*4.1.6 Techniques based on drop generation method*

Mechanical means are used for droplets generation during co-extrusion and has given rise to many modern encapsulation techniques. These all depend on the dripping and jet break up principle for droplet generation at an orifice or from a laminar jet. A droplet that forms at an orifice and is discharged is a result of a formation process that depends on the interplay of surface tension of the extruded liquid, velocity of extrusion, gravitational force, impulse and frictional forces. Configurations for droplet generation are based on five mechanisms.


**15**

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic…*

E. C could also result in droplet formation because of high frictional forces when

Encapsulation methods based on these mechanisms and on the droplet genera-

Generally, in microfluidic devices, several configurations are obtainable for droplet generation and include co-flowing, T-junction and fluid focusing [49]. The fluid focusing configuration is particularly advantageous since it is passive and droplet generation and cell encapsulation depends on hydrodynamically pumping fluid adjacent to an outflowing cell and can therefore prove useful for sensitive materials such as living cells for probiotics [46, 50]. In fluid focusing, the focused fluid (disperse phase) is introduced into a capillary tube enclosed in a chamber containing the focusing fluid (carrier/ continuous phase) which exerts pressure on the focused fluid as it exits the orifice facing the feeding tube. The pressure exerted on the disperse phase, compounded by fluid instabilities, is sufficient to cause it to break into droplets as it squeezes pass the orifice [51]. The droplet size of the internal phase does not depend on the orifice diameter. Device geometry, fluid properties and the process parameters such as flow rate and

pressure drop determine what happens as the internal phase emerges.

found application in the facile preparation of double emulsions.

For microencapsulation applications requiring the generation of microsized droplets of narrow size distribution, it is important that these parameters be tuned such that the carrier phase acts as micro tweezers that pressure the tip of the disperse phase meniscus at the orifice causing it to break into a microjet that eventually breaks into homogenous small droplets [52]. Microfluidic devices have

Spinning disc: In this technique, also known as centrifugal suspension separation, drops are generated when coated particles are flung off a rotating disc by the generated centrifugal force. The core material usually in solid form is suspended in a viscous coating liquid and poured on top of the rotating disc. The suspension

Vibrating nozzle/jet: This encapsulation technique, commercialized by Inotech Biotechnoly Ltd. and Nisco Ltd., makes use of permanent vibrational or sinusoidal frequencies of definite amplitude to break up a laminar jet into equally sized droplets stabilized by electrostatic repulsion and achieved by application of an electric field [45]. Two variations are the vibrating nozzle and the vibrating chamber techniques. The size distribution of the droplets is narrow and the size generated with a given amplitude is in the range of 0.10–1.50 mm and depends on the nozzle diameter, jet velocity, rheology and surface tension of the liquid [46]. It is predominantly used in cell immobilization with Newtonian systems. Dorati and coworkers undertook an assessment of the vibrating nozzle technique combined with freeze drying technique in the encapsulation of a model hydrophilic molecules in a hydrophilic polymer, alginate. They concluded that vibrating nozzle technique is an easy and scalable process for microencapsulation of hydrophilic drugs [47]. Simple dripping: This simple method involves the free formation of a droplet at the orifice. The drop continues to increase in volume until the weight of the liquid just exceed the capillary force. The drop detaches and forms a sphere due to surface tension. This method of low droplet production rate is mostly applicable to laboratory encapsulation with droplets sizes approximately 1000 μm. It has found use in microfluidic devices and in porous membranes such as Shirasu porous glass in which high pressure is applied to cause the droplet generation of a disperse phase directly into the continuous phase held in the membrane [48]. These membranes are used for generation of emulsions and mini emulsions on a lab scale. The membrane pore size directly controls the droplet size. The membranes are hydrophilic and therefore, favors oil-in- water emulsions formation. For an oil continuous phase, there may be a need to coat the membrane using silicone resins [46].

*DOI: http://dx.doi.org/10.5772/intechopen.94856*

a jet is sprayed.

tion method are elucidated below.

**Figure 5.** *A schematic representation of the centrifugal extrusion device.*

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.94856*

E. C could also result in droplet formation because of high frictional forces when a jet is sprayed.

Encapsulation methods based on these mechanisms and on the droplet generation method are elucidated below.

Vibrating nozzle/jet: This encapsulation technique, commercialized by Inotech Biotechnoly Ltd. and Nisco Ltd., makes use of permanent vibrational or sinusoidal frequencies of definite amplitude to break up a laminar jet into equally sized droplets stabilized by electrostatic repulsion and achieved by application of an electric field [45]. Two variations are the vibrating nozzle and the vibrating chamber techniques. The size distribution of the droplets is narrow and the size generated with a given amplitude is in the range of 0.10–1.50 mm and depends on the nozzle diameter, jet velocity, rheology and surface tension of the liquid [46]. It is predominantly used in cell immobilization with Newtonian systems. Dorati and coworkers undertook an assessment of the vibrating nozzle technique combined with freeze drying technique in the encapsulation of a model hydrophilic molecules in a hydrophilic polymer, alginate. They concluded that vibrating nozzle technique is an easy and scalable process for microencapsulation of hydrophilic drugs [47].

Simple dripping: This simple method involves the free formation of a droplet at the orifice. The drop continues to increase in volume until the weight of the liquid just exceed the capillary force. The drop detaches and forms a sphere due to surface tension. This method of low droplet production rate is mostly applicable to laboratory encapsulation with droplets sizes approximately 1000 μm. It has found use in microfluidic devices and in porous membranes such as Shirasu porous glass in which high pressure is applied to cause the droplet generation of a disperse phase directly into the continuous phase held in the membrane [48]. These membranes are used for generation of emulsions and mini emulsions on a lab scale. The membrane pore size directly controls the droplet size. The membranes are hydrophilic and therefore, favors oil-in- water emulsions formation. For an oil continuous phase, there may be a need to coat the membrane using silicone resins [46].

Generally, in microfluidic devices, several configurations are obtainable for droplet generation and include co-flowing, T-junction and fluid focusing [49]. The fluid focusing configuration is particularly advantageous since it is passive and droplet generation and cell encapsulation depends on hydrodynamically pumping fluid adjacent to an outflowing cell and can therefore prove useful for sensitive materials such as living cells for probiotics [46, 50]. In fluid focusing, the focused fluid (disperse phase) is introduced into a capillary tube enclosed in a chamber containing the focusing fluid (carrier/ continuous phase) which exerts pressure on the focused fluid as it exits the orifice facing the feeding tube. The pressure exerted on the disperse phase, compounded by fluid instabilities, is sufficient to cause it to break into droplets as it squeezes pass the orifice [51]. The droplet size of the internal phase does not depend on the orifice diameter. Device geometry, fluid properties and the process parameters such as flow rate and pressure drop determine what happens as the internal phase emerges.

For microencapsulation applications requiring the generation of microsized droplets of narrow size distribution, it is important that these parameters be tuned such that the carrier phase acts as micro tweezers that pressure the tip of the disperse phase meniscus at the orifice causing it to break into a microjet that eventually breaks into homogenous small droplets [52]. Microfluidic devices have found application in the facile preparation of double emulsions.

Spinning disc: In this technique, also known as centrifugal suspension separation, drops are generated when coated particles are flung off a rotating disc by the generated centrifugal force. The core material usually in solid form is suspended in a viscous coating liquid and poured on top of the rotating disc. The suspension

*Nano- and Microencapsulation - Techniques and Applications*

for the probiotic provided additional stability.

*4.1.6 Techniques based on drop generation method*

droplet generation are based on five mechanisms.

spiral symmetrical vibrations.

*A schematic representation of the centrifugal extrusion device.*

vibration.

forming polymer which is usually cured by ionic interactions with divalent cations. Silva and colleagues [44] compared the extrusion technique with co-extrusion for the encapsulation of probiotic, *Lactobacillus acidophilus* LA3 using a blend of alginate and shellac. They found that co-extrusion using sunflower oil as a carrier

Centrifugal extrusion: This variation of co-extrusion is a liquid extrusion technique that makes use of a spinning extrusion head that carries the concentric nozzles. The concentric feeding tube serves as a tributary to the many concentric nozzles located at the surface of the device. As the spinning head rotates, the inner core and the outer shell material are extruded in flow that break into droplets as it makes its way from the nozzles (**Figure 5**). The particle size of extrudates can be as small as 150 μm. The

Mechanical means are used for droplets generation during co-extrusion and has given rise to many modern encapsulation techniques. These all depend on the dripping and jet break up principle for droplet generation at an orifice or from a laminar jet. A droplet that forms at an orifice and is discharged is a result of a formation process that depends on the interplay of surface tension of the extruded liquid, velocity of extrusion, gravitational force, impulse and frictional forces. Configurations for

A. At extremely low velocity, single droplet form at the orifice. The drop detaches under gravity as gravity overcomes surface tension and frictional forces.

B. As the velocity increases, the number of drops ejected from the orifice increases marginally, leading to increased coalescence and polydispersity of the drops.

C. Co-axial flow: A higher increase in velocity results in the formation of a continuous liquid laminar jet that breaks by surface tension and axial symmetrical

D. Further increase in velocity causes normal distribution of droplets because of

particles harden by solvent evaporation as they take flight from the device.

**14**

**Figure 5.**

spreads out to form a thin film on the disc and subsequently gathers momentum as it moves towards the edge of the disc. At the edge, the droplets hold unto the rim due to interfacial tension and viscosity. It is detached when the centrifugal force overcomes the interfacial tension. The drop is detached an angle to the disc and to a distance from the disc depending on their size. This separation by distance traveled as per size is used to sort different size ranges enabling the collection of monodispersed capsules in the solidifying chamber. The rotary speed and geometry of the disc, alongside the viscosity of the suspension and the feed flow rate determine the size of the droplet which usually ranges from 1 to 200 μm [45].

Spinning disc method represented in **Figure 6**, is an easily scalable method for producing large quantities of spherical beads with a narrow size distribution, using liquids of varying viscosities, in minutes. However, product recovery usually requires large space for the gelling bath which makes sterilization difficult. Even though it is amenable to continuous manufacturing, it is expensive comparatively. The coating materials are usually meltable waxes, diglycerides that solidify on cooling.

Electrospraying (Electrohydrodynamic atomization)/Electrospinning: This technique of droplet generation depends on electrohydrodynamics which deals with interaction of fluid and electric field. Electrospraying depends on the principle of charged droplet which states that when an electric field is applied to a drop of liquid, it acquires an electrostatic force which competes with the cohesive force due to surface tension. If this coulomb force is large enough to overcome the surface tension force, the drop detaches and breaks up into submicron droplets which quickly solidify into self-dispersing nano and micro capsules with limited agglomeration and

**17**

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic…*

be prevented by a secondary voltage set up known as corona neutralizer.

ing technique can yield site specific delivery.

Other electrospraying modes, other than the Taylor's cone, is possible as the applied voltage gradually increases. The dripping mode gives way to micro dripping, then spindle, Taylor cone jet, and multiplet mode. Obtaining a continuous jet is important for determining the droplet size and morphology which depends on interplay of factors related to the polymer liquid such as density, concentration, surface tension, conductivity [55], molecular weight, viscosity and solvent; and process parameters such as gravity, applied voltage, flow rate, capillary diameter, and distance of the collector from the capillary tip [56]. Among natural polymers that have been applied in electrospraying are chitosan, cellulose and alginate [57]. Different configurations have been used in electrospraying due to the wide range of factors that needs optimization for droplet size and morphology. One of these is the coaxial assembly which uses two concentric capillaries, the inner and the outer for pushing two different liquid compositions. A typical coaxial set up is shown in **Figure 7**. Shams and colleagues [58] developed pH responsive prednisolone loaded Eudragit L100–55 microparticles for colon specific delivery using single and coaxial electrospraying. In vitro assessment of the five developed formulations showed that careful selection of polymeric system alongside process parameters in electrospray-

Yuan and colleagues [59] also used the coaxial electrospray assembly in the fabrication of curcumin-loaded microcapsules aimed at improving the release profile of curcumin. The improved coaxial electrospray was able to generate stable Taylor's cone mode under a variety of operating condition that yielded an obvious core-shell structure of targeted size and morphology [59]. Likewise, Gómez-Mascaraque and colleagues [60], for the first time, encapsulated probiotic, *Lactobacillus plantarium* with a whey protein inner core and a gelatin outer shell using acetic acid as an external gelling agent. They found that the application of high voltage alongside the presence of acetic acid negatively impacted the viability of the probiotic [60]. Another configuration that has been explored to overcome the low throughput of the stable Taylor's cone mode in electrospray is the multiple capillary assembly. Though not without challenges, Parhizkar and colleagues [55] designed and tested two multiple needle electrospraying geometries with each consisting of four needles. The challenge was to operate all four needles at stable cone mode. Higher particles

coagulation. The technique just like its better applied counterpart, electrospinning, is a facile relatively inexpensive, flexible, easy-to set-up and versatile (in terms of processable materials, set up configuration) process that is amenable to continuous manufacture of tunable compositions, and customized properties of size, and morphology [53]. Droplet generation starts with pushing the liquid in the syringe to flow through the nozzle to the metallic capillary which is connected to the collector through a voltage generating unit. As the liquid passes the electric field, electric charges are inducted leading to the formation of a conically shaped lower meniscus (also known as the Taylor's cone) at which tip the acquired charges are concentrated as a result of equilibrium between capillary forces and electrodynamics [54]. The liquid then accelerates away from the nozzle in a tiny thread tip leading to formation of a jet with high charge density. What happens next determines whether electrospinning or electrospraying will occur. The former occurs if the jet experiences sufficiently high axial tension such that the jet undergoes a whipping instability and elongates to reach the collector instead of breaking up. This high axial tension usually results from a high concentration of high molecular weight polymers. Alternatively, the liquid jet breaks up into primary droplets that could experience the so-called Coulombs fission on their way to the collector. This occurs due to solvent evaporation, droplet shrinkage and subsequent break up again into submicron encapsulation due to charge density. Subsequent break ups that could lead to polydispersity could

*DOI: http://dx.doi.org/10.5772/intechopen.94856*

**Figure 6.** *Schematic representation of the spinning disc assembly.*

#### *Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.94856*

coagulation. The technique just like its better applied counterpart, electrospinning, is a facile relatively inexpensive, flexible, easy-to set-up and versatile (in terms of processable materials, set up configuration) process that is amenable to continuous manufacture of tunable compositions, and customized properties of size, and morphology [53]. Droplet generation starts with pushing the liquid in the syringe to flow through the nozzle to the metallic capillary which is connected to the collector through a voltage generating unit. As the liquid passes the electric field, electric charges are inducted leading to the formation of a conically shaped lower meniscus (also known as the Taylor's cone) at which tip the acquired charges are concentrated as a result of equilibrium between capillary forces and electrodynamics [54]. The liquid then accelerates away from the nozzle in a tiny thread tip leading to formation of a jet with high charge density. What happens next determines whether electrospinning or electrospraying will occur. The former occurs if the jet experiences sufficiently high axial tension such that the jet undergoes a whipping instability and elongates to reach the collector instead of breaking up. This high axial tension usually results from a high concentration of high molecular weight polymers. Alternatively, the liquid jet breaks up into primary droplets that could experience the so-called Coulombs fission on their way to the collector. This occurs due to solvent evaporation, droplet shrinkage and subsequent break up again into submicron encapsulation due to charge density. Subsequent break ups that could lead to polydispersity could be prevented by a secondary voltage set up known as corona neutralizer.

Other electrospraying modes, other than the Taylor's cone, is possible as the applied voltage gradually increases. The dripping mode gives way to micro dripping, then spindle, Taylor cone jet, and multiplet mode. Obtaining a continuous jet is important for determining the droplet size and morphology which depends on interplay of factors related to the polymer liquid such as density, concentration, surface tension, conductivity [55], molecular weight, viscosity and solvent; and process parameters such as gravity, applied voltage, flow rate, capillary diameter, and distance of the collector from the capillary tip [56]. Among natural polymers that have been applied in electrospraying are chitosan, cellulose and alginate [57].

Different configurations have been used in electrospraying due to the wide range of factors that needs optimization for droplet size and morphology. One of these is the coaxial assembly which uses two concentric capillaries, the inner and the outer for pushing two different liquid compositions. A typical coaxial set up is shown in **Figure 7**. Shams and colleagues [58] developed pH responsive prednisolone loaded Eudragit L100–55 microparticles for colon specific delivery using single and coaxial electrospraying. In vitro assessment of the five developed formulations showed that careful selection of polymeric system alongside process parameters in electrospraying technique can yield site specific delivery.

Yuan and colleagues [59] also used the coaxial electrospray assembly in the fabrication of curcumin-loaded microcapsules aimed at improving the release profile of curcumin. The improved coaxial electrospray was able to generate stable Taylor's cone mode under a variety of operating condition that yielded an obvious core-shell structure of targeted size and morphology [59]. Likewise, Gómez-Mascaraque and colleagues [60], for the first time, encapsulated probiotic, *Lactobacillus plantarium* with a whey protein inner core and a gelatin outer shell using acetic acid as an external gelling agent. They found that the application of high voltage alongside the presence of acetic acid negatively impacted the viability of the probiotic [60].

Another configuration that has been explored to overcome the low throughput of the stable Taylor's cone mode in electrospray is the multiple capillary assembly. Though not without challenges, Parhizkar and colleagues [55] designed and tested two multiple needle electrospraying geometries with each consisting of four needles. The challenge was to operate all four needles at stable cone mode. Higher particles

*Nano- and Microencapsulation - Techniques and Applications*

size of the droplet which usually ranges from 1 to 200 μm [45].

spreads out to form a thin film on the disc and subsequently gathers momentum as it moves towards the edge of the disc. At the edge, the droplets hold unto the rim due to interfacial tension and viscosity. It is detached when the centrifugal force overcomes the interfacial tension. The drop is detached an angle to the disc and to a distance from the disc depending on their size. This separation by distance traveled as per size is used to sort different size ranges enabling the collection of monodispersed capsules in the solidifying chamber. The rotary speed and geometry of the disc, alongside the viscosity of the suspension and the feed flow rate determine the

Spinning disc method represented in **Figure 6**, is an easily scalable method for producing large quantities of spherical beads with a narrow size distribution, using liquids of varying viscosities, in minutes. However, product recovery usually requires large space for the gelling bath which makes sterilization difficult. Even though it is amenable to continuous manufacturing, it is expensive comparatively. The coating

Electrospraying (Electrohydrodynamic atomization)/Electrospinning: This technique of droplet generation depends on electrohydrodynamics which deals with interaction of fluid and electric field. Electrospraying depends on the principle of charged droplet which states that when an electric field is applied to a drop of liquid, it acquires an electrostatic force which competes with the cohesive force due to surface tension. If this coulomb force is large enough to overcome the surface tension force, the drop detaches and breaks up into submicron droplets which quickly solidify into self-dispersing nano and micro capsules with limited agglomeration and

materials are usually meltable waxes, diglycerides that solidify on cooling.

**16**

**Figure 6.**

*Schematic representation of the spinning disc assembly.*

#### **Figure 7.**

*(a) Coaxial assembly for electrospraying. (b) Inner and outer coaxial needles [59].*

recovery rate was recorded for the assemble comparatively for the same collection time with no significant changes in size and morphology [55]. Also, Lee and coworkers [61] designed functionable poly-styrene-random-glycidyl methacrylate that was used to fabricate microparticles via electrospraying. They further studied the influence of both polymer factors and process parameters on the size and morphology of the fabricated microparticles. Their results showed that polymer structure and properties can be used to tune the structural parameters of the capsules [61].

Jet cutting method for droplet generation: This technique commercialized by geniaLab is a rarely used but cost effective technique that depends on a set of cutting wires that serve as a cutting tool for a jet of liquid as it rotates about its axis to generate uniformly sized droplets that is shaped as a result of surface tension. It is suited for cutting high viscosity liquids that harden on cooling or by ionotropic gelation. The drops generated are generally in the size range of 120 μm to 3 mm. Paulo and colleagues [62] recently x-rayed the process parameters requisite for the generation of optimally suited calcium alginate beads using the jet cutter. A maximum flow rate of 49 mL/ minute yielded beads of about 2 mm size. Increasing the rotational speed of the cutter decreased the bead size by 50% though increased the tangential velocity of the droplets leading to a larger space requirement for product collection [62]. Other parameters such as gravitational force, surface tension, viscosity and flow rate were also noted. A major limitation is the cutting loss occurring with each cut of the liquid jet.

#### *4.1.7 Phase inversion*

Phase inversion and separation occurs in a system due to mass transfer. Usually, for phase inversion to be induced, a polymer solution is exposed to a miscible nonsolvent. When a polymer solution is exposed to its non-solvent, the solvent molecules would move out of the polymer while the non-solvent will move in. The first step in the process is to dissolve a polymer in its solvent. The second step is to cast the polymer solution. The third step is to initiate phase separation by immersion of the cast polymer in a coagulation bath containing the non-solvent. Other methods that have been used to induce phase separation is non-solvent vapor [63]. Ammendola and colleagues [63] used the phase inversion technique to prepare fragrance loaded cellulose acetate microcapsules. They then compared the vapor induced phase separation with immersion induced phase separation. Their study showed that the

**19**

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic…*

relatively uncommon vapor induced phase separation yielded microcapsules with

Chemical methods of encapsulation generally depend on chemical interactions

In this technique, the wall material is made to form at the oil-in-water interface

This technique is very much like interfacial polymerization. The difference is that the polymerization occurs entirely in the one phase. This term includes suspension polymerization, emulsion polymerization, and dispersion polymerization. In a typical process, the wall forming monomer or pre-polymer is dissolved in the continuous phase and used to emulsify the external phase under high pressure homogenization. Thereafter, an initiator for polycondensation soluble in the continuous phase is added to initiate polycondensation. Acids are normally added to reduce pH and trigger polycondensation which leads to crosslinking and the deposition of crosslinked wall material round the oil drops [66]. Material used, stirring speed, pH, and curing temperature are some of the factors for optimization. Ureaformaldehyde and melamine—formaldehyde are well known examples developed with this method. Ishizuka and colleagues [67] recently prepared microcapsules by this technique with an amphiphilic macro RAFT wall material they synthesized. Their procedure eliminated the use of toxic solvents. The wall monomer was introduced into the rice bran oil continuous phase which was then emulsified with the aqueous phase containing sodium chloride in a shirasu porous glass membrane.

of dispersed oil drops. Monomers of the wall forming polymer (usually multifunctional) is first dissolved in the core material and then emulsified in the aqueous continuous phase containing other polymerization reactant. Polymerization ensues right after on both sides of the interface of the dispersed oil drops with water leading to the formation of rigid capsule walls [64]. Particle sizes as low as 3 μm can be achieved though most commercialized capsules from this technique are in the range of 20–60 μm. This technique can also be employed for reverse emulsions. The polymerization occurs across the interface of the droplets. Four major groups of polymers have been employed and include polyamides, polyurea, polyurethane and polyesters in applications that spans the fields of agriculture, pharmaceutics, cosmetics, and energy storage materials. Interfacial polymerization is a well-controlled technique capable of delivering targeted sizes and morphology. An interfacial polymerization approach has been developed that makes use of safer polymers for cosmetic and internal use is the transacylation interfacial polymerization. In this approach, biodegradable oligosaccharides, polysaccharides such as acacia; and polyethylene glycol, and alginate are used in the internal and external phases respectively or vice versa. On mixing the two phases, acacia reacts with the carboxylic acid group of the propylene glycol leading to the overall attachment of alginate and release of polyethylene glycol. The operational shell material is made up of acacia-alginate polymer that does not require further crosslinking [65].

for encapsulation to occur. These involve predominantly polymerization reactions involving monomer dispersions. The major chemical methods are interfacial polymerization, interfacial polycondensation polymerization, emulsion polymer-

*DOI: http://dx.doi.org/10.5772/intechopen.94856*

ization and in-situ polymerization.

*4.2.1 Interfacial polymerization*

*4.2.2 In-situ polymerization*

more controllable characteristics in terms of structure.

**4.2 Encapsulation techniques based on chemical mechanisms**

relatively uncommon vapor induced phase separation yielded microcapsules with more controllable characteristics in terms of structure.
