**1. Introduction**

Encapsulation remains a fundamental and consistent approach of fabrication of drug and diagnostic delivery systems in the health space and natural polymers such as polysaccharides and proteins continue to play significant roles. Natural polymers (polysaccharides, proteins, and lipids), the macromolecules found in nature are integral in the existence of living organisms especially polysaccharides and proteins. Natural polymers constitute a huge portion of the earth's organic matter because they are synthesized by living organisms such as plants, animals, bacteria, and fungi during their entire life cycle. Polysaccharides also referred to as glycans are the most abundant organic compounds on earth. Polysaccharides are involved in many vital functions in nature such as provision of support and stability for cells and tissues, facilitation of cell communications, storage of energy, protection, lubrication, and cell recognition. Proteins are regarded as the 'workhorses' of cells being involved in many processes necessary for life including being the expressions of genetic information.

that can enhance targeting and delivery properties. Some of the derivatives of polysaccharides called semi-synthetic polysaccharides include carboxymethylcellulose, starch acetate, methylcellulose, ethylpullulan, and chitosan sulfate. Polysaccharides and their derivatives enhance delivery and diagnostic properties such as mechanical strength, stability, protection, solubility, targeting, stimuli responsiveness, controlled release, self-regulation, adhesion, bioimaging, labeling, sitespecificity and multifuntionality which equip them to respond to stimuli, diagnose, image, target and treat as single devices [12]. These potentials of polysaccharides make them convenient materials for encapsulation conferring on the encapsulated

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

Drug delivery systems make use of a wide range of polysaccharide-based deliv-

Polysaccharides used for micro- or nanoencapsulation include varied types of polysaccharides and utilization of specific polysaccharides for encapsulation is dependent on its chemical non reactivity with active pharmaceutical ingredient, chemical compatibility as well as stability target-selected delivery [20]. Micro and nanoencapsulation are achieved majorly via chemical (emulsification, polymerization, and liposomes) or physical (freeze-drying/lyophilization, spray drying, co-crystallization, fluidized-bed coating encapsulation processes). Drugs with low solubility and high permeability i.e. BCS class 2 drugs are usually formulated via encapsulation to optimize bioavailability, stability, and controlled release of

Despite the many advances of encapsulation process, relatively few of these products have been made commercially available due to many reasons which include degradation of highly temperature sensitive compounds, difficulty in controlling the particle size especially as the size of the yield is usually small [21]. Long processing times, expensive costs of production and storage of these dosage forms

Mankala *et al.,* [19] incorporated aceclofenac, a nonsteroidal anti-inflammatory

are also of concern. **Table 1** shows varying polysaccharides utilized on the encapsulation process stating the merits and demerits of each application.

drug (NSAID) with biological half-life of 4.3 h and a BCS class 2 drug into

ery systems from plant (khaya gum, starch and cellulose) [13, 14], animal (chitosan) [15], algae (alginate and carrageenan) and microbial (dextran and xanthan gum) sources [16, 17] and cyclodextrins [18]. These polysaccharides delivery systems can interact with bioactive compounds ensuring that they act as innate drug carriers which bind and encapsulate hydrophilic and hydrophobic functional compounds [19]. The size, shape, and internal structure of these polysaccharide delivery systems differ depending on several factors including the method of formulation and the polysaccharide used in formulation [9]. Size is an integral component of drug delivery systems since it affects their physicochemical stability, encapsulation and release characteristics, and biological activity [9]. Encapsulation of bioactive drug compounds can be achieved via a single or a combination of

product the various benefits of encapsulation.

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

**2.2 Merits and demerits in therapeutic delivery**

**2.3 Case studies/applications**

*2.3.1 Encapsulation of small molecules*

polysaccharides.

drugs [20].

**57**

**2.1 Polysaccharides in micro- and nanoencapsulation**

Polysaccharides, proteins, and lipids interact in nature to enhance day to day functions in living organisms. Polysaccharides bind with protein and lipids to form glycoproteins and glycolipids respectively which can be used for cell communications. Other processes modulated by glycoconjugates (glycoproteins and glycolipids) include molecular targeting, cell migration, cell–cell interactions, immune responses, and blood clotting. Polysaccharides influence how proteins function and how cells respond to stimuli. The behavior of a protein is affected by which glycan is attached to it. Glycoproteins are abundant in the cells where they can serve as regulatory switches.

Since natural polymers are biogenic, when used for therapeutic applications, the body would usually identify with them and not treat as foreign bodies thereby bypassing the body's defense mechanisms leading to long circulation of the delivery system and possible targeting to the site of action. The biological properties of polysaccharides and proteins such as cell recognition and interactions, enzymatic degradability, semblance to extracellular matrix and their chemical flexibility [1] make them materials of choice for encapsulation of drugs and diagnostics. In addition, they are preferred to synthetic polymers because they are less toxic, ecofriendly, biodegradable, biocompatible and renewable. Polysaccharides and proteins are used for micro- and nanoencapsulation because they exhibit good process efficiency, are modifiable and can be tailored to target the desired site of action, have good rheological and emulsification/emulsion stabilizing properties, gelling and film forming [2, 3]. In addition, use of polysaccharides and proteins align with the interest and advocacy for 'green' production of drugs and diagnostics. This chapter reviews the applications of polysaccharides and proteins as preferred encapsulating materials in micro- and nanoencapsulation of therapeutics and diagnostics.

#### **2. Polysaccharide-based encapsulation**

Carbohydrate monosaccharide molecules that are cohesively bound together by glycosidic chains are termed polysaccharides [4]. The nature, sequence, and glycosidic monosaccharide chains inherent in polysaccharides influence the molecular and structural properties of polysaccharides. Water retention ability, digestibility, gelation, and solubility properties of polysaccharides are dependent on the composition of their glycosidic monosaccharide chains [5–7]. Polysaccharides are usually obtained through low cost production techniques with raw materials obtained from natural sources. These polymers possess essential properties critical for drug delivery systems [8–11]. Polysaccharides are macromolecules structured in a linear or branched pattern extensively used in both conventional and advanced drug delivery systems as carriers, building blocks, bioactive materials and excipients. Due to their flexibility, they can be derivatized and tailored to achieve certain functionalities

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

that can enhance targeting and delivery properties. Some of the derivatives of polysaccharides called semi-synthetic polysaccharides include carboxymethylcellulose, starch acetate, methylcellulose, ethylpullulan, and chitosan sulfate. Polysaccharides and their derivatives enhance delivery and diagnostic properties such as mechanical strength, stability, protection, solubility, targeting, stimuli responsiveness, controlled release, self-regulation, adhesion, bioimaging, labeling, sitespecificity and multifuntionality which equip them to respond to stimuli, diagnose, image, target and treat as single devices [12]. These potentials of polysaccharides make them convenient materials for encapsulation conferring on the encapsulated product the various benefits of encapsulation.

#### **2.1 Polysaccharides in micro- and nanoencapsulation**

Drug delivery systems make use of a wide range of polysaccharide-based delivery systems from plant (khaya gum, starch and cellulose) [13, 14], animal (chitosan) [15], algae (alginate and carrageenan) and microbial (dextran and xanthan gum) sources [16, 17] and cyclodextrins [18]. These polysaccharides delivery systems can interact with bioactive compounds ensuring that they act as innate drug carriers which bind and encapsulate hydrophilic and hydrophobic functional compounds [19]. The size, shape, and internal structure of these polysaccharide delivery systems differ depending on several factors including the method of formulation and the polysaccharide used in formulation [9]. Size is an integral component of drug delivery systems since it affects their physicochemical stability, encapsulation and release characteristics, and biological activity [9]. Encapsulation of bioactive drug compounds can be achieved via a single or a combination of polysaccharides.

Polysaccharides used for micro- or nanoencapsulation include varied types of polysaccharides and utilization of specific polysaccharides for encapsulation is dependent on its chemical non reactivity with active pharmaceutical ingredient, chemical compatibility as well as stability target-selected delivery [20]. Micro and nanoencapsulation are achieved majorly via chemical (emulsification, polymerization, and liposomes) or physical (freeze-drying/lyophilization, spray drying, co-crystallization, fluidized-bed coating encapsulation processes). Drugs with low solubility and high permeability i.e. BCS class 2 drugs are usually formulated via encapsulation to optimize bioavailability, stability, and controlled release of drugs [20].

#### **2.2 Merits and demerits in therapeutic delivery**

Despite the many advances of encapsulation process, relatively few of these products have been made commercially available due to many reasons which include degradation of highly temperature sensitive compounds, difficulty in controlling the particle size especially as the size of the yield is usually small [21]. Long processing times, expensive costs of production and storage of these dosage forms are also of concern. **Table 1** shows varying polysaccharides utilized on the encapsulation process stating the merits and demerits of each application.

#### **2.3 Case studies/applications**

#### *2.3.1 Encapsulation of small molecules*

Mankala *et al.,* [19] incorporated aceclofenac, a nonsteroidal anti-inflammatory drug (NSAID) with biological half-life of 4.3 h and a BCS class 2 drug into

(polysaccharides, proteins, and lipids), the macromolecules found in nature are integral in the existence of living organisms especially polysaccharides and proteins. Natural polymers constitute a huge portion of the earth's organic matter because they are synthesized by living organisms such as plants, animals, bacteria, and fungi during their entire life cycle. Polysaccharides also referred to as glycans are the most abundant organic compounds on earth. Polysaccharides are involved in many vital functions in nature such as provision of support and stability for cells and tissues, facilitation of cell communications, storage of energy, protection, lubrication, and cell recognition. Proteins are regarded as the 'workhorses' of cells being involved in

*Nano- and Microencapsulation - Techniques and Applications*

many processes necessary for life including being the expressions of genetic

Polysaccharides, proteins, and lipids interact in nature to enhance day to day functions in living organisms. Polysaccharides bind with protein and lipids to form glycoproteins and glycolipids respectively which can be used for cell communications. Other processes modulated by glycoconjugates (glycoproteins and glycolipids) include molecular targeting, cell migration, cell–cell interactions, immune responses, and blood clotting. Polysaccharides influence how proteins function and how cells respond to stimuli. The behavior of a protein is affected by which glycan is attached to it. Glycoproteins are abundant in the cells where they can serve as

Since natural polymers are biogenic, when used for therapeutic applications, the

Carbohydrate monosaccharide molecules that are cohesively bound together by glycosidic chains are termed polysaccharides [4]. The nature, sequence, and glycosidic monosaccharide chains inherent in polysaccharides influence the molecular and structural properties of polysaccharides. Water retention ability, digestibility, gelation, and solubility properties of polysaccharides are dependent on the composition of their glycosidic monosaccharide chains [5–7]. Polysaccharides are usually obtained through low cost production techniques with raw materials obtained from natural sources. These polymers possess essential properties critical for drug delivery systems [8–11]. Polysaccharides are macromolecules structured in a linear or branched pattern extensively used in both conventional and advanced drug delivery systems as carriers, building blocks, bioactive materials and excipients. Due to their flexibility, they can be derivatized and tailored to achieve certain functionalities

body would usually identify with them and not treat as foreign bodies thereby bypassing the body's defense mechanisms leading to long circulation of the delivery system and possible targeting to the site of action. The biological properties of polysaccharides and proteins such as cell recognition and interactions, enzymatic degradability, semblance to extracellular matrix and their chemical flexibility [1] make them materials of choice for encapsulation of drugs and diagnostics. In addition, they are preferred to synthetic polymers because they are less toxic, ecofriendly, biodegradable, biocompatible and renewable. Polysaccharides and proteins are used for micro- and nanoencapsulation because they exhibit good process efficiency, are modifiable and can be tailored to target the desired site of action, have good rheological and emulsification/emulsion stabilizing properties, gelling and film forming [2, 3]. In addition, use of polysaccharides and proteins align with the interest and advocacy for 'green' production of drugs and diagnostics. This chapter reviews the applications of polysaccharides and proteins as preferred encapsulating materials in micro- and nanoencapsulation of therapeutics and

information.

regulatory switches.

diagnostics.

**56**

**2. Polysaccharide-based encapsulation**


**Polysaccharide**

**59**

**type**

**Encapsulation**

**API**

**Significance**

agent against *E. coli* O157: H7 was entrapped in 0.2% w/v

sodium alginate Alg NPs prepared from 0.2%w/v stock solution could be

appropriate

IgY through the

Microencapsulation

 Paclitaxel

 Alginate emulsification

cytotoxicity

was assessed using human non-small cell lung cancer cell

lines (A549 and Calu-6). Results showed that exposure

of cells to pure paclitaxel and paclitaxel loaded

microparticles

and Calu-6 cells similarly in a

dependent manner thus its use in

primary or metastatic lung cancer.

> 3. Hyaluronate

Nanoencapsulation

Recombinant

The formulation

 of sodium hyaluronate

neuroglobin,

 showed that the formulation

nanoparticles

The nano particles must be preserved at low

[30]

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

 is

temperatures.

neuroglobin

loaded with highly compatible

a delivery system to transport neuroglobin blood. After a stroke, the sodium hyaluronate

nanoparticles

intravenously

cross the BBB. They quickly reached the damaged nerve

cells, being detected inside cytoplasm. This delivery

system successfully

sensing protein NGB to the damaged ischemic brain

after 2 hr. and was retained after 24 hr. of reperfusion.

Microencapsulation

—

Sodium hyaluronate inhalation due to its therapeutic

formulation

target lung

 excipient or drug carrier, and ability to

inflammation

 and cancer.

 was evaluated as a material for

The

time and incurred expensive process costs.

microparticles

 was fabricated over long processing

[31]

 potential, utility as a

 carried the

neuroprotective

 oxygen-

 at the onset of the reperfusion

 loaded with neuroglobin

 injected

 period, can

 for

pharmaceutical

 use and may act as

 within the

 effectively inhibited the growth of A549

concentration-

 and time-

management

 of

 activity of paclitaxel loaded

 technique and

microparticles

 were fabricated by an

characterized.

 The *in vitro*

The yield was small, and the formulation

at high

temperatures.

 Clearance of the

 was not stable

[29]

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

microparticles

 by

microparticles

macrophages

overall

bioavailability.

 is expected to be high. This will alter its

gastrointestinal

 tract.

 candidates for efficient and safe delivery of

nanoparticles

 by ionic gelation method.

 **of the study**

**Demerits of the** 

**encapsulation**

**process/study**

 **set back Reference**


*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

**Polysaccharide**

**58**

**type**

1. Chitosan

Microencapsulation

 Isoniazid and

Spray-dried

 chitosan

microparticles

demonstrating

antibacterial

 activity *in vitro.* Overall, the

 that

microencapsulation

the yield was extremely small. Control of the particle

size during the production

 process was difficult.

 inhibited bacterial

Microparticles

 were sensitive to high

temperatures

 and

[22]

rifabutin

growth by 96%,

preserved drug obtained data suggest the potential of chitosan

microparticles

Nanoencapsulation

 Tamoxifen

 Compared to tamoxifen citrate suspension, of the drug permeated using the nano formulation

increased from 1.5 to 90 times, in absence or in presence

of pancreatin or lipase. The

in

lecithin/chitosan

metabolized

tissue via paracellular

Nanoencapsulation

 Gallic acid

 Gallic acid (GA) loaded chitosan

treated mice, reversed the scopolamine

in mice which may be attributed to its antioxidant

properties and improved cholinergic

effects were significantly

administration

administration,

for GANP.

> 2. Alginate

Microencapsulation

 Gallic acid

calcium alginate for use in

provided alginate beads with a size of 200 μm - 1.3 mm.

Loading capacity acid varied from 7 to 12 g/100 g. There

was a faster release profile in simulated intestinal fluid

than in gastric fluid.

Nanoencapsulation

 IgY

Controlled delivery of therapeutic

nanoparticles

organ. Some therapeutic

not tolerate severe conditions in the

tract.

Concentrations

 of a specific IgY as a

 agents such as proteins could

gastrointestinal

prophylactic

 became an attractive issue in the gastric

 agents by alginate

The yield was small, and the formulation

at high temperature-

because of the

nanoencapsulation

 process.

 sensitive compounds

temperatures.

 Degradation

 of highly

 was also experienced

 was not stable

[28]

Electro-spraying

microencapsulation

 of Gallic acid

Difficulty in controlling particle size, moderate yields

for small batches was handling and storage conditions was required.

experienced.

 The need for special

[26, 27]

management

 of dementia

 but no significant change was observed

 of GANP compared with pure GA

 transport. nanoparticles

 induced amnesia

 functions. These

 increased by the

 (GANP)

The enhance the stability of the product. The yield of the

nanoparticles

 was extremely small.

nanoparticles

 required coating with Tween 80 to

[24, 25]

 drug passing through the rat intestinal

nanoparticles

 improved the non-

encapsulation

 of tamoxifen

 for inhalable lung tuberculosis

 therapy.

 the amount

The effect of enzymes on intestinal permeation

 of

[23]

*Nano- and Microencapsulation - Techniques and Applications*

tamoxifen-loaded

 was

tamoxifen was shown only when

nanoparticles

surface.

The nanoparticle

 yield was extremely small.

 were in intimate contact with the mucosal

**Encapsulation**

**API**

**Significance**

 **of the study**

**Demerits of the** 

**encapsulation**

**process/study**

 **set back Reference**


**Table 1.** *Polysaccharides used in encapsulation of various active pharmaceutical ingredients showing the merits and demerits in therapeutic delivery.* polymeric microcapsules. Aceclofenac-loaded microcapsules was formulated using ionic gelation technique employing sodium alginate as the coat material in combination with some mucoadhesive polysaccharide derivatives such as hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC) and methylcellulose. The microcapsules were spherical (**Figure 1**) with microencapsulation efficiency of 83.25–99.94%, good mucoadhesive property to enhance bioavailability and ensured over 15 hr. sustained release of aceclofenac via zero order kinetic super case 2 transport [19]. The formulation composition of drug:sodium alginate:HPMC in the ratio of 2:4:1 displayed a sustained release of up to 24 hr. In another study using aceclofenac, Dharmendra *et al.,* [20] developed a LbL self-assembly which was utilized to make aceclofenac single bilayer microcapsules produced by sequential adsorption of positively charged chitosan and negatively charged pectin, a polysaccharide on the external surface of negatively charged aceclofenac micro-

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

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

microencapsulated using polysaccharide coat comprising alginate alone or in combination with chitosan via ionotropic gelation process [35]. Abdelbary *et al.,* [35] showed that the microencapsulated glipizide enhanced drug bioavailability causing significant hypoglycaemic activity compared to innovator product. Microencapsulation provides a physical barrier against digestive enzymes, whilst offering protection against the acidic gastric environment. Cholesterol-lowering efficacy of yoghurt formulation containing microencapsulated bile salt hydrolase (BSH)-active *Lactobacillus reuteri* for management of hypercholesterolaemia adults was evaluated by Martoni *et al.* [36]. Microencapsulation of bile salt hydrolase-active *Lactobacillus reuteri* using sodium alginate showed superiority over traditional probiotic therapy and may be an exceptional choice as a cholesterol-lowering agent to be administered

Karan *et al.,* [37] developed novel polymeric microspheres of 5-fluorouracil (5- FU) using natural polysaccharide gum katira via microencapsulation to obtain an optimal therapeutic response at the colon. This controlled release delivery system of 5FU released the chemotherapeutic agent at a controlled rate whilst retarding gastric degradation of the drug. Utilization of natural polysaccharides in microencapsulation of 5FU via optimized katira gum microsphere ensured that a micro-carrier

*(a) SEM images of aceclofenac mucoadhesive microcapsules formulated with HPMC; (b) SEM images of*

crystals. This enabled targeted release of aceclofenac in the colon. Glipizide an antidiabetic agent with short biological half-life was

alone or in combination with other cholesterol-lowering agents [36].

for efficient colon drug targeting was developed.

*aceclofenac mucoadhesive microcapsules formulated with SCMC [19].*

**Figure 1.**

**61**

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

polymeric microcapsules. Aceclofenac-loaded microcapsules was formulated using ionic gelation technique employing sodium alginate as the coat material in combination with some mucoadhesive polysaccharide derivatives such as hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC) and methylcellulose. The microcapsules were spherical (**Figure 1**) with microencapsulation efficiency of 83.25–99.94%, good mucoadhesive property to enhance bioavailability and ensured over 15 hr. sustained release of aceclofenac via zero order kinetic super case 2 transport [19]. The formulation composition of drug:sodium alginate:HPMC in the ratio of 2:4:1 displayed a sustained release of up to 24 hr. In another study using aceclofenac, Dharmendra *et al.,* [20] developed a LbL self-assembly which was utilized to make aceclofenac single bilayer microcapsules produced by sequential adsorption of positively charged chitosan and negatively charged pectin, a polysaccharide on the external surface of negatively charged aceclofenac microcrystals. This enabled targeted release of aceclofenac in the colon.

Glipizide an antidiabetic agent with short biological half-life was microencapsulated using polysaccharide coat comprising alginate alone or in combination with chitosan via ionotropic gelation process [35]. Abdelbary *et al.,* [35] showed that the microencapsulated glipizide enhanced drug bioavailability causing significant hypoglycaemic activity compared to innovator product. Microencapsulation provides a physical barrier against digestive enzymes, whilst offering protection against the acidic gastric environment. Cholesterol-lowering efficacy of yoghurt formulation containing microencapsulated bile salt hydrolase (BSH)-active *Lactobacillus reuteri* for management of hypercholesterolaemia adults was evaluated by Martoni *et al.* [36]. Microencapsulation of bile salt hydrolase-active *Lactobacillus reuteri* using sodium alginate showed superiority over traditional probiotic therapy and may be an exceptional choice as a cholesterol-lowering agent to be administered alone or in combination with other cholesterol-lowering agents [36].

Karan *et al.,* [37] developed novel polymeric microspheres of 5-fluorouracil (5- FU) using natural polysaccharide gum katira via microencapsulation to obtain an optimal therapeutic response at the colon. This controlled release delivery system of 5FU released the chemotherapeutic agent at a controlled rate whilst retarding gastric degradation of the drug. Utilization of natural polysaccharides in microencapsulation of 5FU via optimized katira gum microsphere ensured that a micro-carrier for efficient colon drug targeting was developed.

#### **Figure 1.**

*(a) SEM images of aceclofenac mucoadhesive microcapsules formulated with HPMC; (b) SEM images of aceclofenac mucoadhesive microcapsules formulated with SCMC [19].*

**Polysaccharide**

**60**

**type**

Nanoencapsulation

—

Tempo-oxidized

alginate (SA) composites

properties of hydrogel for cell

compression

TOBC/SA composites SA hydrogel, which indicated that TOBC performed an

important function in enhancing the structural,

mechanical

Nanoencapsulation

 Celecoxib

 Amorphous celecoxib were prepared using ethyl cellulose.

Nanoparticles

higher crystalline drug. for several days and can be spray-dried

powders nanoparticles

absorption and increased

drugs.

Novel

carboxymethyl

cellulose-chitosan

 hybrid

The

microparticles

environmental

 stresses i.e. heat

 produced must be protected from

[34]

Microencapsulation

 Probiotic

bacteria

microparticles

delivery of probiotic bacteria. The model probiotic

bacteria encapsulated

with acceptable viability count for its potential delivery

in the intestinal tract with the purpose of modulating

microbiota and improving human health.

> **Table 1.**

*Polysaccharides*

 *used in* 

*encapsulation*

 *of various active* 

*pharmaceutical*

 *ingredients*

 *showing the merits and demerits in therapeutic*

 *delivery.*

 in

(*Lactobacillus*

 *rhamnosus*)

carboxymethyl

 cellulose based particles

 gut

 was successfully

 were prepared in aqueous media for

bioavailability

 of BCS Class II

 are well suited for providing rapid oral

resuspendable

 in water.

dissolved-drug

Nanoparticle

 suspensions

 were stable

 to form dry

Drug/polymer

concentrations

 than micronized

 released celecoxib rapidly and provided

drug/polymer

nanoparticles

 containing

Absorption

formulation

to avoid dose dumping.

 showed enhanced

bioavailability

 of the API

[33]

*Nano- and Microencapsulation - Techniques and Applications*

 profile must be monitored as the

 and chemical stability of the composites.

 were increased compared with the

 strength and chemical stability of the

 bacterial cellulose (TOBC) and sodium

 were prepared to improve the

encapsulation.

 The

4. Cellulose derivatives

**Encapsulation**

**API**

**Significance**

 **of the study**

**Demerits of the**  Nanocellulose-alginate

are not easily adapted for

encapsulation

 process is time consuming.

 hydrogel for cell

encapsulation

 of biologics. The

encapsulation

[32]

**encapsulation**

**process/study**

 **set back Reference**

The use of polysaccharides for microencapsulation of bicalutamide was utilized by Tekade *et al.,* [38] to develop bicalutamide microspheres using guar gum as a polymer via oil-in-water emulsion solvent diffusion method. The microencapsulated dosage forms containing 2.5% of guar gum, 0.25% span 80 as dispersing agent showed the optimum drug release of 94.22% within 24 hrs, devoid of drug excipient interactions. The potential to enhance bicalutamide bioavailability and sustained release especially due to the polymorphic nature of the drug could be further optimized using nanoencapsulation. This will create a channel for precision targeting of the nonsteroidal antiandrogen to a greater extent than microencapsulation [39] especially where these microencapsulated solid dosage forms have not met the pharmacological need of the patient.

experienced by the patients when on immediate release formulation [44, 45]. It is envisaged the metformin HCl-loaded tamarind seed polysaccharide-alginate encapsulated beads fabricated by Nayak and co-workers [46] will control the release of metformin HCl, improve GI tolerability and more. The drug release was pH sensitive as less than 20% of metformin released in two hours while in acidic medium. Most of the drug was released in pH 7.4, suggesting the maximal absorption may occur in the duodenum and jejunum and possibly increasing the bioavailability of metformin. The release of metformin followed a zero-order pattern which suggests that metformin will be released at a constant rate thereby maximizing its therapeu-

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

In a study undertaken by Sari and colleagues [47], chitosan was used as adjuvant

dimensional microenvironment provided by the alginate microbeads) cells showed a significant increase in expression of pro-angiogenic genes hypoxia-inducible factor-1 (HIF-1 – 80.4%) and VEGF (74%). Seven paracrine signaling factors such as VEGF, TGF-β, TNF-α, IFN-γ, IL-10, IL-6, and IL-1β were secreted. There was an indication that the 3D microenvironment could enhance pluripotency of MSCs. The alginate beads facilitated proper growth and viability of MSCs contributing to the higher therapeutic efficiency of MSCs in vivo. Encapsulated MSCs exhibited anticancer activity against breast cancer stem cells, suppressed cancer-associated genes, inhibited migration and angiogenesis of breast CSCs among other activities making encapsulated MSCs a promising cell-based therapy for targeting cancer cells and

Insulin-loaded arabinoxylan microspheres were fabricated by crosslinking of arabinoxylan employing enzymatic reaction and characterized [51]. Insulin solution was prepared in 0.25 mM HCl and thereafter, glutamic acid was added, and pH

and encapsulating material for the formulation of an anti-botulism single shot vaccine. The toxoids type C and D were encapsulated in chitosan by coacervation method using sodium sulfate as the precipitating agent. The toxoids-loaded chitosan microspheres were compared with the conventional method of mixing the toxoids with aluminum hydroxide which served as an adjuvant. The protein encapsulation efficiency obtained was 41.03% for toxoid C and 32.3% for toxoid D. It is envisaged that modulation of parameters such as protein and chitosan concentration may enhance the encapsulation efficiency. The comparative vaccination in guinea pigs and the neutralization bioassay indicated that the animals were able to develop titers of 10 and 2 IU/mL against *C. botulinum* type C and D respectively for both toxoid-loaded chitosan microspheres and the conventional method of delivery. However, aluminum hydroxide is fraught with adverse reactions such as local pain, swelling, irritation at the injection site, erythema, subcutaneous nodules, contact hypersensitivity and granuloma and allergic reactions [48, 49] making chitosan as an adjuvant a better alternative. Chitosan enables humoral and cellular immune responses and so it is efficient and safe compared to aluminum hydroxide [47]. Sodium alginate was used as an encapsulating material for the encapsulation of mesenchymal stem cells (MSCs), a promising cell-based therapeutic agent for the treatment of cancers, tissue injury, immune disorders, cardiovascular and neurological diseases [50]. MSCs were mixed with alginate solution and cell-encapsulated alginate beads were fabricated by ionotropic gelation with calcium chloride as the crosslinking agent. The cell-encapsulated beads were characterized to assess the ability of the cells to execute their functions despite encapsulation. MSCs were able to proliferate within the alginate beads at different times. Expression of genes proceeded unhindered. In comparison to 2D cultured cells, the 3D (three-

tic efficacy and minimizing adverse effects.

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

*2.3.2 Encapsulation of biologics*

reducing the burden of cancer.

**63**

Molecular Envelope Technology (MET) nanoparticles fabricated from complex polysaccharide chains of N-palmitoyl-N monomethyl- N, N-dimethyl-N,N,Ntrimethyl-6-O-glycol chitosan, a self-assembling polymer amphiphile has been utilized in delivery of formulations to target sites of action across the blood brain barrier [40]. Fisusi *et al.,* [40] developed drug loaded MET formulations containing Lomustine for management of Glioblastoma multiforme. The MET envelope utilizing the complex polysaccharide for nanoencapsulation optimized biodistribution and pharmacodynamics whilst reducing the toxic effects of the active drug, lomustine thereby providing better outcomes for patients managed for brain cancer. As the active pharmaceutical ingredient is protected from degradation, the MET envelope ensures targeted drug delivery due to PEGylation of the polysaccharide to facilitate extended circulation time within the body [40, 41]. Lekshmi *et al.,* [42] prepared and characterized repaglinide loaded ethylcellulose nanoparticles by the solvent evaporation method for the management of type 2 diabetes. The polysaccharide encapsulated nanoparticles showed high encapsulation efficiency suggesting that nanoencapsulation of repaglinide in biodegradable, biocompatible polymer was able to improve its pharmacological activity via modification of surface function and charge to promote cell entry.

Di Martino and co-workers fabricated polysaccharide-based polyelectrolyte nanocomplexes which exhibited the several benefits of encapsulation [43]. Polyelectrolyte nanocomplexes were formed between chitosan (CS) and alginic acid (ALG) and then chitosan and polygalacturonic acid (PGA). Solutions of the polycation (CS in acidic medium) and the polyanions (ALG and PGA in alkaline medium separately) were prepared. The drugs, temozolomide (TMZ) and fluorouracil (5-FU) were dissolved in aqueous solution and added to the separate solutions of the polyanions. The drug(s)-polyanions solutions were added dropwise into increasing concentrations of CS. Characterization of the encapsulated product revealed spherical nanoparticles with diameters within 100–200 nm, increased encapsulation efficiency with increasing concentration of CS, controlled release of drugs, pH sensitivity making it a possible system for colon delivery, stability of drugs especially TMZ. A setback is the burst release which may be due to several factors such as drying method (freeze drying), and degree of complexation. This setback can be modulated by adjusting the ratio of CS:ALG or CS:PGA, harvesting of the nanoparticles early from the fabrication medium. Spray drying may be an alternative to freeze drying to reduce migration of the drugs to the surface during drying. In addition, derivatization of the polysaccharides without loss of their polyelectrolytic nature may increase their mechanical strength, reduce pores within and enhance entrapment.

Gastrointestinal intolerance of metformin HCl may be reduced by encapsulation which provides controlled release of the drug ensuring therapeutic efficacy and minimizing adverse effects. Extended release formulation became necessary to improve patient adherence and reduce gastrointestinal intolerance (GI)

#### *Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

experienced by the patients when on immediate release formulation [44, 45]. It is envisaged the metformin HCl-loaded tamarind seed polysaccharide-alginate encapsulated beads fabricated by Nayak and co-workers [46] will control the release of metformin HCl, improve GI tolerability and more. The drug release was pH sensitive as less than 20% of metformin released in two hours while in acidic medium. Most of the drug was released in pH 7.4, suggesting the maximal absorption may occur in the duodenum and jejunum and possibly increasing the bioavailability of metformin. The release of metformin followed a zero-order pattern which suggests that metformin will be released at a constant rate thereby maximizing its therapeutic efficacy and minimizing adverse effects.

#### *2.3.2 Encapsulation of biologics*

The use of polysaccharides for microencapsulation of bicalutamide was utilized by Tekade *et al.,* [38] to develop bicalutamide microspheres using guar gum as a polymer via oil-in-water emulsion solvent diffusion method. The microencapsulated dosage forms containing 2.5% of guar gum, 0.25% span 80 as dispersing agent showed the optimum drug release of 94.22% within 24 hrs, devoid of drug excipient interactions. The potential to enhance bicalutamide bioavailability and sustained release especially

Molecular Envelope Technology (MET) nanoparticles fabricated from complex

polysaccharide chains of N-palmitoyl-N monomethyl- N, N-dimethyl-N,N,Ntrimethyl-6-O-glycol chitosan, a self-assembling polymer amphiphile has been utilized in delivery of formulations to target sites of action across the blood brain barrier [40]. Fisusi *et al.,* [40] developed drug loaded MET formulations containing Lomustine for management of Glioblastoma multiforme. The MET envelope utilizing the complex polysaccharide for nanoencapsulation optimized biodistribution and pharmacodynamics whilst reducing the toxic effects of the active drug, lomustine thereby providing better outcomes for patients managed for brain cancer. As the active pharmaceutical ingredient is protected from degradation, the MET envelope ensures targeted drug delivery due to PEGylation of the polysaccharide to facilitate extended circulation time within the body [40, 41]. Lekshmi *et al.,* [42] prepared and characterized repaglinide loaded ethylcellulose nanoparticles by the solvent evaporation method for the management of type 2 diabetes. The polysaccharide encapsulated nanoparticles showed high encapsulation efficiency suggesting that nanoencapsulation of repaglinide in biodegradable, biocompatible polymer was able to improve its pharmacological activity via modification of sur-

Di Martino and co-workers fabricated polysaccharide-based polyelectrolyte nanocomplexes which exhibited the several benefits of encapsulation [43]. Polyelectrolyte nanocomplexes were formed between chitosan (CS) and alginic acid (ALG) and then chitosan and polygalacturonic acid (PGA). Solutions of the polycation (CS in acidic medium) and the polyanions (ALG and PGA in alkaline medium separately) were prepared. The drugs, temozolomide (TMZ) and fluorouracil (5-FU) were dissolved in aqueous solution and added to the separate solutions of the polyanions. The drug(s)-polyanions solutions were added dropwise into increasing concentrations of CS. Characterization of the encapsulated product revealed spherical nanoparticles with diameters within 100–200 nm, increased encapsulation efficiency with increasing concentration of CS, controlled release of drugs, pH sensitivity making it a possible system for colon delivery, stability of drugs especially TMZ. A setback is the burst release which may be due to several factors such as drying method (freeze drying), and degree of complexation. This setback can be modulated by adjusting the ratio of CS:ALG or CS:PGA, harvesting of the nanoparticles early from the fabrication medium. Spray drying may be an alternative to freeze drying to reduce migration of the drugs to the surface during drying. In addition, derivatization of the polysaccharides without loss of their polyelectrolytic nature may increase their mechanical strength, reduce pores within

Gastrointestinal intolerance of metformin HCl may be reduced by encapsulation which provides controlled release of the drug ensuring therapeutic efficacy and minimizing adverse effects. Extended release formulation became necessary to improve patient adherence and reduce gastrointestinal intolerance (GI)

due to the polymorphic nature of the drug could be further optimized using nanoencapsulation. This will create a channel for precision targeting of the nonsteroidal antiandrogen to a greater extent than microencapsulation [39] especially where these microencapsulated solid dosage forms have not met the pharmacological

*Nano- and Microencapsulation - Techniques and Applications*

need of the patient.

face function and charge to promote cell entry.

and enhance entrapment.

**62**

In a study undertaken by Sari and colleagues [47], chitosan was used as adjuvant and encapsulating material for the formulation of an anti-botulism single shot vaccine. The toxoids type C and D were encapsulated in chitosan by coacervation method using sodium sulfate as the precipitating agent. The toxoids-loaded chitosan microspheres were compared with the conventional method of mixing the toxoids with aluminum hydroxide which served as an adjuvant. The protein encapsulation efficiency obtained was 41.03% for toxoid C and 32.3% for toxoid D. It is envisaged that modulation of parameters such as protein and chitosan concentration may enhance the encapsulation efficiency. The comparative vaccination in guinea pigs and the neutralization bioassay indicated that the animals were able to develop titers of 10 and 2 IU/mL against *C. botulinum* type C and D respectively for both toxoid-loaded chitosan microspheres and the conventional method of delivery. However, aluminum hydroxide is fraught with adverse reactions such as local pain, swelling, irritation at the injection site, erythema, subcutaneous nodules, contact hypersensitivity and granuloma and allergic reactions [48, 49] making chitosan as an adjuvant a better alternative. Chitosan enables humoral and cellular immune responses and so it is efficient and safe compared to aluminum hydroxide [47].

Sodium alginate was used as an encapsulating material for the encapsulation of mesenchymal stem cells (MSCs), a promising cell-based therapeutic agent for the treatment of cancers, tissue injury, immune disorders, cardiovascular and neurological diseases [50]. MSCs were mixed with alginate solution and cell-encapsulated alginate beads were fabricated by ionotropic gelation with calcium chloride as the crosslinking agent. The cell-encapsulated beads were characterized to assess the ability of the cells to execute their functions despite encapsulation. MSCs were able to proliferate within the alginate beads at different times. Expression of genes proceeded unhindered. In comparison to 2D cultured cells, the 3D (threedimensional microenvironment provided by the alginate microbeads) cells showed a significant increase in expression of pro-angiogenic genes hypoxia-inducible factor-1 (HIF-1 – 80.4%) and VEGF (74%). Seven paracrine signaling factors such as VEGF, TGF-β, TNF-α, IFN-γ, IL-10, IL-6, and IL-1β were secreted. There was an indication that the 3D microenvironment could enhance pluripotency of MSCs. The alginate beads facilitated proper growth and viability of MSCs contributing to the higher therapeutic efficiency of MSCs in vivo. Encapsulated MSCs exhibited anticancer activity against breast cancer stem cells, suppressed cancer-associated genes, inhibited migration and angiogenesis of breast CSCs among other activities making encapsulated MSCs a promising cell-based therapy for targeting cancer cells and reducing the burden of cancer.

Insulin-loaded arabinoxylan microspheres were fabricated by crosslinking of arabinoxylan employing enzymatic reaction and characterized [51]. Insulin solution was prepared in 0.25 mM HCl and thereafter, glutamic acid was added, and pH

adjusted to 4. The insulin solution was added to a solution of arabinoxylan in 0.1 M acetate buffer and then agitated. The enzyme, laccase was added as the crosslinking agent and dropwise extrusion into a hydrophobic liquid, and the microspheres were formed and harvested after 6 hr. The insulin-loaded arabinoxylan microspheres were characterized extensively in vitro and in vivo in diabetic induced Wistar rats. Average size of the spherical shaped and smooth surfaced microspheres was 322 μm having irregular pore sizes and geometries. Insulin aggregates in the microspheres were stabilized by presence of glutamic acid yielding a homogenously distribution of insulin. However, at higher insulin/arabinoxylan mass ratio, micro-phase separation occurred. Arabinoxylan microspheres minimized release of insulin in the gastric and small intestine facilitating delivery of insulin to the colon and limiting degradation by the digestive enzymes. Controlled release of insulin over 10 hr. was observed in vitro. For in vivo studies, insulin was first labeled with RITC before encapsulation. It was observed that the insulin-RITC-loaded arabinoxylan microspheres were relatively intact in the upper GIT releasing about 13–21% of the total RITC load. Maximum amount of RITC was found in the colon, about 78.8% after 8 hr. possibly due to the degradation of arabinoxylan microspheres by the colonic microflora. The blood glucose in the diabetic induced rats decreased by 70% between 9 and 12 hr. after three treatments orally while hyperglycemia was sustained in the control groups. Arabinoxylan microspheres protected insulin from enzymatic degradation, retained a high percentage of insulin for delivery and release in the colon exerting significant hypoglycemic effect.

effect were determined with mice. Average etanercept per microneedle was 42.72 5.81 μg which was sufficient for in vivo evaluation. The microneedles exhibited sufficient mechanical strength, complete dissolution of microneedles in the skin after 90 min, quick recovery of skin after 120 min, good biocompatibility, little interference with bioactivity of etanercept and high anti-inflammatory efficacy. There was evidence of reduction of TNF-α and IL-6 in serum, protection of the joint from erosion and microneedle system showed good bioequivalence to the

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

*Schematic illustration of microneedle-assisted transdermal delivery of etanercept from application on the dorsal*

Hydrogel encapsulation systems compare better than use of autologous chondrocytes and the marrow stimulating technique for cartilage repair because hydrogel encapsulation systems do not just encapsulate chondrocytes but also maintain both cell viability and phenotype and support neocartiliage formation [53]. Fenbo and co-workers [54] fabricated chondrocytes-loaded alginatechondroitin sulfate hydrogel beads by mixing solutions of sodium alginate and chondroitin sulfate and chondrocytes was added to the mixture which was transferred dropwise into a solution of strontium chloride with a syringe. The beads were harvested, rinsed to remove excess strontium chloride, and then cultured. Characterization of the chondrocytes-loaded hydrogel beads showed that low molecular weight alginate-chondroitin sulfate hydrogel promoted high cell viability and upregulated the expression of collagen II and B cell leukemia 2 (Bcl-2). The study suggests that low molecular weight alginate-chondroitin sulfate hydrogel beads promotes cartilage formation and decreases inflammation and may be a promising system for cartilage tissue repair. The study observed that molecular weight of encapsulating materials is an important parameter in tissue engineering.

Encapsulation of biomarkers such as microRNAs (miRNAs or miRs) confer stability on them. MiRNAs are a class of small endogenous non-coding RNAs comprising 18–22 nucleotides regulating various biological processes by preventing expression of target genes [55, 56]. MiRNAs have been suggested and explored as therapeutics and biomarkers for various diseases such as cancer, diabetes, cardiovascular diseases, and other diseases whose etiology is related to atypical gene expression [57]. MiRNAs can be employed in disease environment for diagnosis, treatment, and reoccurrence prediction. Moraes and colleagues [57] modified pullulan by linking quaternized ammonium groups to its backbone. The pullulan

derivative interacted with miRNA to form stable polyplexes which were

classical subcutaneous route.

*skin of the mouse to binding of etanercept to TNF [52].*

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

**Figure 3.**

*2.3.3 Encapsulation of diagnostics*

**65**

Microneedle technology, an encapsulation technology used for biologics and small molecules as an alternative to hypodermic injection and implantation was used to encapsulate etanercept, for transdermal delivery for rheumatoid arthritis [52]. Microneedles fabricated are microscopic needles of lengths 50–900 μm (**Figure 2**) which pierces the stratum corneum barrier generating transient microchannels for delivery of encapsulated biologic or small molecule without triggering the nerves and injuring the blood vessels. Etanercept is a human dimeric fusion protein which is fully soluble and is a tumor necrosis factor (TNF) inhibitor as it binds to TNF preventing the activation of the inflammatory cascade. It is a fusion protein of recombinant human TNF-receptor p75 fused with the Fc domain of human Immunoglobulin G1 (IgG1).

Acrylate modified-hyaluronic acid was used to fabricate the microneedles and on application to the skin released etanercept which was absorbed by the blood capillary and etanercept was transported to the arthritic tissue where it exerted therapeutic effect by binding to TNF (**Figure 3**). The etanercept-loaded microneedles were fabricated by micromoulding method. Thereafter, the microneedles were detached from the mold and crosslinked by exposing to UV light to enhance mechanical strength. Drug loading and in vitro bioactivity were evaluated. Skin penetration, microneedle dissolution and skin recovery, therapeutic

#### **Figure 2.**

*Images of microneedles: (A) microscopic image – 500 μm, (B) microscopic image – 2 mm, (C) scanning electron microscopic (SEM) image – 500 μm, and (D) SEM image – 100 μm [52].*

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

#### **Figure 3.**

adjusted to 4. The insulin solution was added to a solution of arabinoxylan in 0.1 M acetate buffer and then agitated. The enzyme, laccase was added as the crosslinking agent and dropwise extrusion into a hydrophobic liquid, and the microspheres were formed and harvested after 6 hr. The insulin-loaded arabinoxylan microspheres were characterized extensively in vitro and in vivo in diabetic induced Wistar rats. Average size of the spherical shaped and smooth surfaced microspheres was 322 μm having irregular pore sizes and geometries. Insulin aggregates in the microspheres were stabilized by presence of glutamic acid yielding a homogenously distribution of insulin. However, at higher insulin/arabinoxylan mass ratio, micro-phase separation occurred. Arabinoxylan microspheres minimized release of insulin in the gastric and small intestine facilitating delivery of insulin to the colon and limiting degradation by the digestive enzymes. Controlled release of insulin over 10 hr. was observed in vitro. For in vivo studies, insulin was first labeled with RITC before encapsulation. It was observed that the insulin-RITC-loaded arabinoxylan microspheres were relatively intact in the upper GIT releasing about 13–21% of the total RITC load. Maximum amount of RITC was found in the colon, about 78.8% after 8 hr. possibly due to the degradation of arabinoxylan microspheres by the colonic microflora. The blood glucose in the diabetic induced rats decreased by 70% between 9 and 12 hr. after three treatments orally while hyperglycemia was sustained in the control groups. Arabinoxylan microspheres protected insulin from enzymatic degradation, retained a high percentage of insulin for delivery and

*Nano- and Microencapsulation - Techniques and Applications*

release in the colon exerting significant hypoglycemic effect.

of human Immunoglobulin G1 (IgG1).

**Figure 2.**

**64**

Microneedle technology, an encapsulation technology used for biologics and small molecules as an alternative to hypodermic injection and implantation was used to encapsulate etanercept, for transdermal delivery for rheumatoid arthritis [52]. Microneedles fabricated are microscopic needles of lengths 50–900 μm (**Figure 2**) which pierces the stratum corneum barrier generating transient microchannels for delivery of encapsulated biologic or small molecule without triggering the nerves and injuring the blood vessels. Etanercept is a human dimeric fusion protein which is fully soluble and is a tumor necrosis factor (TNF) inhibitor as it binds to TNF preventing the activation of the inflammatory cascade. It is a fusion protein of recombinant human TNF-receptor p75 fused with the Fc domain

Acrylate modified-hyaluronic acid was used to fabricate the microneedles and on application to the skin released etanercept which was absorbed by the blood capillary and etanercept was transported to the arthritic tissue where it exerted therapeutic effect by binding to TNF (**Figure 3**). The etanercept-loaded microneedles were fabricated by micromoulding method. Thereafter, the

microneedles were detached from the mold and crosslinked by exposing to UV light to enhance mechanical strength. Drug loading and in vitro bioactivity were evaluated. Skin penetration, microneedle dissolution and skin recovery, therapeutic

*Images of microneedles: (A) microscopic image – 500 μm, (B) microscopic image – 2 mm, (C) scanning electron*

*microscopic (SEM) image – 500 μm, and (D) SEM image – 100 μm [52].*

*Schematic illustration of microneedle-assisted transdermal delivery of etanercept from application on the dorsal skin of the mouse to binding of etanercept to TNF [52].*

effect were determined with mice. Average etanercept per microneedle was 42.72 5.81 μg which was sufficient for in vivo evaluation. The microneedles exhibited sufficient mechanical strength, complete dissolution of microneedles in the skin after 90 min, quick recovery of skin after 120 min, good biocompatibility, little interference with bioactivity of etanercept and high anti-inflammatory efficacy. There was evidence of reduction of TNF-α and IL-6 in serum, protection of the joint from erosion and microneedle system showed good bioequivalence to the classical subcutaneous route.

Hydrogel encapsulation systems compare better than use of autologous chondrocytes and the marrow stimulating technique for cartilage repair because hydrogel encapsulation systems do not just encapsulate chondrocytes but also maintain both cell viability and phenotype and support neocartiliage formation [53]. Fenbo and co-workers [54] fabricated chondrocytes-loaded alginatechondroitin sulfate hydrogel beads by mixing solutions of sodium alginate and chondroitin sulfate and chondrocytes was added to the mixture which was transferred dropwise into a solution of strontium chloride with a syringe. The beads were harvested, rinsed to remove excess strontium chloride, and then cultured. Characterization of the chondrocytes-loaded hydrogel beads showed that low molecular weight alginate-chondroitin sulfate hydrogel promoted high cell viability and upregulated the expression of collagen II and B cell leukemia 2 (Bcl-2). The study suggests that low molecular weight alginate-chondroitin sulfate hydrogel beads promotes cartilage formation and decreases inflammation and may be a promising system for cartilage tissue repair. The study observed that molecular weight of encapsulating materials is an important parameter in tissue engineering.

#### *2.3.3 Encapsulation of diagnostics*

Encapsulation of biomarkers such as microRNAs (miRNAs or miRs) confer stability on them. MiRNAs are a class of small endogenous non-coding RNAs comprising 18–22 nucleotides regulating various biological processes by preventing expression of target genes [55, 56]. MiRNAs have been suggested and explored as therapeutics and biomarkers for various diseases such as cancer, diabetes, cardiovascular diseases, and other diseases whose etiology is related to atypical gene expression [57]. MiRNAs can be employed in disease environment for diagnosis, treatment, and reoccurrence prediction. Moraes and colleagues [57] modified pullulan by linking quaternized ammonium groups to its backbone. The pullulan derivative interacted with miRNA to form stable polyplexes which were

characterized for physicochemical properties and cellular uptake. Elemental analysis, SEC-MALLS analysis, and IR and NMR spectra confirmed the modification of pullulan. Average size of polyplexes was 130 � 30 nm, zeta potential was �12 � 5 mV and morphology study showed homogenous spherical particles. Agarose gel electrophoresis confirmed the presence of miRNA within the polyplexes and loading efficiency was 80%. There was no indication of degradation or fragmentation of miRNA suggesting that cationic quarternized pullulan could protect miRNA. The polyplexes were found to be stable, cytocompatible, and the complexation of miRNA with quartenized pullalan facilitated the uptake of miRNA into the cells.

(CUR) as a fluorescence probe, polyelectrolyte, chitosan oligosaccharide lactate (COL) as the encapsulating material and silica nanoparticles as the core (**Figure 4**). The nanohybrid particles were fabricated by precipitation technique. Solutions of

Decreased urea content is indicative of pregnancy, low protein diet, overhydration, advanced liver disease and reduced urea synthesis. While there are several techniques for determining urea content, there is a growing need for easy to fabricate, easy to use and cheap diagnostic tools. Khattab and co-workers [61] fabricated crosslinked calcium alginate microcapsules containing urease and tricyanofuran hydrazone fixed on cotton fibers to create a colorimetric cotton strip as a sensor for determining urea content. Solutions of sodium alginate, urease and tricyanofuran hydrazone was mixed and overlaid on cotton fiber strips and dried. Thereafter the dried cotton fiber strips were immersed in a solution of calcium choride for the crosslinking process. The microcapsules were characterized, and the sensor was used to determine urea content. The urea content assay using the sensor fabricated, displayed a visual color change from light yellow to purple indicating the presence

*Schematic illustration of the fabrication of nanohybrid particles and the fluorescence intensity in the presence of*

**Figure 4.**

*cholesterol.*

**67**

COL and curcumin were mixed under agitation and a dispersion of silica nanoparticles was added dropwise and agitated overnight. The nanocapsules formed were harvested and characterized. When not aggregated, the sizes of the spherical particles were 25–35 nm. In the presence of cholesterol, a large blue shift ˃ 100 nm was observed in the fluorescence intensity of nanohybrid particles. The fluorescence intensity of the nanohybrid particles were not affected by interfering substances such as ascorbic acid, uric acid and glucose indicating specificity, selectivity and sensitivity of cholesterol determination using the fabricated nano-sensor. Urea is a waste product of metabolism and is eliminated from the body through the kidney. Evaluation of urea content is used to assess kidney function and other possible implications. Increased level of urea in the urine and blood indicates the presence of some acute and chronic diseases such as kidney failure, and myocardial infarction, or dehydration, gastrointestinal hemorrhage, high protein diet, aging and catabolic states such as trauma, severe infection, starvation and drugs.

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

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

Cellulose nanofibers are appealing cargo carriers due the unique barrier, chemical, interfacial, mechanical, and optical properties of nanocellulose [58]. Cellulose nanofibers-based microcapsules were fabricated as a diagnostic device with glucose oxidase encapsulated within for glucose monitoring [58]. Cellulose nanofibers (CNF), apple pectin (AP) and xyloglucan-amyloid (XyG) were used to fabricate the microcapsules using layer by layer ((LbL - CNF/XyG/CNF/AP)2CNF) technique on top of fluorescein isothiocyanate (FITC) – labeled glucose oxidase-loaded calcium carbonate particles to build the capsule wall. The FITC-glucose oxidase-CaCO3 particles were crosslinked with glutaraldehyde forming the templates on which LbL microcapsules were fabricated. After LbL fabrication, calcium carbonate was removed with 100 mM EDTA in water. The microcapsules collapsed on drying after removal of the CaCO3 core. The glucose oxidase-loaded microcapsules fabricated were porous, spherical, uniform and structurally stable, and the encapsulation efficiency of glucose oxidase was 68 � 2%. The microcapsules were used to monitor/measure glucose. An interaction of glucose oxidase and glucose produced hydrogen peroxide which was transported through channels to an external flow-cell where hydrogen peroxide was oxidized electrically producing current that was recorded and used to determine the concentration of glucose. The microcapsules immobilized the enzymes as well as provided a favorable microenvironment for the sustained biocatalytic activity of glucose oxidase. The nanocellulose microcapsules show promise as a device for in vivo monitoring of analytes.

A glucose biosensor fabricated based on gum tragacanth was tested on actual blood samples. Cadmium Telluride Quantum Dots (CdTe QDs) and glucose oxidase were encapsulated in tragacanth gum for glucose detection [59]. Tragacanth gum nanohydrogels were prepared by adjustment of pH, sonication followed by precipitation. Modified tragacanth gum was prepared by radial graft copolymerization of acrylic acid (AA: 0–5 mL), using N,N<sup>0</sup> -methylenebisacrylamide (MBA: 0.1–0.3 g) as a crosslinker in potassium persulfate solution (initiator) followed by precipitation after agitation for 4 hr. at 70°C. The nanohydrogels were characterized and the composition with the desired mechanical strength and highest swelling ratio was used in fabrication of superabsorbent biosensor nanohydrogels. The biosensor nanohydrogels were tested for leakage of CdTe QDs and glucose oxidase, encapsulation efficiency and glucose detection. The QDs leakage was about 1.48% for 2 hr. There was insignificant change in fluorescence intensity of QDs after 45 days at 4°C and 17% decrease in fluorescence intensity at ambient temperature after 45 days. Enzyme was stable at 4°C and unstable with time at ambient temperature. Fluorescence intensity decreased significantly with increase in hydrogen peroxide concentration indicating the encapsulated glucose oxidase was able to catalyze the oxidation of glucose to hydrogen peroxide and gluconic acid.

Enzymatic reaction, base stacking (aptamers) and antigen–antibody linkers are possible approaches to cholesterol detection; however, they are fraught with some limitations and foreign interference [60]. Chebi and co-workers [60] fabricated a nano-sensor for cholesterol sensing without enzymatic reaction using curcumin

*Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

(CUR) as a fluorescence probe, polyelectrolyte, chitosan oligosaccharide lactate (COL) as the encapsulating material and silica nanoparticles as the core (**Figure 4**). The nanohybrid particles were fabricated by precipitation technique. Solutions of COL and curcumin were mixed under agitation and a dispersion of silica nanoparticles was added dropwise and agitated overnight. The nanocapsules formed were harvested and characterized. When not aggregated, the sizes of the spherical particles were 25–35 nm. In the presence of cholesterol, a large blue shift ˃ 100 nm was observed in the fluorescence intensity of nanohybrid particles. The fluorescence intensity of the nanohybrid particles were not affected by interfering substances such as ascorbic acid, uric acid and glucose indicating specificity, selectivity and sensitivity of cholesterol determination using the fabricated nano-sensor.

Urea is a waste product of metabolism and is eliminated from the body through the kidney. Evaluation of urea content is used to assess kidney function and other possible implications. Increased level of urea in the urine and blood indicates the presence of some acute and chronic diseases such as kidney failure, and myocardial infarction, or dehydration, gastrointestinal hemorrhage, high protein diet, aging and catabolic states such as trauma, severe infection, starvation and drugs. Decreased urea content is indicative of pregnancy, low protein diet, overhydration, advanced liver disease and reduced urea synthesis. While there are several techniques for determining urea content, there is a growing need for easy to fabricate, easy to use and cheap diagnostic tools. Khattab and co-workers [61] fabricated crosslinked calcium alginate microcapsules containing urease and tricyanofuran hydrazone fixed on cotton fibers to create a colorimetric cotton strip as a sensor for determining urea content. Solutions of sodium alginate, urease and tricyanofuran hydrazone was mixed and overlaid on cotton fiber strips and dried. Thereafter the dried cotton fiber strips were immersed in a solution of calcium choride for the crosslinking process. The microcapsules were characterized, and the sensor was used to determine urea content. The urea content assay using the sensor fabricated, displayed a visual color change from light yellow to purple indicating the presence

#### **Figure 4.**

*Schematic illustration of the fabrication of nanohybrid particles and the fluorescence intensity in the presence of cholesterol.*

characterized for physicochemical properties and cellular uptake. Elemental analysis, SEC-MALLS analysis, and IR and NMR spectra confirmed the modification of

polyplexes and loading efficiency was 80%. There was no indication of degradation or fragmentation of miRNA suggesting that cationic quarternized pullulan could protect miRNA. The polyplexes were found to be stable, cytocompatible, and the complexation of miRNA with quartenized pullalan facilitated the uptake of

Cellulose nanofibers are appealing cargo carriers due the unique barrier, chemical, interfacial, mechanical, and optical properties of nanocellulose [58]. Cellulose nanofibers-based microcapsules were fabricated as a diagnostic device with glucose oxidase encapsulated within for glucose monitoring [58]. Cellulose nanofibers (CNF), apple pectin (AP) and xyloglucan-amyloid (XyG) were used to fabricate the microcapsules using layer by layer ((LbL - CNF/XyG/CNF/AP)2CNF) technique on top of fluorescein isothiocyanate (FITC) – labeled glucose oxidase-loaded calcium carbonate particles to build the capsule wall. The FITC-glucose oxidase-CaCO3 particles were crosslinked with glutaraldehyde forming the templates on which LbL microcapsules were fabricated. After LbL fabrication, calcium carbonate was removed with 100 mM EDTA in water. The microcapsules collapsed on drying after removal of the CaCO3 core. The glucose oxidase-loaded microcapsules fabricated were porous, spherical, uniform and structurally stable, and the encapsulation efficiency of glucose oxidase was 68 � 2%. The microcapsules were used to monitor/measure glucose. An interaction of glucose oxidase and glucose produced hydrogen peroxide which was transported through channels to an external flow-cell where hydrogen peroxide was oxidized electrically producing current that was recorded and used to determine the concentration of glucose. The microcapsules immobilized the enzymes as well as provided a favorable microenvironment for the sustained biocatalytic activity of glucose oxidase. The nanocellulose microcapsules

pullulan. Average size of polyplexes was 130 � 30 nm, zeta potential was �12 � 5 mV and morphology study showed homogenous spherical particles. Agarose gel electrophoresis confirmed the presence of miRNA within the

*Nano- and Microencapsulation - Techniques and Applications*

show promise as a device for in vivo monitoring of analytes.

acrylic acid (AA: 0–5 mL), using N,N<sup>0</sup>

**66**

A glucose biosensor fabricated based on gum tragacanth was tested on actual blood samples. Cadmium Telluride Quantum Dots (CdTe QDs) and glucose oxidase were encapsulated in tragacanth gum for glucose detection [59]. Tragacanth gum nanohydrogels were prepared by adjustment of pH, sonication followed by precipitation. Modified tragacanth gum was prepared by radial graft copolymerization of

a crosslinker in potassium persulfate solution (initiator) followed by precipitation after agitation for 4 hr. at 70°C. The nanohydrogels were characterized and the composition with the desired mechanical strength and highest swelling ratio was used in fabrication of superabsorbent biosensor nanohydrogels. The biosensor nanohydrogels were tested for leakage of CdTe QDs and glucose oxidase, encapsulation efficiency and glucose detection. The QDs leakage was about 1.48% for 2 hr. There was insignificant change in fluorescence intensity of QDs after 45 days at 4°C and 17% decrease in fluorescence intensity at ambient temperature after 45 days. Enzyme was stable at 4°C and unstable with time at ambient temperature. Fluorescence intensity decreased significantly with increase in hydrogen peroxide concen-

tration indicating the encapsulated glucose oxidase was able to catalyze the

Enzymatic reaction, base stacking (aptamers) and antigen–antibody linkers are possible approaches to cholesterol detection; however, they are fraught with some limitations and foreign interference [60]. Chebi and co-workers [60] fabricated a nano-sensor for cholesterol sensing without enzymatic reaction using curcumin

oxidation of glucose to hydrogen peroxide and gluconic acid.


miRNA into the cells.


*Nano- and Microencapsulation - Techniques and Applications*

**Table 2.** **Nano-encapsulating**

**69**

**Polysaccharide**

Chitosan derivative

Ethyl cellulose

Chitosan/alginic

Pullulan derivative Cellulose/pectin/xyloglucan

Tragacanth gum

Chitosan Auricularia auricular polysaccharide/chitosan

Pectin-chitosan Depolymerized

glycosaminoglycan

Mango gum Azivash gum Cashew gum Chitosan-fucoidan

Cactus mucilage (*Opuntia monacantha*)

**Table 3.** *Polysaccharide-based*

*nano-encapsulating*

 *carriers for delivery of bioactive compounds.*

Polyvinyl alcohol

N-isopropylacrylamide

 holothurian

oligosaccharide

 lactate

Silica

Acrylic acid

 Glucose

oxidase/cadmium

quantum dots

Curcumin

Doxorubicin

 HCl

> Nisin

Doxorubicin

Donezepil

Catechin Epirubicin Red ginseng extract

Zeaxanthin

Nano

emulsification

Antioxidant

Emulsion crosslinking

Electrospinning

Self-assembly Nanoprecipitation/ionic

 gelation

CNS - dementia

Antioxidant

Cancer Thrombosis

63.67

 1.4%

22.63

[78]

40.13

96.57

 [79]

 [77]

85

 2.14

87.4–99

 [76]

 [75]

Polyelectrolyte

complexation

Cancers

Polyelectrolyte

complexation

Antimicrobial

42.3

65.9

 6.1

55–64.2

 [74]

 3.7–

[73]

Polyelectrolyte

complexation

CANCER

74.1

 2.2

 [72]

 telluride

 acid

**Other materials**

 **materials**

**Active compounds**

Lomustine Repaglinde Temozolomide/fluorouracil

miRNA Glucose oxidase

Polyelectrolyte

Polyelectrolyte

Layer by layer Copolymerization-precipitation

Precipitation

Cholesterol detection

 Glucose detection

complexation

 Gene Glucose detection

delivery/biomarker

80

68

 2

 [58] [59]

[60]

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

 [57]

complexation

Cancer

43

 3–76

 8 [43]

*DOI: http://dx.doi.org/10.5772/intechopen.95402*

Molecular Envelope Technology/

Treatment of Glioblastoma

multiforme

Diabetes Mellitus

 58.8 86.4

 0.31

 0.724–

[42]

Probe sonication

Solvent evaporation

**Micro-encapsulation**

 **techniques**

**Applications**

**EE %**

 **Ref.** [40]

*Polysaccharide-based micro-encapsulating carriers for delivery of bioactive compounds.*


#### *Natural Polymers in Micro- and Nanoencapsulation for Therapeutic and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.95402*

**Table 3.**

*Polysaccharide-based nano-encapsulating carriers for delivery of bioactive compounds.*

**Micro-encapsulating**

**68**

**Polysaccharide**

Gum katira

Guar gum Tamarind seed

Arabinoxylan Hyaluronic acid

Alginate Gellan gum Esterified Agave Fructans

Chitosan Galactomannan *(Delonix regia)*

β-glucan psyllium husk mucilage

Cyanobacterial

Pectin-Alginate

β-cyclodextrin Sodium alginate

**Table 2.** *Polysaccharide-based*

*micro-encapsulating*

 *carriers for delivery of bioactive compounds.*

polysaccharide

pluronic® F127

Methacrylic anhydride

Eudragit E100

Eudragit L30D-55

polysaccharide/alginate

**Other materials**

 **materials**

**Active compounds**

5-fluorouracil Bicalutamide Metformin HCl

Insulin Etanercept Indomethacin Methotrexate

Ibuprofen

Astragalus

Polysaccharide

Riboflavin Anthocyanins

Curcumin Vitamin B12

Vitamin E

Caffeine Urease/tricyanofuran

 hydrazone

 Ionotropic gelation

Emulsification-ionic

Freeze drying

 gelation

Spray drying

Cell metabolism,

 blood cell

production

Antioxidant Psychostimulant

Urea detection

52.91

74 38.5–55.5

 [61]

[71]

[70]

Spray-drying Precipitation

Antioxidant,

 antifungal and

antibacterial

Antioxidant/therapeutic

45 56

[68]

[69]

[67]

Ultrasonic atomization/

polyelectrolyte

Emulsion solvent diffusion

Cancers and auto-immune

 diseases

 50.78–84.8

 [63]

method

Coacervation Spray-drying Spray-drying

Energy conversion, growth of cells

and health of eyes and skin

Inflammation,

Allergic rhinitis

 pain

0.8–21.5

 [64] [65]

87.14–88.53

 [66]

 complexation

Emulsion solvent diffusion

Ionotropic gelation Enzymatic crosslinking

Micromoulding

Emulsion solvent evaporation

 Treatment of colon cancer

 Treatment of prostate cancer

Diabetes Diabetes

Rheumatoid arthritis

Inflammation,

 pain

 59.45 

 69.43 1.06–74 1.07 [38]

94.86 3.92

—

—

74 1

[62]

*Nano- and Microencapsulation - Techniques and Applications*

[52]

[51]

 [46]

3.18–79.25 4.25 [37]

**Micro-encapsulation**

 **technique**

**Application**

**EE %**

**Ref.**

of urea. The detection ability of the sensor was determined by the color strength and the International Commission on Illumination – CIE L\*, a⁎, and b⁎ color coordinates. The dye and enzyme-loaded crosslinked alginate microparticles coated cotton sensor strips were effectively employed to determine unknown concentrations of urea. The spectroscopic parameters indicated the microencapsulated sensor displayed a detection range of 0.1 ppm to 250 ppm. **Tables 2** and **3** indicate applications based on micro- and nano-encapsulation utilizing natural polysaccharides as encapsulating materials.
