The authors gratefully acknowledge the partial funding from the TecNM under the Call for Scientific Research, Technological Development, and Innovation (16898.23-P) for the Institutos Tecnologicos Federales. They also appreciate the support from the Instituto de Ciencia, Tecnolog&#237;a e Innovaci&#243;n del Estado de Michoac&#225;n de Ocampo (FCCHTI23_ME-4.1.-0001).

1. Culture media preparation and inoculum preparation
<ol>
	<li>Prepare 1 L of 3N-BBM+ V(CCAP) growth medium (Macronutrients: NaNO3 [0.75 g L-1], CaCl2 [0.019 g L-1], MgSO4 [0.019 g L-1], K2HPO4 [0.057 g L-1], NaCl [0.025 g L-1], KH2PO4 [0.175 g L-1]; micronutrients: Na2 EDTA [0.0186 g L-1], FeCl3 [0.0024 g L-1], MnCl2 [0.001 g L-1], ZnCl2 [1.25 x 10-4 g L-1], CoCl2 [5 x 10-5 g L-1], Na2MoO4 [9.98 x 10-5 g L-1])8 and sterilize it for 15 min at 121 &#176;C and 1.5 psi.</li>
	<li>Inoculate a flask with 1 L of growth medium at an approximate concentration of 3 x 106 cells mL-1 for the microalga C. sorokiniana; or add 5% to 10% (v/v) of inoculum to the growth medium. 
	NOTE: To maintain sterile conditions, it is recommended to use a laminar flow hood, gloves, lab coat, and face mask.</li>
	<li>Incubate the inoculated flask under red light sources with an approximate intensity of 3,600 to 4,200 lux, at a temperature between 22-26 &#176;C, with a photoperiod of 12 h light and 12 h darkness for C. sorokiniana and photoperiod of 16 h light and 8 h darkness for H. pluvialis, and manually agitate every 24 h, approximately for 7 days for both strains. 
	NOTE: Maintain under growth conditions for at least 7-10 days (until obtaining an intense green color) for C. sorokiniana and 10-12 days for H. pluvialis. To obtain the microalgae concentrate that will serve as the inoculum in the kinetics, it is necessary to remove the supernatant medium from the produced algae.</li>
	<li>In a sterile environment, fill sterile 15 mL conical centrifugation tubes with approximately 10 mL of inoculated medium and centrifuge at 3,000 x g for 20 min under 25-30 &#176;C.</li>
	<li>Under sterile conditions, remove the supernatant using a micropipette or by pouring and concentrating the biomass in a sterile 50 mL conical centrifugation tube.</li>
	<li>Repeat this process as necessary until the required volume of inoculum for the kinetics is obtained.</li>
	<li>Measure the optical density (OD550&#160;nm) of the inoculum to determine the cell concentration of the microalgae and store it at a temperature of 3-4 &#176;C until ready for use. 
	NOTE: Direct cellular counting was initially performed to correlate cell concentration with absorbance. Once counted, the inoculum can be stored under refrigeration for no more than 4 days.</li>
</ol>
2. Study of the growth kinetics
<ol>
	<li>Prepare the required volume of 3N-BBM+ V(CCAP) suitable for each experiment (8 L for photobioreactor level tests for C. sorokiniana and 0.5 L for the flask-level tests for H. pluvialis) according to standard laboratory procedures, ensuring proper sterilization.</li>
	<li>For both photobioreactor and flasks, configure the light source to the desired color spectrum (Blue at 460 nm, purple, mix of 460 nm and 630 nm for C. sorokiniana and red at 630 nm for H. pluvialis); cover both systems to avoid external light interference. 
	NOTE: Due to the variation in wavelength emitted from each light-emitting diode, spectroradiometry was performed using a spectroradiometer on the light sources to determine the wavelength and emission power of each diode9. Purple light was achieved by combining red and blue LEDs, resulting in wavelengths of 460 nm with an emission power of 1.65 W nm-1&#160;for the blue LEDs and 630 nm with an emission power of 0.6 W nm-1&#160;for the red LEDs. These measurements were used for the tests conducted solely under blue and red light.</li>
	<li>Program the timer to alternate between 12 h light and 12 h dark cycles for C. sorokiniana and 16 h light and 8 h dark cycles for H. pluvialis.</li>
	<li>Connect the CO2 aeration system to the photobioreactor culture vessel to provide carbon dioxide supply in intervals of 12 h for C. sorokiniana.</li>
	<li>Inoculate the culture vessel with a sufficient volume of pre-inoculum to achieve a starting cell density of 3 x 105 cells mL-1 or its equivalent in optical density (OD550&#160;nm) = 0.180. 
	NOTE: Ensure aseptic technique during inoculation to prevent contamination.</li>
	<li>Sample the culture at regular intervals of 12 h for C. sorokiniana and 8 h for H. pluvialis to monitor cell growth and physiological parameters. Collect samples using sterile techniques to maintain the integrity of the culture.</li>
</ol>
3. Quantification of chlorophylls
NOTE: Data for cellular quantification is provided in Supplementary File 1.
<ol>
	<li>Place 40 mL of the sample into plastic conical centrifugation tubes.</li>
	<li>Centrifuge at 3,000 x g for 20 min at 15 &#176;C and discard the supernatant by decantation or using a Pasteur pipette.</li>
	<li>Transfer the cell pellet into a glass tube covered with aluminum foil and a screw cap to prevent oxidation.</li>
	<li>Resuspend the cell pellet with 3 mL of 90% pure acetone solution using a vortex.</li>
	<li>Sonicate the suspended sample in an ice bath for two cycles of 5 min each and let it rest at 4 &#176;C for 16 h10.</li>
	<li>After 16 h, sonicate again under the same conditions mentioned in the previous step (step 3.5) and centrifuge again under the previously described conditions (step 3.2).</li>
	<li>Separate the pigment extract using a Pasteur pipette and transfer it to another clean tube protected from light.</li>
	<li>Place the pigment extract in a quartz cell to read in a spectrophotometer at 630 nm, 647 nm, and 664 nm wavelengths. 
	NOTE: Calibrate the spectrophotometer beforehand with 90% acetone.</li>
	<li>To calculate chlorophyll concentrations, use the following equations11: 
	Chlorophyll a = 11.93 A664 - 1.93 A647 
	Chlorophyll b = 20.36 A647 - 5.50 A664 
	NOTE: Results are expressed in &#181;g mL-1 of extract, which are multiplied by the extract volume and divided by the number of mL of sample to obtain chlorophyll concentrations in &#181;g mL-1 of culture.</li>
	<li>To obtain values in &#181;g chl cell-1, use the following formula: 
	&#956;g chl cell-1 = &#181;g chl in 1 mL of culture / [cells mL-1] cells concentration in the culture.</li>
</ol>

Enhancing Chlorophyll Production in Microalgae via Growth Factor Optimization

<ol>
	<li>Khan, M. I., Shin, J. H., Kim, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promising+future+of+microalgae:+Current+status,+challenges,+and+optimization+of+a+sustainable+and+renewable+industry+for+biofuels,+feed,+and+other+products.">The promising future of microalgae: Current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products.</a> Microb Cell Fact. 17 (1), 36 (2018).</li><li>Otero-Paternina, A. S. M., Cruz-Casallas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+hydrocarbon+phenanthrene+on+Chlorella+vulgaris+(Chlorellaceae)+growth.">Effect of the hydrocarbon phenanthrene on Chlorella vulgaris (Chlorellaceae) growth.</a> Acta Biol Col. 18 (1), 87-98 (2013).</li><li>Pancha, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salinity-induced+oxidative+stress+enhanced+the+biofuel+production+potential+of+microalgae+Scenedesmus+sp.+CCNM+1077.">Salinity-induced oxidative stress enhanced the biofuel production potential of microalgae Scenedesmus sp. CCNM 1077.</a> Bioresour Techno. 189, 341-348 (2015).</li><li>Ortiz, M. L., Cort&#233;s, C. E., S&#225;nchez, J., Padilla, J., Otero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+microalgae+Chlorella+sorokiniana+growth+in+different+culture+mediums+in+autotrophic+and+mixotrophic+conditions.">Evaluating microalgae Chlorella sorokiniana growth in different culture mediums in autotrophic and mixotrophic conditions.</a> Orinoquia. 16 (1), 11-20 (2012).</li><li>Camacho Kurmen, J. E., Gonz&#225;lez, G., Klotz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astaxanthin+production+in+Haematococcus+pluvialis+under+different+stress+conditions.">Astaxanthin production in Haematococcus pluvialis under different stress conditions.</a> Nova. 11 (19), 94-104 (2013).</li><li>. Production of chlorophyll and astaxanthin from Haematococcus pluvialis under nitrogen deficiency stress in a 5-liter Biostat Aplus bioreactor Available from: <a target="_blank" href="https://repositorio.unicolmayor.edu.co/handle/unicolmayor/2735">https://repositorio.unicolmayor.edu.co/handle/unicolmayor/2735</a> (2019)</li><li>Bischoff, H. W., Bold, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycological+studies+IV.+Some+soil+algae+from+Enchanted+Rock+and+related+algal+species.">Phycological studies IV. Some soil algae from Enchanted Rock and related algal species.</a> University of Texas. 6318, 95 (1963).</li><li>Cerezo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+characterization+through+the+use+and+programming+of+a+spectrometer.">LED characterization through the use and programming of a spectrometer.</a> Universidad Polit&#233;cnica de. 22323, (2013).</li><li>Couso, I., Vila, M., Vigara, J., Cordero, B. F., Vargas, M. &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+carotenoids+and+regulation+of+the+carotenoid+biosynthesis+pathway+in+response+to+high+light+stress+in+the+unicellular+microalga+Chlamydomonas+reinhardtii.">Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii.</a> Eur J Phycol. 47 (3), 223-232 (2012).</li><li>Vera-L&#243;pez, F., Mart&#237;nez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigments+in+microalgae:+Functions,+applications,+and+overproduction+techniques.">Pigments in microalgae: Functions, applications, and overproduction techniques.</a> BioTechnology. 25 (5), 35-51 (2021).</li></ol>

The authors have nothing to disclose.

The comparative study between H. pluvialis and C. sorokiniana revealed significant differences in chlorophyll production dynamics. While H. pluvialis exhibited a decrease in chlorophyll concentration throughout the experiment, C. sorokiniana showed a steady increase. Additionally, there was initially a lower proportion of chlorophyll a in both species, but this ratio reversed in particular growth conditions, which may give indications of an induction of the production of said ...

In recent years, the growing environmental problems caused by anthropogenic activities and their adverse effects on the health and balance of ecosystems have driven the search for more efficient and environmentally friendly production systems. This has accelerated processes in industries and fostered the implementation of bioremediation treatments and the development of bio compounds to mitigate these harmful effects1.
This context has led to a significant growth in the study of microalgae, driven by the need to find innovative solutions to current environmental and economic challenges. Microalgae thrive in aquatic environments, using sunlight and carbon dioxide as sources of energy and carbon, respectively. This characteristic makes them a sustainable and promising alternative for producing various valuable products. Research in this field has focused on understanding the physiology and metabolism of these cells, as well as developing efficient technologies for their cultivation and processing2.
There is a clear need to develop accessible and reliable tools for studying microalgae to accelerate research processes and deepen the understanding of their physiology, metabolism, and potential applications. These tools should enable rapid analysis of the effect of environmental and production factors on key parameters, such as chlorophyll concentration, which is a fundamental indicator of the health and development of these organisms. A decrease in chlorophyll content may indicate environmental stress, nutritional deficiencies, or diseases.
These green pigments play a crucial role in photosynthesis, capturing sunlight energy to convert carbon dioxide and water into glucose and oxygen and constitute between 0.5% and 5% of the biomass of microalgae3. Beyond their essential role in sustaining life processes, chlorophylls have found applications in various industries. Chlorophyll extracts are utilized in food and beverage processing as natural colorants, imparting vibrant green hues to products while also providing antioxidant properties. Moreover, chlorophyll-based supplements are gaining popularity in the health and wellness sector due to their purported detoxifying and anti-inflammatory effects. By harnessing the multifaceted properties of chlorophyll, industries can develop innovative products that contribute to both visual appeal and consumer well-being1.
In this regard, one microalgae of great biotechnological relevance is C. sorokiniana. This microorganism stands out for its rapid growth rate, making it highly efficient in biomass production. Additionally, C. sorokiniana exhibits a diverse content of highly nutritional compounds, including proteins, lipids, and vitamins, which renders it valuable for various applications in food, feed, and biofuel production2. Moreover, this microalgae species has been found to produce extracellular enzymes with diverse functions, opening up possibilities for biotechnological applications such as wastewater treatment, bioremediation, and pharmaceuticals4. In addition to its rapid growth and versatile applications, C. sorokiniana also demonstrates significant potential for chlorophyll production. As a photosynthetic microorganism, C. sorokiniana possesses the machinery necessary for the synthesis of chlorophyll, which constitutes between 0.5% and 5% of the biomass of microalgae3. This capability makes C. sorokiniana an attractive candidate for commercial-scale chlorophyll production and promises to be a sustainable solution to urgent environmental and nutritional challenges5.
On the other hand, another microalgae species of significant interest is H. pluvialis. This microalga is renowned for its production of astaxanthin, a potent antioxidant pigment with numerous industrial applications. Astaxanthin serves as a protective mechanism for H. pluvialis photosystem, shielding it from oxidative stress induced by environmental factors. This pigment is highly sought after in cosmetics, nutraceuticals, and aquaculture industries, owing to its antioxidant properties and potential health benefits. With its abundant ability to produce astaxanthin, H. pluvialis presents a promising avenue for developing innovative products catering to various industrial and consumer needs6.
Recent studies have also highlighted H. pluvialis1 potential for chlorophyll production, further enhancing its industrial significance. For instance, a study investigated the chlorophyll content of H. pluvialis under varying growth conditions and found that under optimal cultivation parameters, H. pluvialis exhibited remarkable chlorophyll production rates, surpassing those of other microalgae species7. This finding underscores the potential of H. pluvialis as a sustainable source of chlorophyll, offering novel opportunities for its utilization in various industrial sectors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C3H6O</td>
			<td>Meyer</td>
			<td>67-64-1</td>
			<td>Acetone 90%</td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Biologix</td>
			<td>10-9502</td>
			<td>Test tube</td>
		</tr>
		<tr>
			<td>2510-DTH</td>
			<td>Branson</td>
			<td>D-73595</td>
			<td>Sonicator</td>
		</tr>
		<tr>
			<td>5 mL screw cap test tube</td>
			<td>Kimax</td>
			<td>45066-13100</td>
			<td>Test tube</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Biologix</td>
			<td>10-9151</td>
			<td>Test tube</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>611 standard, 12&#34; x 1000 feet</td>
			<td>Test tube cover&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Meyer</td>
			<td>0925-250</td>
			<td>Calcium Chloride</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Dynamica</td>
			<td>14 R</td>
			<td>Centrifuge Refrigerated</td>
		</tr>
		<tr>
			<td>CoCl2</td>
			<td>Merck</td>
			<td>1057-100</td>
			<td>Cobalt dichloride</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>Merck</td>
			<td>157740</td>
			<td>Iron(III) Chloride</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Meyer</td>
			<td>2051-250</td>
			<td>Dipotassium Phosphate</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Meyer</td>
			<td>2055-250</td>
			<td>Monopotassium Phosphate</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Meyer</td>
			<td>1605-250</td>
			<td>Magnesium Sulphate</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>LabNet</td>
			<td>Model Beta-Pette</td>
			<td>Micropipette</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Merck</td>
			<td>429449</td>
			<td>Manganese(II) Chloride&#160;</td>
		</tr>
		<tr>
			<td>Na2 EDTA&#160;</td>
			<td>Merck</td>
			<td>200-449-4</td>
			<td>Edatamil, Edetato Disodium Salt Dihydrate</td>
		</tr>
		<tr>
			<td>Na2MoO4</td>
			<td>Merck</td>
			<td>243655</td>
			<td>Sodium Molybdate</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Meyer</td>
			<td>2365-500</td>
			<td>Sodium Chloride</td>
		</tr>
		<tr>
			<td>NaNO3</td>
			<td>Meyer</td>
			<td>2465-250</td>
			<td>Sodium Nitrate</td>
		</tr>
		<tr>
			<td>RGB LED stripe</td>
			<td>Steren</td>
			<td>GAD-LED2</td>
			<td>Light source</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>PerkinElmer</td>
			<td>Model Lambda35</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>spectroradiometer</td>
			<td>Gigahertz-Optik</td>
			<td>model BTS256</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>Vortex-Genie&#174; 2</td>
			<td>Vortex</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Merck</td>
			<td>208086</td>
			<td>Zinc Chloride</td>
		</tr>
	</tbody>
</table>

evaluation of the effect of growth factors on chlorophylls a and b production from microalgae

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it into chemical energy to facilitate the synthesis of organic compounds. This pigment serves as a valuable primary source for biotechnological input products in the food, pharmaceutical, and cosmetic industries due to its high antioxidant properties and coloring capabilities. The objective of this research was to evaluate the effect of growth factors (CO2 concentration, light color, and light intensity) through a Taguchi L4 experimental design on cell growth and the cellular content of chlorophyll a and b in Chlorella sorokiniana, followed by validation of the method using Haematococcus pluvialis microalgae as an additional study model. Cell growth was quantified using the optical density spectrophotometric technique at a wavelength of 550 nm. For the quantification of chlorophylls, a cell extract was obtained using a 90% pure acetone solution, and subsequently, the concentrations of chlorophylls a and b were quantified using spectrophotometric techniques at wavelengths of 647 nm and 664 nm, according to the method described by Jeffrey and Humphrey. The experimental results indicated that controlling conditions of low CO2 addition, purple light, and low light intensity increases both cell growth and the concentration of chlorophylls a and b within the cells. The implementation of this chlorophyll quantification method allows for quick, simple, and precise determination of chlorophyll content, as the wavelengths used are at the absorbance peaks of both types of chlorophylls, making this technique easily reproducible for any microalgae under study.

To observe the efficiency of the technique detecting variations in chlorophyll cellular concentration and evaluate the effect of growth factors in C. sorokiniana, a Taguchi L4 experimental design was established, evaluating CO2 volume addition, light color, and light intensity. Each factor was assessed at low and high levels, as shown in Table 1, under the conditions defined by the experimental design in Table 2.
Once the experim...

Author Spotlight: Optimizing Growth Factors for Production of Biotechnologically Relevant Secondary Metabolites

The present study highlights the advantages of employing the method developed by Jeffrey and Humphrey for extracting and quantifying fat-soluble pigments from microalgae. This method serves as a valuable tool for assessing the influence of growth factors on chlorophyll production and cellular content in these organisms.

Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it ...

evaluation-effect-growth-factors-on-chlorophylls-b-production-from

Research

JoVE Journal

Biochemistry

2.0K Views.  National Institute of Technology of Mexico. The present study highlights the advantages of employing the method developed by Jeffrey and Humphrey for extracting and quantifying fat-soluble pigments from microalgae. This method serves as a valuable tool for assessing the influence of growth factors on chlorophyll production and cellular content in these organisms.

Video: Author Spotlight: Optimizing Growth Factors for Production of Biotechnologically Relevant Secondary Metabolites

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...