anchored on the membrane of MSCs were used to examine the effects of CNTs on MSC proliferation and migration. Our transwell migration assay showed that 4.26 ng CNT per cell is the threshold value that would not affect the migration speed of CNT-tagged MSCs toward the established gradient of chemoattractant SDF-1α.

Enhanced proliferation and differentiation of mesenchymal stem cells by astaxanthin-encapsulated polymeric micelles.

Astaxanthin is a highly potent antioxidant which can be extracted from Haematococcus pluvialis when cultivated and induced at high stress conditions. Due to astaxanthin's hydrophobicity, methoxypolyethylene glycol-polycaprolactone (mPEG-PCL) copolymer was synthesized to form polymeric micelles for the encapsulation of astaxanthin. Astaxanthin-loaded polymeric micelles were then used to examine the effects on the proliferation and differentiation of human mesenchymal stem cells (MSCs). Dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR) confirmed astaxanthin was encapsulated into mPEG-PCL micelles. Astaxanthin loading and encapsulation efficiency, determined by UV/Vis spectroscopy, were 3.27% and 96.67%, respectively. After 48 h, a total of 87.31% of astaxanthin was released from the polymeric micelles. The drug release profile was better fit by the Michaelis-Menten type model than the power law model. The MSC culture results showed that culture medium supplemented with 0.5 µg/mL astaxanthin-encapsulated polymeric micelles led to a 26.3% increase in MSC proliferation over an 8-day culture period. MSC differentiation results showed that 20 ng/mL astaxanthin-encapsulated polymeric micelles enhanced adipogenesis, chondrogenesis, and osteogenesis of MSCs by 52%, 106%, and 182%, respectively.

Extraction of cobalt(ii) by methyltrioctylammonium chloride in nickel(ii)-containing chloride solution from spent lithium ion batteries.

Spent lithium batteries contain valuable metals such as cobalt, copper, nickel, lithium, etc. After pretreatment and recovery of copper, only cobalt, nickel and lithium were left in the acid solution. Since the chemical properties of cobalt and nickel are similar, separation of cobalt from a solution containing nickel is technically challenging. In this study, Co(ii) was separated from Ni(ii) by chelating Co(ii) with chlorine ions, Co(ii) was then extracted from the aforementioned chelating complexes by methyltrioctylammonium chloride (MTOAC). The effects of concentrations of chlorine ions in the aqueous phase ([Cl-]aq), MTOAC concentrations in organic phase ([MTOAC]org), ratios of organic phase to aqueous phase (O/A), and the initial aqueous pH on cobalt separation were studied. The results showed that [Cl-]aq had a significant impact on cobalt extraction efficiency with cobalt extraction efficiency increasing rapidly with the increase in [Cl-]aq. The effect of initial pH on cobalt extraction efficiency was not significant when it varied from 1 to 6. Under the condition of [Cl-]aq = 5.5 M, [MTOAC]org = 1.3 M, O/A = 1.5, and pH = 1.0, cobalt extraction efficiency reached the maximum of 98.23%, and nickel loss rate was only 0.86%. The stripping rate of cobalt from Co(ii)-MTOAC complexes using diluted hydrochloric acid was 99.95%. By XRD and XRF analysis, the recovered cobalt was in the form of cobalt chloride with the purity of cobalt produced reaching 97.7%. The mode of cobalt extraction was verified to be limited by chemical reaction and the kinetic equation for cobalt extraction was determined to be: R (Co) = 4.7 × 10-3[MTOAC](org) 1.85[Co](aq) 1.25.

Enhanced astaxanthin accumulation in Haematococcus pluvialis using high carbon dioxide concentration and light illumination.

In this study, an economical two-stage method was proposed for the production of natural astaxanthin from Haematococcus pluvialis without a medium replacement step. In stage 1, H. pluvialis were grown under low light illumination until they reached optimal biomass. In stage 2, cells were switched to astaxanthin induction conditions utilizing the combination of high light illumination and elevated carbon dioxide levels (5 or 15%). The introduction of CO2 altered the C/N balance creating a nutrient deficiency without a change of media. The resulting astaxanthin yield was 2-3 times that of using either stressor alone. This astaxanthin induction method has many advantages over current methods including no medium replacement and a short induction time of less than four days.