**2. Method of preparation**

Different methods have been employed for the fabrication of hybrid nanocarriers depending upon their chemical composition and applications. The lipid-polymer hybrid, polymerinorganic hybrid, metal (gold, silver or iron) polymer, silica (SiO2 ) based hybrid nanosystems and hybrid polymeric nanocarriers have been most widely investigated [18]. Most of these hybrid carriers utilized two distinctive fabrication approaches. First, a two-step conventional approach process, in which the inner core and outer shell are prepared separately and then are coincubated for the formation of hybrid nanoparticle. The second approach is the single step, in which various state-of-the art techniques of the self-assembling are being incorporated. These processes are further modified with different chemical moieties to obtain versatile hybrid nanoparticles meeting specific need of therapy [19]. In the present chapter, we will focus on the two step conventional as well as single step formulation approaches along with recent innovations have been presented in order to prepare the hybrid nanocarriers with versatile characteristics.

## **2.1. Conventional two-step method**

It was the first technique employed for the fabrication of hybrid nanocarriers. The inner core and outer shell components are prepared in two separate steps employing suitable polymers and chemicals and are then combined to form the hybrid nanoparticle [17]. The foremost type of core shell hybrid nanoparticles contained a core of the polymeric nanoparticles and an outer shell of preformed lipid component such as liposome or lipoparticles in appropriate ratios [20]. Further, the single or multilayered shell is prepared with other techniques such as sonication [21], extrusion or high pressure homogenization and vortexing [22]. The polymeric core is prepared by emulsification-solvent evaporation or solvent diffusion [23], desolvation [24], nanoprecipitation [25, 26], sonication [27] and high pressure homogenization [28] depending upon the hydrophobicity of the loading drugs, their applications [29] and the size of the core.

The single step method is applied when the core materials such as polymers, silica and organic substances are miscible with the drug payload and also are solubilized in the organic solvent [30, 31]. The double emulsification step is employed when the compound is immiscible with the organic solvents and does not form covalent linkage with the core material. As this method requires multiple steps for mixing of different components, relatively larger hybrid nanoparticles are produced [32]. Further, any of the suitable technique such as ultrasonication or extrusion by high pressure homogenization also reduces the particle size as the polymer solution is passed through the nozzle under high pressure. Furthermore, the freeze drying or cooling at normal temperature produced free flowing characteristic particles [33, 34]. Another recent innovation is the application of nanoprecipitation method for the preparation of polymeric core. The polymer is dissolve in the suitable solvent and then precipitated by using the nonsolvent component [26].

The formed polymeric core and lipid vesicles are mixed by vortexing, extrusion, film hydration and ultrasonication techniques in order to formulate the hybrid nanoparticles. The mixing processes provide the energy for the fusion or adsorption of the shell on the inner core material. Additionally, the electrostatic forces among these components also play their role for fabrication of hybrid nanoparticles [35]. It is worth mentioning here that mixing process must be carried out above the phase transition temperature of the lipid component. The formed hybrid nanoparticles are separated by the ultracentrifugation process [36, 37]. Different investigators such as Liang et al. [38] and Zhao et al. [39] prepared the hybrid nanoparticles and nanocells by the emulsification solvent evaporation technique employing the paclitaxel loaded polymeric nanoparticles as core and the PEG or folic acid conjugated octadecyl-quaternary lysine-modified chitosan and cholesterol as lipid shell [38, 39].

## **2.2. Modified two-step methods**

In this chapter, the different types of hybrid nanocarriers have been described with particular emphasis on the brief rationale for the development of these hybrid nanocarriers along with different fabrication approaches with greater emphasize on the lipid polymer hybrid nanoparticles. A brief description factors governing the optimized response characteristics

**Figure 1.** Structure of lipid-polymer hybrid nanoparticles; (a) polymer core-lipid shell hybrid, (b) 3 layers polymer-lipid hybrid nanoparticles consisting of polymeric core (1) and two lipid layers (2,3) shell, (c) 4 layers hollow core lipidpolymer hybrid, consisting of hollow core (1) covered by reverse surfactant layer (2), polymeric shell (3), and outer shells of two lipids (4). (d) organic core-inorganic shell and inorganic core-organic shell hybrid, (e) inorganic (metallic)-protein

Different methods have been employed for the fabrication of hybrid nanocarriers depending upon their chemical composition and applications. The lipid-polymer hybrid, polymer-

and hybrid polymeric nanocarriers have been most widely investigated [18]. Most of these hybrid carriers utilized two distinctive fabrication approaches. First, a two-step conventional approach process, in which the inner core and outer shell are prepared separately and then are coincubated for the formation of hybrid nanoparticle. The second approach is the single step, in which various state-of-the art techniques of the self-assembling are being incorporated. These processes are further modified with different chemical moieties to obtain versatile hybrid nanoparticles meeting specific need of therapy [19]. In the present chapter, we will focus on the two step conventional as well as single step formulation approaches along

) based hybrid nanosystems

and their potential application of these hybrid nanoparticles are also presented.

hybrid nanoflowers, and (f) graphene oxide coated mesoporous silica-inorganic hybrid nanoparticles.

inorganic hybrid, metal (gold, silver or iron) polymer, silica (SiO2

**2. Method of preparation**

56 Advanced Technology for Delivering Therapeutics

The modifications to the conventional two step method such as spray drying and lithographic molding processes have also been employed for fabrication of hybrid nanoparticles [29]. The inner core is prepared by the spray drying which is dispersed in an appropriate solvent containing the lipid, polymer or any inorganic material. The spray dried lipid coated core shell hybrid nanoparticles were collected after the completion [17].

Freeze or spray dried inhalation hybrid nanoparticles of levofloxacin, ciprofloxacin and isoniazid coated with multiple layers of the lipids were prepared using double emulsion solvent evaporation technique. These hybrid nanoparticles showed better inhalation efficiency, emitted particle size and diameter compared to the conventional two step methods [37, 40]. Another investigations employed nanospray drying for fabrication of hybrid nanoparticles using polyglutamic acid, poly lysine nanoparticles coated with the lipid materials [41]. Recently, Keloglu et al. [42] employed jet spray drying technique for the fabrication of hybrid microfibers-nanoparticles having low density and greater strength using PLGA and poly lactic acid (PLA) [42].

A soft lithography particle molding technique was also utilized for the preparation of hybrid nanocarriers for the delivery of genes to various diseases. De Simon and his coworkers prepared the nanosized particles using the particle replication approach on the silicon wafers. The technique was referred to as Particle Replication in Nonwetting Templates (PRINT) [43]. The process involve the dissolution of the polymer (e.g., PLGA, PLA) in an organic solvents such as dimethyl formamide, methyl acetate and/or dimethyl sulfoxide along with the material to be encapsulated. The PRINT molding device was employed to fabricate the nanoparticles which later were harvested with the help of polyethylene terephthalate sheet [43]. It produces the particles of different shapes and a wide size range depending upon the size of the molding cavities [44].

## **2.3. Single-step preparation methods**

The low encapsulation efficiency due to the leakage of the drugs from the inner core during second step, batch variability and large time consumption are the common problems associated with the conventional two step methods [45]. These constraints can be overcome by designing the simple method that utilized the single step approach and also provide better control on the content uniformity, reproducibility and other characteristics of the system. The method involves the mixing of two different solutions containing the polymer and lipid that self-assembled to form the particles with the core shell hybrid structure [46]. The polymer is dissolved in an appropriate organic solvent while the lipid solution is prepared in the water that may utilize the small fraction of organic solvent as solubilizing agent. The solution containing polymer is added to the lipid phase where the polymer precipitate to formed the nanoparticles and the lipid is self-assembled at the surface to form the hybrid nanoparticles. Single-step preparation is usually achieved by nanoprecipitation, emulsification-solvent evaporation and solvent diffusion methods. These methods and their appropriate modifications are discussed here.

## *2.3.1. Emulsification solvent evaporation method*

Emulsification solvent evaporation method is the most commonly employed single step approach for the fabrication of hybrid nanocarriers. The single emulsification solvent evaporation [47] and double emulsification solvent evaporation (DESE) techniques are employed depending upon the nature and solubility of encapsulating drug. In the ESE method, the oil phase is formed by dissolving the polymer and the drug in the water immiscible organic solvent. The aqueous solution containing the lipid portion which act as a stabilizer itself during the selfassembling process [48, 49]. The organic phase is then added dropwise into the aqueous phase under the sonication or stirring at the constant speed that results in the formation oil in water emulsion. During the emulsification process, the hydrophobic part of the lipid is adsorbed on the inner core material while the hydrophilic parts arrange themselves toward the aqueous medium forming the lipid coated hybrid nanoparticles [45, 50].

The single ESE method is employed for the encapsulation of hydrophobic drugs with low aqueous solubility [51]. Recently, the folate conjugated lipid polymer hybrid nanoparticles have been prepared by the emulsification solvent diffusion method for the targeted delivery of the doxorubicin using phosphatidylcholine (lecithin 99%) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DPSE)-PEG-COOH as lipid portion and PLGA as a polymeric portion [52]. The ESE method was also employed to formulate duel ligand hybrid nanocarriers for the targeted delivery of docetaxel. The hybrid nanoparticles possessed a uniform monolayer of the lipid over the polymeric core. The cell interaction studies revealed better endocytosis profile with sustained release of the drug by preventing the diffusion of the aqueous medium in the polymeric core. However, the particle were relative larger compared to that prepared by the nanoprecipitation method. This might be attributed to higher drug loading that maintained the therapeutic concentration for the longer period of time [53].

The double emulsification solvent evaporation (DESE) has been employed for the hydrophilic drugs and nucleic acid such as siRNA (small interfering ribonucleic acid) which are not dissolved in different organic solvents along with the other suitable polymers or the core/shell materials [54]. The aqueous solution of desired substance is prepared and is then emulsified in the organic/oil phase containing the lipid and polymer. The resultant primary emulsion is again added to another aqueous solution containing the lipid (lecithin, phosphatidylcholine or DSPE) or surface ligand (PEG, half antibodies, aptamer) and a water-in-oil-in-water (w/o/w) multiple emulsion is prepared. The evaporation of the organic phase results in the formation of hybrid nanoparticles [55]. The particles with hollow core covered with an appropriate shell provide the space for the internalization of hydrophilic and small molecules. The evaporation of the organic solvent provides the multilayered shell which has larger size as compared to the other methods [17].

Su et al. [56] prepared the reduction sensitive hybrid nanoparticles of doxorubicin using chitosan with the sodium dodecyl sulfate employing the double emulsification solvent evaporation method. The amphiphilic chitosan and lipid base micelles core provided a unique nanoconfiguration that is enveloped by the triglycerides which enhanced the loading efficiency and provided the drug release profile up to eight folds [56].

## *2.3.2. Nanoprecipitation*

solvent containing the lipid, polymer or any inorganic material. The spray dried lipid coated

Freeze or spray dried inhalation hybrid nanoparticles of levofloxacin, ciprofloxacin and isoniazid coated with multiple layers of the lipids were prepared using double emulsion solvent evaporation technique. These hybrid nanoparticles showed better inhalation efficiency, emitted particle size and diameter compared to the conventional two step methods [37, 40]. Another investigations employed nanospray drying for fabrication of hybrid nanoparticles using polyglutamic acid, poly lysine nanoparticles coated with the lipid materials [41]. Recently, Keloglu et al. [42] employed jet spray drying technique for the fabrication of hybrid microfibers-nanoparticles having low density and greater strength using PLGA and poly lactic acid (PLA) [42].

A soft lithography particle molding technique was also utilized for the preparation of hybrid nanocarriers for the delivery of genes to various diseases. De Simon and his coworkers prepared the nanosized particles using the particle replication approach on the silicon wafers. The technique was referred to as Particle Replication in Nonwetting Templates (PRINT) [43]. The process involve the dissolution of the polymer (e.g., PLGA, PLA) in an organic solvents such as dimethyl formamide, methyl acetate and/or dimethyl sulfoxide along with the material to be encapsulated. The PRINT molding device was employed to fabricate the nanoparticles which later were harvested with the help of polyethylene terephthalate sheet [43]. It produces the particles of different shapes and a wide size range depending upon the size of the molding cavities [44].

The low encapsulation efficiency due to the leakage of the drugs from the inner core during second step, batch variability and large time consumption are the common problems associated with the conventional two step methods [45]. These constraints can be overcome by designing the simple method that utilized the single step approach and also provide better control on the content uniformity, reproducibility and other characteristics of the system. The method involves the mixing of two different solutions containing the polymer and lipid that self-assembled to form the particles with the core shell hybrid structure [46]. The polymer is dissolved in an appropriate organic solvent while the lipid solution is prepared in the water that may utilize the small fraction of organic solvent as solubilizing agent. The solution containing polymer is added to the lipid phase where the polymer precipitate to formed the nanoparticles and the lipid is self-assembled at the surface to form the hybrid nanoparticles. Single-step preparation is usually achieved by nanoprecipitation, emulsification-solvent evaporation and solvent diffusion methods. These methods and their appropriate modifica-

Emulsification solvent evaporation method is the most commonly employed single step approach for the fabrication of hybrid nanocarriers. The single emulsification solvent evaporation [47] and double emulsification solvent evaporation (DESE) techniques are employed depending upon the nature and solubility of encapsulating drug. In the ESE method, the oil phase is formed by dissolving the polymer and the drug in the water immiscible organic solvent.

core shell hybrid nanoparticles were collected after the completion [17].

**2.3. Single-step preparation methods**

58 Advanced Technology for Delivering Therapeutics

tions are discussed here.

*2.3.1. Emulsification solvent evaporation method*

This method is also known as salting out method. It is a well known method for fabrication of hybrid nanoparticles of size less than 100 nm. This method employs two miscible solvents with different solubilizing capacity for the polymer. First, the polymer core is formed by solubilizing in solvent of greater solubility designated as good solvent which is then added to less soluble solvent designated as poor solvent. The two solutions are mixed by dropwise addition, stirring or sonication. Good solvent being miscible with poor solvent diffuses into later, leaving behind the core nanoparticles due to the precipitation of the polymer [19].

The core forming polymer and lipophilic drug are solubilized in a water-miscible organic solvent like acetone, acetonitrile or ethanol [57]. The lipids, inorganic salts or silica are dispersed in water with moderate heating (~60–75°C) and/or addition of hydroalcoholic mixtures for proper dispersion of the lipids.

The hydrophilic drugs are added to the aqueous phase containing dispersed lipids [58]. The polymer containing organic phase is then added dropwise to lipid dispersion with continuous stirring to precipitate the polymer into nanoparticles. The monodispersed hybrid nanoparticles are collected after suitable application of vortexing, homogenization or ultrasonication [55, 59]. Concurrent to the precipitation process, the self-assembly of lipid molecules around the polymer molecules occurs due to the hydrophobic interactions. The polymer core captures the hydrophobic tails of lipid while the heads are facing toward the aqueous phase [17, 60]. Continuous stirring of dispersion for several hours is helpful in uniform lipid coating of hybrid nanoparticles and to ensure the complete removal of organic solvent [55]. Rotary evaporator may also be helpful for the removal of organic solvents [58].

The literature suggests 10% ethanolic solution is employed for solubilization of lipids and PEG may enhance the stability of hybrid nanoparticles [61]. According to the study of Ling et al. [58], dextran sulfate and lecithin/PEG-PLGA hybrid nanoparticles can entrap higher amounts of hydrophilic moiety, the vincristine.

Wang et al. [62] developed PLGA/TPGS-lecithin hybrid nanoparticles using a modified nanoprecipitation method. The PLGA was dissolved in acetone while lipids were dispersed in either aqueous or 4% ethanolic aqueous solution. An inverse-phase nanoprecipitation method (i.e. aqueous phase was added dropwise into organic phase consisting of acetone, the PLGA and the paclitaxel). Initially, the formation of hybrid nanoparticles was slow due to the higher proportion of organic phase in the mixture. Continuous stirring and addition of water boosted the diffusion which leads to solidification of the hybrid nanoparticles. A stable hybrid nanoparticle formulation with low value of PDI (~0.1) was observed at 5:1 aqueous to organic phase ratio [62].

## *2.3.3. Sonication*

Sonication is a fast technique for the fabrication of hybrid nanoparticles which utilizes ultrasonic waves rather than vortexing, solvent evaporation or heating. In this method, the two solutions designated as organic and aqueous phases lead to formation of inner core (polymer) and outer shell or coating materials (lipids), respectively. The sonication has been employed by Fang et al. [63] for the fabrication of hybrid nanoparticles of lecithin-PEG and PLGA by using this approach. The PLGA was dissolved in acetonitrile while the lecithin and the PEG were added in 4% ethanol solution. The former solution was carefully pipetted into the hydro alcoholic solution (aqueous to organic ratio was kept as 10:1). The hybrid nanoparticles were produced as this 'cocktail' mixture was placed in sonicator bath for five minutes at a frequency of 42 kHz and a power of 100 W. The main advantage of this technique is the formation of stable hybrid nanoparticles with short processing time and production rate is 20 times than other processes [63].

The sonication technique has been employed for PLGA and docetaxel hybrid nanoparticles by Liu et al. [64]. In another study, Mandal et al. [65] developed erlotinib loaded hybrid nanoparticles of PCL in which erlotinib and PCL were dissolved in acetone and added to the aqueous phase containing lipids. Hybrid nanoparticles were produced after sonication for 10 minutes at a frequency of 67 kHz and a power of 200 W [65].

A unique method using the combination of modified nanoprecipitation and sonication methods is presented for the fabrication of hybrid nanoparticles. In this method, the lipids melt was mixed with ethanolic solution of Elacridar, a chemosensitizer, and placed in vacuum oven until complete removal of solvents. The doxorubicin being hydrophilic drug was added in water with surfactant (Pluronic-F68) and heated (72–74°C). The drug and surfactant dispersion was mixed with Elacridar-lipid mixture. The whole mixture was stirred for 10 min and then ultrasonicated for two cycles of three minutes. It produced submicron sized lipid emulsion which was dispersed in 4–9 times higher volume of cold water (maintained 4°C) which leads to the formation of hybrid nanoparticles [66, 67].
