**4.1. Lipid polymer hybrid nanoparticles (LPHN)**

LPHN consists of a drug containing polymeric core which is coated by a lipid shell. In these systems, inner polymer core contains drug and lipid shell is used to enhance penetration through biological membranes and to control drug release. Polymeric core can be made from hydrophilic or hydrophobic polymer. Term lipid-polymer hybrid is also used for systems that contain polymer core with lipid coating. Lipid is preferred carrier material for hydrophobic drugs due to higher encapsulation efficiency and extended release pattern. A polymeric coating is applied over lipid core to impart certain characteristics required for novel biomedical applications.

In addition of polymeric and lipid layers, surface of LPHN may be modified with different materials. In one study, a hydrophobic drug was loaded in a hydrophobic biodegradable polymer to enhance encapsulation efficiency of a hydrophobic drug. Then, a lipid layer is applied to stabilize core and shell, and to prolong drug release. Finally, hydrophilic polymeric layer, consisting of DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Ncarboxy (polyethylene glycol)2000) was applied to improve pharmacokinetics of LPHN. The three-layer LPHN showed high encapsulation and sustained release of hydrophilic drug [59]. A hydrophilic polymer monolayer may be applied to LPHN to escape phagocytosis and early removal from body. Generally, polyethylene glycol (PEG) is used to provide this stealth property and enhance circulation time of nanoparticles. PEG will attract water to make an aqueous layer which protect LPHN from attachment of opsonin proteins and let it escape the uptake by reticuloendothelial (RES) system. Hydrophilic polymer layer can also enhance colloidal stability of LPHN due to steric hindrance effect [5]. As stealth layer can also hinder interaction with target cells, PEG can be conjugated with other monomers or polymers to form block copolymers that are specific to certain stimuli. This approach enables long circulating LPHN that can shed stealth layer when come in contact with target cells. The stimuli could be intracellular and extracellular protease enzymes, low pH or reducing agents [103].

Selection of polymeric matrix plays a major role in drug delivery properties of LPHN. LPHN are commonly used for poor water soluble or hydrophobic drugs. A hydrophobic polymer core can encapsulate higher amount of hydrophobic drug and vice versa. Two or more drugs could also be loaded into the core of LPHN. On the other hand, LPHN with hydrophilic and hydrophobic drug could be made to contain one drug in core and the other in shell. Wong et al. [67] prepared LPHN containing lipid core to encapsulate hydrophobic drug Elacridar (GG918) and hydrophilic shell of hydrolyzed polymer of epoxidized soybean oil (HPESO) to encapsulated doxorubicin. They found that both drugs were released in sustained manner for more than 72 hours (**Figure 2**). Simultaneous delivery of chemosensitizer GG918 was able to revert multidrug resistance to anticancer drug doxorubicin. These simultaneously loaded LPHN showed better efficacy than free drug solution or LPHN of any of the two drugs.

not improve or impart new functional aspect of nanoparticles. Thus, many researchers do not regard PEG coated as hybrid systems. Similarly, nanoparticles conjugated with targeting

LPHN consists of a drug containing polymeric core which is coated by a lipid shell. In these systems, inner polymer core contains drug and lipid shell is used to enhance penetration through biological membranes and to control drug release. Polymeric core can be made from hydrophilic or hydrophobic polymer. Term lipid-polymer hybrid is also used for systems that contain polymer core with lipid coating. Lipid is preferred carrier material for hydrophobic drugs due to higher encapsulation efficiency and extended release pattern. A polymeric coating is applied over lipid core to impart certain characteristics required for novel biomedical

In addition of polymeric and lipid layers, surface of LPHN may be modified with different materials. In one study, a hydrophobic drug was loaded in a hydrophobic biodegradable polymer to enhance encapsulation efficiency of a hydrophobic drug. Then, a lipid layer is applied to stabilize core and shell, and to prolong drug release. Finally, hydrophilic polymeric layer, consisting of DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Ncarboxy (polyethylene glycol)2000) was applied to improve pharmacokinetics of LPHN. The three-layer LPHN showed high encapsulation and sustained release of hydrophilic drug [59]. A hydrophilic polymer monolayer may be applied to LPHN to escape phagocytosis and early removal from body. Generally, polyethylene glycol (PEG) is used to provide this stealth property and enhance circulation time of nanoparticles. PEG will attract water to make an aqueous layer which protect LPHN from attachment of opsonin proteins and let it escape the uptake by reticuloendothelial (RES) system. Hydrophilic polymer layer can also enhance colloidal stability of LPHN due to steric hindrance effect [5]. As stealth layer can also hinder interaction with target cells, PEG can be conjugated with other monomers or polymers to form block copolymers that are specific to certain stimuli. This approach enables long circulating LPHN that can shed stealth layer when come in contact with target cells. The stimuli could be intracellular and extracellular protease enzymes, low pH or reducing

Selection of polymeric matrix plays a major role in drug delivery properties of LPHN. LPHN are commonly used for poor water soluble or hydrophobic drugs. A hydrophobic polymer core can encapsulate higher amount of hydrophobic drug and vice versa. Two or more drugs could also be loaded into the core of LPHN. On the other hand, LPHN with hydrophilic and hydrophobic drug could be made to contain one drug in core and the other in shell. Wong et al. [67] prepared LPHN containing lipid core to encapsulate hydrophobic drug Elacridar (GG918) and hydrophilic shell of hydrolyzed polymer of epoxidized soybean oil (HPESO) to encapsulated doxorubicin. They found that both drugs were released in sustained manner for more than 72 hours (**Figure 2**). Simultaneous delivery of chemosensitizer GG918 was able to revert multidrug resistance to anticancer drug doxorubicin. These simultaneously loaded LPHN showed better efficacy than free drug solution or LPHN of any of the two drugs.

ligands cannot be regarded as hybrid system.

70 Advanced Technology for Delivering Therapeutics

applications.

agents [103].

**4.1. Lipid polymer hybrid nanoparticles (LPHN)**

**Figure 2.** Drug release from gold nanoparticles containing solid lipid nanoparticles. F1 (5mg diacerein) and F2 (10 mg diacerein) show sustained release of drug for 39 and 48 hours. However, F3 had same composition as F2 but drug release was conducted at 40°C. F5 and F6 contained increasing amount of hydrophilic gold nanoparticles with 5 mg diacerein. F7 contained gold nanoparticles same as F6 but 10 mg diacerein. Finally, F8 contained lipophilic gold nanoparticles with 10 mg diacerein. Drug release was faster at higher temperature (F3) as compared to 37°C. Furthermore, hydrophilic gold nanoparticles containing formulations F5, F6 and F7 released drug in less than 5 hours, whereas hydrophobic gold nanoparticles showed prolonged release up to 25 hours.

As a wide variety of polymers and lipids are available, LPHN can be prepared to theoretically load any therapeutic moiety. Nucleic acid based therapeutics i.e. plasmid DNA, antisense oligonucleotide, small interfering RNA and small hairpin RNA, have shown promise to cure many diseases. LPHN have emerged as nonviral carriers for nucleic acid products with low toxicity, immunogenicity and cost of production. Cationic polymers and lipids have been widely investigated for this purpose. Cationic groups can bind negatively charged nucleic acid molecules and deliver to target cells. Zhong et al. [104] prepared LPHN with biodegradable PLGA and two cationic lipids i.e. 1, 2-dioleoyl-3-trimethyammonium-propane (DOTAP) or 3β-[N-(N′,N′-dimethylaminoethane)-carbamyl]cholesterol (DC-Chol). LPHN were prepared by two either with cationic lipid core so that DNA is loaded inside core or with cationic lipid shell so that DNA is loaded on surface. The *in vitro* evaluation was done in human embryonic kidney cells. They found that LPHN with DNA on surface showed higher transfection efficiency than those with DNA inside core. Next, they prepared LPHN with polymer both inside core and on the surface which showed efficiency similar to that of LPHN with DNA on surface. This study concluded that LPHN can show transfection efficiency about 600 times higher than unbound DNA. However, cationic lipids and polymers may have some problems on their own. They may interact with biological components, be nonbiodegradable or toxic after systemic administration. These factors are controlled by hydrophobic chain length, nature of cation group and linkage. To solve these problems, Shi et al. [54] prepared novel LPHN with four distinct layers. First is a hollow core i.e. aqueous droplet containing nucleic acid which is coated by an inner lipid layer of cationic lipid ethylphosphocholine.

The cationic lipid orients itself in such a way that cationic group faces inward and its hydrophobic chain faces outward. Third layer is formed by ester terminated PLGA. It is a hydrophobic polymer that intermingles with protruding chains of cationic lipid. Finally, self-assembled lecithin and DSPE-PEG form outer coating to facilitate transfection and to impart stealth property to LPHN. This LPHN system release loaded siRNA in sustained manner up to 6 days and enhanced gene expression in mice.

A recent trend in drug delivery research has focused on the development of human-like vesicular drug delivery system. This concept emerged when exosome were found to be responsible for cell to cell communication in tumors and regulate tumor microenvironment. It was believed that exosomes isolated from patients may be filled with antitumor drugs and injected back to the patients for personalized treatment. As isolation of exosomes from patients is complicated and very costly, this dream was realized by synthesizing surface antigens of exosomes by genetic engineering and grafting on the surface of drug containing liposomes or other vesicular systems [5]. In addition to this, many bacterial and viral antigens have been used. These antigens are used for the delivery of vaccine and act as immune adjuvant i.e. enhance immune response to vaccine. Moreover, polymeric core produce better adjuvant effects than lipid core. Bershteyn et al. [105] prepared PLGA core and phospholipid bilayer coated LPHN that were stabilized by PEG for simultaneous loading of antigen and adjuvant. The protein adjuvant was covalently bonded on surface and lipophilic adjuvants, such as monophosphoryl lipid A and α-galactosylceramide, which were loaded in lipid bilayer. Immune response was shown at dose as low as 2.5 ng which was detectable after 100 days. It was also found that α-galactosylceramide shows rapid rise in antibody titer whereas monophosphoryl lipid A produced response in sustained manner. Interestingly, coloading of both adjuvants with antigen further increased antigen titer by 12 fold. These results show that LPHN can reduce dose of antigen to reduce cost and side effects.

Term LPHN may also be extended to nanoparticle systems consisting of two or more polymer at least one of which is lipophilic. A hydrophilic shell may be applied to drug containing hydrophobic (or lipophilic) polymeric core to impart mucoadhesion or to make them stealth. For example, PEG or chitosan coating has been widely used to improve circulation life of sustained release solid lipid nanoparticles [106]. On the other hand, a hydrophobic polymer shell may be formed over hydrophilic polymer core to enhance LPHN absorption through biological membranes. This approach is especially useful for oral administration of therapeutic macromolecules [107]. Recently, Liu et al. has synthesized supramolecular vectors for gene delivery. First, adamantyl-terminated polyethyleneimine was admixed with β-cyclodextrin to encapsulate nucleic acid, i.e., DNA or siRNA which was further coated with adamantyl-PEG. The supramolecular vector was stabilized by host-guest interaction. This LPHN system showed low toxicity and high transfection efficiency during in vitro experiments. Graphene is another two-dimensional framework of carbon atoms that is investigated for hybrid applications. When treated with suitable reagents, it can be oxidized, hydroxylated, carboxylated or halogenated. These functional groups can be conjugated with different materials desired for biomedical applications [108].

## **4.2. Inorganic/organic hybrid nanoparticles (IOHN)**

IOHN are synthesized from organic and inorganic materials. Most commonly, core is made of inorganic materials and the shell of an organic material is applied to improve its pharmacokinetic parameters. On the other hand, inorganic shell may be applied to the core of organic materials to impart different properties. IOHN are interesting because they offer properties of both materials. Like organic polymer, they can be functionalized with different groups. Like metallic nanoparticles, inorganic shell provides physical and chemical stability to polymeric core. Generally, the inorganic portion is developed by reduction of metal ions to zerovalent state. Inorganic core is synthesized by mixing metal ions solution with a reducing agent with or without heating. However, inorganic shell may be synthesized either by reduction of metal ions on polymeric core or by deposition of preformed metal colloids on organic core.

A recent trend in drug delivery research has focused on the development of human-like vesicular drug delivery system. This concept emerged when exosome were found to be responsible for cell to cell communication in tumors and regulate tumor microenvironment. It was believed that exosomes isolated from patients may be filled with antitumor drugs and injected back to the patients for personalized treatment. As isolation of exosomes from patients is complicated and very costly, this dream was realized by synthesizing surface antigens of exosomes by genetic engineering and grafting on the surface of drug containing liposomes or other vesicular systems [5]. In addition to this, many bacterial and viral antigens have been used. These antigens are used for the delivery of vaccine and act as immune adjuvant i.e. enhance immune response to vaccine. Moreover, polymeric core produce better adjuvant effects than lipid core. Bershteyn et al. [105] prepared PLGA core and phospholipid bilayer coated LPHN that were stabilized by PEG for simultaneous loading of antigen and adjuvant. The protein adjuvant was covalently bonded on surface and lipophilic adjuvants, such as monophosphoryl lipid A and α-galactosylceramide, which were loaded in lipid bilayer. Immune response was shown at dose as low as 2.5 ng which was detectable after 100 days. It was also found that α-galactosylceramide shows rapid rise in antibody titer whereas monophosphoryl lipid A produced response in sustained manner. Interestingly, coloading of both adjuvants with antigen further increased antigen titer by 12 fold. These results show that LPHN can reduce

Term LPHN may also be extended to nanoparticle systems consisting of two or more polymer at least one of which is lipophilic. A hydrophilic shell may be applied to drug containing hydrophobic (or lipophilic) polymeric core to impart mucoadhesion or to make them stealth. For example, PEG or chitosan coating has been widely used to improve circulation life of sustained release solid lipid nanoparticles [106]. On the other hand, a hydrophobic polymer shell may be formed over hydrophilic polymer core to enhance LPHN absorption through biological membranes. This approach is especially useful for oral administration of therapeutic macromolecules [107]. Recently, Liu et al. has synthesized supramolecular vectors for gene delivery. First, adamantyl-terminated polyethyleneimine was admixed with β-cyclodextrin to encapsulate nucleic acid, i.e., DNA or siRNA which was further coated with adamantyl-PEG. The supramolecular vector was stabilized by host-guest interaction. This LPHN system showed low toxicity and high transfection efficiency during in vitro experiments. Graphene is another two-dimensional framework of carbon atoms that is investigated for hybrid applications. When treated with suitable reagents, it can be oxidized, hydroxylated, carboxylated or halogenated. These functional groups can be conjugated with different materials desired for biomedical applications [108].

IOHN are synthesized from organic and inorganic materials. Most commonly, core is made of inorganic materials and the shell of an organic material is applied to improve its pharmacokinetic parameters. On the other hand, inorganic shell may be applied to the core of organic materials to impart different properties. IOHN are interesting because they offer properties of both materials. Like organic polymer, they can be functionalized with different groups. Like metallic nanoparticles, inorganic shell provides physical and chemical stability

dose of antigen to reduce cost and side effects.

72 Advanced Technology for Delivering Therapeutics

**4.2. Inorganic/organic hybrid nanoparticles (IOHN)**

Methods for synthesis of organic core and shell have already been discussed in detail in a chapter. We have prepared IOHN consisting of gold core with fatty acid shell. First, gold nanoparticles were synthesized with lecithin bilayer (hydrophilic surface) and lecithin monolayer by acid treatment (hydrophobic surface). The gold nanoparticles were added to molten fatty acids and emulsified with aqueous surfactant solution. Upon cooling, we found that gold nanoparticles with hydrophobic surface are more stable as compared to gold nanoparticles with hydrophilic surface [109]. The presence of gold nanoparticles in core enhanced drug release rate from lipid nanoparticles. This can be attributed to the presence of gold nanoparticles that push drug toward periphery and reduce diffusion path length (**Figure 1**). In another study, we prepared an organic core of lecithin and inorganic shell of gold nanoparticles. First, lecithin nanoparticles were prepared and loaded with drug. Next, preformed gold nanoparticles were adsorbed on its surface. We found that drug release was controlled by both gold nanoparticles. Gold nanoparticles retard release of drug due to physical barrier. Lecithin controlled release of anti-inflammatory drug from core in pH-dependent manner [110]. Gold is also known to possess anti-inflammatory effect. In this study, gold shell was found to synergize anti-inflammatory effect of encapsulated drug diacerein by many folds (**Figure 3**).

Various organic materials have been used to prepare IOHN to improve their performance. The materials that are used to synthesize or stabilize nanoparticles may impart specific function. The most pronounced function is enhanced penetration inside target cells which in turn controls toxicity of IOHN. Freese et al. [47] studied toxicity of gold nanoparticles with different organic coatings with neutral, positive and negative charge. The results showed that IOHN with positive charge coating shows more internalization in cells, and thus, higher toxicity. The cell membrane has a negative charge, whereas the IOHN are positively charged particles. This charge difference triggers the rapid binding to the cell surface and internalization of these IOHNs. As gold can cause toxicity at higher dose, higher internalization in cell will lead to high toxicity [111].

Metallic nanoparticles smaller than 100 nm are usually responsive to different stimuli, a technique that has been widely employed in diagnosis and therapy. IOHN with metallic core can be used for thermotherapy of cancer whereby IOHN produces heat when exposed to external magnetic field. Similarly, metallic moieties, i.e., nanoparticles or tagged polymers, can be bound to core of organic materials. These nanoparticles will be targeted to cancerous tissues and magnetic moieties will produce hyperthermia under external stimuli. When core of organic material is loaded with drug, inorganic part can release the drug by hyperthermia-mediated degradation of core after reaching the target site [5]. In addition to magnetic field, inorganic nanoparticles are also responsive to infrared and ultrasound waves. This makes IOHN interesting candidates for biomedical imaging of targeted tis-

**Figure 3.** Efficacy of anti-inflammatory drug encapsulated in lecithin core-gold shell hybrid nanoparticles; (A) Antiinflammatory effect of diacerein is synergized in the presence of gold as compared to pure drug, diacerein. PEG-AuNP = PEG coated gold nanoparticles, LD-NP = diacerein loaded lecithin nano[articles, L PEG-AuNP = Lecithin nanoparticles surface coated with PEG coated gold nanoparticles, L Cit-AuNP = Lecithin nanoparticles surface coated with citrate coated gold nanoparticles, L B-AuNP = Lecithin nanoparticles surface coated with sodium borohydrate coated gold nanoparticles, LD PEG-AuNP = L PEG-AuNP loaded with diacerein, LD Cit-AuNP = L Cit-AuNP loaded with diacerein, LD B-AuNP = L B-AuNP loaded with diacerein. (B) represents decrease in swelling as measured by Vernier caliper before (a, b, c, d, e, f, g) and 3 h after (b´,c´, d´, e´, f´, g´) from untreated (b), diacerein (c), PEG-Au NPs (d), LD PEG-Au NPs (e), LD Cit-Au NPs (f), and LD B-Au NPs (g) treatments groups, while a is normal rat paw.

sues. More recently, multimodal IOHN have ensured imaging and drug release from the same system after systemic administration. This target can be achieved in two ways. First, magnetic field of low frequency or intensity is applied for imaging of IOHN. Once in cancer tissue, intensity or frequency is increased to produce hyperthermia-based cell killing or drug release [112]. Secondly, inorganic materials responsive to more than one stimulus can be used. One stimulus aids in imaging, whereas second stimulus will lead to drug release or thermotherapy [113].

IOHN have also been prepared with hollow core enclosed inside a hybrid shell. Hollow core IOHN can be prepared by many ways. First strategy is to make layer of inorganic or organic material which is then stabilized by other component of IOHN system. Similarly, it can consist of a mixed shell of inorganic and organic materials enclosing hollow core. Metal-tagged polymers with amphiphilic nature self-assemble to form micelles in aqueous solution or after reaching the target microenvironment [114]. Whole virus or virus capsid has been investigated as drug delivery systems by many researchers due to its inherent high penetration in cells.

Portney et al. [115] hybridized virus capsid with quantum dots and single-wall carbon nanotubes to yield hybrid structures that can find various applications. These hybrid structures are very stable to chemical and mechanical stress. IOHN with metallic core and organic shells have been widely investigated for diagnostic application. Although, organic shell usually employed to improve the pharmacokinetics and targeting properties of the metallic nanoparticles but may be beneficial by enhancing the diagnostic efficiency of the system. The most prominent example is nucleic acid-based biosensors with metallic core. When metallic nanoparticles aggregate, they show blue shift due to increase in size. Metallic core is coated with single-stranded DNA (ssDNA) that can identify specific sequence on target DNA and bind it. In bioassay, when metallic nanoparticle conjugated ssDNA start bind target DNA, they come close to each other and test solution color changes from red to blue. This indicates the presence of target DNA as visualized by naked eye or through UV-visible spectrophotometer [116].

## **4.3. Metalloprotein hybrid nanoflowers (MPHNs)**

sues. More recently, multimodal IOHN have ensured imaging and drug release from the same system after systemic administration. This target can be achieved in two ways. First, magnetic field of low frequency or intensity is applied for imaging of IOHN. Once in cancer

LD Cit-Au NPs (f), and LD B-Au NPs (g) treatments groups, while a is normal rat paw.

74 Advanced Technology for Delivering Therapeutics

**Figure 3.** Efficacy of anti-inflammatory drug encapsulated in lecithin core-gold shell hybrid nanoparticles; (A) Antiinflammatory effect of diacerein is synergized in the presence of gold as compared to pure drug, diacerein. PEG-AuNP = PEG coated gold nanoparticles, LD-NP = diacerein loaded lecithin nano[articles, L PEG-AuNP = Lecithin nanoparticles surface coated with PEG coated gold nanoparticles, L Cit-AuNP = Lecithin nanoparticles surface coated with citrate coated gold nanoparticles, L B-AuNP = Lecithin nanoparticles surface coated with sodium borohydrate coated gold nanoparticles, LD PEG-AuNP = L PEG-AuNP loaded with diacerein, LD Cit-AuNP = L Cit-AuNP loaded with diacerein, LD B-AuNP = L B-AuNP loaded with diacerein. (B) represents decrease in swelling as measured by Vernier caliper before (a, b, c, d, e, f, g) and 3 h after (b´,c´, d´, e´, f´, g´) from untreated (b), diacerein (c), PEG-Au NPs (d), LD PEG-Au NPs (e), Although MPHN can be categorized as inorganic-organic hybrid NP, they are discussed here separately due to difference in structure and many fold increased surface area. The flower-like structure of MPHN is due to the presence of proteins that stabilize metallic crystals in the structure. Proteins act as glue and hold metallic crystals in a pattern which mimics flower petals. Unlike inorganic-organic hybrids, synthesis of MPHN occurs in three stages. First stage is the growth stage in which metal ions bond with proteins through amide bond. This acts as nucleation site leading to growth of primary crystals. In the second stage, metalloprotein crystals aggregate to form larger structures bearing primary petals like structures. Finally, anisotropic growth on metalloprotein aggregates leads to formation of complete petals. Generally, their size lies in the range of 2–30 μm which is another reason to differentiate MPHN from OIHN. MPHN is mostly used for bioassay whereby desired enzyme is conjugated with metallic part. Encapsulation efficiency of enzymes in MPHN has been achieved up to 66%. Enzyme loading above or below this limit decreases encapsulation efficiency. Nevertheless, enzyme efficiency of MPHN varies between 85% and 1000%. Enzyme efficiency higher than free enzyme is due to many reasons. MPHN shows high surface area due to petal-like projections. The petals also have hole-like spaces between them that may be up to 100 nm in diameter. It is also observed that immobilized enzyme shows cooperative interaction to enhance enzyme efficiency. Similarly, metal ions, such as copper, calcium and manganese, may also help enzyme in catalysis. Copper (Cu2+) is the most widely used metal with different enzymes. Cu2+ and laccase enzyme MPHN have been developed for detection of phenols. The prepared MPHN was adsorbed on filter, and a mixture of phenol and 4-aminoantipyrine was added to it. Laccaseassisted reaction of both compounds produced red antipyrine dyes in 5 minutes. The changes in color will be visible with the naked eye, and UV-visible spectrophotometer can be used for quantitative detection. The MPHN-coated filters are reusable and are much faster than chromatography and mass spectrometry based methods. Likewise, MPHN of Cu2+ and horseradish peroxidase was prepared for detection of phenol and hydrogen peroxide. This MPHN was able to detect very low amounts of phenol (1 μM) and hydrogen peroxide (0.5 μM) as change in color was observed with the naked eye. It has been found that hydrogen peroxide induces cell death at concentration higher than 50 μM and the limit of detection of free enzyme is around 20 μM. Thus, these MPHNs will be very efficient to detect slight changes in hydrogen peroxide efficiently even below its threshold level. Cu2+ and trypsin MPHN have been used to carry out proteolysis which is an important step in protein identification. The enzyme efficiency of proteolytic MPHN is similar or superior to free enzyme but are fast and reusable.

Another form of nanoflowers is synthesized using deoxyribonucleic acid (DNA) which, like proteins, possesses high number of nitrogen molecules and serves as a template for nanoflowers. In one study, a drug and a dye molecule was bonded to DNA that was used to synthesize nanoflowers. These nanoflowers showed multimodel property of drug delivery and imaging by using FRET technology. More recently, capsular MPHNs have been prepared with improved characteristics. This technique involved coating of MPHN with protamine and silica. Then, metallic core is removed from capsular MPHN system. Capsular nanoflowers show higher enzyme efficiency and improved stability in harsh environmental conditions.

## **4.4. Mesoporous silica hybrid nanoparticles**

Silica has been widely used in drug delivery due to its nontoxic and biocompatible nature. Silica shell has been applied to metallic nanoparticles to reduce their toxicity in various biomedical applications. Mesoporous silica nanoparticles (MSNPs) are silica materials with mesopores of up to 50 nm. They are also termed as hollow mesoporous silica nanoparticles due to the fact that mesopores are hollow. The advantages of MSNP are enhanced surface area and that hollow mesopores can be loaded with therapeutic molecules. First, MSNPs were loaded with drugs. Later, MSNPs were used for the delivery of different dyes and macromolecules such as enzymes. MSNP hybrids have been prepared with both organic and inorganic materials. One problem with the use of MSNP is the leakage of drugs from pores. Sreejith et al. [117] used graphene oxide (GO) coating on MSNP to prevent leakage of drugs. After drug loading, GO coating is applied which acts as blanket to physically block the pores. GO coating also prevents encapsulated drug from environmental degradation. In addition to applications in drug delivery, MSNPs are also used for diagnosis and imaging.

Maji et al. [118] prepared MSNP-GNP (gold nanoparticle) hybrids for detection of hydrogen peroxide. They coated MSNP with graphene oxide, and GNPs were coated on this surface. The hybrids were first used for electrochemical detection of hydrogen peroxide in the presence of other biological molecules. Later, MSNP-GNPs were successfully used for in vivo imaging in mice. MSNP surface can be modified with different functional groups that provide opportunities to form hybrid with different materials [118].
