**3.1.4 Animal proteins**

Animal proteins are amphiphilic compounds because they compose of block copolymers with both hydrophilic and hydrophobic amino acid residues. There are various types of animal proteins such as milk proteins: whey proteins and casein, gelatin, and bovine serum albumin which can be used in micro- and nanoencapsulation processes as wall materials either alone or in combination with other biopolymers: proteins (as soy protein) and polysaccharides (as chitosan) [91, 129]. Also, they possess many advantages more than plant protein as good wall materials as summarized below:


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

#### *3.1.4.1 Milk proteins*

minority fractions. Pea legumin protein (denoted 11S globulin) has molecular weight ranged 350–400 KDa while convicilin and vicilin (denoted 7S globulin) have a molecular weight of about 150 KDa [98]. Pea proteins have interesting emulsifying and gel-forming properties, so they are used alone or in combination with either proteins or polysaccharides. This interaction creates a stable emulsion that improves the efficiency of a micro/nanoencapsulation technique as it gives good particle size distribution. Also, encapsulation occurs without chemical or enzymatic modification, due to the surfactant, foaming, and solubility properties. Besides, they are

Chickpea protein possesses many excellent advantages such as low cost, biodegradable, biocompatible, and non-toxic, hence, its use in the encapsulation field. Moreover, it is generally safe for use in the food industry because of little or no toxicity and side reactions [118–120]. Chickpea protein contains glutein, albumin, prolamine, and globulin with different percentage contents as the main composition: 3.12–6.89%, 8.39–12.31%, 19.38–24.40%, and 53.44–60.29%, respectively, and represents about 28.6% of total chickpea crops [121, 122]. It is a low-cost wall material and possesses high encapsulating ability, emulsifying properties, nutritional value, and beneficial health effects. Additionally, it can form thick viscoelastic films around oil droplets thus enhancing their stability through processing. The chickpea protein is widely used in different culinary applications because it has sustained nutritional

benefits, and it is used in stews, soups, and salads [84, 93, 123, 124].

used as a wall material to form stable capsules with high EE % [128].

• More soluble than plant proteins over the wide pH range,

Lentil crops are implanted in over 48 countries around the world and they contain soluble and dietary fiber more than in both pea and chickpea crops, besides, they are rich in protein sources which ranged from 20.6–31.4% of the total lentil plant. Lentil protein comprises globulins (Mwt 320–380 KDa), albumin (Mwt20 KDa), glutelins (Mwt 17–46 KDa), and prolamins (Mwt 16–64 kDa) with different percentages: 70%, 16%, 11%, and 3%, respectively [125–127]. It has good properties such as good solubility, drying, and emulsifying properties that lead to being widely

Animal proteins are amphiphilic compounds because they compose of block copolymers with both hydrophilic and hydrophobic amino acid residues. There are various types of animal proteins such as milk proteins: whey proteins and casein, gelatin, and bovine serum albumin which can be used in micro- and nanoencapsulation processes as wall materials either alone or in combination with other biopolymers: proteins (as soy protein) and polysaccharides (as chitosan) [91, 129]. Also, they possess many advantages more than plant protein as good wall materials

• Lower Mwt than plant proteins (e.g., soy protein has Mwt: 350 kDa, while

cheap and highly nutritious [116, 117].

*Nano- and Microencapsulation - Techniques and Applications*

*3.1.3.8 Chickpea protein*

*3.1.3.9 Lentil protein*

**3.1.4 Animal proteins**

as summarized below:

• More flexible.

**76**

casein protein has Mwt: 20 kDa)

Milk proteins can be divided into two groups: casein and whey proteins which can bind their hydrophilic and hydrophobic moieties with different substances with various affinities [130]. They are considered a good choice for micro- and nanoencapsulation of bioactive materials as wall materials due to their physicochemical properties. They are available commercial products, they are flexible materials to encapsulate hydrophilic, hydrophobic and viable bioactive compounds, and they are rich bioactive peptide sources of various physiological effects. Also, they have a variety of characteristics including pH-responsiveness, self-assembly, and gel swelling behavior that lead to their use as good candidates for bioactive delivery systems [91, 131, 132].

### *3.1.4.2 Whey proteins*

Whey proteins are produced from the manufacture of either cheese or casein as the dairy byproduct. They compose of a mixture of β-lactoglubulin, α-lactalbumin, and serum albumin which are water-soluble, so they have a variety of applications [133]. They are considered complete proteins because they have nine essential amino acids, in addition, low in lactose content. The three forms of whey protein are:


Whey proteins are widely used as good wall coating materials in micro- and nanoencapsulation processes for the controlled release of different bioactive materials such as oils/fats, vitamins, and volatile compounds because of the high encapsulation efficiency and stability during storage [134–137].

#### *3.1.4.3 Casein proteins*

Casein is a major amphiphilic milk protein (it makes up about 80% of total milk protein) which is an essential part of the global daily diet. It has a variety of interesting physicochemical properties such as its availability, low-cost, nontoxicity, high stability, biocompatibility, biodegradability, binding of small and ions molecules, excellent emulsification, and self-assembly that increase its efficacy in both encapsulation and loading efficiency of the loaded bioactive ingredients [130, 138]. Casein protein's composition is 94% protein and 6% low Mwt colloidal calcium phosphate. There are four different casein fractions: αS1-, αS2-, β, and κ-casein which are amphiphilic structures in proportions of 4:1:4:1 by weight, respectively. Mwt is ranged from 19 kDa to 25 kDa [139–141]. Casein micro- and nanoencapsulation carrier systems have attracted attention in recent years for controlled and sustained release delivery of bioactive compounds because of the following advantages: their cheap price, digestibility, good dispersibility in an aqueous system, good amphiphilicity, the capability to encapsulate a variety of drug and nutrients, and form uniform spherical structures [142–144].

#### *3.1.4.4 Bovine serum albumin*

Bovine serum albumin (BSA) is a globular natural albumin protein. Its origin is either bovine serum or milk which is transferred between bovine plasma and milk through the lactating cells [131, 145]. Its structure composes a single chain of 583 amino acids with Mwt of 62.2 kDa and contains three domains: I, II, and III which are divided into two helical subdomains (A and B which bond through 17 disulfide bridges) which is specified to bind lipid, nucleotide, and metal ion [130, 146, 147]. Additionally, BSA is negatively charged at physiological pH (pH 7.4). Moreover, BSA is one of the most common protein plasma that is widely used in many applications such as drug and antigen delivery and food industry because it has good features: biocompatibility, biodegradability, non-toxicity, no immunogenicity, good stability, low cost, abundance, ease of purification and unusual ligand-binding. Consequently, its microand nano-capsule carriers have gained traction in recent years [145, 148].

**3.3 Case studies/applications**

*3.3.1.1 Hydrophobic compounds*

*3.3.1 Encapsulation of small molecules*

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

It is used with hydrophilic compounds to improve stability and bioavailability of certain compounds such as lipid vitamins (eg: A, D, E, and K). Vitamin A (VA) and VE were also successfully incorporated into biodegradable gelatin nanofibers. Curcumin is a fat-soluble polyphenol that possesses significant antioxidant and anticarcinogenic activities [158]. The release profile showed sustained release behavior of curcumin for over 7 days (around 75%) without significant burst effect when curcumin was encapsulated within amaranth protein isolate (API)/pullulan nanofibers. Dextran and whey protein concentrate (WPC) and chitosan were used as matrix materials to encapsulate lycopene by emulsion electrospinning. WPC afforded the greatest EE (around 75%), and it was also able to protect lycopene against moisture and thermal degradation [158]. Zein nanoparticles were used as delivery nano-system to enhance the oral bioavailability of quercetin (3,3<sup>0</sup>

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

pentahydroxyflavone), which is found in tea, red wine, fruits, and some vegetables. Zein is a protein extracted from corn (with molecular weight usually from 22 to 27 kDa). It has a high content of hydrophobic amino acids, such as proline, gluta-

mine, and asparagine. The encapsulation of quercetin ameliorates its antiinflammatory effect on endotoxemia was studied in a mouse model [159]. Okagu et al., [160] studied the encapsulation of hydrophobic nutraceuticals (curcumin) by biopolymer nano-complexes based on insect proteins as uncoated or coated with chitosan. The authors explained the interaction between curcumin and insect via hydrophobic forces. They observed under gastrointestinal conditions, over 90% of

the nutraceutical was released. Hu et al. [161] formed biopolymer-based

ratio of 6% loading ratio and high efficiency of encapsulation.

100 nm. The efficiency of D-limonene encapsulation was about 88%.

*3.3.1.2 Hydrophilic compounds*

**79**

nanoparticles through ionic gelation between stearic acid-chitosan conjugate (SA-CS) and sodium caseinate (NaCas) and cross-linked using oxidized dextran (Odex) via Schiff base reaction, as shown in **Figure 8**. The prepared nanoparticles were used to encapsulate Astaxanthin (ASTX) to improve its bioavailability and solubility in an aqueous medium. The authors successfully prepared nanoparticles with a diameter of 120 nm with good dispersity. They estimated the capability of loading

Xiang et al. [162] formed nanocomplexes composed of ovalbumin (OVA) and methoxy pectin (PEC) to encapsulate Vitamin D3 (VD3). Vitamin D3 is fat-soluble and readily degrades under acidic conditions. The authors observed the efficiency of VD3 encapsulation up to 96.37%. Whey proteins (positive proteins) (4% w/w), and pectin (negatively charged polysaccharides) (1% w/w) were used to form nanocomplexes which were used to encapsulate D-limonene [163]. Resulted nanocomplexes have spherical shaped nanoparticles with an average diameter of

Hydrophilic compounds are encapsulated to prevent their interactions with different compounds or to guarantee a certain release pattern. A study was undertaken to assess the release kinetics of prepared nano-encapsulated folic acid using a double W1/O/W2 emulsion [164]. Initially, loaded W1/O nano-emulsions with folic acid were formed and then re-emulsified into an aqueous stage (W2) having a concentrate on a single whey protein (WPC) layer or double-layered complex of WPC-pectin for W1/O/W2 emulsions formation. Single-layer WPC encapsulated

,4<sup>0</sup> ,5,7-

#### *3.1.4.5 Gelatin protein*

Gelatin protein is not available in nature, but it is extracted from partially hydrolyzed collagen which is the most abundant protein in the skin and bones of the animal bovine or fish. Also, it is a linear denatured protein which is carrying dual charges: positively charged (when it is extracted with acid hydrolysis of collagen, it is known as type –A gelatin and its pI is ranged 7–9.4) and negatively charged (if it is extracted with alkaline hydrolysis, it is known as type –B gelatin and its pI is ranged 4.8–5.5) and its thermal denaturation temperature is about 40°C [149, 150]. In addition, gelatin protein is a good coating material due to its amphoteric nature, and so it is widely used as coating materials in combination with different polysaccharides such as chitosan, pectin, and alginate to form hard and soft capsules (in range of micro- and nanoscale) in food and pharmaceutical applications [151–154].

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

The link of a drug with a delivery system is named "controlled drug delivery". This link can control drug pharmacokinetics. Various delivery systems were aroused. Among them, we can cite nanoparticles, liposomes, surface-modified nanoparticles, and solid-lipid nanoparticles. Among nanoparticles, protein-based nanoparticles (PBNs) have special merits because they are metabolizable, biodegradable, and can be easily controlled as there are different chances for surface improvement for drug fixation [155].

Many proteins have functional merits making them suitable for encapsulation of bioactive agents, such as pharmaceuticals and nutraceuticals. Natural proteins are biological polymers composed of amino acid chains linked together via peptide bonds, which serve in important biological functions, such as enzyme catalysis, signaling, transport, and structure formation [156]. Some of the proteins' chemical and physical characteristics can be used to construct encapsulation and carrier systems. Proteins used in the nanoparticles field can be classified as animal proteins and plant proteins, and both have advantages and disadvantages.

Low toxicity of the end product using animal proteins gives them an advantage over synthetic polymers. The major drawback of animal proteins is the risk of infection from any pathogenic contamination, although, it is not important as animal proteins can be disinfected. As for plant proteins, their hydrophobic character is the main advantage compared to animal proteins. This could lead to avoiding toxic chemical cross-linkers [157]. Besides, plant proteins are also cheaper than animal proteins.

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

#### **3.3 Case studies/applications**

*3.1.4.4 Bovine serum albumin*

*Nano- and Microencapsulation - Techniques and Applications*

*3.1.4.5 Gelatin protein*

Bovine serum albumin (BSA) is a globular natural albumin protein. Its origin is either bovine serum or milk which is transferred between bovine plasma and milk through the lactating cells [131, 145]. Its structure composes a single chain of 583 amino acids with Mwt of 62.2 kDa and contains three domains: I, II, and III which are divided into two helical subdomains (A and B which bond through 17 disulfide bridges) which is specified to bind lipid, nucleotide, and metal ion [130, 146, 147]. Additionally, BSA is negatively charged at physiological pH (pH 7.4). Moreover, BSA is one of the most common protein plasma that is widely used in many applications such as drug and antigen delivery and food industry because it has good features: biocompatibility, biodegradability, non-toxicity, no immunogenicity, good stability, low cost, abundance, ease of purification and unusual ligand-binding. Consequently, its micro-

and nano-capsule carriers have gained traction in recent years [145, 148].

**3.2 Merits and demerits in therapeutic delivery**

improvement for drug fixation [155].

**78**

Gelatin protein is not available in nature, but it is extracted from partially hydrolyzed collagen which is the most abundant protein in the skin and bones of the animal bovine or fish. Also, it is a linear denatured protein which is carrying dual charges: positively charged (when it is extracted with acid hydrolysis of collagen, it is known as type –A gelatin and its pI is ranged 7–9.4) and negatively charged (if it is extracted with alkaline hydrolysis, it is known as type –B gelatin and its pI is ranged 4.8–5.5) and its thermal denaturation temperature is about 40°C [149, 150]. In addition, gelatin protein is a good coating material due to its amphoteric nature, and so it is widely used as coating materials in combination with different polysaccharides such as chitosan, pectin, and alginate to form hard and soft capsules (in range of micro- and nanoscale) in food and pharmaceutical applications [151–154].

The link of a drug with a delivery system is named "controlled drug delivery".

Many proteins have functional merits making them suitable for encapsulation of bioactive agents, such as pharmaceuticals and nutraceuticals. Natural proteins are biological polymers composed of amino acid chains linked together via peptide bonds, which serve in important biological functions, such as enzyme catalysis, signaling, transport, and structure formation [156]. Some of the proteins' chemical and physical characteristics can be used to construct encapsulation and carrier systems. Proteins used in the nanoparticles field can be classified as animal proteins

Low toxicity of the end product using animal proteins gives them an advantage over synthetic polymers. The major drawback of animal proteins is the risk of infection from any pathogenic contamination, although, it is not important as animal proteins can be disinfected. As for plant proteins, their hydrophobic character is the main advantage compared to animal proteins. This could lead to avoiding toxic chemical cross-linkers [157]. Besides, plant proteins are also cheaper than animal proteins.

This link can control drug pharmacokinetics. Various delivery systems were aroused. Among them, we can cite nanoparticles, liposomes, surface-modified nanoparticles, and solid-lipid nanoparticles. Among nanoparticles, protein-based nanoparticles (PBNs) have special merits because they are metabolizable, biodegradable, and can be easily controlled as there are different chances for surface

and plant proteins, and both have advantages and disadvantages.

#### *3.3.1 Encapsulation of small molecules*

#### *3.3.1.1 Hydrophobic compounds*

It is used with hydrophilic compounds to improve stability and bioavailability of certain compounds such as lipid vitamins (eg: A, D, E, and K). Vitamin A (VA) and VE were also successfully incorporated into biodegradable gelatin nanofibers. Curcumin is a fat-soluble polyphenol that possesses significant antioxidant and anticarcinogenic activities [158]. The release profile showed sustained release behavior of curcumin for over 7 days (around 75%) without significant burst effect when curcumin was encapsulated within amaranth protein isolate (API)/pullulan nanofibers. Dextran and whey protein concentrate (WPC) and chitosan were used as matrix materials to encapsulate lycopene by emulsion electrospinning. WPC afforded the greatest EE (around 75%), and it was also able to protect lycopene against moisture and thermal degradation [158]. Zein nanoparticles were used as delivery nano-system to enhance the oral bioavailability of quercetin (3,3<sup>0</sup> ,4<sup>0</sup> ,5,7 pentahydroxyflavone), which is found in tea, red wine, fruits, and some vegetables. Zein is a protein extracted from corn (with molecular weight usually from 22 to 27 kDa). It has a high content of hydrophobic amino acids, such as proline, glutamine, and asparagine. The encapsulation of quercetin ameliorates its antiinflammatory effect on endotoxemia was studied in a mouse model [159]. Okagu et al., [160] studied the encapsulation of hydrophobic nutraceuticals (curcumin) by biopolymer nano-complexes based on insect proteins as uncoated or coated with chitosan. The authors explained the interaction between curcumin and insect via hydrophobic forces. They observed under gastrointestinal conditions, over 90% of the nutraceutical was released. Hu et al. [161] formed biopolymer-based nanoparticles through ionic gelation between stearic acid-chitosan conjugate (SA-CS) and sodium caseinate (NaCas) and cross-linked using oxidized dextran (Odex) via Schiff base reaction, as shown in **Figure 8**. The prepared nanoparticles were used to encapsulate Astaxanthin (ASTX) to improve its bioavailability and solubility in an aqueous medium. The authors successfully prepared nanoparticles with a diameter of 120 nm with good dispersity. They estimated the capability of loading ratio of 6% loading ratio and high efficiency of encapsulation.

Xiang et al. [162] formed nanocomplexes composed of ovalbumin (OVA) and methoxy pectin (PEC) to encapsulate Vitamin D3 (VD3). Vitamin D3 is fat-soluble and readily degrades under acidic conditions. The authors observed the efficiency of VD3 encapsulation up to 96.37%. Whey proteins (positive proteins) (4% w/w), and pectin (negatively charged polysaccharides) (1% w/w) were used to form nanocomplexes which were used to encapsulate D-limonene [163]. Resulted nanocomplexes have spherical shaped nanoparticles with an average diameter of 100 nm. The efficiency of D-limonene encapsulation was about 88%.

#### *3.3.1.2 Hydrophilic compounds*

Hydrophilic compounds are encapsulated to prevent their interactions with different compounds or to guarantee a certain release pattern. A study was undertaken to assess the release kinetics of prepared nano-encapsulated folic acid using a double W1/O/W2 emulsion [164]. Initially, loaded W1/O nano-emulsions with folic acid were formed and then re-emulsified into an aqueous stage (W2) having a concentrate on a single whey protein (WPC) layer or double-layered complex of WPC-pectin for W1/O/W2 emulsions formation. Single-layer WPC encapsulated

#### *Nano- and Microencapsulation - Techniques and Applications*

albumin, soy protein, and milk protein. Various methods used for the formulation include emulsification, desolvation, electrospray, and coacervation. Characterization parameters of these nano-formulations involve morphology of particle, size of a particle, their surface charge, entrapment of drug, loading of a drug, structure of the particle, and in vitro drug release. Different methods for the application of route

of administration for protein nanoparticles have been studied by renowned researchers [170]. In nature, particular proteins have a self-assembling property inside cells leading to the formation of nanoscale particles (called "proteinticles") with constant surface topology and structure [171]. Unlike chemically synthesized nano-formulations (e.g., various carbon, metal and polymer nanoparticles), a set of effective proteinticles can be easily produced through genetic modulation of the proteinticles surface, i.e., by inserting or adding specified peptides/proteins to the C- or -N-terminus or the internal region of the modified protein. Proteins/peptides were presented that were proven to recognize specific antibodies in certain diseases that were recognized on the outer surface of human ferritin based proteinticles purposed at accurate 3D diagnosis of human infectious and autoimmune diseases. The surface exhibited the extracellular domain of myelin oligodendrocyte glycoprotein (MOG) with native conformation successfully differentiated between autoantibodies to denatured or native MOG, leading to an accurate diagnosis of multiple sclerosis. Different antigenic peptides from the hepatitis C virus (HCV) were displayed simultaneously on the same proteinticles surface with modification of the composition of each peptide. Proteinticles having heterogeneous peptide surfaces were detected with anti-HCV antibodies in patient serum with 100% accuracy. The desired method of proteinticles engineering can be used in general to the specific

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

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

and sensitive diagnosis of several human diseases [171].

medicine [170].

**81**

sensitivity of oral cancer detection [172].

The aptamer is defined as an oligonucleotide-based nano-formulation. Unique characteristics of aptamer exhibit specificity and high-binding affinity with target molecules both intra- and extracellular. It functions as an agonist or antagonist in a biological system [170]. Recently, numerous aptamers were used for the detection of disease, with curative purposes under development for the identification of different molecules of HCC (hepatocellular carcinoma). The aptamer has been proved to improve the effectiveness of conventional chemotherapies and decrease the growth of HCC cells in vitro. Aptamer was proved to elicit antitumor activity and cell death in vivo. The overall data showed that aptamer possessed reduced toxicity levels. Moreover, it may provide a safer base in the field of personalized

Tumor Necrosis Factor-α (TNF-α) by gold protein chip was sensed using a total internal reflection fluorescence microscopy (TIRFM) as a detection method for a nano-based single biomarker for oral cancer diagnosis. Authors observed this method which is an attomolar (aM) concentration level leading to a higher

Apoferritin Ferritin is a complex of an iron-containing protein having 24 selfassembled polypeptide subunits with external and internal diameters of 12 and 7.6 nm, respectively [173]. These protein-based cage-like networks show three characteristic interfaces, the interior, exterior, and the interface present between the subunits, which exhibit functionalization. When the iron core from the inner cavity is removed, it results in a hollow protein cage-like called the Apoferritin nanocage, which is subjected to assembling and disassembling as a result of the change in the environment surrounding the molecule. Apoferritin nanocage can be utilized to insert inorganic metals inside its cavity purposing at scavenging ROS which are generated during several mechanisms in the cellular environment. This

character has been used as a template for the synthesis of an array of

nanocomposites for theragnostic applications in cancer treatment. Apoferrtin

#### **Figure 8.**

powders was the best model that fits for folic acid release pattern observed with the highest R2 . Enzymes are particularly important in therapeutics because of their catalytic activity and specificity. The challenge in the intracellular delivery of enzymes is that enzymes are unstable and have a huge size. Estrada et al. [165] developed β-Galactosidase delivery nanoparticles based on a protein. β-Galactosidase (β-gal) is an important enzyme, and its deficiency leads to several lysosomal storage disorders. The authors observed that protein-enzyme nanoparticles showed internalization in multiple cell lines in vitro higher than soluble enzyme. Authors based on the result concluded that protein nanoparticles are a biocompatible and display good efficiency for active enzyme therapeutics delivery.

#### *3.3.2 Encapsulation of biologics*

Noorani et al. [166] fabricated albumin nanoparticles enhancing anticancer efficiency of albendazole in the xenograft model of ovarian cancer. Nanoparticles based albumin was formulated with the diameter in the range of 7 to 10nm. Loaded albendazole onto albumin nanoparticles showed the highest killing effect with specificity against ovarian cancer cells studied ex vivo [167].

Trafani de Melo et al. [168] studied the design of whey protein drug delivery system for a photoactive compound, aluminum phthalocyanine chloride for targeting of glioblastoma brain cancer. Nanoparticles were fabricated by spray drying technique with particle size between 100 and 300 nm. Authors based on their results concluded that a combination of hydrophobic drugs and irradiation achieve efficient treatment.

Stein et al. formulated mTHPC-albumin nanoparticles using nab-technology [169]. Nanoparticles showed colloidal stability over a wide range of pH and in physiological NaCl with different concentrations. The authors observed cell culture uptake of mTHPC in a cholangiocarcinoma cell line (TFK-1).

#### *3.3.3 Encapsulation of diagnostics*

Nanoscale materials permit nanodevices to enter novel scientific and technological frontiers in different diseases especially cancer diagnosis. Proteinticles are nanoscale protein particles designated by engineering, which are very useful in changing different properties based on surface area and size of various conventional things [170]. Combined detection of two serum biomarkers is also feasible through multiplexed viral detection. The disease detection method is imperative in diseases such as AIDS and hepatitis. Such a protocol is based on lateral flow assay (LFA) for protein nanoparticles. Proteinticles were found to have better biocompatibility and biodegradability with compliance with surface modifications. These nanoparticles are formed by using different proteins like elastin, gliadin, gelatin, zein, legumin,

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

albumin, soy protein, and milk protein. Various methods used for the formulation include emulsification, desolvation, electrospray, and coacervation. Characterization parameters of these nano-formulations involve morphology of particle, size of a particle, their surface charge, entrapment of drug, loading of a drug, structure of the particle, and in vitro drug release. Different methods for the application of route of administration for protein nanoparticles have been studied by renowned researchers [170]. In nature, particular proteins have a self-assembling property inside cells leading to the formation of nanoscale particles (called "proteinticles") with constant surface topology and structure [171]. Unlike chemically synthesized nano-formulations (e.g., various carbon, metal and polymer nanoparticles), a set of effective proteinticles can be easily produced through genetic modulation of the proteinticles surface, i.e., by inserting or adding specified peptides/proteins to the C- or -N-terminus or the internal region of the modified protein. Proteins/peptides were presented that were proven to recognize specific antibodies in certain diseases that were recognized on the outer surface of human ferritin based proteinticles purposed at accurate 3D diagnosis of human infectious and autoimmune diseases. The surface exhibited the extracellular domain of myelin oligodendrocyte glycoprotein (MOG) with native conformation successfully differentiated between autoantibodies to denatured or native MOG, leading to an accurate diagnosis of multiple sclerosis. Different antigenic peptides from the hepatitis C virus (HCV) were displayed simultaneously on the same proteinticles surface with modification of the composition of each peptide. Proteinticles having heterogeneous peptide surfaces were detected with anti-HCV antibodies in patient serum with 100% accuracy. The desired method of proteinticles engineering can be used in general to the specific and sensitive diagnosis of several human diseases [171].

The aptamer is defined as an oligonucleotide-based nano-formulation. Unique characteristics of aptamer exhibit specificity and high-binding affinity with target molecules both intra- and extracellular. It functions as an agonist or antagonist in a biological system [170]. Recently, numerous aptamers were used for the detection of disease, with curative purposes under development for the identification of different molecules of HCC (hepatocellular carcinoma). The aptamer has been proved to improve the effectiveness of conventional chemotherapies and decrease the growth of HCC cells in vitro. Aptamer was proved to elicit antitumor activity and cell death in vivo. The overall data showed that aptamer possessed reduced toxicity levels. Moreover, it may provide a safer base in the field of personalized medicine [170].

Tumor Necrosis Factor-α (TNF-α) by gold protein chip was sensed using a total internal reflection fluorescence microscopy (TIRFM) as a detection method for a nano-based single biomarker for oral cancer diagnosis. Authors observed this method which is an attomolar (aM) concentration level leading to a higher sensitivity of oral cancer detection [172].

Apoferritin Ferritin is a complex of an iron-containing protein having 24 selfassembled polypeptide subunits with external and internal diameters of 12 and 7.6 nm, respectively [173]. These protein-based cage-like networks show three characteristic interfaces, the interior, exterior, and the interface present between the subunits, which exhibit functionalization. When the iron core from the inner cavity is removed, it results in a hollow protein cage-like called the Apoferritin nanocage, which is subjected to assembling and disassembling as a result of the change in the environment surrounding the molecule. Apoferritin nanocage can be utilized to insert inorganic metals inside its cavity purposing at scavenging ROS which are generated during several mechanisms in the cellular environment. This character has been used as a template for the synthesis of an array of nanocomposites for theragnostic applications in cancer treatment. Apoferrtin

powders was the best model that fits for folic acid release pattern observed with the

*General mechanism of ASTX loaded SA-CS/NaCas/Odex nanoparticles formulation and its application.*

catalytic activity and specificity. The challenge in the intracellular delivery of enzymes is that enzymes are unstable and have a huge size. Estrada et al. [165] developed β-Galactosidase delivery nanoparticles based on a protein. β-Galactosidase (β-gal) is an important enzyme, and its deficiency leads to several lysosomal storage disorders. The authors observed that protein-enzyme nanoparticles showed internalization in multiple cell lines in vitro higher than soluble enzyme. Authors based on the result concluded that protein nanoparticles are a biocompatible and

display good efficiency for active enzyme therapeutics delivery.

*Nano- and Microencapsulation - Techniques and Applications*

specificity against ovarian cancer cells studied ex vivo [167].

uptake of mTHPC in a cholangiocarcinoma cell line (TFK-1).

. Enzymes are particularly important in therapeutics because of their

Noorani et al. [166] fabricated albumin nanoparticles enhancing anticancer efficiency of albendazole in the xenograft model of ovarian cancer. Nanoparticles based albumin was formulated with the diameter in the range of 7 to 10nm. Loaded albendazole onto albumin nanoparticles showed the highest killing effect with

Trafani de Melo et al. [168] studied the design of whey protein drug delivery

Stein et al. formulated mTHPC-albumin nanoparticles using nab-technology [169]. Nanoparticles showed colloidal stability over a wide range of pH and in physiological NaCl with different concentrations. The authors observed cell culture

Nanoscale materials permit nanodevices to enter novel scientific and technolog-

ical frontiers in different diseases especially cancer diagnosis. Proteinticles are nanoscale protein particles designated by engineering, which are very useful in changing different properties based on surface area and size of various conventional things [170]. Combined detection of two serum biomarkers is also feasible through multiplexed viral detection. The disease detection method is imperative in diseases such as AIDS and hepatitis. Such a protocol is based on lateral flow assay (LFA) for protein nanoparticles. Proteinticles were found to have better biocompatibility and biodegradability with compliance with surface modifications. These nanoparticles are formed by using different proteins like elastin, gliadin, gelatin, zein, legumin,

system for a photoactive compound, aluminum phthalocyanine chloride for targeting of glioblastoma brain cancer. Nanoparticles were fabricated by spray drying technique with particle size between 100 and 300 nm. Authors based on their results concluded that a combination of hydrophobic drugs and irradiation

highest R2

**Figure 8.**

*3.3.2 Encapsulation of biologics*

achieve efficient treatment.

*3.3.3 Encapsulation of diagnostics*

**80**


#### *Nano- and Microencapsulation - Techniques and Applications*

**Table 5.**

 *Plant and animal proteins-based micro-encapsulated carriers for delivery of bioactive compounds.* nanoparticles enter the targeted tumor cells via clathrin-mediated endocytosis,

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

**Nanoencapsulation techniques**

— β-carotene Emulsification Inhibiting the DNA

separation

emulsification

precipitation

precipitation

— Resveratrol Coacervation Inflammation and

Modified desolvation

emulsification

precipitation

— Mequindox Spray drying Inhibition of pathogenic

precipitation

Modified desolvation

*Plant and animal proteins-based nano-encapsulated carriers for delivery of bioactive compounds.*

*Ch:Chitosan; CDF: di-fluorinated curcumin; CMCFG: carboxymethylated corn fiber gum.*

*EE: Encapsulation Efficiency; SPI: Soy protein isolate; WPC: Whey protein concentrate; WPI: Whey protein isolate;*

— Celecoxib Coacervation Inflammation treatment 216.1 nm

**Applications Particle**

Cancer Treatment 247 nm

Cancer treatment 200 nm

damage and enhancing the immune system

> Folate deficiency treatment

Satietogenic and inflammation treatment

cancer treatment

Ovarian and cervical Cancer treatment

Deficient Vitamin D treatment

bacteria

Metastatic colorectal cancer

> Ischemic stroke treatment

Inflammation treatment 100 nm

Curcumin Coacervation Cancer treatment 200 nm

**size (nm) and EE %**

90 nm EE: 93.5%

EE: 90.1%

100 nm EE: 86.6%

EE: 83.8%

109 nm EE: 98.5%

175 nm EE: 60%

197.8 nm, EE: 78.4% (CDF) & EE:77.4% (Paclitaxel)

EE: 92.1%

233 nm EE: 96.0%

EE: 99.2%

EE: 90.7%

262.5 nm EE: 72.2%

200 nm EE:11.2%

362.3 nm EE: 95.5% **Ref.**

[186]

[187]

[188]

[189]

[190]

[191]

[192]

[193]

[194]

[195]

[196]

[197]

[198]

[199]

A ferritin-based multifunctional nanomaterial was prepared for MR and fluorescence simultaneous imaging of lung cancer cells. Human H-chain ferritin was engineered with green fluorescent protein aiming at stable fluorescence in the cells. Moreover, arginylglycylaspartic acid peptide was fused on the external surface of the ferritin cage for αvβ3 integrin receptors targeting human tumor cells (human glioblastoma U87MG cells and A549 cells) [173]. Multifunctional nanostructures based on ferritin (RGD-GFP-ferritin [RGF]/Fe3O4, rHF/Fe3O4,

receptor-mediated endocytosis, and macropinocytosis processes.

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

**Active compounds**

— Doxorubicin Phase

Maltodextrin Vitamin B9 Nano-

Folic acid Doxorubicin Nano-

Folic acid Paclitaxel

Starch and chitosan

Ch Trypsin Nano-

and CDF

— Vitamin D Nano-

CMCFG Curcumin Nano-

Gelatin Folic acid Irinotecan Nano-

— 17β-estradiol (E2)

**Nano-encapsulating materials**

**materials**

**Protein Other**

Barley protein

Zein protein

WPC protein

SPI protein

WPI protein

BSA protein

Pea Protein

Casein protein

**Table 6.**

**83**

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

nanoparticles enter the targeted tumor cells via clathrin-mediated endocytosis, receptor-mediated endocytosis, and macropinocytosis processes.

A ferritin-based multifunctional nanomaterial was prepared for MR and fluorescence simultaneous imaging of lung cancer cells. Human H-chain ferritin was engineered with green fluorescent protein aiming at stable fluorescence in the cells. Moreover, arginylglycylaspartic acid peptide was fused on the external surface of the ferritin cage for αvβ3 integrin receptors targeting human tumor cells (human glioblastoma U87MG cells and A549 cells) [173]. Multifunctional nanostructures based on ferritin (RGD-GFP-ferritin [RGF]/Fe3O4, rHF/Fe3O4,


*EE: Encapsulation Efficiency; SPI: Soy protein isolate; WPC: Whey protein concentrate; WPI: Whey protein isolate; Ch:Chitosan; CDF: di-fluorinated curcumin; CMCFG: carboxymethylated corn fiber gum.*

#### **Table 6.**

*Plant and animal proteins-based nano-encapsulated carriers for delivery of bioactive compounds.*

**Micro-encapsulating**

**82**

**Protein** Barley protein Chickpea and lentil proteins

WPI protein Zein and WPI proteins

Casein protein

Gelatin

—

—

Lactose

—

Ethyl cellulose

GPTMS

—

Ch and HA

—

Polyglycerol

polyricinoleate

*3-glycidoxypropyltrimethoxysilane;*

 *WPI: Whey protein isolate; BSA: Bovine serum albumin; Ch: Chitosan; HA: hyaluronic acid.*

 Riboflavin

WPI

BSA protein

Pea protein

*EE:*  **Table 5.** *Plant and animal* 

*proteins-based*

*micro-encapsulated*

 *carriers for delivery of bioactive compounds.*

*Encapsulation*

 *Efficiency; GPTMS:* 

Oil/milkfat

 compounds

Ciprofloxacin

Curcumin Vancomycin

Riboflavin Sorafenib Hemoglobin

Desolvation

 and Spray drying

Complexation Co-precipitation

Emulsification

Hepatocellular

Erythrocytes

 Shortage treatment

Ariboflavinosis

 treatment

 EE: 84.0%

 [185]

 carcinoma treatment

Ariboflavinosis

 treatment

 EE: 96.6%

 EE: 45.6%

 EE: 82.0%

 [184]

 [183]

 [182]

Spray drying

Spray drying

Spray drying Emulsification

Skin and joint infections treatment

Docosahexan-oic

 acid

> β-carotene

Spray drying Spray drying

Improving the

Inhibiting the DNA damage and

enhancing the immune system Reducing the lipid oxidation problem

Respiratory

 tract infection treatment

Bladder cancer treatment

 EE: 95.2%

 EE: 80.0%

—

—

[181]

[180]

 [179]

 [178]

hypertriglyceridemia

 EE: 93.2% EE: 74.0%

 [177]

 [176]

*Nano- and Microencapsulation - Techniques and Applications*

Maltodextrin

Flaxseed oil

Spray drying

**Other materials**

—

Fish oil

Spray drying

Reducing the

improving the

Reducing the coronary heart risks

 EE:88.0%

[175]

(Lentil) &

EE:86.3%

(Chickpea)

inflammation

 and

EE: 92.9%

 [174]

hypertriglyceridemia

 **materials**

**Active compounds**

**Micro-encapsulation**

 **techniques**

**Applications**

**EE %**

 **Ref.** and GFP-rFH/Fe3O4) were prepared by synthesizing iron oxide (Fe3O4) nanoparticles in the previously engineered ferritin cages. Imaging of these cages with fluorescence targeted to αvβ3 integrin-positive A549 and U87MG cells showed higher-intensity fluorescence with RGF, when compared to GFP-rHF control cells. Furthermore, MRI with RGF showed significant enhancement of the signal to facilitate meticulous diagnosis, when compared to GFP-rHF/Fe3O4 or without contrast agent. Therefore, efficient targeting and fluorescence imaging of lung cancer cells utilizing engineered nanocages were proved to be a useful vehicle among the different multifunctional, nanostructured, protein-based tools to be used in fluorescent imaging.

The antioxidant enzymes present normally inside the human body, like catalase, superoxide dismutase (SOD), and peroxidase, fail in the protection of the cells under sudden oxidative damage/stress conditions Thus, further studies have developed artificial antioxidants capable of decreasing oxidative stress during lung cancer treatment [173]. Apoferritin-encapsulated protein nanoparticles have been prepared as artificial antioxidants on account of their peroxidase, catalase, and SOD-mimicking activity. Apoferritin-CeO2 nano-truffle has been used as an artificial redox enzyme owing to its ability to mimic SOD activity. This character can be utilized to combat ROS-mediated lung cancer by scavenging hydrogen superoxide, peroxide, and other small molecules triggered in sudden oxidative damage. Thus, these systems show potential for hopeful application in lung cancer treatment [173]. **Tables 5** and **6** indicate applications based on micro- and nano-encapsulation utilizing animal/plant proteins as encapsulating materials.

**Author details**

Joan O. Erebor<sup>4</sup>

Jos, Jos, Nigeria

Ndidi C. Ngwuluka<sup>1</sup>

Soliman M.A. Soliman<sup>6</sup>

\*, Nedal Y. Abu-Thabit<sup>2</sup>

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

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

, Margaret O. Ilomuanya<sup>5</sup>

College, Jubail Industrial City 31961, Saudi Arabia

Sciences, University of Toledo, Toledo, Ohio, USA

and abuthabit\_nidal@yahoo.com

**85**

provided the original work is properly cited.

\*, Onyinye J. Uwaezuoke<sup>3</sup>

,

, Riham R. Mohamed<sup>6</sup>

, Mahmoud H. Abu Elella<sup>6</sup> and Noura A.A. Ebrahim<sup>7</sup>

1 Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of

2 Department of Chemical and Process Engineering Technology, Jubail Industrial

3 Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria

4 Department of Pharmacy Practice, College of Pharmacy and Pharmaceutical

5 Center for Biomedical Research, Population Council, New York, 10065, USA

7 Pathology Department, National Cancer Institute, Cairo University, Giza, Egypt

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

6 Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt

\*Address all correspondence to: ndidi.ngwuluka@biodrudel.com

,

### **4. Conclusions and future trends**

This chapter describes in details the applications of polysaccharides, and proteins, as natural nanocarriers for encapsulation and safe delivery of various therapeutic, diagnostic and theragnostic agents. The chapter provides detailed discussion with recent examples and case studies for using polysaccharides and proteins as biocompatible, biodegradable nanocarriers for encapsulation and delivery of small molecules, biologics, and diagnostics.

Encapsulation will remain a valuable process in the design and development of drug delivery systems and fabrication of diagnostic tools. Advances in naturapolyceutics and encapsulation technologies will continue to drive the applicability of natural polymers and encapsulation in drug delivery and diagnostics. More of polymer blending or interactions; increasing combination of the classes of natural polymers will be observed to achieve the evolving need to improve on the delivery of existing drugs and drugs in the development pipeline. The desire to enhance selectivity, specificity and sensitivity of biosensors will continue to drive the innovations and applications of natural polymers in diagnostic space. Filling the gaps in patient related therapies will place encapsulation as the main stay technology in solving delivery related problems and diagnostic challenges. The quest for maximization, cost effectiveness, reducing patient complications, and optimization of systems and devices will lead to increased assembling of multifunctional all-in-one devices. Theragnostics has come to stay and will precipitate combination of natural polymers and encapsulation technologies to achieve the desired theragnostics that will detect biomarkers, bioimage; and target, deliver and monitor drugs at the site of action. As drug delivery and diagnostics advance, natural polymers will remain materials of focus due to their biogenicity, biodegradability, biocompatibility, good interactions with living cells, suitability for long circulation and targeting, and cell recognition.

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