**5. Natural polymers in encapsulation**

In 1953, Hermann Staudinger was awarded the Nobel Prize in chemistry for demonstrating the existence of "*Makromoleküle*" macromolecules which led to the birth of the polymer chemistry field [94]. In the past 50 years, various natural, synthetic and semi-synthetic polymers have been investigated for developing diverse nano-, micro-, and macroscale drug delivery system (DDSs) for various therapeutic and diagnostic applications [94–96]. Natural polymers along with their derivatives (semi-synthetic polymers) are the safest micro- and nanocarriers due to their low toxicity, biocompatibility and intrinsic biodegradability by enzymes [97, 98]. This section highlights the main types of natural polysaccharides, proteins and lipids that have been employed as nanocarriers for therapeutic and theranostic applications.

#### **5.1 Polysaccharides**

*Nano- and Microencapsulation - Techniques and Applications*

encapsulate polar materials in their core while keeping hydrophobic materials in their lipid bilayer. Liposomes are traditionally made by the film hydration method with constituents like lipid, cholesterol, and solvent. Film hydration involves the dissolution of the lipid components in a suitable solvent most commonly ethanol and chloroform. The solvent is removed in a rotary evaporator leaving behind a thin film which is rehydrated to yield large multilamellar vesicles liposomes. The size of the liposomes can be reduced by passing through successively smaller sized polycarbonate filters. Ultrasonication method of preparing liposomes involves an aqueous dispersion of lipids using a strong sonicator probe and usually yields small unilamellar vesicles. Reverse phase evaporation is another method for liposome preparation [91]. In this method, a mixture of lipids and cholesterol dissolved in an appropriate solvent is subjected to the rotary evaporator for solvent removal. The residue is dried with hydrogen and resuspended in an organic solvent usually diethyl ether. An aqueous solution of the drug to be encapsulated is added to the lipid solution and sonicated under nitrogen until a homogenous mixture result. The solvent is then removed to yield large unilamelar vesicles usually used to encapsulate large molecular weight biomolecules. Ether vaporization method involves a mixture of lipids dissolved in an organic solvent such as ether and subsequently

injected into a hot aqueous solution resulting in osmotic liposomes [92].

*4.3.8 Molecular inclusion complexes*

Major instability issues with liposomes is related to hydrolysis, oxidation, aggregation, and fusion. Appropriate buffer inclusion is necessary to limit oxidation of liposome phospholipids. Freeze drying has also been used to overcome the effect of temperature on liposomes. Such proliposomes are then reconstituted in water just before use. Research by Gomez and coworkers [91] showed that the encapsulation efficiency of any liposome preparation depend on the encapsulated molecule.

Inclusion complexes are microcapsules made by including a material to be encapsulated into the cavity of cyclodextrin molecule. Cyclodextrins are a family of cyclic oligosaccharides made up of glucopyranosyl linked by α (1,4) bonds. The most common members of the family are α-, β-, and γ- cyclodextrins consisting of 6, 7, and 8 glucopyranose units respectively. The most frequently used is β-cyclodextrin. The unique nature of a cyclodextrin molecule with a hydrophobic cavity enclosed by a hydrophilic container makes them targets for encapsulation of hydrophobic molecules. They serve as host to a great variety of hydrophobic compounds. Materials are enclosed into their cavity through different means. Physical mixing through a kneading action of a solution of guest molecule with a slurry of cyclodextrin. The kneaded paste is dried and washed with a solvent. This is usually reserved for very poorly soluble materials and unsuitable for large scale production. In co-precipitation method, the guest molecule is dissolved is a suitable organic solvent such as diethyl ether, chloroform. Then, an aqueous solution of the cyclodextrin is added under agitation. The complex formed is precipitated out of solution using temperature reduction. The crystals are collected, washed with organic solvent and dried at

50°C. This method is usually reserved for payloads not too soluble in water [93].

dried in vacuum. This method is scalable and gives good yields [93].

Heating can also be used for inclusion complex formation. For this procedure, the physical mixture of the guest and the host can adsorb water and thereafter is heated in an enclosed vessel at a temperature of 40–145°C. This process yields crystalline complexes but can only be used for payloads stable at such temperature range [93]. Freeze drying is usually reserved for heat labile water-soluble cargoes. The required quantities of both guest and host materials are dissolved in water with stirring and then freeze dried. The obtained crystals are then washed with an organic solvent and

**26**

Polysaccharides are the most abundant natural biopolymers derived from diverse bioresources, **Figure 12**. Polysaccharides are different from proteins, nucleic acids,

**Figure 12.**

*Classification of polysaccharides based on their origin [100].*

glycoproteins and glycolipids, in that they contain repetitive structural features [99]. Polysaccharides have been employed as responsive nanocarriers for targeted and controlled gene delivery and drug delivery of small molecules, proteins, peptides, nucleic acids, and antibiotics [100]. Among various polysaccharides, cellulose is the most abundant renewable natural polymer on earth, which is unbranched, linear homopolysaccharide, composed of repeating β-(1 → 4) linked d-glucose units [101]. However, since cellulose is water-insoluble, various water-soluble and hydrophilic cellulose-based derivatives have been used for creating macroscale DDSs and devices for oral drug delivery to the gastrointestinal (GI) tract due to their good compression characteristics and adequate water-swelling property which allows for controlled release drugs through rapid formation of an external gel layer [102]. Examples of commercialized macroscale DDSs based on cellulose acetate and hydroxypropyl methylcellulose (HPMC) are shown in **Figure 13** (I, II). The details about the former DDSs/devices and the mechanisms of drug release are described by Abu-Thabit and Makhlouf [94].

Another important polysaccharide is starch. Starches are made from 300 to 1000 glucose monomeric units. The main components of starch are amylose (~20%) and amylopectin (~80%) macromolecules. Amylose is unbranched, linear homopolysaccharide, composed of repeating α-(1 → 4) linked d-glucose units. Amylose adapts helical structure due to the formation of hydrogen bonding among D-glucose monomeric units. The helical conformation of amylose provides room to accommodate the iodine molecules in its core, and results in the formation of iodine-amylose complex with the characteristic blue-violet color as a strong indication for the presence of tiny amounts of starch. Amylopectin is a branched polysaccharide that is composed of repeating

#### **Figure 13.**

*(I) And (II) represent the chemical structures and examples of macroscale-based DDSs using HPMC, and cellulose acetate; reproduced with permission from ref. [94]. (III) schematic illustration of (a) structure of cyclodextrin polymer; diversity of using cyclodextrins for drug delivery systems via (b) host-guest interactions; (c) formation of supramolecular inclusion complexes (e.g. with PEG); and (d) cyclodextrindrug conjugates; (IV) conventional representation for native cyclodextrins (CDs) as a truncated cone with "hydrophobic" cavity (blue color) that can accommodate hydrophobic drugs; reproduced with permissions from ref. [103].*

**29**

**5.2 Proteins**

proteins including gelatin, casein, and albumin.

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

α-(1 → 4) linked d-glucose units with occasional α-1,6-glycosidic bonds, which are responsible for the branching. The helical structure of amylopectin is disrupted by the available branched side chains which yield less intense reddish-brown color for the formed amylopectin-iodine complex instead of intense blue-violet color. Another class of polysaccharides nanocarriers is cyclodextrins (CDs) which are crystalline cyclic oligosaccharides consisting of α-1,4-glycosidic bonded D-glycopyranose units with glucose units arranged in a donut shape ring [103]. CDs are produced by enzymatic degradation of starch. CDs are classified as cage molecules with hydrophilic exterior and hydrophobic inner cavity which enables the formation of inclusion complexes with a variety of hydrophobic drug molecules [103], **Figure 13** (III). CDs are categorized based on the number of glucose residues in their structure, for example, CD with the glucose hexamer is named as α-CD, the heptamer as β-CD and the octomer as γ-CD [104]. The designs of supramolecular systems with CD are very diverse; since CDs can be used alone, grafted to other molecules or linked to each other [105], **Figure 13** (IV). Chitin is the second most abundant natural polysaccharide which can be described

as cellulose with one hydroxyl group on each monomer replaced by acetyl amine group. Chitin is abundant in invertebrates, mollusks, the cell walls of fungi, and the exoskeletons of arthropods. Like cellulose, chitin is a hydrophobic and water-insoluble biopolymer with limited application for fabricating DDSs. Chitosan, which is prepared by alkaline or enzymatic hydrolysis of chitin, is considered as the most important derivative of chitin due to its biocompatibility, biodegradability and non-toxic nature [106]. Unlike most anionic polysaccharides, chitosan is classified as a cationic polymer due to the presence of amine group which can be protonated upon dissolving chitosan in dilute acidic solutions such as acetic acid or hydrochloric acid. This unique character allowed chitosan to be used for fabricating various DDSs, such as micro/nanoparticles and hydrogels, *via* formation of polyelectrolyte complexes with various anionic polysaccharides [107, 108]. Another naturally occurring linear polysaccharide is hyaluronic acid (HA) (also called hyaluronan) which is composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide. HA acts as an anionic polyelectrolyte at neutral pH, as the pKa of the carboxylic acid groups is ≈ 3–4, which makes HA highly hydrophilic and superabsorbent for water, with ability to expand up to 1000 times its solid volume, leading to loose, hydrated network [97]. Dextran is a water-soluble branched polysaccharide with varied chain lengths and molecular weight in the range of 30,000 – 2,000,000 g/mole [106]. In 1861, Louis Pasteur isolated dextran from wine as a microbial product [109]. Dextran has been applied in nanomedicine, a novel discipline that applies submicron particles for therapeutic and diagnostic purposes. Dextran has been applied in nanomedicine as encapsulating matrix for therapeutic and diagnostic purposes [94], and as an alternative to PEGylation to avoid nanoparticles (NPs) [95] and opsonin interactions [110]. Dextran has been employed for preparing pH-sensitive NPs by polyelectrolyte complexation between dextran sulfates and chitosan [108, 111].

The term protein *was coined in 1838 by the* Swedish chemist Jöns Jacob Berzelius, which was derived from the Greek *prōteios*, meaning "holding first place" [112]. Proteins are versatile biomacromolecules with large and diverse functions in living organisms such as transcription, translation, transport and metabolism [113]. Proteins are key class of biopolymers that have been extensively used as nanocarriers for therapeutic and diagnostic drug delivery applications [114]. Proteins can be classified based on their origin as plant-based proteins and animal-based proteins, **Figure 14**. Detailed description and characteristic for each protein type is provided in the next chapter on proteins. This section provides brief idea about animal-based

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

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

α-(1 → 4) linked d-glucose units with occasional α-1,6-glycosidic bonds, which are responsible for the branching. The helical structure of amylopectin is disrupted by the available branched side chains which yield less intense reddish-brown color for the formed amylopectin-iodine complex instead of intense blue-violet color. Another class of polysaccharides nanocarriers is cyclodextrins (CDs) which are crystalline cyclic oligosaccharides consisting of α-1,4-glycosidic bonded D-glycopyranose units with glucose units arranged in a donut shape ring [103]. CDs are produced by enzymatic degradation of starch. CDs are classified as cage molecules with hydrophilic exterior and hydrophobic inner cavity which enables the formation of inclusion complexes with a variety of hydrophobic drug molecules [103], **Figure 13** (III). CDs are categorized based on the number of glucose residues in their structure, for example, CD with the glucose hexamer is named as α-CD, the heptamer as β-CD and the octomer as γ-CD [104]. The designs of supramolecular systems with CD are very diverse; since CDs can be used alone, grafted to other molecules or linked to each other [105], **Figure 13** (IV).

Chitin is the second most abundant natural polysaccharide which can be described as cellulose with one hydroxyl group on each monomer replaced by acetyl amine group. Chitin is abundant in invertebrates, mollusks, the cell walls of fungi, and the exoskeletons of arthropods. Like cellulose, chitin is a hydrophobic and water-insoluble biopolymer with limited application for fabricating DDSs. Chitosan, which is prepared by alkaline or enzymatic hydrolysis of chitin, is considered as the most important derivative of chitin due to its biocompatibility, biodegradability and non-toxic nature [106]. Unlike most anionic polysaccharides, chitosan is classified as a cationic polymer due to the presence of amine group which can be protonated upon dissolving chitosan in dilute acidic solutions such as acetic acid or hydrochloric acid. This unique character allowed chitosan to be used for fabricating various DDSs, such as micro/nanoparticles and hydrogels, *via* formation of polyelectrolyte complexes with various anionic polysaccharides [107, 108]. Another naturally occurring linear polysaccharide is hyaluronic acid (HA) (also called hyaluronan) which is composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide. HA acts as an anionic polyelectrolyte at neutral pH, as the pKa of the carboxylic acid groups is ≈ 3–4, which makes HA highly hydrophilic and superabsorbent for water, with ability to expand up to 1000 times its solid volume, leading to loose, hydrated network [97]. Dextran is a water-soluble branched polysaccharide with varied chain lengths and molecular weight in the range of 30,000 – 2,000,000 g/mole [106]. In 1861, Louis Pasteur isolated dextran from wine as a microbial product [109]. Dextran has been applied in nanomedicine, a novel discipline that applies submicron particles for therapeutic and diagnostic purposes. Dextran has been applied in nanomedicine as encapsulating matrix for therapeutic and diagnostic purposes [94], and as an alternative to PEGylation to avoid nanoparticles (NPs) [95] and opsonin interactions [110]. Dextran has been employed for preparing pH-sensitive NPs by polyelectrolyte complexation between dextran sulfates and chitosan [108, 111].

#### **5.2 Proteins**

*Nano- and Microencapsulation - Techniques and Applications*

glycoproteins and glycolipids, in that they contain repetitive structural features [99]. Polysaccharides have been employed as responsive nanocarriers for targeted and controlled gene delivery and drug delivery of small molecules, proteins, peptides, nucleic acids, and antibiotics [100]. Among various polysaccharides, cellulose is the most abundant renewable natural polymer on earth, which is unbranched, linear homopolysaccharide, composed of repeating β-(1 → 4) linked d-glucose units [101]. However, since cellulose is water-insoluble, various water-soluble and hydrophilic cellulose-based derivatives have been used for creating macroscale DDSs and devices for oral drug delivery to the gastrointestinal (GI) tract due to their good compression characteristics and adequate water-swelling property which allows for controlled release drugs through rapid formation of an external gel layer [102]. Examples of commercialized macroscale DDSs based on cellulose acetate and hydroxypropyl methylcellulose (HPMC) are shown in **Figure 13** (I, II). The details about the former DDSs/devices and the mecha-

nisms of drug release are described by Abu-Thabit and Makhlouf [94].

Another important polysaccharide is starch. Starches are made from 300 to 1000 glucose monomeric units. The main components of starch are amylose (~20%) and amylopectin (~80%) macromolecules. Amylose is unbranched, linear homopolysaccharide, composed of repeating α-(1 → 4) linked d-glucose units. Amylose adapts helical structure due to the formation of hydrogen bonding among D-glucose monomeric units. The helical conformation of amylose provides room to accommodate the iodine molecules in its core, and results in the formation of iodine-amylose complex with the characteristic blue-violet color as a strong indication for the presence of tiny amounts of starch. Amylopectin is a branched polysaccharide that is composed of repeating

*(I) And (II) represent the chemical structures and examples of macroscale-based DDSs using HPMC, and cellulose acetate; reproduced with permission from ref. [94]. (III) schematic illustration of (a) structure of cyclodextrin polymer; diversity of using cyclodextrins for drug delivery systems via (b) host-guest interactions; (c) formation of supramolecular inclusion complexes (e.g. with PEG); and (d) cyclodextrindrug conjugates; (IV) conventional representation for native cyclodextrins (CDs) as a truncated cone with "hydrophobic" cavity (blue color) that can accommodate hydrophobic drugs; reproduced with permissions* 

**28**

**Figure 13.**

*from ref. [103].*

The term protein *was coined in 1838 by the* Swedish chemist Jöns Jacob Berzelius, which was derived from the Greek *prōteios*, meaning "holding first place" [112]. Proteins are versatile biomacromolecules with large and diverse functions in living organisms such as transcription, translation, transport and metabolism [113]. Proteins are key class of biopolymers that have been extensively used as nanocarriers for therapeutic and diagnostic drug delivery applications [114]. Proteins can be classified based on their origin as plant-based proteins and animal-based proteins, **Figure 14**. Detailed description and characteristic for each protein type is provided in the next chapter on proteins. This section provides brief idea about animal-based proteins including gelatin, casein, and albumin.

#### **Figure 14.**

*Classification of proteins based on their origin with the list of the main components.*

Collagen is the most abundant protein in mammals which forms 30% of all vertebrate body protein with a majority in bone and skin. Gelatin is a denatured collagen which is obtained by either acid hydrolysis (gelatin type A with isoelectric point ≈ 7–9), or alkaline hydrolysis (gelatin type B with isoelectric point ≈ 4.8–5) [115]. Gelatin is biocompatible and biodegradable with high physiological tolerance and low immunogenicity. Gelatin is classified as "Generally Recognized as Safe" (GRAS) by the US Food and Drug Administration (FDA). Therefore, gelatin have been used for vitamin preparation, drug capsules, scaffolding materials to promote cell migration, wound healing, tissue regeneration and as a nanocarrier for drug and gene delivery [106, 115]. Casein and whey proteins are important protein sources for human nutrition. Casein is one of the oldest natural polymers, used for adhesives, dating back to thousands of years [116]. In contrast to whey proteins, caseins are water-insoluble and account for 80% of total bovine milk proteins. Casein protein is found in milk which serves biologically to transfer nutrients from mother to her offspring. Hence, it can be used as a carrier depot for delivery of drugs. Casein has four constituent phosphate-rich sub-units, which are amphiphilic and self-assemble into a micellar structure in the size range 50–300 nm, held together by calcium phosphate nanoclusters acting as bridges connecting these subunits [117]. Although the word "albumin" is usually associated to serum albumin, it is also employed to describe a family of proteins characterized by their solubility in water [114]. Human serum albumin (HSA) and lactalbumin (known as whey protein) are the most popular albumin proteins employed for drug delivery applications [114]. Besides that, albumins can be found in foods, particularly in seeds and nuts. Serum albumin is the major protein constituent in the blood plasma of all vertebrates. The two main exponents are human (HSA) and bovine serum albumin (BSA). Albumin has diverse physiological functions such as maintaining the pH and colloidal osmotic pressure of plasma, its antioxidant effect by trapping free radicals, and its reversible binding ability to variety of important exogenous and endogenous [118, 119]. Albumin has the ability to bind with positively and negatively charged

**31**

**Figure 15.**

*Classification of lipids based on their origin.*

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

as nanocarrier for drugs, vaccines and genes delivery [120–123].

hydrophobic organic anions such as bilirubin and long-chain fatty acids and divalent cations such as calcium and magnesium [119]. Albumin can bind to different types of compounds including drugs, bile acids, copper, zinc, and even compounds with specific serum binders such as vitamin D and thyroxin [119]. The binding feature of albumin reduces the free concentration of compounds, and hence, limiting their biologic activity, distribution, and rate of clearance [119]. Therefore, HSA is considered an ideal protein for the production of parenteral medications, which has been employed

In 1972, the first protein-based nanoparticle (human serium albumin (HSA) microspheres) was prepared [124]. In January 2005, the first nanotechnology-based drug product, called as Abraxane®, was approved for treatment of metastatic breast cancer [125]. The anticancer and water-insoluble paclitaxel chemotherapeutic agent was easily encapsulated in a shell of protein albumin, where the cancer cells are tricked by the albumin coating into taking the nanospheres embedded with the active cancer-fighting paclitaxel molecules [125]. The encapsulation of paclitaxel drug inside the albumin protein biopolymer provided a harmless way for drug administration as compared to the use of toxic solvents like polyhydroxylated castor oil (Cremophor EL or CrEL), which requires patients to receive premedication for elimination of the allergic reactions and solvent-related hypersensitivity side effects [126]. The FDA approved use of Abraxane® was extended for treatment of non-small-cell lung carcinoma (NSCLC) in 2012, followed by the FDA approval in 2013 for use in treating advanced pancreatic cancer as less toxic alternative to FOLFIRINOX [94]. Gas microbubbles have been encapsulated in the elastic shell of HSA which served as ultrasound contrast agents (e.g. Albunex™ and Optison™

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

products) for diagnostic applications [127].

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

hydrophobic organic anions such as bilirubin and long-chain fatty acids and divalent cations such as calcium and magnesium [119]. Albumin can bind to different types of compounds including drugs, bile acids, copper, zinc, and even compounds with specific serum binders such as vitamin D and thyroxin [119]. The binding feature of albumin reduces the free concentration of compounds, and hence, limiting their biologic activity, distribution, and rate of clearance [119]. Therefore, HSA is considered an ideal protein for the production of parenteral medications, which has been employed as nanocarrier for drugs, vaccines and genes delivery [120–123].

In 1972, the first protein-based nanoparticle (human serium albumin (HSA) microspheres) was prepared [124]. In January 2005, the first nanotechnology-based drug product, called as Abraxane®, was approved for treatment of metastatic breast cancer [125]. The anticancer and water-insoluble paclitaxel chemotherapeutic agent was easily encapsulated in a shell of protein albumin, where the cancer cells are tricked by the albumin coating into taking the nanospheres embedded with the active cancer-fighting paclitaxel molecules [125]. The encapsulation of paclitaxel drug inside the albumin protein biopolymer provided a harmless way for drug administration as compared to the use of toxic solvents like polyhydroxylated castor oil (Cremophor EL or CrEL), which requires patients to receive premedication for elimination of the allergic reactions and solvent-related hypersensitivity side effects [126]. The FDA approved use of Abraxane® was extended for treatment of non-small-cell lung carcinoma (NSCLC) in 2012, followed by the FDA approval in 2013 for use in treating advanced pancreatic cancer as less toxic alternative to FOLFIRINOX [94]. Gas microbubbles have been encapsulated in the elastic shell of HSA which served as ultrasound contrast agents (e.g. Albunex™ and Optison™ products) for diagnostic applications [127].

**Figure 15.** *Classification of lipids based on their origin.*

*Nano- and Microencapsulation - Techniques and Applications*

Collagen is the most abundant protein in mammals which forms 30% of all vertebrate body protein with a majority in bone and skin. Gelatin is a denatured collagen which is obtained by either acid hydrolysis (gelatin type A with isoelectric point ≈ 7–9), or alkaline hydrolysis (gelatin type B with isoelectric point ≈ 4.8–5) [115]. Gelatin is biocompatible and biodegradable with high physiological tolerance and low immunogenicity. Gelatin is classified as "Generally Recognized as Safe" (GRAS) by the US Food and Drug Administration (FDA). Therefore, gelatin have been used for vitamin preparation, drug capsules, scaffolding materials to promote cell migration, wound healing, tissue regeneration and as a nanocarrier for drug and gene delivery [106, 115]. Casein and whey proteins are important protein sources for human nutrition. Casein is one of the oldest natural polymers, used for adhesives, dating back to thousands of years [116]. In contrast to whey proteins, caseins are water-insoluble and account for 80% of total bovine milk proteins. Casein protein is found in milk which serves biologically to transfer nutrients from mother to her offspring. Hence, it can be used as a carrier depot for delivery of drugs. Casein has four constituent phosphate-rich sub-units, which are amphiphilic and self-assemble into a micellar structure in the size range 50–300 nm, held together by calcium phosphate nanoclusters acting as bridges connecting these subunits [117]. Although the word "albumin" is usually associated to serum albumin, it is also employed to describe a family of proteins characterized by their solubility in water [114]. Human serum albumin (HSA) and lactalbumin (known as whey protein) are the most popular albumin proteins employed for drug delivery applications [114]. Besides that, albumins can be found in foods, particularly in seeds and nuts. Serum albumin is the major protein constituent in the blood plasma of all vertebrates. The two main exponents are human (HSA) and bovine serum albumin (BSA). Albumin has diverse physiological functions such as maintaining the pH and colloidal osmotic pressure of plasma, its antioxidant effect by trapping free radicals, and its reversible binding ability to variety of important exogenous and endogenous [118, 119]. Albumin has the ability to bind with positively and negatively charged

*Classification of proteins based on their origin with the list of the main components.*

**30**

**Figure 14.**

#### **5.3 Lipids**

Lipids are heterogenous polymers of fatty acids and in nature occur as fats if solid at ambient temperature, oils if liquids at ambient temperature, fatty acid derivatives, and sterols. A major division among lipids irrespective of their categorization but pertinent for their role in encapsulation and based on polarity divides lipids into polar and non-polar lipids with all types occurring in nature. Polar lipids form aqueous phases with water and occur in nature as constituents of the cell membrane where they form a barrier between the cell and the external water environment. Except for cholesterol, polar lipids have a polar head and a long non-polar tail that aligns itself in a bilayer and include lipids such as glycerophospholipids, sphingolipids and monoglycerides. On the other hand, non-polar lipids such as triglycerides, waxes, are used as energy store and form a solvent for many lyophilic compounds during formulation. Lipids are classified based on origin as shown in **Figure 15**.

## **6. Lipid-based encapsulation**

Lipids are a group of hydrocarbons based organic macromolecules that are rather soluble in non-polar and organic solvents instead of water. Though like carbohydrates in terms of elemental constituents of carbon, hydrogen and oxygen, they differ in containing considerably lower levels of oxygen often attached as part of a single carboxylic acid group at the end of a long hydrocarbon chain. Non-saponifiable lipids such as triglycerides, waxes and phospholipids cannot be hydrolyzed by acid or bases but lipids such as steroids, prostaglandins and terpenes are easily hydrolyzed due to the presence of ester groups. Lipid based encapsulates include liposomes, nanoliposomes, proliposomes, self-assembled micelles, nanostructured lipid carriers, Solid lipid nanoparticles, solid lipid microparticles, liquid lipid nano and micro particles, nanoemulsion, microemulsions, emulsions, nanosuspensions, lipid nanotubes, lipid-polysaccharide complexes and hybrids (**Figure 16**). Some other lipid-based drug delivery systems such as the self-emulsifying microemulsions are not included here because often, they are only encapsulated after ingestion.

A wide range of architectures can be realized depending on the nature and composition of lipids, encapsulation technique, among others. Tuning of process parameters such as pH, temperature, nature and composition of lipid, presence of other constituents such as electrolytes, buffers and sugars will usually determine the size and morphology of the resulting capsules or vesicles. The morphology could be micelles, vesicles, or bilayer sheets. Specific applications will demand specific manipulation of charge, size, pegylation, functionalization, phase transition temperature and drug loading mechanism.

Size: For applications targeting the delivery of macromolecules and tissue penetration, the required size should be below 100 nm but above 5 nm to prevent filtration through the kidney. Multilamellar vesicles are particularly good for depot and sustained release injections while small unilamellar vesicles are good for systemic injections.

Charge: Neutral vesicles usually result in long circulation time and minimal effect of the reticuloenthothelial system. Cationic vesicles however undergo aggregation due to interaction with body protein while anionic vesicles are easily taken up by the liver and spleen.

#### **6.1 Lipids in micro- and nanoencapsulation**

Encapsulation processes employed in the use of lipids for formation of micro and nano capsules are similar in principle for both nanocapsules and microcapsules.

**33**

medicines.

**Figure 16.**

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

Lipids of diverse characteristics and functionalities have in contemporary times remained the focus of hope for the delivery of over 90% new chemical entities in development pipeline that may encounter bioavailability challenges due to their lipophilicity [129]. Likewise, drug delivery systems based on lipids have almost taken center stage in many pharmaceutical companies due to potential profit, both financial and otherwise, accruable to lipid-based reformulation of existing

*Classes of lipid-based drug delivery system. Adapted from [128].*

Lipids play a major role in many encapsulation processes as either membrane/ shell components, core/carrier component, water-insoluble or water-soluble surfactant or as a hydrophilic cosolvent. The type of lipid used for an encapsulation process may depend on several factors that include the target application, size range required for the application, physicochemical properties of the material to be encapsulated. Lipids involved in encapsulation vary widely depending on specific

Homolipids: These are also known as simple lipids and are formed by an esterification action of an alcohol with fatty acids which can be short chain (less than 6 carbon atoms), medium chain (6–12 carbons) or long chain (14–24 carbon atoms). Their elemental composition is just carbon, hydrogen, and oxygen. The constituent fatty acid chain may contain a double bond which always occurs in the cis configuration. Examples are naturally occurring glycerides such as fats and oil (coconut

application. There are also a variety of classification system available.

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

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

#### **Figure 16.**

*Nano- and Microencapsulation - Techniques and Applications*

**6. Lipid-based encapsulation**

because often, they are only encapsulated after ingestion.

temperature and drug loading mechanism.

**6.1 Lipids in micro- and nanoencapsulation**

by the liver and spleen.

Lipids are heterogenous polymers of fatty acids and in nature occur as fats if solid

Lipids are a group of hydrocarbons based organic macromolecules that are rather soluble in non-polar and organic solvents instead of water. Though like carbohydrates in terms of elemental constituents of carbon, hydrogen and oxygen, they differ in containing considerably lower levels of oxygen often attached as part of a single carboxylic acid group at the end of a long hydrocarbon chain. Non-saponifiable lipids such as triglycerides, waxes and phospholipids cannot be hydrolyzed by acid or bases but lipids such as steroids, prostaglandins and terpenes are easily hydrolyzed due to the presence of ester groups. Lipid based encapsulates include liposomes, nanoliposomes, proliposomes, self-assembled micelles, nanostructured lipid carriers, Solid lipid nanoparticles, solid lipid microparticles, liquid lipid nano and micro particles, nanoemulsion, microemulsions, emulsions, nanosuspensions, lipid nanotubes, lipid-polysaccharide complexes and hybrids (**Figure 16**). Some other lipid-based drug delivery systems such as the self-emulsifying microemulsions are not included here

A wide range of architectures can be realized depending on the nature and composition of lipids, encapsulation technique, among others. Tuning of process parameters such as pH, temperature, nature and composition of lipid, presence of other constituents such as electrolytes, buffers and sugars will usually determine the size and morphology of the resulting capsules or vesicles. The morphology could be micelles, vesicles, or bilayer sheets. Specific applications will demand specific manipulation of charge, size, pegylation, functionalization, phase transition

Size: For applications targeting the delivery of macromolecules and tissue penetration, the required size should be below 100 nm but above 5 nm to prevent filtration through the kidney. Multilamellar vesicles are particularly good for depot and sustained release injections while small unilamellar vesicles are good for systemic injections. Charge: Neutral vesicles usually result in long circulation time and minimal effect of the reticuloenthothelial system. Cationic vesicles however undergo aggregation due to interaction with body protein while anionic vesicles are easily taken up

Encapsulation processes employed in the use of lipids for formation of micro and nano capsules are similar in principle for both nanocapsules and microcapsules.

at ambient temperature, oils if liquids at ambient temperature, fatty acid derivatives, and sterols. A major division among lipids irrespective of their categorization but pertinent for their role in encapsulation and based on polarity divides lipids into polar and non-polar lipids with all types occurring in nature. Polar lipids form aqueous phases with water and occur in nature as constituents of the cell membrane where they form a barrier between the cell and the external water environment. Except for cholesterol, polar lipids have a polar head and a long non-polar tail that aligns itself in a bilayer and include lipids such as glycerophospholipids, sphingolipids and monoglycerides. On the other hand, non-polar lipids such as triglycerides, waxes, are used as energy store and form a solvent for many lyophilic compounds during formulation. Lipids are classified based on origin as shown in **Figure 15**.

**5.3 Lipids**

**32**

*Classes of lipid-based drug delivery system. Adapted from [128].*

Lipids of diverse characteristics and functionalities have in contemporary times remained the focus of hope for the delivery of over 90% new chemical entities in development pipeline that may encounter bioavailability challenges due to their lipophilicity [129]. Likewise, drug delivery systems based on lipids have almost taken center stage in many pharmaceutical companies due to potential profit, both financial and otherwise, accruable to lipid-based reformulation of existing medicines.

Lipids play a major role in many encapsulation processes as either membrane/ shell components, core/carrier component, water-insoluble or water-soluble surfactant or as a hydrophilic cosolvent. The type of lipid used for an encapsulation process may depend on several factors that include the target application, size range required for the application, physicochemical properties of the material to be encapsulated. Lipids involved in encapsulation vary widely depending on specific application. There are also a variety of classification system available.

Homolipids: These are also known as simple lipids and are formed by an esterification action of an alcohol with fatty acids which can be short chain (less than 6 carbon atoms), medium chain (6–12 carbons) or long chain (14–24 carbon atoms). Their elemental composition is just carbon, hydrogen, and oxygen. The constituent fatty acid chain may contain a double bond which always occurs in the cis configuration. Examples are naturally occurring glycerides such as fats and oil (coconut

oil), cerides such as waxes (beeswax and carnauba wax), and sterides such as esters of fatty acids and cholesterol.

Heterolipids or compound lipids: These contain an additional nitrogen atom or phosphorus atom. They include phospholipids, sulfolipids, and glycolipids (when conjugated with a sugar moiety). Two classes of phospholipids occur in abundance naturally and include sphingolipids such as ceramide and phosphoglycerides. They abound in nature as structural components of membranes.

Complex lipids: These include overly complex lipids such as lipoprotein (when conjugated with protein and are responsible for the transport of cholesterol and other molecules) and chylomicrons.

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

Challenges abound in the drug delivery terrain particularly for new chemical entities in development pipeline majority of whom are poorly soluble molecules. In addition, better understanding of the molecular basis of diseases is yielding treatment options such as large proteins that pose challenges for delivery. For instance, proteins are easily degraded when administered even parenterally necessitating frequent administrations that contribute to patient cost, side effects and compliance issues. Moreover, often they are large molecules. At the nanosized level, most nanoparticles are easily removed from the circulation by the endoplasmic reticulum. Encapsulation in lipids can solve a great number of these issues. **Figure 17** captures

**35**

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

functionalization with specific ligands can be modulated for specific tasks.

and may result in toxicity or loss of efficacy.

**6.3 Case studies/applications**

*6.3.1 Encapsulation of small molecules*

Most lipids used for encapsulation are relatively cheap, biocompatible, biodegradable and exhibit low toxicity and allergenicity. The use of organic solvents is limited in their preparation. Ease of formulation, ease of characterization, sterilization and scale up, and amenability to delivery by various routes all contribute to their versatility. In spite of the plethora of advantages, lipid-based system has a few challenges. Digestibility of lipids and drug leakage: Most lipids used for encapsulation and other lipid-based delivery systems are natural. Lipids therefore have a natural propensity to be digested and degraded in the body by enzymes [133]. Digestion of one or more components will break up the membrane or shell which may result in supersaturation, precipitation or dilution depending on the route of administration

Drug loading: Passive drug loading may lead to low efficiency, but active drug loading ensures high efficiency depending on the API and the other excipients. Uncontrolled precipitation and aggregation: many of the lipid systems are prone to physical destabilization of their membranes requiring extra effort to stabilize them. The nanocapsules are prone to aggregation which may lead to non-uniformity of doses.

Small molecules are low molecular weight compounds that include drugs, xenobiotics, lipids, metabolites, metal ions, monosaccharides, second messenger, etcetera. Encapsulation of small molecules using lipids predominantly aims to solubilize poorly soluble molecules, target or control release of the medicament. Lipids

some of the benefits of lipid encapsulation to drug delivery. The merits of a lipidbased system for drug delivery may vary slightly depending on the type of lipid

Modulation of bioavailability: Irrespective of the route of administration or lipid system involved, lipid-based systems have been employed to modulate rate and extent of absorption of active ingredient. They bring about an increase in surface area available for dissolution thereby increasing absorption. In oral delivery, lipid systems have predominantly been used to improve solubilization of poorly soluble solids thereby increasing bioavailability. Solubilization in lipid systems also greatly diminishes intra and inter subject variability enabling caregivers to better adjust dosing to individual needs. Lipid based delivery also reduces the hepatic first pass metabolism for susceptible drugs. The overall improvement may lead to a reduction in the required dose and a proportional decrease in the accompanying side effects and toxicities which may translate to better compliance. An example is the formulation of amphotericin B initially as fungizone with high toxicity as compared to the lipid particle formulation, Abelcet [131]. In addition, existence of areas of opposite polarity within the same systems opens the possibility of delivering 2 physically different compounds through one system. Lipid based systems in the form of micro and nano particulate systems modulate biodistribution [132]. They are usually used to sustain drug release and target drugs to specific sites. Lipids have been used to deliver large protein macromolecules to specific sites through lipid-drug conjugates. Encapsulation in lipid bilayer membrane spares the drug the attack of the reticuloendothelial system or shield a drug from detection by the immune cells since they have similar membrane. Lipid systems improve or maintain the chemical and physical stability of the included API. They also effectively mask taste, and odor. Formulation efforts are also targeted towards stabilizing the API both during storage and from endogenous enzymes and chemicals until it arrives its site of action. Physicochemical properties of vesicles such as size, charge or surface

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

system and the route of administration.

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

some of the benefits of lipid encapsulation to drug delivery. The merits of a lipidbased system for drug delivery may vary slightly depending on the type of lipid system and the route of administration.

Modulation of bioavailability: Irrespective of the route of administration or lipid system involved, lipid-based systems have been employed to modulate rate and extent of absorption of active ingredient. They bring about an increase in surface area available for dissolution thereby increasing absorption. In oral delivery, lipid systems have predominantly been used to improve solubilization of poorly soluble solids thereby increasing bioavailability. Solubilization in lipid systems also greatly diminishes intra and inter subject variability enabling caregivers to better adjust dosing to individual needs. Lipid based delivery also reduces the hepatic first pass metabolism for susceptible drugs. The overall improvement may lead to a reduction in the required dose and a proportional decrease in the accompanying side effects and toxicities which may translate to better compliance. An example is the formulation of amphotericin B initially as fungizone with high toxicity as compared to the lipid particle formulation, Abelcet [131]. In addition, existence of areas of opposite polarity within the same systems opens the possibility of delivering 2 physically different compounds through one system.

Lipid based systems in the form of micro and nano particulate systems modulate biodistribution [132]. They are usually used to sustain drug release and target drugs to specific sites. Lipids have been used to deliver large protein macromolecules to specific sites through lipid-drug conjugates. Encapsulation in lipid bilayer membrane spares the drug the attack of the reticuloendothelial system or shield a drug from detection by the immune cells since they have similar membrane. Lipid systems improve or maintain the chemical and physical stability of the included API. They also effectively mask taste, and odor. Formulation efforts are also targeted towards stabilizing the API both during storage and from endogenous enzymes and chemicals until it arrives its site of action. Physicochemical properties of vesicles such as size, charge or surface functionalization with specific ligands can be modulated for specific tasks.

Most lipids used for encapsulation are relatively cheap, biocompatible, biodegradable and exhibit low toxicity and allergenicity. The use of organic solvents is limited in their preparation. Ease of formulation, ease of characterization, sterilization and scale up, and amenability to delivery by various routes all contribute to their versatility.

In spite of the plethora of advantages, lipid-based system has a few challenges.

Digestibility of lipids and drug leakage: Most lipids used for encapsulation and other lipid-based delivery systems are natural. Lipids therefore have a natural propensity to be digested and degraded in the body by enzymes [133]. Digestion of one or more components will break up the membrane or shell which may result in supersaturation, precipitation or dilution depending on the route of administration and may result in toxicity or loss of efficacy.

Drug loading: Passive drug loading may lead to low efficiency, but active drug loading ensures high efficiency depending on the API and the other excipients.

Uncontrolled precipitation and aggregation: many of the lipid systems are prone to physical destabilization of their membranes requiring extra effort to stabilize them. The nanocapsules are prone to aggregation which may lead to non-uniformity of doses.

#### **6.3 Case studies/applications**

#### *6.3.1 Encapsulation of small molecules*

Small molecules are low molecular weight compounds that include drugs, xenobiotics, lipids, metabolites, metal ions, monosaccharides, second messenger, etcetera. Encapsulation of small molecules using lipids predominantly aims to solubilize poorly soluble molecules, target or control release of the medicament. Lipids

*Nano- and Microencapsulation - Techniques and Applications*

abound in nature as structural components of membranes.

**6.2 Merits and demerits in therapeutic delivery**

of fatty acids and cholesterol.

other molecules) and chylomicrons.

oil), cerides such as waxes (beeswax and carnauba wax), and sterides such as esters

Heterolipids or compound lipids: These contain an additional nitrogen atom or phosphorus atom. They include phospholipids, sulfolipids, and glycolipids (when conjugated with a sugar moiety). Two classes of phospholipids occur in abundance naturally and include sphingolipids such as ceramide and phosphoglycerides. They

Complex lipids: These include overly complex lipids such as lipoprotein (when conjugated with protein and are responsible for the transport of cholesterol and

Challenges abound in the drug delivery terrain particularly for new chemical entities in development pipeline majority of whom are poorly soluble molecules. In addition, better understanding of the molecular basis of diseases is yielding treatment options such as large proteins that pose challenges for delivery. For instance, proteins are easily degraded when administered even parenterally necessitating frequent administrations that contribute to patient cost, side effects and compliance issues. Moreover, often they are large molecules. At the nanosized level, most nanoparticles are easily removed from the circulation by the endoplasmic reticulum. Encapsulation in lipids can solve a great number of these issues. **Figure 17** captures

*Some factors contributing to efficiency of lipid encapsulation to therapeutic drug delivery [130].*

**34**

**Figure 17.**

being major constituents of the cell membranes can ferry included cargo through the tightly controlled formidable barrier and through various ports of entry such as the stratum corneum, the ocular cornea, parenteral or oral route.

Cantón and colleagues [134] recently reported the preparation of SN-38-βcyclodextrin complex in solid lipid nanoparticles. The aim was to develop a delivery system that will deliver, stabilize, and protect the FDA approved drug for colorectal cancer. This was necessary since SN-38 is highly insoluble and unstable at physiological pH and easily converts to the carboxylate form that has higher binding affinity to serum and is more stable at the basic pH of the GIT. Initial attempt to include SN-38 -cyclodextrin inclusion complex to a liposome lead to the disassembly of the liposome and the formation of solid lipid nanoparticles. The lipids used were hydrogenated L-α-phosphatidylcholine, 1,2-distearoyl-*sn*-glycero-3- phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin), 1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N- [folate(polyethylene glycol)-5000] (DSPE-PEG-folate) 1,2-dihexadecanoyl-*sn*-glycero-3-phosphoethanolamine (DHPE-TR). Evaluation of the stability by determining the presence of the inactive form was undertaken using the size exclusion chromatography. Ultimately, stable, solid lipid particles containing the SN-38 cyclodextrin complex was prepared even though the concentration of the encapsulated drug was narrow.

Photodynamic therapy (PDT) is a treatment modality for cancers that involve the use of reactive oxygen species, photosensitizers, and light for destruction of cancer cells. However, PDT is limited by the inability of high energy light used in PDT to penetrate tissues, and the ability of the body to disperse used photosensitizers systemically [135]. Unfortunately, too, the internal tumor environment is hypoxic, the low oxygen content limiting the efficacy of PDT and being responsible for angiogenesis and subsequent metastasis within cancer cells. Therefore, to improve the outcomes of PDT, particularly for solid tumors, there is a need for the presence of oxygen generator within the tumor cells and the presence of the photosensitizer near the targeted tumor cell. Liu and colleagues [136] developed a calcium peroxide/methylene blue-loaded liposome as an oxygen generating species which targets a photosensitizer, methylene blue in PDT therapy. Many of the oxygen generated previously studied were encapsulated in hydrophobic polymers that had limited capacity for hydrophilic cargo and delayed the generation of oxygen. The use of liposomes provided a hydrophobic shell that served to carry the photosensitizer and a means to penetrate the cell membrane while carrying a hydrophilic cargo in its core. On irradiation, the phospholipid bilayer is easily disrupted causing the release of calcium peroxide which reacts with water rapidly to generate oxygen. On subsequent irradiation, the generated oxygen potentiates the effect of PDT on tumor hypoxia. The first step involved the preparation of calcium peroxide nanoparticles and further encapsulation of the nanoparticles in a pegylated liposome. Composed of DSPE-PEG, DPPC-egg lecithin, and cholesterol. In-vivo tests in a mouse model of mammary cell carcinoma demonstrated the efficacy of the system to limit hypoxia in treated animals when compared to untreated animals.

Kenechukwu and colleagues [137] prepared a lipid matrix made up of sun seed oil: Softisan® in the ratio 1:9 and PEG 4000 by a melt homogenization process for the intravaginal delivery of a poorly soluble drug, Miconazole. The concentration of PEG was varied giving rise to different formulations. The PEG content consequently affected the particle sizes, the encapsulation efficiency, and the loading capacity. The optimum concentration of PEG 4000 according to their study was 40% w/w.

Stella and colleagues [138] investigated the possibility of delivering a doxorubicin pro-drug, squalenoyl-derivative through entrapment in solid lipid nanoparticles. The highly reduced cardiotoxicity of liposomal doxorubicin catapulted the search for other lipid-based carrier systems that will also help in mitigating the resistance to doxorubicin. Squalenoyl derivative is highly lipophilic derivative that has shown capacity to form

**37**

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

effectively transport the curcumin and methotrexate to the tumor site.

Biologics are large high molecular weight proteins, nucleic acids, monoclonal antibodies, vaccines, and enzymes. The delivery of macromolecular proteins is

Synthetic small interfering RNA, siRNA are nucleic acid fragments that can modify the activities of mRNA when they enter the cell. Many diseases are due to certain abnormality or malefaction at the genetic level and hence the silencing of the specific mRNA can translate to cure. The problem of delivering such proteins are multiple and varied. One of such is the rapid clearance of such protein on systemic administration due to nuclease activity and renal filtration and the induction of immunogenic reactions. In addition, siRNA, and its like are rarely able to diffuse into the cell, hence requiring the complex generation of multiply functionalized systems. Patisiran is the first FDA approved siRNA formulation prepared using lipid nanoparticulate platform for delivery to hepatocytes [140]. It is a double stranded siRNA which degrades 3'untranslated region of the wild type transthyretin by RNA interference [141]. Hereditary transthyretin mediated amyloidosis is a disorder resulting from deposition of abnormal form of the protein produced in liver cells. The entrapment of nucleic acids in lipid nanoparticles require the presence of a cationic lipid to trap the negatively charged nucleic acid. Secondly, the pKa of the lipid should be such that at physiological pH, there is a net neutral charge. The lipid should also display a positive charge when in the endosome environment and finally,

*6.3.2 Encapsulation of biologics*

particularly challenging.

very stable self-assembles in water. Their absorption in the body had been shown to be mediated by endogenous low-density lipoproteins. Therefore, the researchers initially prepared squalenoyl derivative self-assembly in water using the nanoprecipitation technique. The solid lipid particles were prepared by complex coacervation using fatty acids that were precipitated by acidification and stabilized with poly vinyl alcohol. Targeting cytotoxic drugs to the tumor environment has always been both desirable and a challenge due to severe side effects to normal cells and the peculiarities of the tumor microenvironment. For instance, multi drug resistance to drugs like doxorubicin has been associated to the hypoxia encountered in tumors. In this study, Xie and colleagues [139] aimed to use methotrexate conjugated with a polymer-lipid hybrid through an imine linkage, as both a targeting moiety and as the drug targeted to the cancer cells, through the nanoplatform of self-assembled lipid micelles also incorporating curcumin. In addition, pH responsiveness and prodrug status were built into the platform using an imine crosslinker. The intent was that at a particular pH unique to the tumor environment, the acid responsive imine aldehyde linkage will be disrupted leading to the release of the active methotrexate. The methotrexate with its strong resemblance to folate will be used as a target to the folate receptors on the tumor. Curcumin, a well-known naturally occurring polyphenol with strong antiinflammatory and antiproliferative properties was assembled into the hydrophobic core of the resulting lipid-polymer hybrid micelles to forestall drug resistance. In their study, lipid-polymers such as DSPE-PEG and DSPE-Mpeg were used. The first step was the formation of the prodrug complex, DSPE-MPEG-imine- methotrexate, by the conjugation of methotrexate to DSPE-PEG through a Schiff base reaction between the aldehyde group of the polymer and the aromatic group of methotrexate. The resulting prodrug complex was later self-assembled in the presence of the poorly soluble and unstable curcumin. One pot ultra-sonification with solvent evaporation was the method of micelle formation for both drug-loaded and unloaded micelles. The animal studies carried out on HeLA tumor bearing BALB/c nude mice demonstrated the workability of the concept and confirmed the ability of the lipid carrier system to

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

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

very stable self-assembles in water. Their absorption in the body had been shown to be mediated by endogenous low-density lipoproteins. Therefore, the researchers initially prepared squalenoyl derivative self-assembly in water using the nanoprecipitation technique. The solid lipid particles were prepared by complex coacervation using fatty acids that were precipitated by acidification and stabilized with poly vinyl alcohol.

Targeting cytotoxic drugs to the tumor environment has always been both desirable and a challenge due to severe side effects to normal cells and the peculiarities of the tumor microenvironment. For instance, multi drug resistance to drugs like doxorubicin has been associated to the hypoxia encountered in tumors. In this study, Xie and colleagues [139] aimed to use methotrexate conjugated with a polymer-lipid hybrid through an imine linkage, as both a targeting moiety and as the drug targeted to the cancer cells, through the nanoplatform of self-assembled lipid micelles also incorporating curcumin. In addition, pH responsiveness and prodrug status were built into the platform using an imine crosslinker. The intent was that at a particular pH unique to the tumor environment, the acid responsive imine aldehyde linkage will be disrupted leading to the release of the active methotrexate. The methotrexate with its strong resemblance to folate will be used as a target to the folate receptors on the tumor. Curcumin, a well-known naturally occurring polyphenol with strong antiinflammatory and antiproliferative properties was assembled into the hydrophobic core of the resulting lipid-polymer hybrid micelles to forestall drug resistance. In their study, lipid-polymers such as DSPE-PEG and DSPE-Mpeg were used. The first step was the formation of the prodrug complex, DSPE-MPEG-imine- methotrexate, by the conjugation of methotrexate to DSPE-PEG through a Schiff base reaction between the aldehyde group of the polymer and the aromatic group of methotrexate. The resulting prodrug complex was later self-assembled in the presence of the poorly soluble and unstable curcumin. One pot ultra-sonification with solvent evaporation was the method of micelle formation for both drug-loaded and unloaded micelles. The animal studies carried out on HeLA tumor bearing BALB/c nude mice demonstrated the workability of the concept and confirmed the ability of the lipid carrier system to effectively transport the curcumin and methotrexate to the tumor site.

#### *6.3.2 Encapsulation of biologics*

Biologics are large high molecular weight proteins, nucleic acids, monoclonal antibodies, vaccines, and enzymes. The delivery of macromolecular proteins is particularly challenging.

Synthetic small interfering RNA, siRNA are nucleic acid fragments that can modify the activities of mRNA when they enter the cell. Many diseases are due to certain abnormality or malefaction at the genetic level and hence the silencing of the specific mRNA can translate to cure. The problem of delivering such proteins are multiple and varied. One of such is the rapid clearance of such protein on systemic administration due to nuclease activity and renal filtration and the induction of immunogenic reactions. In addition, siRNA, and its like are rarely able to diffuse into the cell, hence requiring the complex generation of multiply functionalized systems. Patisiran is the first FDA approved siRNA formulation prepared using lipid nanoparticulate platform for delivery to hepatocytes [140]. It is a double stranded siRNA which degrades 3'untranslated region of the wild type transthyretin by RNA interference [141]. Hereditary transthyretin mediated amyloidosis is a disorder resulting from deposition of abnormal form of the protein produced in liver cells. The entrapment of nucleic acids in lipid nanoparticles require the presence of a cationic lipid to trap the negatively charged nucleic acid. Secondly, the pKa of the lipid should be such that at physiological pH, there is a net neutral charge. The lipid should also display a positive charge when in the endosome environment and finally,

*Nano- and Microencapsulation - Techniques and Applications*

being major constituents of the cell membranes can ferry included cargo through the tightly controlled formidable barrier and through various ports of entry such as

Cantón and colleagues [134] recently reported the preparation of SN-38-βcyclodextrin complex in solid lipid nanoparticles. The aim was to develop a delivery system that will deliver, stabilize, and protect the FDA approved drug for colorectal cancer. This was necessary since SN-38 is highly insoluble and unstable at physiological pH and easily converts to the carboxylate form that has higher binding affinity to serum and is more stable at the basic pH of the GIT. Initial attempt to include SN-38 -cyclodextrin inclusion complex to a liposome lead to the disassembly of the liposome and the formation of solid lipid nanoparticles. The lipids used were hydrogenated L-α-phosphatidylcholine, 1,2-distearoyl-*sn*-glycero-3- phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin), 1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N- [folate(polyethylene glycol)-5000] (DSPE-PEG-folate) 1,2-dihexadecanoyl-*sn*-glycero-3-phosphoethanolamine (DHPE-TR). Evaluation of the stability by determining the presence of the inactive form was undertaken using the size exclusion chromatography. Ultimately, stable, solid lipid particles containing the SN-38 cyclodextrin complex was prepared even

Photodynamic therapy (PDT) is a treatment modality for cancers that involve the use of reactive oxygen species, photosensitizers, and light for destruction of cancer cells. However, PDT is limited by the inability of high energy light used in PDT to penetrate tissues, and the ability of the body to disperse used photosensitizers systemically [135]. Unfortunately, too, the internal tumor environment is hypoxic, the low oxygen content limiting the efficacy of PDT and being responsible for angiogenesis and subsequent metastasis within cancer cells. Therefore, to improve the outcomes of PDT, particularly for solid tumors, there is a need for the presence of oxygen generator within the tumor cells and the presence of the photosensitizer near the targeted tumor cell. Liu and colleagues [136] developed a calcium peroxide/methylene blue-loaded liposome as an oxygen generating species which targets a photosensitizer, methylene blue in PDT therapy. Many of the oxygen generated previously studied were encapsulated in hydrophobic polymers that had limited capacity for hydrophilic cargo and delayed the generation of oxygen. The use of liposomes provided a hydrophobic shell that served to carry the photosensitizer and a means to penetrate the cell membrane while carrying a hydrophilic cargo in its core. On irradiation, the phospholipid bilayer is easily disrupted causing the release of calcium peroxide which reacts with water rapidly to generate oxygen. On subsequent irradiation, the generated oxygen potentiates the effect of PDT on tumor hypoxia. The first step involved the preparation of calcium peroxide nanoparticles and further encapsulation of the nanoparticles in a pegylated liposome. Composed of DSPE-PEG, DPPC-egg lecithin, and cholesterol. In-vivo tests in a mouse model of mammary cell carcinoma demonstrated the efficacy of the system

the stratum corneum, the ocular cornea, parenteral or oral route.

though the concentration of the encapsulated drug was narrow.

to limit hypoxia in treated animals when compared to untreated animals.

optimum concentration of PEG 4000 according to their study was 40% w/w.

Kenechukwu and colleagues [137] prepared a lipid matrix made up of sun seed oil: Softisan® in the ratio 1:9 and PEG 4000 by a melt homogenization process for the intravaginal delivery of a poorly soluble drug, Miconazole. The concentration of PEG was varied giving rise to different formulations. The PEG content consequently affected the particle sizes, the encapsulation efficiency, and the loading capacity. The

Stella and colleagues [138] investigated the possibility of delivering a doxorubicin pro-drug, squalenoyl-derivative through entrapment in solid lipid nanoparticles. The highly reduced cardiotoxicity of liposomal doxorubicin catapulted the search for other lipid-based carrier systems that will also help in mitigating the resistance to doxorubicin. Squalenoyl derivative is highly lipophilic derivative that has shown capacity to form

**36**

it must display the hexagon shape. For proteins, there must be rational design to take into cognizance; linkers, acyl chains and the ionizable groups required.

Vascular endothelial growth factor, VEGF, has been implicated in tumor progression and metastasis. It has been shown that in cancer and some other diseases such as age-related macular degeneration, the expression for VEGF is usually upregulated to promote angiogenesis. The possibility of using RNA interference to silence genes through the interference of siRNA will be a welcome option in the battle against tumors. Important barriers to the use of siRNA is the availability of vectors, low transfection efficiency and stability. Polymeric lipids have been in the lead as a choice for siRNA delivery material. Cationic lipids are of importance due to the need for a positive charge to stabilize the negatively charged nucleic acid and for internalization. Cationic lipids however pose a problem of toxicity and may offer no protection to the nucleic acid. In Chen and co-workers' study [142], polyethyleneimine (PEI) was used for protonation of the polycation liposome while 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine (DOPE), was used as a destabilizer to promote the escape of siRNA into the cytosol. siRNA was conjugated to calcium phosphate nanoparticles which has shown low toxicity, biocompatibility and biodegradability in previous studies involving the delivery of DNA and siRNA. siRNA-calcium phosphate nanoparticles were first prepared and introduced as an aqueous solution into a polycation liposome prepared by the film dispersion method using equimolar concentration of DOPE and PEI-Cholesterol. The in vitro gene silencing assay was performed using human breast adenocarcinoma cell lines while animal studies were done in tumor infected BALB/C-nu female mice. The results obtained showed the superior silencing effect of the siRNA delivered through the core-shell polycationic liposome.

Antibodies can be generated as inhibitory agents to many diseases causing proteins in the cytosol. Unfortunately, this has not been effectively utilized in treatment modalities due to inability of the large antibody proteins to transverse the cytosol. Wang and coworkers [143] developed a lipid carrier for immunoglobulin G, IgG, by first conjugating it with anionic polypeptides and subsequently complexed them (through electrostatic interactions) with cationic lipids previously used for the delivery of nucleic acids. They initially fused the polypeptide to a photoreactive antibody binding domain and subsequently to a chain of IgG without disturbing the IgG binding site and enabling the easy exchange of cargo functionalization of of-the-shelf IgG. The functionalized IgG was subsequently complexed with diverse types of cationic lipids and evaluated comparatively with cell penetrating peptides for cytosolic delivery of about 500 nM of IgG. The lipid complexed IgG was functional and also capable of inhibiting the drug efflux pump MRP I (responsible for multi drug resistance) and the transcription factor NFκB. Results also showed the supremacy of this method over traditional cell penetrating peptide method (delivering small proteins) in terms of delivering very large proteins.

Kose and coworkers [144] developed a lipid nanoparticle encapsulated mRNA encoding the antibody against chikungunya virus. The lipid nanoparticle system was prepared by the ethanol drop nanoprecipitation using ionizable lipid, lipidpolymer hybrid, cholesterol and DSPC in a microfluidic mixer. The lipid nanoparticle system provided approximately 90% encapsulation with particle sizes in the range of 80–100 nm. The protective ability of the developed system was tested in AG129 mice. The study showed that treatment lipid encapsulated mRNA protected the mice in a dose dependent manner.

#### *6.3.3 Encapsulation of diagnostics*

Certain factors can trigger responses in lipid particles of vesicles and include pH, reactive oxygen species, redox agents' presence of biomolecules, as well as certain environmental stimuli such as temperature, and light. Biosensors constitute a receptor

**39**

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

that will interact with the stimuli to be detected and a transducer that will translate the analyte/stimuli -receptor information to measurable signal. Treatment and survival for many terminal and chronic diseases depend on early detection and diagnosis. Fortunately, many biosensing and bioimaging materials are being developed for possible use in diagnosis and treatment. There is a need to transport these nanoplatforms to the targeted site in a non-invasive and hidden manner to avoid destruction by the bodyguards of the body. One way this has been mitigated is by enclosure into vesicles that have a close resemblance to the body's own cells, the liposomes. Encapsulation based on lipid systems is driving the development of bioimaging and biosensing devices towards picogram detection thereby aiding both treatment such as fluorescence guided surgeries and survival. Some of the challenges being mitigated by lipid based or encapsulated devices include targeting, sustained release, and circulation. Photoacoustic tomography (PAT) that makes use of light and sound has been considered a viable alternative to overcoming some of the limitations of conventional imaging systems such as computed tomography (CT) and magnetic resonance imaging (MRI), in early detection of atypical liver cancers that are less than 10 mm diameter. In addition, surgical resection remains about the most viable treatment option. Gold nanorods, due to their easy effusion into solid tumors, biocompatibility, and low toxicity [135] is usually considered good for PAT. It is also possible to move its absorption peak from red to near infrared (NIR) due to its anisotropic shape and enhancing its photoacoustic signal with large absorption cross section. Exploiting these factors, Guan and coworkers [145] developed dual PAT-NIR probe to aid early liver cancer detection and for guided surgical resection. They tapped a sort of synergistic effect of both gold nano rods and indocyanine green, an FDA approved photoacoustic NIR fluorescent dye that has dominated clinical practice for a while, and played down on some limitations of indocyanine green such as aggregation, rapid clearance, low energy conversion efficiency as a dual photoacoustic and fluorescence dye, and fluorescence quenching. In a relatively facile process, indocyanine green liposomes were prepared with phosphatidylcholine and cholesterol using the thin film hydration method. Pegylated gold nanorods were subsequently encapsulated with the indocyanine green liposomes by ultrasonication overnight. The dual system was used for resection surgery in tumor infected laboratory animals and proved successful.

Nucleolin is a nuclear and cytoplasmic protein also expressed on the cell surface and partly responsible for angiogenesis and by extension metastasis and tumor progression. Unlike other markers of tumor progression, that become less prominent as the tumor size increases, nucleolin is detected even in big tumors. It could therefore be mapped as a means of tracking metastasis. In current practice, Nucleolin is tracked using biopsy which is invasive and cannot indicate the full extent of metastasis or spread. Zhang and colleagues [146] developed a nucleolin targeted ultrasound contrast agent for detecting the presence of nucleolin in cells. The contrast agent is microbubble which is an encapsulated air commonly generated by sonicating a polymer solution in the presence of air or by compressing air into a polymer solution and then releasing it through specialized nozzles. In this study, Zhang and colleagues [146] synthesized an F3 peptide that has been shown to target nucleolin and conjugated it to the surface of generated microbubbles. Subsequently, they evaluated the ability of the F3 conjugated nucleolin targeting microbubble to detect the presence of nucleolin non-invasively. They initially synthesized the F3 peptides, lipo-peg peptides. Subsequently the microbubble which was encapsulated in a liposomal shell was synthesized using disteroylphosphatidylcholine (DSPC), 1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K), and lipo-peg-peptide earlier synthesized. The liposomal shell was prepared using the thin film hydration method and the microbubble generated by shaking the liposomal solution in air and used within 2 hours of purification. Two batches were made, one batch without the targeting

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

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

that will interact with the stimuli to be detected and a transducer that will translate the analyte/stimuli -receptor information to measurable signal. Treatment and survival for many terminal and chronic diseases depend on early detection and diagnosis. Fortunately, many biosensing and bioimaging materials are being developed for possible use in diagnosis and treatment. There is a need to transport these nanoplatforms to the targeted site in a non-invasive and hidden manner to avoid destruction by the bodyguards of the body. One way this has been mitigated is by enclosure into vesicles that have a close resemblance to the body's own cells, the liposomes. Encapsulation based on lipid systems is driving the development of bioimaging and biosensing devices towards picogram detection thereby aiding both treatment such as fluorescence guided surgeries and survival. Some of the challenges being mitigated by lipid based or encapsulated devices include targeting, sustained release, and circulation.

Photoacoustic tomography (PAT) that makes use of light and sound has been considered a viable alternative to overcoming some of the limitations of conventional imaging systems such as computed tomography (CT) and magnetic resonance imaging (MRI), in early detection of atypical liver cancers that are less than 10 mm diameter. In addition, surgical resection remains about the most viable treatment option. Gold nanorods, due to their easy effusion into solid tumors, biocompatibility, and low toxicity [135] is usually considered good for PAT. It is also possible to move its absorption peak from red to near infrared (NIR) due to its anisotropic shape and enhancing its photoacoustic signal with large absorption cross section. Exploiting these factors, Guan and coworkers [145] developed dual PAT-NIR probe to aid early liver cancer detection and for guided surgical resection. They tapped a sort of synergistic effect of both gold nano rods and indocyanine green, an FDA approved photoacoustic NIR fluorescent dye that has dominated clinical practice for a while, and played down on some limitations of indocyanine green such as aggregation, rapid clearance, low energy conversion efficiency as a dual photoacoustic and fluorescence dye, and fluorescence quenching. In a relatively facile process, indocyanine green liposomes were prepared with phosphatidylcholine and cholesterol using the thin film hydration method. Pegylated gold nanorods were subsequently encapsulated with the indocyanine green liposomes by ultrasonication overnight. The dual system was used for resection surgery in tumor infected laboratory animals and proved successful.

Nucleolin is a nuclear and cytoplasmic protein also expressed on the cell surface and partly responsible for angiogenesis and by extension metastasis and tumor progression. Unlike other markers of tumor progression, that become less prominent as the tumor size increases, nucleolin is detected even in big tumors. It could therefore be mapped as a means of tracking metastasis. In current practice, Nucleolin is tracked using biopsy which is invasive and cannot indicate the full extent of metastasis or spread. Zhang and colleagues [146] developed a nucleolin targeted ultrasound contrast agent for detecting the presence of nucleolin in cells. The contrast agent is microbubble which is an encapsulated air commonly generated by sonicating a polymer solution in the presence of air or by compressing air into a polymer solution and then releasing it through specialized nozzles. In this study, Zhang and colleagues [146] synthesized an F3 peptide that has been shown to target nucleolin and conjugated it to the surface of generated microbubbles. Subsequently, they evaluated the ability of the F3 conjugated nucleolin targeting microbubble to detect the presence of nucleolin non-invasively. They initially synthesized the F3 peptides, lipo-peg peptides. Subsequently the microbubble which was encapsulated in a liposomal shell was synthesized using disteroylphosphatidylcholine (DSPC), 1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K), and lipo-peg-peptide earlier synthesized. The liposomal shell was prepared using the thin film hydration method and the microbubble generated by shaking the liposomal solution in air and used within 2 hours of purification. Two batches were made, one batch without the targeting

*Nano- and Microencapsulation - Techniques and Applications*

it must display the hexagon shape. For proteins, there must be rational design to take

Vascular endothelial growth factor, VEGF, has been implicated in tumor progression and metastasis. It has been shown that in cancer and some other diseases such as age-related macular degeneration, the expression for VEGF is usually upregulated to promote angiogenesis. The possibility of using RNA interference to silence genes through the interference of siRNA will be a welcome option in the battle against tumors. Important barriers to the use of siRNA is the availability of vectors, low transfection efficiency and stability. Polymeric lipids have been in the lead as a choice for siRNA delivery material. Cationic lipids are of importance due to the need for a positive charge to stabilize the negatively charged nucleic acid and for internalization. Cationic lipids however pose a problem of toxicity and may offer no protection to the nucleic acid. In Chen and co-workers' study [142], polyethyleneimine (PEI) was used for protonation of the polycation liposome while 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine (DOPE), was used as a destabilizer to promote the escape of siRNA into the cytosol. siRNA was conjugated to calcium phosphate nanoparticles which has shown low toxicity, biocompatibility and biodegradability in previous studies involving the delivery of DNA and siRNA. siRNA-calcium phosphate nanoparticles were first prepared and introduced as an aqueous solution into a polycation liposome prepared by the film dispersion method using equimolar concentration of DOPE and PEI-Cholesterol. The in vitro gene silencing assay was performed using human breast adenocarcinoma cell lines while animal studies were done in tumor infected BALB/C-nu female mice. The results obtained showed the superior silencing effect of

into cognizance; linkers, acyl chains and the ionizable groups required.

the siRNA delivered through the core-shell polycationic liposome.

Antibodies can be generated as inhibitory agents to many diseases causing proteins in the cytosol. Unfortunately, this has not been effectively utilized in treatment modalities due to inability of the large antibody proteins to transverse the cytosol. Wang and coworkers [143] developed a lipid carrier for immunoglobulin G, IgG, by first conjugating it with anionic polypeptides and subsequently complexed them (through electrostatic interactions) with cationic lipids previously used for the delivery of nucleic acids. They initially fused the polypeptide to a photoreactive antibody binding domain and subsequently to a chain of IgG without disturbing the IgG binding site and enabling the easy exchange of cargo functionalization of of-the-shelf IgG. The functionalized IgG was subsequently complexed with diverse types of cationic lipids and evaluated comparatively with cell penetrating peptides for cytosolic delivery of about 500 nM of IgG. The lipid complexed IgG was functional and also capable of inhibiting the drug efflux pump MRP I (responsible for multi drug resistance) and the transcription factor NFκB. Results also showed the supremacy of this method over traditional cell penetrating peptide method (delivering small proteins) in terms of delivering very large proteins. Kose and coworkers [144] developed a lipid nanoparticle encapsulated mRNA encoding the antibody against chikungunya virus. The lipid nanoparticle system was prepared by the ethanol drop nanoprecipitation using ionizable lipid, lipidpolymer hybrid, cholesterol and DSPC in a microfluidic mixer. The lipid nanoparticle system provided approximately 90% encapsulation with particle sizes in the range of 80–100 nm. The protective ability of the developed system was tested in AG129 mice. The study showed that treatment lipid encapsulated mRNA protected

Certain factors can trigger responses in lipid particles of vesicles and include pH, reactive oxygen species, redox agents' presence of biomolecules, as well as certain environmental stimuli such as temperature, and light. Biosensors constitute a receptor

**38**

the mice in a dose dependent manner.

*6.3.3 Encapsulation of diagnostics*

peptide contained just the lipids DSPC: DSPE-PEG2K in the ratio of 90:10 while the targeted batch had I % of the lipo-peg-peptide replacing part of the DSPE-PEG2K. Evaluation of the contrast media in tumor infected female FVB mice in vivo and nucleolin expressing breast cancer cell line in vitro demonstrated that the accumulation of the contrast media facilitated the detection of murine breast tumors.

Deng and colleagues [147] recently developed a multicomponent system for diagnosis and treatment of cancers. They combined near infrared luminescence of quantum dots and thermo-sensitivity of magnetic liposomes to encapsulate and control drug localization and release. They prepared thermosensitive liposomes that were eventually loaded with drug, paclitaxel, magnetic nanoparticles and NIR luminescent quantum dots. The liposomes were prepared by the thin film hydration method using dipalmitoylphophatidylcholine, DPPC, 1,2-diaccyl-sn-glycero-3-phosphoethanolamine-N-(methoxy [polyethylene glycol]-2000 (DSPE–MPEG-2000), 1,2-diaccyl-sn-glycero-3-phosphoglycerol sodium (DSPG-Na). Effect of the developed systems were studied on cancer cell MCF-7 and SKOV-3 cell lines and uptake of the drug followed in real time by confocal scanning microscope. **Tables 2** and **3**


*Note: 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid: DMPA, 1,2 Distearoyl-sn-glycero-3-phosphocholine: DSPC, Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000: DSPE-MPEG2000, 1,2 dipalmitoyl-sn-glycero-3-phosphate: DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine: DPPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000]): DSPE-PEG-COOH, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]: DSPE-PEG-PDP, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine: DOPC; EE: Encapsulation Efficiency.*

**41**

**Nano-encapsulating materials**

DPPC, Stearic acid

Lipoid S75–3 DPPC, DSPC, DSPE-PEG 2000

DSPE-PEG2000-amine, DPPC

DSPC, DSPE-PEG

DSPC, DSPE-PEG3400-maleimide,

DSPE-PEG-1000

Phosphatidylcholine, cholesterol.

DPPC, DPPA, DPPE, DSPE-PEG-COOH

DPPC, DSPE-MPEG, DPPA, DPPE

DSPC, DPPE, DSPE-PEG2000-Biotin.

*Encapsulation Efficiency.*

**Table 3.**

*Plant and animal lipids-based nano-encapsulating carriers for diagnostic applications.*

octafluoropropane

Nanobubble *Note: 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid: DMPA, 1,2 Distearoyl-sn-glycero-3-phosphocholine: DSPC, Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000:* 

*DSPE-MPEG2000, 1,2 dipalmitoyl-sn-glycero-3-phosphate: DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine: DPPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene* 

*glycol)-2000]): DSPE-PEG-COOH, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]: DSPE-PEG-PDP, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine: DOPC; EE:* 

pentafluoroctane

Pentafluoroctane

Antibody conjugated

nanobubble

Nanobubble

oil Topotecan perfluoro-15-crown-5-ether

camptothecin-floxuridine

Perfluorohexane (antibody

ligated)

Indocyanine green and gold

Liposomes

nanorods

**Active compounds**

Sulfur hexafluoride

Nanobubble Phase inversion

liposomes Pressure extrusion

Nanoprecipitation/

microbubble

Emulsion

MRI

Focused ultrasound

Isolation of Circulating Tumor Cells.

Photoacoustic and fluorescence imaging

Contrast agent for ultrasound imaging for

ovarian cancer diagnosis

Contrast agent for ultrasound imaging

Ultrasound imaging for cancer detection

97%

[145]

(implied)

Not

[160]

applicable

Not

[161]

applicable

Not

[162]

applicable

—

O2 sensor for MRI

MRI guided focused ultrasound

Ultrasound imaging

**Nano-encapsulation technique**

**Diagnostic applications**

**EE %**

**Ref.**

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

[159]

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

Not Applicable

—

—

—

56.7 ± 2.3

[158]

[155]

[156]

[157]

[154]

#### **Table 2.**

*Plant and animal lipid-based microencapsulating carriers for diagnostic applications.*


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

> **Table 3.**

*Plant and animal lipids-based nano-encapsulating carriers for diagnostic applications.*

*Nano- and Microencapsulation - Techniques and Applications*

**Active compounds**

Indocyanine green liposomes/ Perfluorobutane

DSPC Perfluorobutane Gene loaded

Magnetic iron oxide nanoparticles

peptide contained just the lipids DSPC: DSPE-PEG2K in the ratio of 90:10 while the targeted batch had I % of the lipo-peg-peptide replacing part of the DSPE-PEG2K. Evaluation of the contrast media in tumor infected female FVB mice in vivo and nucleolin expressing breast cancer cell line in vitro demonstrated that the accumulation

Deng and colleagues [147] recently developed a multicomponent system for diagnosis and treatment of cancers. They combined near infrared luminescence of quantum dots and thermo-sensitivity of magnetic liposomes to encapsulate and control drug localization and release. They prepared thermosensitive liposomes that were eventually loaded with drug, paclitaxel, magnetic nanoparticles and NIR luminescent quantum dots. The liposomes were prepared by the thin film hydration method using dipalmitoylphophatidylcholine, DPPC, 1,2-diaccyl-sn-glycero-3-phosphoethanolamine-N-(methoxy [polyethylene glycol]-2000 (DSPE–MPEG-2000), 1,2-diaccyl-sn-glycero-3-phosphoglycerol sodium (DSPG-Na). Effect of the developed systems were studied on cancer cell MCF-7 and SKOV-3 cell lines and uptake of the drug followed in real time by confocal scanning microscope. **Tables 2** and **3**

> **Microencapsulation technique**

Microbubble/ liposomes

Gas microbubble Ultrasound

microbubble decorated with antibody

containing perfluoropentane loaded liposome

microbubble

*Note: 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid: DMPA, 1,2 Distearoyl-sn-glycero-3-phosphocholine: DSPC, Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000: DSPE-MPEG2000, 1,2 dipalmitoyl-sn-glycero-3-phosphate: DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine: DPPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene* 

*glycol)-2000]): DSPE-PEG-COOH, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]: DSPE-PEG-PDP, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine: DOPC; EE: Encapsulation* 

*Plant and animal lipid-based microencapsulating carriers for diagnostic applications.*

Liposome Magnetic

Lipids perfluoropropane microbubble Ultrasound

Perfluorocarbon Emulsion/

Perfluoropentane Emulsion

**Diagnostic applications**

Fluorescence and ultrasound imaging

imaging

imaging

Tumor cell isolation

Ultrasound guided tumor destruction.

Ultrasound guided tumor destruction

resonance imaging contrast agent

Paclitaxel Liposomes NIR imaging 86.46 ± 1.43%. [147]

**EE % Ref.**

— [148]

Not applicable [146]

Not applicable [149]

— [150]

— [151]

— [152]

— [153]

of the contrast media facilitated the detection of murine breast tumors.

**40**

*Efficiency.*

**Table 2.**

**Microencapsulating materials**

DPPC, CHOLESTEROL, DSPE-PEG-MALEIMIDE, DSPE-PEG-PDP.

DSPC, DSPE-PEG2K

DSPC, PEG40 stearate, DSPE-PEG3400 maleimide

DMPA, DPPC, DPPA, Cholesterol

Cholesterol, DDSPC, DSPE-PEG and DOPC

DPPC, DSPE– MPEG-2000 and DSPG-Na.

indicate diagnostic applications based on micro- and nano-encapsulation utilizing animal/plant lipids as encapsulating materials.
