Name: preparation of cross-linked sodium alginate microspheres with different metal ions using the microfluidic electrospray technology
Uploaded: 2024-06-07T14:40:00.000+00:00
Duration: 7 min 24 s
Description: Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

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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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><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&amp;nbsp; 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&amp;nbsp; 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&amp;nbsp; 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&amp;nbsp;</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.

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...

Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

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.

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 ...

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

Research

JoVE Journal

Bioengineering

1.6K 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.

Video: Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Chemistry

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicine

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...