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

Abstract

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.

Introduction

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.

Protocol

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

1. Weigh ALG and dissolve in ultrapure water by stirring in a water bath at 50 °C to obtain 2% ALG solution.
2. 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.

2. Microfluidic electrospray device

1. Take a glass capillary tube and place it over a blowtorch flame to soften, then stretch it to various diameters (50 µm-500 µm). 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.
2. 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.

3. ALG microspheres preparation

1. 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).
2. Switch on the high voltage power switch and turn the knob to set the appropriate voltage (5-7 kV).
3. 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).
4. 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.
5. Take a small amount of the prepared microspheres, disperse them as evenly as possible in PBS, and place them under a microscope for visualization.
6. 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.

4. Antimicrobial performance test

1. 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.
2. 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 °C and 200 rpm for 12 h. Add an equal amount of saline to the control group.
3. Dilute each bacterial solution to 10⁵ times with sterile water. Use a sterile glass bead to spread 200 µL of bacterial solution on an LB solid medium.
4. 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 °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.

5. Drug release testing

1. To evaluate the drug release ability of different microspheres, use bovine serum albumin (BSA) as a loaded model drug. Add 500 µL of each microsphere (Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS) to 1 mg/mL BSA suspension for 24 h.
2. 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 °C with continuous shaking at 80 rpm.
3. Use 100 µL of the solution and replace the medium with a fresh one at 1, 3, 6, 12, 24, 36, and 48 h.
4. Quantify the supernatant protein concentrations at specific times using a BCA kit per the manufacturer'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%
5. At regular intervals, remove an equal volume of PBS from each well and replace it with an equal volume of fresh medium.

6. Hemolysis test

1. Place the prepared Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS in PBS and incubate them at 37 °C for 24 h to formulate the incubation solution.
2. 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.
3. Flush the red blood cells 2x-3x with PBS and prepare a 10% red blood cell solution with PBS.
4. Preparation of various ALGMS: Take 500 µL 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 µL of blood cell solution.
5. Incubate the samples at 37 °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.
6. 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

7. Cytobiocompatibility test

1. Wash the different ALGMS 2x with PBS and place in a 5 mL Petri dish for 15 min and discard the supernatant.
2. Separately add different ALGMS at 0.2 mL in complete culture medium (DMEM) containing 10% FBS, incubate at 37 °C for 24 h, and filter the solution to obtain the leachate.
3. 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.
4. Remove the cell culture medium and wash the cells with PBS.
5. Assess cell viability using Calcein-AM/PI assay. Take 2.5 µL of Calcein-AM solution (4 mM) and 12.5 µL of PI solution (2 mM) and add them to 5 mL of 1x PBS and mix well to obtain a working solution with 2 µM Calcein-AM and 5 µM PI.
6. Add the working solution to previously seeded cell culture plates at 500 µL per well, incubate at 37 °C under light protection for 15 min, and observe and photograph under an inverted fluorescence microscope. Set up three replicates for each group.
7. Calculate cell viability according to the following formula:
   Cell viability (%) = number of viable cells / (number of viable cells + number of dead cells)

Representative Results

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 Figure 3, the microspheres were generally spherical with well-defined roundness. The surface of Zn-ALGMS was unevenly distributed, appearing rougher with many wrinkles. We performed energy dispersive spectroscopy to determine the content distribution of metal ions involved in the cross-linking reaction in the gel. Notably, the microsphere size can be adjusted by changing parameters such as the collection distance, gel concentration, and electric field voltage¹². In the outlined protocol, by adjusting the parameters of the microfluidic device and the liquid concentration, particles of different sizes can be easily obtained according to specific requirements.

Assessment of antimicrobial properties
We evaluated the antimicrobial capacity of different microspheres using the plate method, as shown in Figure 4. Different microspheres exhibited antibacterial activity against E. coli and S. aureus, with Cu-ALGMS and Zn-ALGMS showing the strongest antibacterial properties. This heightened effectiveness can be attributed to the antimicrobial activity of metals, namely copper (Cu) and zinc (Zn)¹³. Sukhodub et al. demonstrated that Fe³⁺, Zn²⁺, Ca²⁺, and Cu²⁺ exhibited synergistic antibacterial effects with chitosan, while the samples without chitosan showed no such activity, validating the synergistic antibacterial effect of the complexes formed¹⁴. The results obtained align with this study, with Cu-ALGMS and Zn-ALGMS being superior to other hydrogel microspheres in the treatment of bacterial infectious diseases.

Evaluation of drug release properties
The evaluation of the drug release from different metal-based alginate hydrogel microspheres using BSA as the model drug revealed differences in their release profiles. (Supplementary Figure 3). Ions exhibited a better slow drug-release ability than those of other materials.The drug-release rate of Fe²⁺ was relatively faster than that of the other three ions, whereas the drug-release rate of Ca²⁺ and Zn²⁺ was relatively slower. These results highlight the differences in the effects of different ions on drug release. We hypothesize that Fe²⁺ possibly interacts with the drug or binds in a way that makes it easier to release, whereas Ca²⁺ and Zn²⁺ bind to the drug more tightly, or there are other factors that limit the rate of release. This sustained drug release from the hydrogel microspheres may be related to the cross-linking strength between the metal and the alginate polysaccharide. In addition, the difference in the adsorption capacity of different metals compared to that of BSA likely contributed to the differences observed in drug retardation abilities.

Biocompatibility assessment
Good biocompatibility is a prerequisite for drug delivery carriers in clinical applications. Therefore, we evaluated the hemocompatibility of microspheres using an in vitro hemolysis test. We used pure water as the positive control and PBS solution as the negative control. The experimental results are shown in Supplementary Figure 4, revealing that the red blood cells in the suspension remained intact upon contact with different microspheres, indicating minimal hemolysis by the microspheres. Cytobiocompatibility results showed that the microspheres did not affect cellular activity (Supplementary Figure 5). These results indicated that the microspheres have good blood cell compatibility.

Figure 1: Alginate hydrogel microspheres preparation. (A) Installation of microfluidic electrospray technology. (B) The real-time image of the microfluidic electrospray process. (C) The prepared Ca²⁺, Cu²⁺, Zⁿ²⁺, and Fe³⁺ alginate hydrogel microspheres. Please click here to view a larger version of this figure.

Figure 2: Alginate hydrogel microspheres micrograph. Micrograph of (A) Ca-ALGMS, (B) Cu-ALGMS, (C) Zn-ALGMS, and (D) Fe-ALGMS in PBS (pH 7.4). Please click here to view a larger version of this figure.

Figure 3: Scanning electron microscopy and energy dispersive spectroscopy. The images show the characterization of (A) Ca-ALGMS, (B) Cu-ALGMS, (C) Zn-ALGMS, and (D) Fe-ALGMS with i and ii for scanning microscopy data and iii-v for spectroscopy data. The images iii-v show an EDS mapping, in which the EDS selects a face on the sample surface to scan to obtain elemental distribution information over the entire area. Map sweep mode is used in applications for compositional analysis, phase zone analysis, and particle size distribution of materials, where each element is represented by a different color, as shown. Please click here to view a larger version of this figure.

Figure 4. Antimicrobial properties of microspheres. (A) The antimicrobial properties of the groups were tested using the bacterial smear method. (B, C) Quantification of the bacterial smear plate count results for each group. The control samples show colonies grown on LB medium without any addition. The relative colonies for the other bacteria were calculated by taking the control group's clone count as 100% and using it as a baseline. The error bar: standard deviation, n = 3. Please click here to view a larger version of this figure.

Supplementary Figure 1: A glass tube is connected to the syringe through a long rubber tube. Please click here to download this File.

Supplementary Figure 2: Particle size. (A) Zn-ALGMS, (B) Ca-ALGMS, (C) Cu-ALGMS, (D) Fe-ALGMS. A molar concentration of 5% was used for all the samples. Please click here to download this File.

Supplementary Figure 3: Drug release. The drug release curve of Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS in PBS (pH 7.4). The error bar: standard deviation, n = 3 Please click here to download this File.

Supplementary Figure 4: Hemolysis assay of Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS. PC (Positive Control): ddH₂O; NC (Negative Control): PBS. The error bar: standard deviation, n = 3. Please click here to download this File.

Supplementary Figure 5: Cytotoxicity of microspheres cross-linked with different ions. The biocompatibility assessment of Zn-ALGMS, Ca-ALGMS, Cu-ALGMS, and Fe-ALGMS was done. Calcein-AM/PI was used to perform the test, and for the results here, 5 fields of view were randomly selected. ImageJ was used to analyze the ratio of red blood cells to dead cells to obtain relative cell viability. 1.PC, Positive Control, 2.NC, Negative Control, 3. Zn-ALGMS, 4. Ca-ALGMS, 5. Cu-ALGMS, 6. Fe-ALGMS, The error bar: standard deviation, n = 3. Please click here to download this File.

Discussion

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 exhibited significant bacterial inhibitory effects.

Hydrogel microspheres are flexible microstructures with excellent plasticity, enabling effective drug delivery through specific material selection and structural design. Alginate, a natural polysaccharide that gels under mild conditions is biocompatible, biodegradable, and non-toxic. Micron-scale sodium alginate microspheres are widely used in biochemistry and biomedicine, including pharmaceutical research, drug delivery, and tissue engineering. A key advantage of sodium alginate microspheres in drug delivery applications is their ability for sustained drug release, ensuring the maintenance of high concentrations of the active pharmaceutical ingredient over an extended duration¹⁴. Our sustained drug release experiments revealed that ALGMS with different ionic cross-linking have the ability to sustain drug release and can be used for drug delivery applications.

Common fabrication methods for hydrogel microspheres include batch emulsification, mechanical comminution, lithography, microfluidic emulsion, and electrospray^8,15. The advantages of batch emulsification and mechanical crushing methods lie in the simplicity of the fabrication method and high output efficiency. However, it is difficult to control the shape of individual hydrogel microspheres using these methods. Photolithography involves focusing light on a mask plate or mold to cure cross-linking and form hydrogel microspheres. However, the production efficiency of this method is easily affected by the mask plate, light source, and mold. Moreover, the microfluidic emulsion method can better control the molding process of hydrogel microspheres and produce hydrogel microspheres with specified shapes; however, its production efficiency is low¹⁶. Compared to these methods, the microfluidic electrospray method can produce hydrogel microspheres with uniform size, regular shape, and adjustable particle size¹⁷. The key step in this protocol is the preparation of the microspheres. The size and shape of the microspheres may vary depending on the flatness of the glass tube, the voltage value, and the flow rate¹⁸. There are also some limitations in using microfluidic electrospray technology to prepare microspheres, such as the tendency of microchannels to clog and the limited yield, the limited compatibility of materials and the difficulty of process scale-up, and the difficulty of process control and monitoring. This limits their widespread application to some extent¹⁹.

In summary, microfluidic electrospray technology is a mature microsphere preparation method, offering the advantage of one-step synthesis. By optimizing the flow channel size parameters and controlling the material concentration and flow rate, microspheres of various morphologies and sizes can be easily obtained. Notably, the process involves only the aqueous phase and no additional surfactants, rendering it environmentally friendly and simple to operate, avoiding the need for complex washing processes. This method provides a strategy for producing microspheres from hydrogel materials similar to the alginate gelation process.

Materials

Name	Company	Catalog Number	Comments
120 mesh screen	Solarbio,China	YA0946
Alcohol burner	Solarbio,China	YA2320
BALB/c mice	Wukong Biotechnology,China
Bicinchoninic Acid Assay reagent	Meilunbio,China	MA0082
Bovine Serum Albumin	Lablead,China	9048-46-8
CaCl2  powder	Aladdin,China	10043-52-4
Calcein-AM/PI	Biosharp,China	BL130A
Centrifuge tubes	Corning,America	430290
CuSO4  powder	Jnxinyuehuagong,China	7758-99-8
DMEM	Gibicol,China	C11995500BT
FeCl3  powder	Aladdin,China	7705-08-0
Fetal Bovine Serum	HAKATA,China	HN-FBS
Glass tubes	Sartorius,Germany	CC0028
Light microscopy	Evidentscientific,Japan	BX53(LED)
Microfluidic syringe pump	Longerpump,England	LSP01-3A
NIH3T3	HyGyte,China	TCM-C752
Petri dish	Thermofisher,America	150464
Phosphate buffer saline	Thermofisher,America	3002
Scanning electron microscope	Thermofisher,America	Axia ChemiSEM
Sodium alginate powder	Bjbalb,China	Y13095
ZnSO4 powder	Jnxinyuehuagong,China	7733-02-0