Rapamycin-Loaded, CapryolTM 90 and Oleic Acid Mediated Nanoemulsions: Formulation Development, Characterization and Toxicity Assessment.

This study was planned to explore the capability of nanoemulsions (NEs) consisting of CapryolTM 90 and oleic acid for the delivery of rapamycin (RAP). Permeability and cytotoxicity of RAP-loaded NEs were also inspected. Pseudo-ternary phase diagrams were created with oleic acid and CapryolTM 90 (as oil phase) and four surfactants and co-surfactants at various weight ratios (Rsm). Selected NEs from O/W region on the phase diagrams with the drug concentration of 1 mg/mL, were prepared via the spontaneous emulsification technique, characterized for particle size and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release was determined for a period of 48 h using a dialysis sac. The assay of RAP was determined using HPLC technique. Cytotoxicity of NEs was evaluated by MTT assay on breast cancer cell line, namely SKBR-3. The permeability of RAP-loaded NEs across Caco-2 monolayers was assessed by measurement of TEER (transepithelial electrical resistance) value. The intracellular uptake of coumarin 6-loaded NEs by SKBR-3 cells was also investigated using florescence microscopy. NEs containing oleic acid/Tween 20/propylene glycol, CapryolTM 90/Tween 20/iso-propanol, and CapryolTM 90/Cremophor® RH40/Transcutol® P showed more cytotoxicity and permeability compared with the RAP methanolic solution. The minimum toxic concentration of RAP in NE formulations was found to be 7.5 µg/mL. The highest intracellular uptake was observed for the NE composed of CapryolTM 90/Tween 20/iso-propanol which was in consistent with the results obtained from cytotoxicity and permeability tests. The overall results implicated that this novel carrier was effective for enhancing RAP permeation in Caco-2 cell membrane along with enhancement of cytotoxicity.

Challenges to Design and Develop of DNA Aptamers for Protein Targets. II. Development of the Aptameric Affinity Ligands Specific to Human Plasma Coagulation Factor VIII Using SEC-SELEX.

Protein specific aptamers are highly applicable affinity ligands in different fields of research and clinical applications. They have been developed against various targets, in particular, bio-macromolecules such as proteins. Among human proteins, the coagulation factors are the most attractive targets for aptamer selection and their specific aptamers have valuable characteristics in therapeutic and analytical applications. In this study, a plasma derived coagulation factor VIII was considered as the protein target for DNA aptamer selection using size exclusion chromatography-SELEX. Potential aptameric oligonucleotides with high affinity and specificity were achieved during eight rounds of selection. Binding affinity constant of selected aptamer and aptameric enriched pool were in nanomolar range that was comparable to monoclonal antibodies. Further improvement studies can result in aptamers that are more promising as an industrial affinity ligand for the purification of anti-hemophilia factor from plasma source.

Can Aptameric Ligands Specific to Plasma Coagulation Factor VII Bind the Recombinant Form with High Affinity: Affinity Measurement by Fluorescence Method.

BACKGROUND: Among diverse protein purification systems, affinity chromatography is the most attractive one in the purification process of coagulation factors. Coagulation factor VII is a plasma serine protease that has a significant role in natural human hemostasis and its recombinant form such as AryoSeven™, has been applied in clinical treatment of bleeding disorders. Immunoaffinity chromatography is the purification method of choice that is currently applied in the development of coagulation factor VIIa products. Aptamers as nucleic acid based affinity ligands are more promising than monoclonal antibodies. In addition, DNA aptamers are more acceptable than RNA ones in this regard. METHODS: In this study, two of the aptameric DNA oligonucleotides that showed acceptable affinities for purification of coagulation factor VIIa from plasma, were selected to evaluate their affinity against Aryoseven. A serial dilution of fluorescence labeled aptamers was incubated against the concentration of 1 nM from Aryoseven. Then, a fluorescence index was calculated according to the fluorescence intensity data measured from test and control samples. The dissociation constant of aptamers was calculated according to the fluorescence index using Prism5 software. RESULTS: Results showed that the binding affinity of the 44 nucleotide aptamer was more than 81 nucleotide aptamer sequence. As a result, this aptamer could be optimized in order to develop aptamer based affinity chromatography process for this form of recombinant coagulation factor VIIa. DISCUSSION: Aptamers with shorter length of sequence could show higher affinity in target binding, as they could adapt more easily to suitable conformation according to target interaction. However, it should be considered that the selectivity of affinity ligands is also important for target purification and analytical applications.

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

BACKGROUND: In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. METHODS: We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. RESULTS: Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). CONCLUSIONS: Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

Ectoine and hydroxyectoine inhibit thermal-induced aggregation and increase thermostability of recombinant human interferon Alfa2b.

This study is to investigate whether ectoines (ectoine and hydroxyectoine) can reduce aggregation of rhIFNα2b in aqueous solutions on thermal stress. The effect of thermal stress condition on the stability was therefore investigated using size exclusion-high performance liquid chromatography (SE-HPLC), different spectroscopic measurements, dynamic light scattering (DLS), electrophoresis, and differential scanning calorimetry (DSC). All experiments were performed in a sodium phosphate buffer system (100mM, pH7). The protein samples (100µg/ml) were incubated at 50°C for 14days in the absence or presence (1, 10, 20, and 100mM) of ectoines. In summary, thermal-induced aggregation was reduced in the presence of ectoines, regardless of the ectoines concentration in different periods of incubation time by analyzing with SE-HPLC and turbidity measurement. The inhibitory effect of ectoines on the aggregation was shown by other techniques used. The optimal ectoines concentration was 10mM for aggregation reduction, so samples containing of 10mM of ectoines were selected for further evaluation. Secondary structural and conformational stability increased in presence of ectoines as measured by far-UV circular dichroism and fluorescence spectroscopy, respectively. DSC showed slight increase in Tm of interferon in the presence of ectoines. Hydroxyectoine had superior protein-stabilizing properties than ectoine. In conclusion, this study demonstrates that ectoine and hydroxyectoine are highly effective excipients which can significantly reduce the thermal-induced aggregation of rhIFNα2b at low concentration.

L-Asparaginase Activity in Cell Lysates and Culture Media of Halophilic Bacterial Isolates.

The objective of this study was to isolate halophilic bacteria with the ability to produce intracellular or extracellular L-asparaginase. A total number of 120 halophilic bacteria were isolated from 17 different saline habitats of Iran including salt lakes, wetlands, brine springs and deserts. Among these, 68 were able to grow in the presence of 1.5 M NaCl and 52 demonstrated the ability to grow in the selection medium containing 3.5 M NaCl. None of the isolates appeared to produce appreciable amounts of extracellular L-asparaginase. Among the isolates that produced intracellular L-asparaginase, 5 moderate and 1 extreme halophiles were selected for further study based on their observed activity level. The moderately halophilic isolates were shown to belong to the genus Halomonas while the extreme halophile was identified as a member of the genus Aidingimonas.

Laccase Activity in CTAB-Based Water-in-Oil Microemulsions.

The aim of this study was to develop a microemulsion system as a medium for laccase-catalyzed reactions. Phase behavior studies were conducted by constructing partial pseudo-ternary phase diagrams for systems comprising of cetyltrimethylammonium bromide (CTAB), various organic solvents as the oil phase (i.e., hexane, cyclohexane, heptane, octane, isooctane, toluene, isopropyl myristate), two co-surfactants (i.e., 1-butanol and 1-hexanol) and citrate buffer solution, at various surfactant/co-surfactant weight ratios (Rsm). A monophasic, transparent, non-birefringent area (designated as microemulsion domain) was seen to occur in some phase diagrams along the surfactant/organic solvent axis, the extent of which was dependent mainly upon the nature of co-surfactant and Rsm. On each phase diagram, three different water-in-oil (w/o) microemulsion systems with less than 50 wt% surfactant mixture and less than 20 wt% of aqueous phase were selected for laccase loading and activity measurements. Results revealed that the catalytic activity of laccase in CTAB-based w/o microemulsions decreased considerably, compared with its activity in the buffer solution, the extent of which depended upon the type of component and their compositions in the microemulsions. It was suggested that the conformational changes due to the electrostatic interactions between the cationic head group of CTAB and the negative enzyme might be the reason for the reduction of laccase activity, once entrapped in the microemulsion.

Formulation Development and Toxicity Assessment of Triacetin Mediated Nanoemulsions as Novel Delivery Systems for Rapamycin.

The aim of this investigation was to design and develop nanoemulsions (NEs) as novel delivery systems for rapamycin. Phase behavior of quaternary systems composed of Traicetin (as oil), various surfactants and co-surfactants and water at different surfactant/co-surfactant weight ratios was investigated by the construction of phase diagrams. Formulations were taken from the o/w NE region of the phase diagrams, depending upon the extent of NE domain. The spontaneous emulsification method was used to prepare various formulations containing 1 mg/mL of the drug. The NEs were characterized and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release from the selected formulations was determined for a period of 48 h using a dialysis sac. The assay of rapamycin was carried out using an HPLC technique. The effect of NEs on the viability of SKBR-3 cells was evaluated by MTT assay. The integrity of Caco-2 cell monolayers was measured by Transepithelial Electrical Resistance (TEER) and the transport of rapamycin-loaded NEs across Caco-2 cell monolayers was then assessed. The uptake of NEs by SKBR-3 cells was also investigated using florescence microscopy. Maximum drug release was observed in case of 4 formulations prepared with Tween 80 and Tween 20. MTT test results revealed different toxicity of NEs for SKBR-3 cell line and TEER demonstrated that formulations containing Tween 20 caused a more considerable decrease in cell integrity in comparison with those prepared with Tween 80. The results obtained from cellular uptake experiments were in consistent with those obtained from TEER and cytotoxicity experiments.

Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 (DE3) and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.

Study on the effect of solution conditions on heat induced-aggregation of human alpha interferon.

A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b (rhIFNα2b) has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFNα2b. The protein was incubated for 1 h at 40-70 °C and for up to 240 h at 50 °C and its aggregation tendency was then studied using optical density (at 350 nm), SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH (5, 6 and 7) and buffer concentrations (10, 55 and 100 mM) on the aggregation of protein following incubation at 50 °C for 72 h was also evaluated. The results obtained for samples incubated at 50 °C for up to 240 h showed that OD350 and the amount of higher molecular weight aggregates (HMW) increased and the monomer content decreased significantly (p<0.05) as the incubation time increased. Following incubation at various temperatures, a significant increase in OD350, drop in monomer content and increase in the amount of HMW aggregates were observed (p<0.05). Data obtained from incubation of samples at 50 °C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 (p<0.05). At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFNα2b.

Challenges to design and develop of DNA aptamers for protein targets. I. Optimization of asymmetric PCR for generation of a single stranded DNA library.

Aptamers, or single stranded oligonucleotides, are produced by systematic evolution of ligands by exponential enrichment, abbreviated as SELEX. In the amplification and regeneration step of SELEX technique, dsDNA is conversed to ssDNA. Asymmetric PCR is one of the methods used for the generation of ssDNA. The purpose of this study was to design a random DNA library for selection of aptamers with high affinity for protein targets and develop an efficient asymmetric PCR amplification. Thus, the influence of factors including annealing temperature, number of amplification cycles, primer ratio, Mg(2+) concentration and the presence of a PCR enhancer on the amplification of the desired product were evaluated. Results obtained by agarose gel electrophoresis showed that the annealing temperature of 64 °C, Mg(2+) concentration of 0.25 mM, reverse to forward primer ratio of 15:1, amplification cycle of 25 and the presence L-ectoin as a PCR enhancer with the concentration of 0.4 M were the optimal conditions. Our results supported that the yield of this type of ssDNA production is sufficient for combinatorial screening of aptamers.

Challenges to Improve the Stability and Efficacy of an Intravesical BCG Product.

The aim of this investigation was to improve the storage stability and survival rate of an intravesical BCG product, manufactured with an attenuated strain of Mycobacterium bovis (Pasteur strain 1173P2 of BCG) in the presence of sodium glutamate. Formulations with various concentrations of trehalose (a known protectant) were developed as liquid and lyophilized forms. Formulations were evaluated by different methods, including optical density measurement, safety assessment, skin reaction test, moisture content determination, viability assay, bacterial and fungal contaminations and the results were compared with those obtained for sodium glutamate-containing formulations. The stability tests were also carried out in various storage durations and different temperatures. To develop the lyophilization protocol, glass transition temperatures in the presence of both stabilizers were determined using differential scanning calorimetry. In general, results showed that trehalose could considerably increase the stability of the product against freezing and drying processes, increase the survival rate even in the liquid formulations, as well as the production of an acceptable cake. However, further studies are required to optimize the product characteristics.

A reversed phase high performance liquid chromatographic method for determination of rapamycin.

Easily degradating and various isomeric forms of rapamycin (Sirolimus) face the determination of this compound to many challenges. In this study, we developed and validated the isocratic reversed phase high performance liquid chromatographic (RP-HPLC) method for rapamycin. Separation was performed on a C8 column (MZ, 15 × 4.6 mm, 5 µm particle size) using methanol:water (80:20 v/v) as the mobile phase with the flow rate of 1 mL/min. The column temperature was set at 57°C and the detection was carried out at the wavelength of 277 nm. The method was linear over a concentration range of 0.025-2 µg/mL. The coefficient of variation of intra- and inter-day, assessed at three concentration levels of 0.075, 0.3 and 0.900 µg/mL, was less than 2%. Limit of quantification (LOQ) was found 25 ng/mL. The method with high percent recovery and short retention time of rapamycin, was found to be simple, rapid and reproducible.

Halotolerant Ability and α-Amylase Activity of Some Saltwater Fungal Isolates.

Four halotolerant fungal isolates originating from the saltwater Lake Urmia in Iran were selected during a screening program for salt resistance and α-amylase activity. The isolates were identified based on sequencing the ITS region and a part of the ß-tubulin gene, as Penicillium chrysogenum (isolate U1; CBS 132820), Fusarium incarnatum (isolate U2; CBS 132821), and Penicillium polonicum (isolate U3; CBS 132822, and isolate U4; CBS 132823). The growth of these isolates was determined by measuring the colony diameter and mycelia dry weight in Sabouraud dextrose agar and yeast nitrogen base medium supplemented with NaCl, KCl, and LiCl. Isolate U4 showed a growth up in 15% NaCl and U1 was the only isolate that could grow in 20% KCl. None of the strains grew in a media containing LiCl. The salt supplemented medium did not increase the size of colony diameter in all isolates (p > 0.05). The ability of the selected isolates for amylase production was quantitatively tested and showed that P. polonicum isolate U4 was the most potent producer of amylase with a yield of 260.9 U/L after 60 h, whereas P. polonicum isolate U3 was the lowest one with a production level of 97.9 U/L after 48 h. P. polonicum isolate U4 could be a suitable candidate for production of amylase on an industrial scale after optimization.

PEGylated single-walled carbon nanotubes as nanocarriers for cyclosporin A delivery.

Single-walled carbon nanotubes (SWCNTs) have attracted the attention of many researchers due to their remarkable physicochemical features and have been found to be a new family of nanovectors for the delivery of therapeutic molecules. The ability of these nanostructures to load large amounts of drug molecules on their outer surface has been considered as the main advantage by many investigators. Here, we report the development of a PEGylated SWCNT-mediated delivery system for cyclosporin A (CsA) as a potent immunosuppressive agent. The available OH group in the CsA structure was first linked to a bi-functional linker (i.e., succinic anhydride) in order to provide a COOH terminal group. CsA succinylation process was optimized by using the modified simplex method. The resulting compound, CsA-CO-(CH(2))(2)-COOH, was then grafted onto the exterior surface of SWCNTs, previously PEGylated with phospholipid-PEG(5000)-NH(2) conjugates, through the formation of an amide bond with the free amine group of PEGylated SWCNTs. Drug loading, stability of the PEGylated SWCNT-CsA complex, and in vitro release of the drug were evaluated. Loading efficiencies of almost 72% and 68% were achieved by UV spectrophotometry and elemental analysis methods, respectively. It was observed that 57.3% of cyclosporine was released from CsA-Pl-PEG(5000)-SWCNTs after 3 days. In this investigation, we conjugated CsA to an amine-terminated phospholipid-polyethylene glycol chain attached on SWCNTs via a cleavable ester bond and demonstrated the possible potential of PEGylated SWCNT-based systems for CsA delivery.

Immobilization of laccase in alginate-gelatin mixed gel and decolorization of synthetic dyes.

Alginate-gelatin mixed gel was applied to immobilized laccase for decolorization of some synthetic dyes including crystal violet. The immobilization procedure was accomplished by adding alginate to a gelatin solution containing the enzyme and the subsequent dropwise addition of the mixture into a stirred CaCl(2) solution. The obtained data showed that both immobilized and free enzymes acted optimally at 50°C for removal of crystal violet, but the entrapped enzyme showed higher thermal stability compared to the free enzyme. The immobilized enzyme represented optimum decolorization at pH 8. Reusability of the entrapped laccase was also studied and the results showed that ca. 85% activity was retained after five successive cycles. The best removal condition was applied for decolorization of seven other synthetic dyes. Results showed that the maximum and minimum dye removal was related to amido black 10B and eosin, respectively.

Optimization of single-walled carbon nanotube solubility by noncovalent PEGylation using experimental design methods.

In this study, noncovalent functionalization of single-walled carbon nanotubes (SWCNTs) with phospholipid-polyethylene glycols (Pl-PEGs) was performed to improve the solubility of SWCNTs in aqueous solution. Two kinds of PEG derivatives, ie, Pl-PEG 2000 and Pl-PEG 5000, were used for the PEGylation process. An experimental design technique (D-optimal design and second-order polynomial equations) was applied to investigate the effect of variables on PEGylation and the solubility of SWCNTs. The type of PEG derivative was selected as a qualitative parameter, and the PEG/SWCNT weight ratio and sonication time were applied as quantitative variables for the experimental design. Optimization was performed for two responses, aqueous solubility and loading efficiency. The grafting of PEG to the carbon nanostructure was determined by thermogravimetric analysis, Raman spectroscopy, and scanning electron microscopy. Aqueous solubility and loading efficiency were determined by ultraviolet-visible spectrophotometry and measurement of free amine groups, respectively. Results showed that Pl-PEGs were grafted onto SWCNTs. Aqueous solubility of 0.84 mg/mL and loading efficiency of nearly 98% were achieved for the prepared Pl-PEG 5000-SWCNT conjugates. Evaluation of functionalized SWCNTs showed that our noncovalent functionalization protocol could considerably increase aqueous solubility, which is an essential criterion in the design of a carbon nanotube-based drug delivery system and its biodistribution.

An Approach to the Design of a Particulate System for Oral Protein Delivery .II. Preparation and Stability Study of rhGH-Loaded Microspheres in Simulated Gastrointestinal Fluids.

The delivery of therapeutic proteins has gained momentum with development of biotechnology. However, large molecular weight, hydrophilic nature and susceptibility to harsh environment of gastrointestinal tract (GIT) resulted in low absorption. The main objective of this work was the design of a particulate system for oral delivery of recombinant human growth hormone (rhGH) on the basis of particle uptake mechanism in GIT. Biodegradable protein-loaded microspheres were prepared using Resomers (RG207, RG756 and RG505) by double emulsion methods. Aqueous solution of protein and freshly prepared rhGH-zinc complex were used for loading process. Various analytical methods, including fluorescence spectroscopy, SDS-PAGE electrophoresis and reversed-phase chromatography, were set up for the quantification and qualification of rhGH before and after the formulation and fabrication procedures. At the optimum conditions, microspheres were mostly below 10 µm with relatively high protein loading (> 50%). Obtained data showed that the stability of protein did not change during the formulation and microencapsulation processes. Results also showed that the encapsulation process in the presence of zinc caused no detectable change in the protein chemical stability. In-vitro stability study of microspheres in different simulated GI media indicated that the entrapped protein was physically stable. Less than 20% of rhGH was released from the microspheres incubated in both simulated stomach and intestine fluids for 3 and 6 h, respectively.

Preparing Poly (Lactic-co-Glycolic Acid) (PLGA) Microspheres Containing Lysozyme-Zinc Precipitate Using a Modified Double Emulsion Method.

Lysozyme, as a model protein, was precipitated through the formation of protein-Zn complex to micronize for subsequent encapsulation within poly (lactic-co-glycolic acid) (PLGA) microspheres. Various parameters, including pH, type and concentration of added salts and protein concentration, were modified to optimize the yield of protein complexation and precipitation. The resulting protein particles (lysozyme-Zn complex as a freshly prepared suspension or a freeze-dried solid) were then loaded into PLGA (Resomer(®) 503H) microspheres, using a double emulsion technique and microspheres encapsulation efficiency and their sizes were determined. It was observed that salt type could significantly influence the magnitude of protein complexation. At the same conditions, zinc chloride was found to be more successful in producing pelletizable lysozyme. Generally, higher concentrations of protein solution led also to the higher yields of complexation and at the optimum conditions, the percentage of pelletizable lysozyme reached to 80%. Taking advantage of this procedure, a modified technique for preparation of protein-loaded PLGA microspheres was established, although it is also expected that this technique increases the protein drugs stabilization during the encapsulation process.

Green synthesis of gold nanoparticles by the marine microalga Tetraselmis suecica.

The application of green-synthesis principles is one of the most impressive research fields for the production of nanoparticles. Different kinds of biological systems have been used for this purpose. In the present study, AuNPs (gold nanoparticles) were prepared within a short time period using a fresh cell extract of the marine microalga Tetraselmis suecica as a reducing agent of HAuCl4 (chloroauric acid) solution. The UV-visible spectrum of the aqueous medium containing AuNPs indicated a peak at 530 nm, corresponding to the surface plasmon absorbance of AuNPs. The X-ray diffraction pattern also showed a Bragg reflection related to AuNPs. Fourier-transform infrared spectroscopy was performed for analysis of surface functional groups of AuNPs. Transmission electron microscopy and particle-size-distribution patterns determined by the laser-light-scattering method confirmed the formation of well-dispersed AuNPs. The most frequent size of particles was 79 nm.