This work was supported by a Zhejiang Shuren University research project (2023R053 and 2023KJ237).

The blood used in the experiments was obtained from SPF-grade BALB/c female mice weighing 20-25 g and approximately 7 weeks old. The Animal Experimentation Ethics Committee of Zhejiang Shuren College approved all animal care and experimental procedures.
1. Solution preparation
<ol>
	<li>Weigh ALG and dissolve in ultrapure water by stirring in a water bath at 50 &#176;C to obtain 2% ALG solution.</li>
	<li>Separately prepare mass fraction with 5% (or molar mass concentration is 0.3 M) solutions of CaCl2, FeCl3, ZnSO4, and CuSO4 in ultrapure water. Pour each solution separately into a collecting dish.</li>
</ol>
2. Microfluidic electrospray device
<ol>
	<li>Take a glass capillary tube and place it over a blowtorch flame to soften, then stretch it to various diameters (50 &#181;m-500 &#181;m). Cut off the fine parts with a glass cutter and gently polish the tip port with high-quality silicon carbide sandpaper. After cleaning, connect the thick end of the capillary tube to a long tubing and the other end of the tubing to a dispensing needle and a 5 mL syringe (Supplementary Figure 1). 
	NOTE: Sharp edges of glass tube cuts are difficult to insert into the hose; thus, the break should be rounded at the flame edge.</li>
	<li>Attach the syringe to the microfluidic syringe pump and the capillary tubing at one end to the holder. Connect the red high-voltage end clip of the high-voltage power supply to the dispensing needle of the syringe and place the silver clip in the collection fluid. This configuration creates a simple microfluidic electrospray device (Figure 1A). 
	NOTE: Place the capillary tube perpendicular to the table.</li>
</ol>
3. ALG microspheres preparation
<ol>
	<li>Place the separate collection dishes containing different metal ions directly under the capillary tube. Turn on the microfluidic syringe pump and select Fast Forward to prime the tube with the ALG, then set the appropriate flow rate (2 mL/h).</li>
	<li>Switch on the high voltage power switch and turn the knob to set the appropriate voltage (5-7 kV).</li>
	<li>Add 20 mL solution to each of the 90 mm Petri dishes to collect ALG microspheres to form ALGMS cross-linked with different metal ions (Figure 1B).</li>
	<li>ALG forms microspheres under the action of electric field and gravity in different ion-collecting solutions within a few seconds. Aspirate the microspheres with a sterilized pipette and transfer them to individual 1.5 mL centrifuge tubes to obtain Zn2+, Ca2+, Cu2+, and Fe3+ -ALG hydrogel microspheres (Figure 1C). Label the Zn-ALGMS, Ca-ALGMS, Cu-ALGMS and Fe-ALGMS tubes. 
	NOTE: Microspheres of different diameters can be obtained by adjusting parameters such as voltage, tip diameter, flow rate, and solution concentration.</li>
	<li>Take a small amount of the prepared microspheres, disperse them as evenly as possible in PBS, and place them under a microscope for visualization.</li>
	<li>Randomly selected 10 observation fields in the same batch of microspheres, import the images to be measured into the ImageJ software, convert the images to the appropriate grayscale mode, and use the threshold segmentation technology to accurately define the area of the target particles and start the function of analyzing the particles so that ImageJ can identify and measure the target particles. Obtain data on the microspheres and then import the data into the Origin software for analysis and particle size mapping.</li>
</ol>
4. Antimicrobial performance test
<ol>
	<li>Prepare 5 mL suspensions of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) with an initial bacterial turbidity of 0.2 mcF using a liquid Luria-Bertani (LB) medium.</li>
	<li>Add 10 mL of each bacterial solution to 4 individual 15 mL sterile centrifuge tubes. Then, add 0.5 mL of Zn-ALGMS, Ca-ALGMS, Cu-ALGMS, and Fe-ALGMS ALG to each tube and place them in a constant-temperature shaker at 37 &#176;C and 200 rpm for 12 h. Add an equal amount of saline to the control group.</li>
	<li>Dilute each bacterial solution to 105 times with sterile water. Use a sterile glass bead to spread 200 &#181;L of bacterial solution on an LB solid medium.</li>
	<li>Spread the solution quickly and evenly on the coating plate, avoiding back and forth in the same area. Then, place the plates in an incubator at 37 &#176;C for 12 h. Observe the colonies of different groups. 
	NOTE: All aseptic procedures should be performed near an alcohol lamp. Sterilize microbial culture vessels, inoculation utensils, and culture media.</li>
</ol>
5. Drug release testing
<ol>
	<li>To evaluate the drug release ability of different microspheres, use bovine serum albumin (BSA) as a loaded model drug. Add 500 &#181;L of each microsphere (Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS) to 1 mg/mL BSA suspension for 24 h.</li>
	<li>For the drug release assay, add each saturated sample to 5.0 M of phosphate-buffered saline (PBS; pH 7.4), followed by agitation for 15 min at 37 &#176;C with continuous shaking at 80 rpm.</li>
	<li>Use 100 &#181;L of the solution and replace the medium with a fresh one at 1, 3, 6, 12, 24, 36, and 48 h.</li>
	<li>Quantify the supernatant protein concentrations at specific times using a BCA kit per the manufacturer&#39;s instructions. Quantitatively measure the released drug by enzyme marker to obtain the corresponding absorbance and convert the measured value to actual drug concentration according to the standard curve. Calculate the rate of drug release using the following formula: 
	Percentage of drug release = (concentration of drug released at a specific time point / initial drug concentration) x 100%</li>
	<li>At regular intervals, remove an equal volume of PBS from each well and replace it with an equal volume of fresh medium.</li>
</ol>
6. Hemolysis test
<ol>
	<li>Place the prepared Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS in PBS and incubate them at 37 &#176;C for 24 h to formulate the incubation solution.</li>
	<li>Obtain whole blood from healthy BALB/c mice by ocular phlebotomy, collected in anticoagulated tubes containing sodium citrate. Vortex and centrifuge at 1509 x g for 15 min to obtain red blood cells.</li>
	<li>Flush the red blood cells 2x-3x with PBS and prepare a 10% red blood cell solution with PBS.</li>
	<li>Preparation of various ALGMS: Take 500 &#181;L of each microsphere for the experimental group, 1 mL ultrapure water (ddH2O) for the positive control group, and 1 mL of PBS solution for the negative control group and mix each with 20 &#181;L of blood cell solution.</li>
	<li>Incubate the samples at 37 &#176;C for 4 h, centrifuge at 1509 x g, 15 min. Keep the supernatant aside and photograph the red blood cells in each group after hemolysis.</li>
	<li>Take the hemolysis rate of the positive control group as 100% and the negative control group as 0%. Measure the absorbance values of the supernatants in each group and compute the hemolysis rate by using the formula: 
	Hemolysis (%) = (Absorbance values of the supernatants of each group- 
	Absorbance values of distilled water alone) / (Absorbance values of the positive control group- Absorbance values of distilled water alone) x 100</li>
</ol>
7. Cytobiocompatibility test
<ol>
	<li>Wash the different ALGMS 2x with PBS and place in a 5 mL Petri dish for 15 min and discard the supernatant.</li>
	<li>Separately add different ALGMS at 0.2 mL in complete culture medium (DMEM) containing 10% FBS, incubate at 37 &#176;C for 24 h, and filter the solution to obtain the leachate.</li>
	<li>Culture NIH3T3 cells to approximately 70% cell density in 24-well plates, treat the NIH3T3 cells with microspheres leaching solution, and continue to culture with complete medium for another 24 h.</li>
	<li>Remove the cell culture medium and wash the cells with PBS.</li>
	<li>Assess cell viability using Calcein-AM/PI assay. Take 2.5 &#181;L of Calcein-AM solution (4 mM) and 12.5 &#181;L of PI solution (2 mM) and add them to 5 mL of 1x PBS and mix well to obtain a working solution with 2 &#181;M Calcein-AM and 5 &#181;M PI.</li>
	<li>Add the working solution to previously seeded cell culture plates at 500 &#181;L per well, incubate at 37 &#176;C under light protection for 15 min, and observe and photograph under an inverted fluorescence microscope. Set up three replicates for each group.</li>
	<li>Calculate cell viability according to the following formula: 
	Cell viability (%) = number of viable cells / (number of viable cells + number of dead cells)</li>
</ol>

Developing Sodium Alginate Hydrogel Microspheres for Biomedical Use

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S., Hashem, A. H., Khattab, T. A., Kamel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+antibacterial+hydrogels+based+on+sodium+alginate.">New antibacterial hydrogels based on sodium alginate.</a> Int. J Biol Macromol. 248, 125872 (2023).</li><li>Olukman &#350;ahin, M., &#350;anl&#305;, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+5-fluorouracil+release+properties+investigation+from+pH+sensitive+sodium+alginate+coated+and+uncoated+methyl+cellulose/chitosan+microspheres.">In vitro 5-fluorouracil release properties investigation from pH sensitive sodium alginate coated and uncoated methyl cellulose/chitosan microspheres.</a> Int J Biol Macromol. 258 (1), 128895 (2024).</li><li>Chi, H., Qiu, Y., Ye, X., Shi, J., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+strategy+of+hydrogel+microsphere+and+its+application+in+skin+repair.">Preparation strategy of hydrogel microsphere and its application in skin repair.</a> Front Bioeng Biotechnol. 11, 1239183 (2023).</li><li>Yang, L., 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=Bio-inspired+dual-adhesive+particles+from+microfluidic+electrospray+for+bone+regeneration.">Bio-inspired dual-adhesive particles from microfluidic electrospray for bone regeneration.</a> Nano Res. 16 (4), 5292-5299 (2022).</li><li>Zheng, H., 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=Celastrol-encapsulated+microspheres+prepared+by+microfluidic+electrospray+for+alleviating+inflammatory+pain.">Celastrol-encapsulated microspheres prepared by microfluidic electrospray for alleviating inflammatory pain.</a> Biomater Adv. 149, 213398 (2023).</li><li>Yang, L., Yang, W., Xu, W., Zhao, Y., Shang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+Janus+microcarriers+with+sequential+actives+release+for+bone+regeneration.">Bio-inspired Janus microcarriers with sequential actives release for bone regeneration.</a> Chem Eng J. 476, 146797 (2023).</li><li>Yang, L., Sun, L., Zhang, H., Bian, F., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-inspired+lubricated+drug+delivery+particles+from+microfluidic+electrospray+for+osteoarthritis+treatment.">Ice-inspired lubricated drug delivery particles from microfluidic electrospray for osteoarthritis treatment.</a> ACS Nano. 15 (12), 20600-20606 (2021).</li><li>Li, S., 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=Ultra-flexible+stretchable+liquid+metal+circuits+with+antimicrobial+properties+through+selective+laser+activation+for+health+monitoring.">Ultra-flexible stretchable liquid metal circuits with antimicrobial properties through selective laser activation for health monitoring.</a> Chem Eng J. 482, 149173 (2024).</li><li>Sukhodub, L., Kumeda, M., Sukhodub, L., Bielai, V., Lyndin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ions+doping+effect+on+the+physicochemical,+antimicrobial,+and+wound+healing+profiles+of+alginate-based+composite.">Metal ions doping effect on the physicochemical, antimicrobial, and wound healing profiles of alginate-based composite.</a> Carbohydr Polym. 304, 120486 (2023).</li><li>Daly, A. C., Riley, L., Segura, T., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+microparticles+for+biomedical+applications.">Hydrogel microparticles for biomedical applications.</a> Nat Rev Mater. 5 (1), 20-43 (2020).</li><li>Bertsch, P., Diba, M., Mooney, D. J., Leeuwenburgh, S. C. 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A., Shum, 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=Development+and+future+of+droplet+microfluidics.">Development and future of droplet microfluidics.</a> LOC. 24 (5), 1135-1153 (2024).</li></ol>

No conflicts of interest are to be disclosed.

In this protocol, we present a method for preparing ALGMS based on microfluidic electrospray technology. The method is simple to operate and yields a large number of microspheres with uniform roundness and controllable diameter. This approach offers convenience to researchers and can promote the research and application of hydrogel microspheres. In addition, by cross-linking with different metal ions, the stability and bioactivity of the ALGMS were improved. In the antimicrobial experiments, Cu-ALGMS and Zn-ALGMS exhibit...

Drug delivery systems are a research hotspot in the field of bio-tissue engineering, aiming to improve drug delivery efficiency and efficacy and reduce adverse reactions and side effects1. Among these systems, hydrogel microspheres, characterized by good biocompatibility, tunable mechanical properties, and functional plasticity, are one of the most commonly used vehicles for drug loading and delivery2. They can be used for both slow and controlled release of drugs, provide good protective effects for drugs, avoid or minimize non-specific effects of drugs in other tissues, and target drug delivery to specific tissue structures3. Therefore, hydrogel microspheres have become a new and efficient drug delivery system, with research in this field gradually emerging4.
Hydrogel microspheres are typically synthesized from biodegradable materials, including polysaccharides, proteins, and natural polymers5. Among them, ALG is a biocompatible, biodegradable polysaccharide extracted from marine brown algae6. Its molecular chain contains free hydroxyl and carboxyl groups that can crosslink with most divalent or multivalent cations to form a water-insoluble hydrogel structure with a three-dimensional network5. The hydrogel microspheres formed by ALG can be converted into negatively charged polyelectrolytes in neutral and alkaline solutions. This repulsion between negative charges causes the microspheres to swell, allowing the release of the encapsulated active ingredient or drug. These properties have led to the consideration of ALG microspheres as promising drug carriers widely used for drug loading and controlled release7.
Various methods exist for the preparation of hydrogel microspheres. Traditional ALGMS preparation methods usually include the sol-gel method or the emulsion-sol method. These methods involve steps such as precipitation, co-precipitation, and gelation reactions to obtain the target microspheres8. In recent years, with the continuous development of microfluidic technology, the microfluidic electrospray method has gradually become an efficient and precise microsphere preparation method9. This method utilizes microfluidic technology to electrospray a polymer solution through a microfine nozzle to form micrometer-sized droplets and microspheres during the subsequent curing or cross-linking process10. Compared to the traditional method, microfluidic electrospray offers precise control of microsphere particle size and morphology by adjusting parameters such as solution flow rate, voltage, and fine nozzle size11. It also enables high-speed continuous preparation of microspheres, improving preparation efficiency and maintaining mild reaction conditions. In addition, ALGMS can be prepared to possess various functions, such as controlled-release drugs and loaded catalysts, enabling their easy application in various fields.
Here, we present a protocol for the preparation of ALG microspheres using the microfluidic electrospray method. The process involves passing an ALG solution through a microfluidic device and subjecting it to electrospray. The resulting droplets were collected in the solution containing different metal ions (Ca2+, Cu2+, Zn2+, and Fe3+) to initiate the cross-linking reaction. This reaction improves the stability and adhesion of the microspheres and endows them with different functionalities. This method is easy to perform, and the synthesized microspheres exhibit good size uniformity in their morphology. In addition, we investigated their antimicrobial properties, slow drug-release ability, and biocompatibility. This protocol will be useful for further drug development and production.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>120 mesh screen</td>
			<td>Solarbio,China</td>
			<td>YA0946</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol burner</td>
			<td>Solarbio,China</td>
			<td>YA2320</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Wukong Biotechnology,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchoninic Acid Assay reagent</td>
			<td>Meilunbio,China</td>
			<td>MA0082</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Lablead,China</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#160; powder</td>
			<td>Aladdin,China</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM/PI</td>
			<td>Biosharp,China</td>
			<td>BL130A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Corning,America</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4&#160; powder</td>
			<td>Jnxinyuehuagong,China</td>
			<td>7758-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibicol,China</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#160; powder</td>
			<td>Aladdin,China</td>
			<td>7705-08-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HAKATA,China</td>
			<td>HN-FBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td>Sartorius,Germany</td>
			<td>CC0028</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscopy</td>
			<td>Evidentscientific,Japan</td>
			<td>BX53(LED)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic syringe pump</td>
			<td>Longerpump,England</td>
			<td>LSP01-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH3T3</td>
			<td>HyGyte,China</td>
			<td>TCM-C752</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermofisher,America</td>
			<td>150464</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Thermofisher,America</td>
			<td>3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Thermofisher,America</td>
			<td>Axia ChemiSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate powder&#160;</td>
			<td>Bjbalb,China</td>
			<td>Y13095</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4 powder</td>
			<td>Jnxinyuehuagong,China</td>
			<td>7733-02-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of cross-linked sodium alginate microspheres with different metal ions using the microfluidic electrospray technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within polymers. In the field of biomedicine, hydrogel microspheres have been extensively studied for their application as drug carriers owing to their ability to reduce the frequency of drug administration, minimize side effects, and improve patient compliance. Sodium alginate (ALG) is a naturally occurring linear polysaccharide with three backbone glycosidic linkages. There are two auxiliary hydroxyl groups present in each of the moieties of the polymer, which have the characteristics of an alcohol hydroxyl moiety. The synthetic ALG units can undergo chemical cross-linking reactions with metal ions, forming a cross-linked network structure of polymer stacks, ultimately forming a hydrogel. Hydrogel microspheres can be prepared using a simple process involving the ionic cross-linking properties of ALG. In this study, we prepared ALG-based hydrogel microspheres (ALGMS) using a microfluidic electrodeposition strategy. The prepared hydrogel microspheres were uniformly sized and well-dispersed, owing to accurate control of the microfluidic electrospray flow. ALGMS cross-linked with different metal ions were prepared using a microfluidic electrospray technique combining microfluidic and high electric field, and its antimicrobial properties, slow drug release ability, and biocompatibility were investigated. This technology holds promise for application in advanced drug development and production.

Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

Hydrogen microspheres are characterized by good compatibility, design flexibility, and high degree of customization. They have a broad application protector in biomedical fields such as therapeutic drug delivery and the construction of TC repair discoveries. In this study, we prepared hydrogen microspheres using a simple microfluidic device.Recently we have been trying to use hydrogen microspheres loaded with some small molecules from traditional Chinese medicine to promote all the healing. In this study, we found that zinc and copper ion cross-link the sodium alginate microspheres have good anti-microbial properties and thus copper ARGMS, and zinc ARGMS are suitable for the treatment of bacterial infectious diseases.

Characterization of ALGMS cross-linked with different metal ions 
The optical morphology of Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS is shown in Figure 2, exhibiting good sphericity, smooth surface, uniform particle size distribution (Supplementary Figure 2) and excellent monodispersity. We further performed microscopic characterization using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. As shown in <strong clas...

The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

preparation-cross-linked-sodium-alginate-microspheres-with-different

Research

JoVE Journal

Bioengineering

Creating Alginate Hydrogel Microspheres Using Microfluidic Electrospray for Controlled Drug Release

To begin, mix ALG with ultrapure water in a water bath, tempered at 50 degrees Celsius. Separately, prepare mass fraction with 5%solutions of calcium chloride, ferric chloride, zinc sulfate, and copper sulfate in ultrapure water. Pour each solution separately into a collecting dish.Place a glass capillary tube over a blowtorch flame to soften it and stretch it to various diameters. With a glass cutter, cut off the fine parts and gently polish the tip port with silicon carbide sandpaper. Connect the thick end of the capillary tube to a long tubing and the other end to a dispensing needle in a five-milliliter syringe.Attach the syringe to the microfluidic syringe pump and the capillary tubing at one end to the holder. Connect the red high-voltage end clip of the high-voltage power supply to the dispensing needle of the syringe and place the silver clip in the collection fluid. This configuration creates a simple microfluidic electrospray device.Place the collection dishes containing different metal ions directly under the capillary tube. Turn on the microfluidic syringe pump. Select fast forward to prime the tube with the ALG and set the flow rate to two milliliters per hour.Switch on the high-voltage power switch and turn the knob to set the voltage to five to seven kilovolts. Collect ALG microspheres cross-linked with different metal ions. After the formation of ALG hydrogel microspheres, transfer them to individual labeled 1.5-milliliter centrifuge tubes.The micrographs of ALG microspheres showed good sphericity, smooth surface, and uniform particle size distribution. Scanning electron microscopy showed that the microspheres were generally spherical with well-defined roundness.

Biocompatibility Tests to Assess the Use of Sodium Alginate Hydrogel Microspheres for Drug Delivery

To begin, prepare zinc, calcium, copper, and ferric alginate hydrogel micro-spheres. For the antimicrobial performance test, add 10 milliliters of each of the two bacterial solutions into a 15 milliliter sterile centrifuge tube. Then incubate the cultures with 0.5 milliliters of hydrogel microspheres in a constant temperature shaker.Dilute each bacterial solution, 10 to the power of five times, with sterile water. Using sterile glass beads, evenly spread 200 microliters of bacterial solution on an LB solid medium. Place the plates in an incubator at 37 degrees Celsius for 12 hours to assess colony formation.Add one milligram per milliliter of bovine serum albumin or BSA suspension, to 500 microliters of each microsphere and incubate for 24 hours. Add each saturated sample to one milliliter PBS, and agitate for 15 minutes at 37 degrees Celsius with continuous shaking at 80 revolutions per minute. After aliquoting, replace the medium with a fresh one at one, three, six, 12, 24, 36, and 48 hours.Quantify the supernatant protein concentrations at specific times. Add the prepared ALG microspheres to PBS, and incubate them at 37 degrees Celsius for 24 hours. Obtain whole blood from healthy mice in anticoagulation tubes containing sodium citrate.After vortexing centrifuge the tube for 15 minutes at 1, 500 revolutions per minute to obtain red blood cells. After flushing the cells, prepare a 10%red blood cell solution with PBS, and add 20 microliters of blood cell solution to the microsphere experimental and control groups. Incubate the samples at 37 degrees Celsius for four hours, and centrifuge them at 1, 500 RPM for 15 minutes.After keeping the supernatant aside, photograph the red blood cells in each group following hemolysis. Finally, measure the absorbance values of the supernatants in each group and compute the hemolysis rate using the formula. Different microspheres exhibited antibacterial activity against staphylococcus aureus and escherichia coli with copper and zinc, ALG microspheres, showing the strongest antibacterial properties.The drug release assay revealed that the release rate of the iron microsphere was relatively faster than that of the other three ions. Biocompatibility assessment showed that the red blood cells in the suspension remained intact upon contact with different microspheres, indicating minimal hemolysis by the microspheres.

891 Views.  Zhejiang Shuren University.  The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Developing Sodium Alginate Hydrogel Microspheres for Biomedical Use

Summary