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

Summary

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

Abstract

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 ± 0.01 mm and spherical shape of 0.03 ± 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% ± 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.

Introduction

Black seed, and more especially BSO, has been used for ages to cure a wide range of illnesses due to its well-established medicinal properties. Thymoquinone is perhaps one of the most important phytochemicals found in BSO¹. In recent years, researchers have studied thymoquinone's potential therapeutic benefits in vivo and in vitro, producing empirical evidence to support the use of BSO. Antihypertensive, antibacterial, antihistaminic, antifungal, analgesic, antidiabetic, lipid-lowering, and anti-inflammatory properties have all been demonstrated by these studies for BSO, which may be used to treat symptoms such as eczema, high blood pressure, asthma, cough, headache, influenza, fever, anticancer, dizziness, and activity^2,3.

Applying relatively thin coverings on small droplets of liquids and dispersions, or particles of solid material, is known as microencapsulation. When it comes to oil, microencapsulated oil is usually quite valuable because some forms of oil, like BSO, are regarded as nutritious foods and offer medicinal advantages⁴. However, adding oils directly to the food's matrix may lead to volatilization, which can quickly cause activities to disappear as a result of exposure to oxygen and UV light⁵. Furthermore, the lack of control over the oils' rate of release results in an immediate and transient effect. Creating a polymeric coating around the essential oil by microencapsulation or microspherification is one method to get past these drawbacks⁶.

Microcapsules, also known as microspheres, shield the oils from harmful environmental conditions⁷. This process has been widely used to increase drug efficacy, preserve drug contents, enable time-released tablets, improve taste masking, reduce flavor loss during product shelf life, prolong mouthfeel, and separate incompatible ingredients in a single dosage⁸. Microencapsulation also aids in maintaining metabolic absorption, controlling the rate of oil release, and maintaining the appropriate concentrations to yield the intended result at a particular location⁹.

Electro-hydrodynamic encapsulation is a straightforward and adaptable method. The active substance is housed in the inner core of a microcapsule, which is composed of an exterior shell. In this regard, it features a fairly strong matrix to guarantee that the active component can be disseminated more effectively rather than a clearly defined nucleus. Prior to sphericyclation, the active substance and polymer solution must be combined to produce the microspheres⁹. On the other hand, because oil is volatile, microencapsulating it can be extremely difficult and requires careful temperature control.

There are various methods for encapsulating oils. For example, certain oils need to be encapsulated at low temperatures to prevent the breakdown or volatilization of their bioactive components. To create micro- and nano-sized structures, electrohydrodynamic atomization (EHDA) has been extensively studied by researchers¹⁰. In this sense, the processing conditions, which include flow rate, applied voltage, and nozzle size, as well as the collection distance properties of the polymeric solution, are the two primary factors that must be taken into account to produce the desired particle size or morphology^11,12.

In this investigation, alginates -- a type of naturally occurring polysaccharides fit for oral ingestion -- were used to encapsulate the BSO. Brown seaweeds contain alginate, an anionic polymer that occurs naturally. It is made up of two monomeric structures: α-L-guluronic (G) and 1-4βD-mannuronic (M) acid¹³. Its polymer is non-toxic¹⁴, has a high degree of biocompatibility, is inexpensive, and degrades effectively¹⁵. It is, therefore, frequently employed in the biotechnology and engineering sectors.

Alginates are the material of choice for encapsulation by ionic gelation because they may create a cross-linked structure between the G groups of various alginate chains by forming ionic connections with divalent cations like Sr²⁺, Ca²⁺, or Zn²⁺ ions. The gelation process can be adequately characterized by the egg-box model, which limits the divalent cation to two carboxyl groups on the side-by-side alginate molecules. It has been suggested that the hydrogel characteristics of sodium alginate beads may regulate the release of macromolecules and small molecules. The sodium alginate beads may cling to the intestinal mucosa for an extended length of time due to their muco-adhesive qualities. Moreover, alginate offers a shield that may shield oils from external elements such as acidic media¹⁶ and transfers oils into the delivery channels of the gastrointestinal tract¹⁷. It has since been employed in research to aid in the site-specific administration of medication to mucosal tissues^18,19.

The electro-hydrodynamic approach was used in this study to investigate the viability of emulsifying commercial oils to create capsules²⁰. Here, the electro-hydrodynamic approach was used to generate and analyze alginate-BSO-loaded microspheres²⁰. This study evaluated a number of other factors, including the microspheres' SF, ex-vivo, muco-adhesive properties, EE%, physical appearance, size distribution, and zeta potential; attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was utilized to test for chemical compatibility²⁰.

Protocol

1. Preparation of alginate-BSO emulsion

1. Disperse 10% w/v BSO in 1% w/v sodium alginate solution containing 1, 3, and 5% w/v lecithin in a 50 mL beaker.
2. Obtain a nano-emulsion by using an ultrasonic homogenizer. Set the power level to 20%. Run the homogenizer for 55 s by clicking the start button to complete the process.

2. Characterization of alginate-BSO emulsion

1. Analyze the zeta potential and particle size distribution
   1. Take 0.1 mL of the freshly prepared emulsion in a 25 mL glass beaker and dilute it with 9.9 mL of distilled water.
   2. Take 2.5 mL of this diluted solution into a 3 mL quartz-cuvette and place the cuvette in a measuring chamber.
   3. Open the cover and place the cuvette inside the device, making sure the cuvette is oriented correctly with respect to the path of the light beam. Click the measurement icon.
   4. Take out the cuvette. Retrieve the sample or properly dispose of it.
   5. Save the data as a pdf file in a personal folder for further use.
2. Determine emulsion stability (ES)
   1. Take 5 mL of freshly prepared emulsion in 10 mL centrifuge tubes. Centrifuge the emulsions (n = 3) for 5 min at 894 × g.
   2. Using equation (1), determine the ES based on the phase separation interface position.
          (1)
      Where Vemul is the volume of remaining emulsion after centrifugation and Vinitial is the volume of initial emulsion.
3. Bead preparation
   1. BSO alginate beads
      1. Prepare BSO alginate beads using the electrospray technique called EHDA. Employ a weight by volume (w/v) BSO emulsion that is composed of 10% BSO, 1% sodium alginate, and 3% lecithin solution).
      2. Using a syringe pump to regulate the flow rate, load the emulsion into a 10 mL plastic syringe and push it through a 22 G needle. Attach the needle tip to the positive electrode of a high voltage power supply.
      3. As the collector, use a grounded beaker with 50 mL of 1% w/w calcium chloride (gelling bath) in it. Alternate the dripping flow rate between 1 mL/min and 3 mL/min at voltages of 3, 5, and 7 kV while maintaining a distance of 10 cm above the calcium chloride solution's surface.
      4. To confirm full gelation, leave the beads in the gelling bath for 30 min while agitating them. Use a stainless-steel filter to remove the beads from the gelling bath and wash the gathered beads with ultrapure distilled water.
      5. Let the beads dry for 16 h at room temperature on a lab bench. Use Equation (2) to calculate the percentage yield of beads.
         (2)
   2. BSO-free alginate beads
      1. Prepare a 1% w/v solution of sodium alginate. Add lecithin at concentrations of 1%, 3%, and 5% w/v to the solution. Mix the solution thoroughly until the lecithin is dissolved completely.
      2. Use the solution from step 2.3.2.1 to prepare BSO-free alginate beads as described in steps 2.3.1.2-2.3.1.5. Calculate the yield using equation (2).

3. Bead characterization

1. Determination of size and shape
   1. To ascertain the size and shape of the beads, use an image analyzer. Take photos using a digital camera of the wet and dried beads.
   2. Then, measure bead diameter using the instrument's preinstalled scale bar. Using the diameter values, calculate the SF from the obtained diameter values using equation (3):
      SF =     (3)
      Where Dmax represents the largest diameter that goes through the center of a bead (in mm), while Dper refers to the diameter that is perpendicular to Dmax and passes through the bead's center (in mm).
      NOTE: An SF of zero denotes an ideally spherical bead, with increasing SF values signifying greater deviation from a spherical shape. Furthermore, beads are considered spherical if their SF is 0.05 or lower.

4. Determination of EE%

1. Disintegrate the beads in phosphate-buffered saline (PBS) to return them into emulsion. Measure the absorbance of the resulting emulsion at 600 nm using a UV-vis spectrophotometer.
2. Use the absorbance value to represent the turbidity of the emulsion. Create a standard curve using a known amount of BSO in the emulsion.
3. Calculate the EE% using equation (4):
       (4)

5. Scanning electron microscopy (SEM)

NOTE: Use SEM to observe the microstructure and surface morphology of the BSO alginate beads.

1. To inspect the inside of the dried beads, cut a few of them. Spot the cut beads onto aluminum stubs and paste them using carbon adhesive tapes.
2. Sputter-coat the beads with a carbon sputter module in a vacuum evaporator in an argon atmosphere. Apply a thickness of 100 Å and 50 Å for the carbon coating.
3. Acquire images of the coated beads at high vacuum with a voltage accelerator between 10 kV and 15 kV.

6. Determine drug-excipient interaction using ATR-FTIR

1. Set the instrument wave numbers between 4,000 cm⁻¹ and 400 cm⁻¹ using ambient air as a background and a resolution of 1 cm⁻¹. See Supplemental File 1.
2. Record spectra of BSO, sodium alginate, lecithin, calcium chloride, BSO-free beads, alginate-BSO beads, and physical mixture of active ingredient and excipients (sodium alginate, lecithin, calcium chloride, and BSO) separately.
   1. Place the sample (5-10 mg) on the sampling area. Adjust 20 scans, resolution 4, gauge force 80, pressure arm with a flat tip. Verify that the Auto Increment remains set to the Blank option so that the spectrum is automatically stored in the desired folder.
   2. To begin the sample measurement, click the [Sample] button. After selecting [Sample], since there is no waiting period, be sure to have the sample ready and the pressure clamp reduced. Analyze all the samples individually. Individually analyze all the recorded spectra using spectroscopy software.

7. Differential scanning calorimetry (DSC)

NOTE: The thermal properties and compatibility of the BSO-loaded beads were investigated using DSC (Supplemental File 1).

1. Seal beads weighing ~3.20 mg in a regular aluminum pan. Heat the samples at a rate of 10 °C/min while being analyzed at a temperature range of 50-350 °C under a stream of nitrogen flowing at a rate of 20 L/min.

8. Swelling characteristics of beads

1. Prepare 100 mg of dried alginate-BSO beads.
2. Prepare simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) in a clean, dry mixing vessel of a suitable size-6 L, 10 L or 25 L. Add purified water to approximately 33% of the required volume-2 L, 3 L or 8 L, and transfer the contents of the bottle of concentrate into the vessel. Rinse the bottle with purified water and add the rinses and purified water to the mixing vessel to obtain the required volume; mix thoroughly. Measure the pH and proceed if within specification; adjust the pH if necessary.
3. Submerge the beads in 50 mL of media containing simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). Maintain the conditions for 2 h at 37 ± 0.5 °C.
   1. Remove the inflated beads and filter them through a metal mesh at predefined intervals of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min or more. Use a paper towel to remove extra fluid from the swollen beads.
   2. Measure the weight of wiped beads using an electronic analytical balance. Determine the percentage of swelling index (%SI) using equation (5):
      (5)

Results

Preparation of BSO-loaded alginate microbeads
Figure 1 represents the experimental setup to prepare BSO-loaded alginate microbeads. The amount of lecithin utilized had a considerable impact on the stability of the BSO emulsion. Emulsions made with all three lecithin concentrations were comparatively stable. The centrifugation method (894 × g, 5 min) was used in this experiment to assess the stability of the emulsion²⁰. The res...

Discussion

Using the EHDA process, BSO-loaded alginate microbeads were created as a pH-sensitive carrier. The beads' network exhibited pH-dependent swelling and drug-release behavior due to the abundant presence of carboxylic acid groups. The strong intermolecular hydrogen bonding between the polymer chains was revealed to be the reason behind the decreased swelling character of BSO-loaded beads at pH 1.2. BSO-loaded beads may benefit from this reduced SB for pH-sensitive medication administration. pH had an impact on the BSO r...

Materials

Name	Company	Catalog Number	Comments
10 mL Centrifuge Tubes	Globe Scientific	22-171-624
22 G needle	Sigma-Aldrich (St.Louis, Missouri, USA).	CAD4172
3 mL quartz-cuvette	Sigma-Aldrich (St.Louis, Missouri, USA).	Z276669
50 mL beaker
Aluminum stubs
An electronic analytical balance
ATR-FTIR	Bruker Malaysia Sdn Bhd, Kawasan Perindustrian Temasya, 40150 Shah Alam, Selangor, Malaysia.
Black seed oil	IKOP Pharmaceutical Ltd. (IKOP, Faculty of Pharmacy, IIUM, 25200 Kuantan, Pahang, Malaysia	B182111	Active ingredient
Calcium chloride dehydrate, CaCl2 · 2H2O	Sigma-Aldrich (St.Louis, Missouri, USA).	21074	Gelling agent
Carbon adhesive tapes
Centrifuge
Differential scanning calorimetry
Digital   camera
Grounded beaker
High guluronic acid content Sodium alginate (mw. 97,000) with medium viscosity (40 – 100 mPa s)	Sigma-Aldrich (St.Louis, Missouri, USA).	W201502	Polymer
High voltage power supply
Isopropyl alcohol	Sigma-Aldrich (St.Louis, Missouri, USA).	W292912	ATR-FTIR cleaning purpose
Lecithin	Sigma-Aldrich (St.Louis, Missouri, USA).	P7568	Surfactant
Microscope
Paper towel
Scanning electron microscopy
Simulated gastric fluid	Sigma-Aldrich (St.Louis, Missouri, USA).	1651	Release media and swelling media
Simulated intestinal fluid	Sigma-Aldrich (St.Louis, Missouri, USA).	84082-64-4	Release media and swelling media
Spectroscopy software
Stainless-steel filter
Syringe pump	SEN JIN SDN BHD Malaysia, Taman Desaria, 46150 Petaling Jaya, Selangor Darul Ehsan Malaysia
Ultrapure distilled water	Supplied by institutional lab
Ultrasonic homogenizer	SEN JIN SDN BHD Malaysia, Taman Desaria, 46150 Petaling Jaya, Selangor Darul Ehsan Malaysia
UV-vis spectrophotometer.
Vacuum evaporator	SEN JIN SDN BHD Malaysia, Taman Desaria, 46150 Petaling Jaya, Selangor Darul Ehsan Malaysia
Voltage accelerator	SEN JIN SDN BHD Malaysia, Taman Desaria, 46150 Petaling Jaya, Selangor Darul Ehsan Malaysia
Zetasizer Nano-ZS	(Malvern Zetasizer Nano series Nano-S and Nano-Z, Malvern Instruments Ltd., Worcestershire, UK)