2.1. solid lipid nanoparticles (slns) -...

c h a p t e r - 2 SOLID LIPID NANOPARTICLES: A REVIEW

2.1. Solid lipid nanoparticles (SLNs)

SLNs (Figure 3) were developed in mid 1980s as an alternative system to the existing

traditional carriers (emulsions, liposomes, microparticles and their polymeric

counterparts) when Speiser prepared the first micro and nanoparticles (named

nanopellets) made up of solid lipids for oral administration (Speiser, 1986). SLNs avoid

some of their major disadvantages like cytotoxicity of polymers and the lack of a

suitable large scale production method for polymeric nanoparticles (Smith and

Hunneyball, 1986). SLNs are colloidal carriers made up of lipids that remain solid at

room temperature and body temperature and also offer unique properties such as small

size (50-500 nm), large surface area, high drug loading and the interaction of phases at

the interfaces, and are attractive for their potential to improve performance of

pharmaceuticals, neutraceuticals and other materials (Cavalli et al., 1993). Moreover,

SLN are less toxic than other nanoparticulate systems because of their biodegradable

and biocompatible nature. SLN are capable of encapsulating hydrophobic and

hydrophilic drugs, and they also provide protection against chemical, photochemical or

oxidative degradation of drugs, as well as the possibility of a sustained release of the

incorporated drugs (Estella-Hermoso de et al., 2009).

Figure 3: Schematic representation of solid lipid nanoparticles dispersion

stabilized with surfactant molecules showing entrapment of drug

Some other properties of lipid nanoparticles are- the drug pseudo-zero order kinetics,

the prolonged/sustained/controlled release obtained in vitro for drugs incorporated in

SLN (depending upon the surface properties), their rapid uptake (internalization) by cell

lines (5-10 min), and also the possibility of preparation of stealth SLN by using PEG

molecules so as to avoid the Reticuloendothelial system (RES) (Manjunath, 2005).

10JAMIA HAMDARD

Moreover, the possibility of loading drugs with differing physico-chemical and

pharmacological properties, no requirement of specialized instruments/apparatuses,

preparation without the use of organic solvents (in some methods like self-

emulsification and high pressure homogenization etc.) make SLNs a highly versatile

delivery system (Gasco, 2007). Altering surface characteristics by coating SLN with

hydrophilic molecules improves plasma stability and biodistribution, and subsequent

bioavailability of drugs entrapped (Bocca et al., 1998; Cavalli et al., 1999; Heiati et al.,

1998).

2.2. Advantages of SLNs

SLNs combine the advantages and avoid the drawbacks of several colloidal carriers of

its class. The advantages of SLNs over other carriers are given in Figure 4 (Muller et

al., 1995; Muhlen et al., 1998).

2.3. General excipients used for SLN production

The general ingredients used for preparation of SLNs are solid lipid(s) with relatively

low melting points (solid at room and body temperature), emulsifier(s) and water. An

overview of excipients, which are commonly used for SLNs are listed here with few

examples.

2.3.1. Lipids

• Triacylglycerols: Tricaprin, Trilaurin, Trimyristin, Tripalmitin, Tristearin, etc.

• Hard fats: Witepsol™ (W/H 35, H42, E85)

• Acylglycerols: Glyceryl behenate (Gelucire 50/02, 50/13, 44/14), Glycerol

monostearate (Imwitor 900™), Glycerol behenate tribehenate (Compritol 888

ATO™), Glycerol Palmitostearate (Precirol ATO 5™), Cutina CBS, Glyceryl

tripalmitate (Dynasan® 116), Glyceryl trimyristin (Dynasan® 114), Cetyl

palmitate, Glyceryl tristearin (Dynasan 118), etc.

• Fatty acids: Stearic acid, Palmitic acid, Decanoic acid, Behenic acid, etc.

• Waxes: Cetyl palmitate

• Others: Hydrogenated coco-glycerides, Softisan 154

c h a p t e r - 2 SOLID LIPID NANOPARTICLES: A REVIEW

11JAMIA HAMDARD

CHAPTER - 2 SOL.ID LI^ID NANOPARTICLES: A REVIEW

Figure 4: Advantages of Solid Lipid Nanoparticles over other conventional and novel drug delivery systems

JA M IA HAM D^ RD12

2.3.2. Surfactant/ Co-emulsifier

• Phospholipids: Soybean Lecithin, Egg lecithin, Phosphatidylcholine

• Ethylene/propylene oxide copolymer: Polaxomer 188, 182, 407, 908

• Sorbitan ethylene/propylene oxide copolymer: Polysorbate 20, 60, 80

• Alkylaryl polyether alcohol polymer: Tyloxapol

• Bile salts: Sodium cholate, glycocholate, taurocholate, taurodeoxycholate

• Alcohols/acids: Butanol, Butyric acid, Ethanol, Poly vinyl alcohol

• Others: Hydroxypropyl distarch, Tegocare, Epikuron 200, etc.

2.4. Production methods for SLNs

2.4.1. High pressure homogenization technique

In this technique, lipids are forced through a narrow gap (few micron ranges) with high

pressure (100-200 bars). Disruption of particles into submicron range occurs due to the

shear stress and cavitation (due to sudden decrease in pressure) force. There are two

approaches - hot and cold homogenization techniques. For both the techniques, a

common preparatory step involves the drug incorporation into the bulk lipid by

dissolving/ dispersing/ solubilizing the drug in the lipid being melted at approximately

5-10°C above the melting point (Muller and Runge, 1998).

2.4.1.1. Hot homogenization technique

The melted lipid containing drug is dispersed in the aqueous surfactant solution of

identical temperature under continuous stirring by high shear device. This pre-emulsion

is homogenized by using a piston gap homogenizer and the produced hot oil-in-water

nanoemulsion is cooled down to room temperature. The lipid recrystallizes and leads to

formation of SLNs (Jenning et al., 2000; Sznitowska et al., 2001; Olbrich et al., 2002;

Lim and Kim, 2002; Jee et al., 2006; Kumar et al., 2007; Liu et al., 2007; Attama and

Muller-Goymann, 2007).

Advantages

• Preparation without the use of organic solvent

• Feasibility of large scale production

• High temperature results in lower particle size due to the decreased viscosity of

the inner phase

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13JAMIA HAMDARD

• Suitable for drugs showing some temperature sensitivity because the exposure to

an increased temperature is relatively short

Disadvantages

• Poor technique for hydrophilic drugs

• Drug and carrier degradation is more at high temperature

• Due to small particle size and presence of emulsifier, lipid crystallization may be

highly retarded and the sample remains as super-cooled melt for several months

• Burst release of drugs from SLNs because during heating the drug partitions into

aqueous phase and when cooled, most of drug particles remained at the outer

layer of the SLNs

• Increasing the homogenization pressure or the number of cycles often results in

an increase of the particle size due to particle coalescence which occurs as a result

of the high kinetic energy of the particles

2.4.I.2. Cold homogenization technique

In comparison to above, the cold homogenization technique is carried out with the solid

lipid and represents, therefore, a high pressure milling of a suspension. The drug

containing lipid melt is cooled; the solid lipid ground to lipid microparticles

(approximately 50-100 mm) and these lipid microparticles are dispersed in a cold

surfactant solution yielding a pre-suspension. Then this pre-suspension is homogenized

at or below room temperature, the cavitation forces are strong enough to break the lipid

microparticles directly to solid lipid nanoparticles (Siekmann and Westesen, 1994;

Miglietta et al., 2000).

Advantages

• Avoids temperature induced drug degradation and complexity of the

crystallization step

• Minimizes the melting of lipid and therefore minimizing loss of hydrophilic drug

to aqueous phase

• Water from aqueous phase can be replaced with other media (e.g. oil or PEG 600)

with low solubility for the drug

• Suitable for highly temperature-sensitive compounds

CHAPTE R - 2 SOL.ID LI^ID NANOP.^R ^ LES: A REVIEW

14JAMIA HAMDARD

Disadvantages

• In comparison to hot homogenization, particle size and polydispersity index are

more

• Minimizes the thermal exposure of drug, but does not avoid it completely

• High pressure homogenization increases the temperature of the sample (10-20°C

for each cycle)

2.4.2. High shear homogenization (Ultrasonication) technique

SLNs were also developed by high stirring or ultrasonication, which are known as

dispersing techniques. Ultrasonication is based on the mechanism of cavitation. The

step-wise methodology is given in Figure 5 (Speiser, 1990; Eldem et al., 1991; Mei et

al., 2003; Krzic et al., 2001; Song and Liu, 2005).

Advantages

• Widespread and easy to handle

• Production of SLNs without organic solvents

Disadvantages

• Extra step of filtration in order to remove impurity materials (e. g. metal)

• Broader particle size distribution ranging into micrometre range

• Bigger size lead physical instabilities likes particle growth upon storage

2.4.3. Solvent emulsification-evaporation technique

The solvent emulsification-evaporation technique is a widespread procedure, being

firstly used for SLN preparation (precipitation in o/w emulsion) by Sjostrom and

Bergenstahl (Sjostrom and Bergenstahl, 1992). Lipid and drug are dissolved in a water

immiscible organic solvent by ultrasound and emulsified in an aqueous phase

containing surfactant/ emulsifier using magnetic stirrer (1000 rpm). The organic solvent

was evaporated by mechanical stirring at room temperature and reduced pressure (e.g.

rotary evaporator) leaving lipid precipitates of SLNs (Lemos-Senna et al., 1998; Cortesi

et al 2002; Shahgaldian et al 2003).

Advantages

• Very small particle size

• Suitable for the incorporation of highly thermo-labile drugs

• Avoids temperature-induced drug degradation

CHAPTE R - 2 SOL.ID LI^ID NANOP^ RTI^LES: A REVIEW

15JAMIA HAMDARD

CHAPTER - 2 SOL.ID LI^ID NAN0PA RTIC:LES: A RE VIEW

Figure 5: Step wise procedure for ultrasonication and high shear homogenization

techniques

Disadvantages

• Toxicity of solvent in final product

• Extra step of filtration and evaporation

2.4.4. Solvent emulsification-diffusion technique

The difference in solvent evaporation and solvent diffusion technique is that instead of

taking any solvent in solvent evaporation, partially water miscible solvents (e.g. benzyl

alcohol, butyl lactate, ethyl acetate, isopropyl acetate, methyl acetate) are used in

diffusion technique and this technique can be carried out either in aqueous phase or in

oil (Hu et al., 2005; Pandey et al., 2005; Quintanar-Guerrero et al., 2005).

Water miscible solvent and water are mutually saturated to ensure the thermodynamic

equilibrium of both liquids (Trotta et al., 2003).

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Advantages

• Efficient and versatile

• Easy implementation and scaling up (no need of high energy sources)

• High reproducibility and narrow size distribution

• Less physical stress (short exposure to high temperatures and mechanical

dispersion)

• Suitable for thermo-labile drugs

Disadvantages

• Necessary to dissolve the drug in the melted lipid

• Drug diffusion into aqueous phase leads to low entrapment of drugs in SLN

• Need to clean up and concentrate the SLN dispersion


• Extra step of vacuum distillation or lyophilisation to remove diffused solvent

2.4.5. Microemulsion technique

Gasco and co-workers introduced the concept of microemulsion technique (Gasco,

1993). In this, the drug is partitioned partly in the internal oily/lipidic phase and partly

at the interface between internal and continuous phase, depending on their lipophilicity.

Due to the dilution step; achievable lipid contents are considerably lower compared

with the homogenization based formulations (Mehnert and Mader, 2001).

2.4.5.1. SLNs from warm microemulsion

This is a modified method of microemulsion technique. Warm microemulsions are

prepared at temperature ranging from 60-80°C by using melted lipid and are

subsequently dispersed in cold water. Nanodropletes obtained using this procedure

becomes SLNs, which are successively washed by tangential flow filtration (Gasco,

1997; Mao et al., 2003; Bondi et al., 2010).

Advantages

• Thermodynamically stable system

• Easy to prepare and require no significant energy contribution during

preparation

• High temperature gradients facilitate rapid lipid crystallization and prevent

aggregation


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• Low mechanical energy input

• Theoretical stability

Disadvantages

• Extremely sensitive to change

• Labour intensive formulation work

• Low nanoparticle concentrations

1.4.6. Double emulsion technique

This method is based on the solvent emulsification-evaporation technique in which the

drug is encapsulated with a stabilizer to prevent drug partitioning to external water

phase during solvent evaporation in the external water phase of w/o/w double emulsion.

Primary emulsion was formed by dissolving the hydrophilic drug in aqueous solution,

followed by emulsification in melted lipid containing surfactant/stabilizer. The primary

emulsion then dispersed in aqueous phase containing hydrophilic emulsifier forming the

double emulsion (Jaganathan et al., 2005).

1.4.6.1. Reverse micelle-double emulsion technique

Due to the rapid migration in double emulsion technique, the average particle size

comes in the micrometer range (Cortesi et al., 2002), and the drug loading capacity of

the carriers becomes low and therefore, drug loss into the external aqueous phase. This

led to a modified method of double emulsion technique reported by Liu and co-workers

(2007), in which sodium cholate-phosphatidylcholine based mixed micelles, were used.

Here, the drug is encapsulated with a stabilizer to prevent the partitioning of drug in to

external water phase during solvent evaporation in the external water phase of w/o/w

double emulsion (Liu et al., 2007).

Advantages

• Possibility of lowering the fat content

• Entrapping (and releasing) therapeutic, nutritional or odourous compounds in

the internal water droplets

• Separation of incompatible substances

Disadvantages

• Formation of high percentage of microparticles

CHAPTE R - 2 SOL.ID LI^ID NANOP.^R ^ LES: A RE VIEW

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1.4.7. Melting dispersion method (Hot melt encapsulation method)

The drug and solid lipid were melted together in organic solvent forming oily phase and

water phase containing surfactant/emulsifier was also heated at the same temperature.

The emulsion was formed by adding the oil phase in to a small volume of water phase

with stirring at higher rpm for few hours. It was then cooled down to room temperature

to give SLNs. The last step was same as solvent emulsification evaporation method

except the evaporation of solvent (Reithmeier et al., 2001a & 2000b; Cortesi et al.,

2002).

Advantages

• Reproducibility was more than ultra-sonication method

Disadvantages

• Reproducibility was less than that of solvent emulsification-evaporation method


• Not suitable for thermo-labile drugs

1.4.8. Solvent injection/solvent displacement technique

It is a novel approach based on lipid precipitation from the dissolved lipid in solution.

The solid lipid was dissolved in water-miscible solvent forming the organic phase. This

organic phase was rapidly injected through an injection needle in to an aqueous phase

(with or without surfactant) with continuous stirring. The resulted dispersion was then

filtered with a filter paper in order to remove any excess lipid. The presence of

emulsifier within the aqueous phase helps to produce lipid droplets at the site of

injection and stabilize SLN until solvent diffusion was complete by reducing the surface

tension between water and solvent (Schubert and Muller-Goymann, 2003; Shah and

Pathak, 2010).

Advantages

• Use of pharmacologically acceptable organic solvent

• Easy handling

• Fast production process

• No use of technically sophisticated equipment


• Small and uniform particle size distribution


19JAMIA HAMDARD

• Avoidance of any thermal stress

• Avoids temperature induced drug degradation

Disadvantages

• Extra step of filtration


1.4.9. Precipitation technique

The drug and lipid are dissolved in an organic solvent and the solution will be

emulsified in an aqueous phase with continuous stirring. The organic solvent is

evaporated and the lipid containing drug will be precipitated out forming nanoparticles.

The obtained SLNs are washed with distilled water. SLNs obtained by this technique

are comparable to the production of nanoparticles by solvent evaporation method

(Siekmann and Westesen, 1996).

Advantages

• No use of sophisticated instrument

• Easy to prepare

• Suitable for thermo-labile substances

Disadvantages

• Bigger particle size

• Use of organic solvents

• Problem of solvent evaporation in scaling up process

1.4.10. Complex coacervation technique

Solution of polymeric stabilizer was prepared by heating the polymer in water and it

was then cooled at room temperature. Lipid was dispersed in polymeric stabilizer stock

solution and the mixture was heated under stirring just above the Kraft point of lipid to

obtain a clear solution. Drug solution was added to the warm aqueous lipid solution. An

acidifying solution (coacervating solution) was added drop wise until the required pH

was reached. The obtained suspension was immediately cooled in a water bath with

continuous stirring till temperature reaches to 15°C (Battaglia et al., 2011; Francesco

and Francesco, 2011; Hao et al., 2012).


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Advantages

• Solvent free technique

• No need of complex machines

• Avoids high temperatures to melt the lipid matrix

• Easy to prepare

• Suitable for thermo-labile substances

• Applied successfully to hydrophobic ion pairs of hydrophilic drugs

Disadvantages

• Difficulties in scaling-up

• Use of large amount of organic solvent


1.4.11. Phase inversion technique

This method was introduced by Shinoda and Saito (1968 & 1969), using poly-

ethoxylated non-ionic surfactants to undergo a phase inversion following a variation of

temperature (Shinoda and Saito, 1968; Shinoda and Saito, 1969). The phase inversion

occurs, when the relative affinity of the surfactant for the different phases is changed

and controlled by the temperature at which the hydrophilic and lipophilic properties of a

nonionic surfactant just balance. Temperatures at which the nonionic surfactants show

very close affinities for the two phases, the ternary system shows an ultra-low

interfacial tension and curvature, typically creating nanoscaled systems. This is

followed by rapid cooling at phase inversion temperature or by a sudden dilution in

water/oil, which will immediately generate nanoparticles (Anton et al., 2007; Anton et

al., 2008; Montenegro et al., 2011).

Advantages

• Easy and few steps for preparation

• No need of complex machines

Disadvantages

• Difficulties in scaling-up


• Bigger particle size


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21JAMIA HAMDARD

1.4.12. Membrane contactor technique

Lipid particles are formed by this novel technique using microchannels, microfluidic

arrays or microreactors (Sugiura et al., 2000; Zhang et al., 2008a & b; Czermak et al.,

2010). This method is also known as membrane emulsification/liquid flow-focusing and

gas displacing technique.

In this technique, the liquid phase containing lipid is pressed through the membrane

pores at a temperature above the melting point of lipid forming the small droplets. A

solvent phase containing dissolved lipid in a water-miscible solvent is injected into the

inner capillary, and the aqueous phase containing surfactant/emulsifier is injected

through the outer capillary simultaneously. When both the phases come in contact with

each other in the outer capillary, the solvent phase diffuses rapidly into the aqueous

phase, resulting in the formation of SLNs due to super-saturation of lipid. Both the

phases were placed in the thermostated bath to maintain the required temperature and

nitrogen is used for creating the pressure for the liquid phase (Charcosset et al., 2005;

El-Harati et al., 2006).

Advantages

• Scaling up ability

• Small diameter and narrow size distribution

• Effective mass transfer efficiency and precise-controlled operation conditions

• Continuous production process

Disadvantages

• High speed, high temperature/pressure, toxicological solvents-challenging

parameters

• Toxicity of solvents in final product


2.4.14. Supercritical flu id technology

This is a novel technique which is applied for the production of SLNs (Kaiser et al.,

2001; Elvassore et al., 2003; Date and Patravale, 2004; Won et al., 2005; Chen et al.,

2006; Chattopadhyay et al., 2007; Almeida and Souto, 2007; Pasquali et al., 2008;

Yasuji et al., 2008; Salmaso et al., 2009). A substance whose temperature and pressure

are simultaneously higher than at the critical point is referred to as a supercritical fluid


22JAMIA HAMDARD

(SCF). Carbon dioxide (99.99%) is the good choice as a solvent for this method. The

advantages of using CO2 for SCF technique are: safe, easily accessible critical point

[31.5°C, 75.8 bar), not causes oxidation of drug, leaves no traces behind after the

process, inexpensive, non-inflammable, environmentally acceptable and easy to recycle

or to dispose-off. Several modifications of this technique are used for production of

nanoparticles like rapid expansion of supercritical solution (RESS), particles from gas

saturated solution (PGSS), gas/supercritical antisolvent (GAS/SAS), aerosol solvent

extraction solvent (ASES), solution enhanced dispersion by supercritical fluid (SEDS),

supercritical fluid extraction of emulsions (SFEE) (Jung and Perrut 2001; Santos et al.,

2002; Jovanovic et al., 2004; He et al, 2004; Bouchard et al., 2007; Chattopadhyay et

al., 2007).

Advantages

• Solvent-less processing

• Single step generation of particles

• Faster method

• Narrow size distribution

• Yielding very small and regularly sized particles

• Particles are obtained as a dry powder, instead of suspensions

• Mild pressure and temperature conditions

• Carbon dioxide solution is the good choice as a solvent for this method


Disadvantages

• Carbon dioxide, which is the only supercritical fluid that is preferentially used in

pharmaceutical processes, is not a good solvent for many Active Pharmaceutical

Ingredients (API)

• Solubilisation limitation

• Scale up problem

• Costlier than other methods

2.4.14.1. Rapid expansion o f supercritical solutions (RESS)

RESS involves saturation of the SCF (CO2) with a solid substrate. Then, the

depressurization of the solution through a heated nozzle into a low pressure vessel

CHAPTE R - 2 SOL.ID LI^ID NANOP^ R ^ LES: A REVIEW

23JAMIA HAMDARD

produces a rapid nucleation of the substrate in the form of very small particles that

are collected from the gaseous stream (Turk et al., 2002; Knez and Weidner, 2003;

Vezzu et al., 2009).

2.4.14.2. Rapid expansion o f a supercritical solution into a liquid solvent (RESOL V)

A variation of the RESS process is the RESOLV that consists of spraying the

supercritical solution into a liquid, often containing a stabilizer. In this manner, it

should be possible to quench particles growth in the precipitator, thus improving the

RESS process performance (Ganapathy et al., 2008; Sane and Limtrakul, 2009).

2.4.14.3. Particles from gas saturated solutions (PGSS)

This technique is used for making particles of materials that absorb supercritical

fluids at high concentrations; taking advantage of the fact that polymers/lipid can be

saturated with carbon dioxide decreasing their melting temperature (Sampaio de

Sousa et al., 2007; Cocero et al., 2009; Sampaio de Sousa et al., 2009; Garc^a-

Gonzalez et al., 2010).

2.4.14.4. Supercritical (fluid) assisted atomization (SAA)

In this process, SCF/CO2 acts as an atomizing medium and the process is based on

the two mechanisms of atomization (Adami et al., 2011):

Pneumatic atomization:, a high-speed fluid flow pushes the solution into the

injector.

Decompressive atomization: The decompressive atomization is caused by the

mixing between CO2 and the solvent, and the efficiency of the atomization is

due to the fast release of CO2.

2.4.14.5. Supercritical anti-solvent (SAS) and gas antisolvent (GAS) micronization

According to its name "supercritical anti-solvent" the SAS technology applies the

supercritical fluid as an anti-solvent for processing solid that are insoluble in SCF.

The only difference between SAS and GAS techniques is the phase in which

precipitation occurs. In case of SAS, the precipitation occurs in liquid phase and it is

a semi-continuous process whereas in GAS, the precipitation occurs in gas phase

and it is a batch process (Chen et al., 2005).


24JAMIA HAMDARD

2.4.14.6. Supercritical flu id extraction o f emulsion (SFEE)

The supercritical fluid extraction of emulsion (SFEE) is a process combining

conventional emulsion techniques with the unique properties of supercritical fluids

to produce tailor-made solid lipid nanoparticles (Chattopadhyay et al., 2007). An

organic solvent is prepared containing dissolved drug and encapsulation material,

which is then mixed with water containing surfactant/ stabilizer forming an o/w

emulsion. This emulsion is sprayed in a vessel continuously purged with

supercritical CO2 , which is used as an anti-solvent and an extraction fluid at the

same time in this process (Chattopadhyay et al., 2004 & 2007).

2.5. Secondary production steps

2.5.1. Sterilization

Sterilization of SLN is an issue in the case of pulmonary or parenteral administration.

There are several procedures to sterilize the SLNs with few advantages as well as some

disadvantages.

2.5.1.1. Autoclaving/Steam sterilization

The result/physical stability of SLNs depends on the temperature condition (121°C for

15 min and 110°C for 15 min) and SLNs composition (type of emulsifier and nature of

drug). Autoclaving is possible for lecithin-stabilized SLNs. The SLN melt during the

autoclaving and recrystallize during the cooling down (Schwarz et al., 1995).

2.5.1.2. Heating/heat sterilization

During heat sterilization chemical composition of drug may also undergo changes and

the incorporated drug leaks out of the carrier, hence it is essential to study the stability

of SLN’s following sterilization.

2.5.1.3. Sterile filtration

SLN dispersions can also be sterilized by filtration if the particle size is less than 200

nm. The sterilization should not change the properties of the SLNs with respect to

physical and chemical stability and the drug release kinetics (Olbrich, 2000).

2.5.1.4. Aseptic production o f SLNs

Sterile SLN’s can be produced in laminar air flow chamber by using aseptic procedures.

Aseptic manufacturing of SLNs includes sterilization of the starting materials or


25J ^ IA HAMDARD

exposure to ethylene oxide gas followed by sterilization of final dispersion by gamma

or e-beam irradiation (Uner and Yener, 2007).

2.5.1.5. y-irradiation

y-irradiation is an alternative method to steam sterilization used for thermo-labile

samples. High energy of the y-rays generates free radicals in all the samples during y-

sterilization. These radicals may either re-combine to form SLNs with no modification

of the sample or it might undergo secondary reactions, which leads to chemical

modifications of the sample (Sculier et al., 1986).

2.5.2. Lyophilization/ Freeze drying

Freeze-drying/ lyophilization/ cryodesiccation, is a dehydration process used to remove

water for the improvement of physical and chemical stability of SLNs. This is based on

freezing the material and then reducing the surrounding pressure to allow the frozen

water in the material to sublimate directly from the solid phase to the gas phase. It can

be divided into three steps: freezing, primary drying and secondary drying (Franks,

1998):

• Two transformations occur during lyophilization, first from aqueous dispersion

to powder and second re-solubilization. The main objectives of lyophilization

are: an elegant lyophilizate, rapid reconstitution time of the suspension,

conservation of the physico-chemical characteristics of the freeze-dried product,

weak residual humidity (<2%), and also good long-term stability of the

formulation (Abdelwahed et al., 2006).

• The process generates various stresses during freezing and drying steps. So,

protectants are usually added to avoid these problems. They decrease the

osmotic activity of water and crystallization and favour the glassy state of the

frozen sample. Typical cryoprotective agents are sorbitol, trehalose, glucose and

polyvinylpyrrolidone (Shulkin et al., 1984; Mobley and Schreier, 1994).

2.5.3. Spray dried SLNs

The cost-effective and alternative to lyophilization, spray-drying technique was

investigated for SLNs. It converts a liquid dispersion of SLNs into a dry system in a

one-step process. The process consists of following steps (Broadhead et al., 1992):

1. Atomization o f the feed into a spray.


26JAMIA HAMDARD

2. Spray-air contact: In the chamber, atomized liquid is brought into contact with

hot gas, resulting in the evaporation of 95% of the water contained in the

droplets in few seconds.

3. Drying o f the spray: Moisture evaporation takes place.

4. Separation: Separation of the dried product from the drying gas.

The lipids having melting point > 70°C are preferred for spray drying process. Elevated

temperature, shear forces and partial melting of the lipid increase the kinetic energy,

which results in particle collision/ aggregation.

2.6. Problems associated with preparation methods

2.6.1. High pressure-induced drug degradation

This problem is associated with the high pressure homogenization technique. The

molecular weight of polymers and the molecular structure are responsible for the drug

degradation. Formation of free radical is responsible for decrease in the molecular

weight of the polymers due to shear stress. Almeida and co-workers (1997) reported

that high pressure homogenization-induced drug degradation will not be a serious

problem for the majority of the drugs (Almeida et al., 1997).

2.6.2. Lipid crystallization

Lipid crystallization is an important point for the performance of the SLN carriers.

Crystalline structure is related to the chemical nature of the lipid, which is a key factor

to decide in determining whether a drug will be expelled or firmly incorporated in the

long-term (Garti and Sato, 1998).

2.6.2.1. Super-cooled melts

Super-cooled melts are usual in SLN systems and represent as emulsions. The lipid

crystallization may not occur although the sample is stored at a temperature below the

melting point of the lipid (Maia et al., 2000). The advantages of SLNs over

nanoemulsions are linked to the solid state of the lipid therefore supercooled melts

should be properly identified and characterized by DSC, NMR and X-ray diffraction

studies. The formation of supercooled melts depends on the crystallization processes.

2.6.2.2. Lipid modifications/polymorphism

The crystallized lipid may be present in several modifications of the crystal lattice.

Lipid molecules have a higher mobility in thermodynamically unstable configurations


27JAMIA HAMDARD

with lower density and ultimately, a higher capability to incorporate guest molecules

(e.g. drugs). During storage, rearrangement of the crystal lattice might occur in favour

of thermo-dynamically stable configurations and this is often connected with expulsion

of the drug molecules (Muller et al., 2000). Thermodynamic stability and lipid packing

density increases with crystal order (supercooled melt < a-modification < P’-

modification < P-modification), on the contrary to the crystallization kinetics (Westesen

et al., 1993).

2.6.2.3. Particle shape

The shape of lipid nanoparticles (platelet form) may significantly differ from a

nanoemulsion (sphere). Lipids have tendency to crystallize in the platelet form; which

are having larger surface areas as compared to spheres; therefore, require higher

amounts of surfactants for stabilization. As a result, much higher amount of drug will be

localized directly on the surface of SLNs, which is in divergence with the general aim

of the SLN systems (drug protection and controlled release due to the incorporation of

the drug in the solid lipid) (Siekmann and Westesen, 1992; Freitas and Muller, 1998).

2.6.2.4. Gelation phenomena

A gelation phenomenon is an irreversible process, in which rapid and unpredictable

transformation of low-viscosity SLN dispersion into a viscous gel occurs. As a result,

the colloidal particle size is lost. In this process increase in particle surface takes place

due to the formation of platelets in P-modification, so that the surfactant molecules no

longer provide coverage of the new surfaces and therefore, particle aggregation is seen

(Siekmann and Westesen, 1994).

2.6.3. Co-existence o f several colloidal species

There exists several colloidal species in SLN dispersion. Stabilizing agents are not

localized exclusively on the lipid surface, but also in the aqueous phase. Therefore,

micelle forming surfactant molecules will be present in three different forms- as

micelle, on the lipid surface and as surfactant monomer. Dynamic phenomena as well

as the presence of several colloidal species are very important to describe the structure

of colloidal lipid dispersions. Drug stabilization is a very challenging task for colloidal

drug carriers, because of the very high surface area and the short diffusion pathways.

CHAPTE R - 2 SO£.ID LI^ID NANOP^ RTI^LES: A REVIEW

28J ^ IA HAMDARD

2.7. Characterization of SLNs

An adequate characterization of the SLN’s is necessary for the control of the quality of

the product. Several parameters have to be considered which have direct impact on the

stability and release kinetics.

2.7.1. Entrapment efficiency and drug loading

Entrapment efficiency describes the efficiency of the preparation method to incorporate

drug into the carrier system. A very important point to judge the suitability of a drug

carrier system is its loading capacity. The loading capacity is generally expressed in

percent related to the lipid phase (matrix lipid: drug). In addition, the amount of drug

entrapped also determines the performance of the drug delivery system since it

influences the rate and extent of drug release from the system. Both drug loading and

entrapment efficiency depends on the physicochemical properties and the interactions

between the drug, carrier matrix and the surrounding medium (Muller et al., 2002;

Uner, 2006).

Entrapment efficiency is determined, as the ratio between actual and theoretical

loading, and drug loading capacity is calculated as drug analyzed in the nanoparticles

versus the total amount of the drug and the excipients added during preparation,

according to the following equations (Subedi et al., 2009):

EE (%) = Ws/ Wrotai X 100

DL (%) = Ws/ WLipd X 100

Where, Ws- amount of drug loaded in the SLNs; Wtotat total drug amount in AD-SLNs

dispersion; Wlipid- weight of the vehicle.

2.7.2. Particle size and distribution

A particle size and distribution analysis is a measurement designed to determine and

report information about the size and range of a set of particles representative of a

material. Particle size and distribution analysis of a sample can be performed using a

variety of techniques.

2.7.2.1. Photon correlation spectroscopy/ dynamic light scattering/ quasi-elastic light

scattering

The nano-dispersed system is placed into the optical path of a Laser. The Laser is then

scattered upon interacting with the particles in the suspension, which are moving by


29JAMIA HAMDARD

brownian motion. The accepted way to report results from DLS is on an Intensity basis

using the Z-Average along with the polydispersity index (PDI). The PDI is an indicator

of the “broadness” of the particle size distribution (Tscharnuter, 2000).

2.7.2.2. Laser diffraction

Laser diffraction, also known as static light scattering, has become one of the most

widely used particle sizing distribution techniques across various industries. Samples

can be analysed on either a liquid suspension or dry dispersion basis (Shekunov et al.,

2007). The final result is reported on an equivalent spherical diameter volume basis.

2.7.2.3. Light obscuration

Light Obscuration, also referred to as Photozone and Single Particle Optical Sensing

(SPOS), is an analytical technique of high resolution used to obtain an overall size

distribution, when operated with proper technique. The results can be reported on a

Number and/or Volume weighted basis, using the classic assumption of Equivalent

Spherical Diameter (Shekunov et al., 2006).

2.7.2.4. Aerodynamic technique

Aerodynamic particle size measurement has a unique and focused application for

inhalation delivery system (Pandey and Khuller, 2005; Grenha et al., 2008; Ru et al.,

2009; Yang et al., 2012). This technique evaluates how the particle (either solid or

droplet) behaves as it settles through air and reports the diameter of a sphere with the

same aerodynamic drag properties.

2.7.3. Surface charge

The surface charge of a dispersed system is best described by measuring the zeta (Z)

potential of the system. This parameter is a useful predictor of the storage stability of

colloidal dispersions. A minimum Z potential of ± 30 mV is considered the benchmark

to obtain a physically stable system. Particle aggregation is less likely to occur for

charged particles (high zeta potential) due to electric repulsion (Heurtault et al., 2003).

Zeta potential helps in designing particles with reduced reticuloendothelial uptake. In

order to divert SLNs away from the RES, the surface of the particles should be

hydrophilic and free from charge.


30JAMIA HAMDARD

2.7.4. Particle morphology and ultra-structure

Particle size results are often validated by Transmission electron microscopy (TEM),

which provides direct information on SLN morphology and ultra-structure. In this, an

electron beam is focused and directed through a sample by several magnetic lenses,

with part of the beam adsorbed or scattered by the sample while the remaining is

transmitted. The transmitted electron beam is magnified and then projected onto a

screen to generate an image of the specimen. The fraction of electrons transmitted

depends on sample density and thickness (typically < 100 nm) (Bunjes, 2005; Bunjes

and Siekmann, 2006; Herrera and Sakulchaicharoen, 2009).

SEM uses a focused electron beam to generate a variety of signals (i.e., backscattered,

or secondary electrons) at the surface of solid specimens. The signals derived from

electron sample interactions reveal information about the sample including morphology,

chemical composition, and potentially crystalline structure (Misra et al., 2004;

Hatziantoniou et al., 2007; Urban-Morlan et al., 2010).

2.7.5. Atomic force microscopy (AFM)

AFM is a new technique to visualize SLN ultrastructure that avoids the high vacuum

required in TEM, and the need for the sample to be conductive as required in SEM. An

atomic force microscope is excellent for visualizing particles with sizes ranging from 1

nm to 10 ^m. AFM provides a three-dimensional surface profile (Zur Muhlen et al.,

1996). AFM allowed observation of SLNs in a hydrated state similar to that of SLN

suspensions while SEM led to observation of SLNs in a less aggregated state (Dubes et

al., 2003).

2.7.6. Polymorphic state (crystalline or amorphous) o f SLNs

Differential scanning colorimetry (DSC), X-ray diffraction (XRD), small-angle x-ray

scattering/diffraction (SAXS/SAXD), and wide-angle x-ray scattering/diffraction

(WAXS/WAXD) have been used in concert with particle size and electron microscopy

techniques to confirm the polymorphic state and properties of SLNs in lipid dispersions

(Muller et al., 2000; Bunjes et al., 2003; Schubert et al., 2006).

2.8. In vitro assessment of drug release

In vitro release studies are generally performed to accomplish the objectives like

(D’Souza and DeLuca, 2006):

CHAPTE R - 2 SO£.ID LI^ID NANOP^ RTI^LES: A REVIEW

31J ^ IA HAMDARD

• Indirect measurement of drug availability, especially in preliminary stages of

product development

• Quality control to support batch release and to comply with specifications of

batches proven to be clinically and biologically effective

• Assess formulation factors and manufacturing methods that are likely to

influence bioavailability

• Substantiation of label claim of the product

• As a compendial requirement

In vitro release study tells about basic information regarding the structure (e.g.,

porosity) and behavior of the formulation at molecular level, possible drug-excipient

interactions and about the factors influencing the rate and mechanism of drug release

(Abdel-Mottaleb and Lamprecht, 2011). Such information facilitates a scientific and

predictive approach to the design and development of sustained delivery systems with

desirable properties. Various methods have been reported to study the in vitro drug

release from colloids and divided into four broad categories:

2.8.1. Membrane diffusion techniques

Membrane diffusion techniques are considered the best for assessing the in vitro drug

release from nanoparticles. In these techniques the colloidal carrier is separated from

the sink release medium by dialysis membrane, which is permeable to the drug.

2.8.1.1. Dialysis membrane method

A suspension of the particles is introduced into the dialysis bag that is sealed and placed

in a vessel containing buffer. Drug diffusion from the dialysis bag into the outer sink

may be increased by agitating the vessel contents, thereby minimizing unstirred water

layer effects. Volume of media inside the dialysis membrane containing the particles is

at least 6-10 folds less than that of the outer bulk, providing a driving force for drug

transport to the outside and maintaining sink conditions (D’Souza and DeLuca, 2006).

The method is widely utilized for the in vitro drug release testing and satisfactory

results are obtained as the drug release experiments can be viewed in most of the cases

as a partitioning phenomenon (Lamprecht et al., 2002; Nounou et al., 2006; Mukherjee

et al., 2007).


32JAMIA HAMDARD

2.8.1.2. Reverse dialysis method

A reverse dialysis technique has been proposed to avoid this criticism where the

colloidal particles suspension can be directly diluted with the release medium and

dialysis bags containing the release medium are then added and analyzed at different

time intervals for drug content (Leavy and Benita, 1990).

2.9. Strategies to enhance anticancer activity based on SLN-based drug delivery

systems

So far, a number of cytotoxic drugs have been successfully incorporated into SLNs.

Preclinical studies involving cell culture or animal models have shown promising

effects involving the improvement in the anticancer activity of cytotoxic drugs, along

with minimizing their severe side effects. The mechanisms for delivering drugs to

tumors by SLNs include passive targeting, active targeting, long circulating and a

combination of strategies (Arias et al., 2011).

2.9.1. Tumor-specific targeting based on SLNdrug delivery systems

Most cytotoxic drugs exhibit narrow therapeutic windows due to a lack of tumor

selectivity. Sometimes, the effective dose is close to the maximum tolerated dose for

the cytotoxic agent. Therefore, the development of drug delivery systems able to target

the tumor site is becoming a real challenge. Recently, SLNs have been shown to exhibit

great potential for tumor targeting. This is based on two different principles, namely,

passive targeting and active targeting (Danhier et al., 2010; Irache et al., 2011).

2.9.1.1. Passive tumor targeting

Tumor vessels are markedly different from normal tissues in their microscopic

anatomical architecture. For instance, the blood vessels in the tumor are irregular,

dilated, leaky or defective, and the endothelial cells are out of order with large

fenestrations. Also, the perivascular cells and the basement membrane are frequently

absent or abnormal in the vascular wall, and the lymphatic network is always impaired

(Iyer et al., 2006). These unique tumor abnormities lead to increased accumulation of

high molecular weight compounds or nanocarriers (such as liposomes, niosomes, and

SLNs) in the tumor tissue, which is called the EPR effect (Figure 6). This effect allows

SLNs to be able to distinguish between normal tissue and tumor tissue. The anti-tumor

effect of SLN is usually better than that obtained with solution preparations.


33JAMIA HAMDARD

Tocotrienol-loaded lipid carriers have been developed to increase their anti-proliferative

effects against neoplastic + SA mammary epithelial cells (Ali et al, 2010). An improved

cytotoxicity against A549 cells has also been obtained by encapsulating docetaxel in

lipid carriers (LCs) (Liu et al., 2011). The inhibition rate of docetaxel LCs was 90.36%,

while that of commercial Duopafei® was only 42.74%, indicating that docetaxel LCs

are more effective inhibitors of tumor growth. However, the pore size of tumor vessels

usually ranges from 100 to 780 nm (Yuan et al., 1995) [40], which sets the upper size

limit for the extravasated LNs (<200 nm). The effects of particle size on the EPR effect

need further study.


Figure 6: Schematic depiction of nanoparticle extravasation into tumor tissue via

the enhanced permeability and retention (EPR) effect

Due to their smaller particle size, SLNs are always concentrated in tissues with a rich

reticuloendothelial system, such as liver, spleen and lymph nodes. This passive

targeting effect is favorable if the tumor is present in the reticuloendothelial system.

Moreover, the toxicity could be reduced because fewer drugs are distributed to other

organs. For example, doxorubicin, a broad-spectrum anti-tumor drug with a therapeutic

effect on lymph tumor, often produces serious cardiac toxicity. Three hours after i.v.

administration of doxorubicin SLNs, the drug concentration was higher in the spleen

and lower in the heart, compared with free drug solution. This indicated that

doxorubicin SLNs exhibit lower drug accumulation in unexpected organs via the

passive targeting effect (Zara et al., 1999).

Amazingly, several studies have found that cytotoxic drugs can be detected in the brain

when loaded into SLNs, an indication that they have bypassed the blood brain barrier

34JAMIA HAMDARD

(BBB) (Yang et al., 1999). This shows that SLNs are tumor targeting system for

antitumor drugs due to the lipophilicity of SLNs. Wang et al. have synthesized 3,5-

dioctanoyl-5-fluoro-2-deoxyuridine (DOFUdR) and incorporated it into SLNs (DO-

FUdR-SLNs) (Wang et al., 2002). After intravenous administration, the brain AUC of

DO-FUdR-SLN was 10.97-and 2.06-fold greater than that of FUdR solution and DO-

FUdR solution, respectively. The overall brain targeting efficiency of DO-FUdR-SLN

was 29.84%, while the corresponding figure for FUdR solution was 11.77%. A

significantly increased brain uptake of drug has also been observed by incorporating

paclitaxel into SLNs (Koziara et al., 2004) [43]. In brief, SLNs can improve the ability

of a drug to cross the BBB and is a promising targeting system for treating brain

tumors.

2.9.I.2. Active tumor targeting

The distribution and permeability of vessels between different tumor sites may not be

the same, and certain tumors do not exhibit an EPR effect (Peer et al., 2007). An

effective method (active targeting) is to attach the ligands, molecules that bind to

specific receptors on the tumor cell surface, to the surface of SLNs to selectively deliver

drugs to specific tumor cells. The ligands commonly used include folate, ferritin,

monoclonal antibody, aptamers, and peptides. Usually, folate receptors are over

expressed in cancer cell membranes, which make it possible to attach folate to the

surface of LNs and, subsequently specifically target tumor cells (Lu & Low, 2003).

Paclitaxel-7-carbonyl-cholesterol (Tax-Chol), a lipophilic prodrug of paclitaxel, has

been incorporated into NLC, which also contained folate-polyethylene

glycolcholesterol (f-PEG-Chol) as a ligand to target tumor marker folate receptors (FR).

In FR (+) KB (a human oral carcinoma cell line) cells, FR-targeted NLCs exhibited a

greater than 4-fold reduction in IC50 compared with nontargeted NLCs (0.61 ^M vs.

2.82 ^M). Furthermore, FR-targeted NLCs showed greater tumor growth inhibition and

animal survival was increased in mice FR (+) M109 tumors, compared with non

targeted NLCs (Stevens et al., 2004a). A similar significantly reduced IC50 in FR-

overexpressing tumor cells was also obtained for FR-targeted SLN as a carrier of a

lipophilic derivative of the photosensitizer hematoporphyrin (Hp) (Stevens et al.,

2004b).


35JAMIA HAMDARD

Transferrin receptor (TFR) is another kind of overexpressing receptor on the cancer cell

membrane and, therefore, TFR may be a suitable target for cancer treatment

(Hogemann-Savellano et al., 2003). Jain et al. prepared 5-fluorouracil-loaded ferritin

coupled SLNs (Fr-SLNs) for tumor targeting (Jain et al., 2008). An in vitro cytotoxicity

assay on Fr-SLN exhibited an IC50 of 1.28 ^M, while a non-targeted SLN had an IC50

of 3.56 ^M.Fr-SLN produced an effective reduction in tumor growth of MDA-MB-468

tumor bearing Balb/c mice compared with free 5-FU.

These findings confirm that ligand linked LNs can efficiently target tumors. However, it

should be noted that the targeted nanoparticles can be removed by the

reticuloendothelial system within minutes before they are able to bind to tumor cells.

2.9.1.3. Long-circulating SLNs to avoid clearance by the reticuloendothelial system

(RES)

Despite the promising effect obtained with conventional LNs, their usefulness is limited

by their rapid blood clearance and recognition by the RES (Wong et al., 2007). For

active targeting SLNs, the brief circulation in the body reduces the opportunity to bind

to specific receptors, and it is hard to achieve an active targeting effect. Therefore, it is

necessary to prolong the circulating time of LNs, and this has been successful to some

extent. The SLNs with a surface modified by particular hydrophilic polymers is not

easily recognized by the RES (Moghimi & Szebeni, 2003). Some hydrophilic materials,

like polyethylene glycol (PEG), poloxamers, or poloxamines, and/or some amphipathic

polymers, the hydrophobic part of which can lock into the inner of the SLNs carrier and

the hydrophilic part of which can cover the surface, are widely used. Some studies have

indicated that there is a close relationship between the circulating time and the length of

the polymer chain. Chen has prepared two kinds of long circulating SLN, Brij78-SLNs

and F68-SLNs (Chen et al., 2001).

Compared with paclitaxel injection, Brij78-SLNs and F68-SLNs exhibited rather slow

drug elimination with a t1/2 of 4.88 h and 10.06 h, respectively. The PEG-DSPE in the

F68-SLNs has a longer hydrophilic chain, which can produce a stronger forbidden force

on the plasma protein and prolong the circulating time. By encapsulating doxorubicin in

PEG 2000-modified SLNs, doxorubicin could still be detected in rabbit blood more

than 6 h after injection, while no doxorubicin was detectable when the drug solution

was administered (Fundaro et al., 2000; Zara et al., 2002). In the case of tamoxifen


36JAMIA HAMDARD

citrate (TC)- loaded SLNs, a long circulating effect was also obtained by an increased

t1/2 and prolonged mean residence time (Reddy et al., 2006). Unfortunately, due to the

lack of studies on tumor bearing-mice, it is still unknown whether this kind of SLNs can

lead to increased tumor drug concentrations.

Recently, more and more attention has been paid to active ligand-conjugated stealth

SLNs. Docetaxel-loaded NLCs modified with an amphiphilic copolymer, folate-

poly(PEG-cyanoacrylate-co-cholesteryl cyanoacrylate) (FA-PEG), was prepared to

produce a long blood circulating effect and improve the targeting ability of antitumor

drugs (Zhao et al., 2011). The AUC was increased, clearance was decreased, and the

MRT was prolonged for the FA-DTX-NLC group. The hydrophilic part PEG chains,

attached to the surface of NLCs, can mask the surface charge, prevent the opsonin-

nanoparticle interaction, and ensure efficient steric stabilization. The interference with

the recognition by opsonin allows the drug carrier to achieve a prolonged blood

circulation time. Su et al., developed a new conjugate, octreotide-polyethylene glycol

(100) monostearate (OPMS), to enhance the targeted delivery of hydroxycamptothecine

(HCPT)-loaded NLCs (Su et al., 2011). Octreotide was chosen to specifically bind to

somatostatin receptors (SSTRSs) overexpressed in some tumors (Lamberts et al., 2002).

The OPMS-modified NLCs exhibited approximately a 3-fold longer t1/2 than non

modified NLCs, and a 10-fold longer t1/2 than free HCPT solution. Furthermore,

florescence microscopy observations also showed the highest uptake for OPMS-

modified NLCs in tumor cells (SMMC-7721). Interestingly, a greater degree of

modification was found to lead to a longer circulating time and a higher drug uptake.

This result can be explained as follows: Firstly, with a high degree of modification,

OPMS modified NLCs exhibited a slower release rate of HCPT. Secondly, a higher

degree of modification resulted in a greater fixed aqueous layer thickness (FALT),

which could improve stability, prevent opsonization and macrophage uptake. Thirdly,

the highly modified NLCs exhibited an increased average surface density of PEG

chains (SDPEG), 0.592 PEG/nm2, and a shorter distance (D), 1.30 nm, between two

neighboring PEG grafting sites. The optimal SDPEG for avoiding complement

consumption should be around 0.5-0.67 PEG/nm2 for one PEG, and a D value around

1-1.5 nm is suggested to avoid the adsorption of proteins (Fang et al., 2006; Hu et al.,

2007). It was appropriate for the highly OPMS modified NLCs to maintain its

resistance to RES, and have a prolonged circulation time. Further studies should be


37JAMIA HAMDARD

carried out on the antitumor effects in vitro and in vivo. Xue et al. recently applied this

integrated approach to deliver small-interfering RNA (siRNA) for treating prostate

cancer in an all-in-one manner (Xue & Wong, 2011). A folate-coated lipid-PEI hybrid

nanocarrier (LPN) achieved a longer circulation time, cancer-specific targeting,

extended release and a less toxic form of siRNA therapy in an “all-in-one” manner. The

surface modification of SLN/NLC, i.e. ligand-conjugated pegylation, avoids clearance

by the RES, prolongs the drug circulation time in blood, increases the exposure time of

tumor cells to drug and enhances the specific targeting effect.

2.9.2. Strategies to overcome MDR based on SLN/NLC drug delivery systems

MDR is one of the most serious challenges to cancer chemotherapy. The increased

efflux rate of drug caused by overexpressing ATP-binding cassette (ABC) transporters,

typically P-gp and MRP1, is one of the major mechanisms of MDR in cancer cells.

Therefore, current research into reversing MDR is mainly focused on blocking specific

drug efflux. Recently, SLNs and NLCs have shown potential promise to reverse MDR.

Doxorubicin-loaded SLNs with a drug encapsulation efficiency of 60-80% and a

particle size of 80-350 nm were prepared by Wong et al. for application to human MDR

breast cancer cells (MDA435/LCC6/MDR1) and a mouse cell line (EMT6/AR1) (Wong

et al., 2006a & b). Both cell lines were characterized by high expression of a classical

membrane transporter, P-gp. Doxorubicin-loaded SLNs showed more than an 8-fold

increase in killing MDR cancer cells but comparable efficacy on a wild-type cell line

compared with doxorubicin solutions at the same drug concentration. A 10-fold greater

cytotoxicity in a P-gp-overexpressing cell line P388/ADR was found for doxorubicin

SLNs with increased entrapment efficiency (>80%) and a smaller particle size (<100

nm) (Ma et al., 2009). After 4 h of efflux, 15-fold more doxorubicin remained in the P

gp cells with doxorubicin SLNs compared with free doxorubicin. Furthermore, mice

bearing P388/ADR murine leukemia tumors, treated with the SLN formulation, had a

longer median survival time (20 days) than animals given free drug solution (14.5 days)

at a dose of 3.5 mg/kg. SLNs exhibited a significantly improved therapeutic effect in

this MDR mouse model. The greater cytotoxicity compared with that in the previous

study of the Wong group may be attributed to the smaller particle size. Since larger

sized particles will usually be taken up by the RES in vivo, particles with size less than

100 nm more easily accumulate in tumors (Brannon-Peppas & Blanchette, 2004).


38JAMIA HAMDARD

Analogous results have also been reported for cytotoxic drug-loaded NLCs. In the study

by Zhang and his group, NLC formulations of paclitaxel and doxorubicin were

developed (Zhang et al., 2008). Compared with taxol and doxorubicin solution, the

paclitaxel- and doxorubicin-loaded NLCs both exhibited high cytotoxicities in

multidrug resistant cells (MCF-7/ADR cells and SK0V3-TR30 cells). The reversal

power of the paclitaxel NLCs for these two kinds of cells was 34.3- and 31.3- fold,

respectively, while that of doxorubicin NLC was 6.4- and 2.2-fold, respectively.

Moreover, the percentage cellular uptake of paclitaxel NLC in MCF-7/ADR cells was

2- to 3- fold higher than that of free drug solution over the same incubation time. These

findings support the hypothesis that LNs (i.e. SLNs and NLCs) may be used to reverse

MDR.

The mechanism of MDR reversal activity of drug-loaded LNs has been investigated and

discussed in several papers. Drug in LNs probably enters tumor cells in two ways, i.e.

simple diffusion and phagocytosis (Wong et al., 2006b). The intracellular drug

internalized via phagocytosis likely remains physically associated with LNs, which

allows the drug to bypass the efflux action handled by the membrane-associated P-gp.

As a result, more drug molecules are trapped within P-gp-overexpressing cells after

treatment with drug-loaded LNs compared with free drug solution. In addition, due to

the membrane affinity of lipid materials and the nano-scale size of LNs, internalization

of drug into tumor cells is enhanced, resulting in an increased intracellular drug

concentration (Miglietta et al., 2000; Serpe et al., 2004). Moreover, it is noteworthy that

some of the lipids and surfactants in LNs possess intrinsic P-gp inhibitory activities

(Bogman et al., 2003).

A P-gp reversing surfactant, Brij 78, was used for preparing doxorubicin and paclitaxel-

loaded LNs (Dong et al., 2009). These drug-loaded nanoparticles exhibited 6- to 9-fold

lower IC50 values than free drugs in P-gp overexpressing human cancer cells. The

tumor volume of NCI/ADR-RES bared mice remained almost unchanged following

treatment with paclitaxel PEGylated Brij 78-based LNs. The enhanced anticancer

efficiency can be partially attributed to the reduced drug efflux rate caused by the

inhibition of P-gp function and ATP transient depletion of Brij 78. This positive finding

(P-gp inhibition and ATP depletion of Brij 78-based LNs) offers a novel therapeutic

strategy to overcome MDR.


39JAMIA HAMDARD

The simple encapsulation of anticancer drugs in LNs was useful but not enough to

overcome the MDR problem. A few other strategies based on the SLN/NLC systems

have been investigated to overcome MDR. Some researchers found that P-gp inhibitors,

sometimes known as “chemosensitizers”, displayed a synergistic action with cytotoxic

drugs. Wong et al. developed a PLN system loaded with doxorubicin and P-gp-specific

inhibitor GG-918 (Wong et al., 2006c). Interestingly, the greatest cytotoxicity against

MDR cells was found after treating with PLNs loaded with two agents, while a lower

cytotoxicity was obtained after co-administration of PLNs loaded with two single

agents. The sequential or concurrent treatment of separate formulations of P-gp

inhibitors and cytotoxic drugs or drug combinations cannot guarantee the co-action of

drugs in the same tumor cells because of their different pharmacokinetics and tissue

distribution. SLN provides a potential approach to loading cytotoxic drugs and

chemosensitizer in the same carrier due to its ability to encapsulate drugs with a variety

of properties (Wong et al., 2004 & 2006c). To maximize the synergistic action, a

chronological or sequential release of chemosensitizers and anticancer agents to the

target site may be an alternative strategy to overcome efflux-mediated resistance. The

modification of doxorubicin-loaded SLN with chitosan oligosaccharide gave the SLN

some positive characteristics, such as improved drug loading, reduced burst drug

release and a delayed release rate (Ying et al., 2011). A novel chitosan-SLN

microparticle (CSM) system was also developed to co-encapsulate phenethyl

isothiocyanate (PEITC), an antitumor agent, and efflux-transporter inhibitors, such as

tamoxifen, verapamil HCl or nifedipine (Dharmala et al., 2008). CSM, a core shell type

delivery system, was incorporated with one fast-releasing efflux inhibitor in the shell

and a slower releasing anti-cancer agent, PEITC, in the core. The PEITC loaded CSM

produced enhanced cytotoxic expression of Calu-3 cells in the presence of the efflux

inhibitors, due to the enhanced accumulation of PEITC through the modulation of the

efflux transporters.

Besides the inhibition of efflux transporters, a few other strategies have also been

exploited to overcome MDR. As reported by Liu et al., (Liu et al., 1999 & 2004), high

glucosylceramide synthase (GCS) activity also contributes to MDR, since GCS can

glycosylate the pro-apoptotic ceramide to glucosylceramide. The accumulation of

glucosylceramide will result in cell proliferation and MDR. Therefore, down-regulation

and/or blockade of GCS have been considered as a promising approach to overcome


40JAMIA HAMDARD

MDR and subsequently improve the cytotoxicity of conventional cytotoxic drugs (Liu

et al., 2004). Mixed backbone antisense glucosylceramide synthase oligonucleotide

(MBO-asGCS), which down-regulates the production of GCS, was loaded in SLNs to

concurrently treat NCI/ADR-RES human ovary cancer cells with free doxorubicin

(Siddiqui et al., 2010). The cell viability significantly decreased to approximately 12%.

This finding also suggested that SLN would be a potential carrier for genetic material.

Furthermore, the aforementioned active targeting SLN/NLCs help overcome MDR by

specifically targeting cancer cells and enhancing endocytosis and, subsequently,

bypassing or avoiding the pump efflux of P-gp. The list of anticancer drugs

incorporated in SLNs is tabulated in table 1.


41JAMIA HAMDARD

Table 1: List of drugs incorporated in SLN used for the treatment of cancer

c h a pte r - 2 SOf ID LIPOID NANOPARTICLES: A REVIEW

S. No. Drug Origin Type of tumor/cell lineRoute of

administratio n

References

1. Methotrexate Synthetic MCF-7 and Mat B-III cells Intra-venous Battaglia et al., 2011

2. Paclitaxel Synthetic Ovarian cancer Intra-venous Xu et al., 2013

3. Paclitaxel Synthetic Breast cancer Intra-venous Videira et al., 2013

4. Artemisinin Natural HER2+ cells In vitro study Zhang et al., 2013a

5. Paclitaxel Synthetic A549 cells In vitro study Zhang et al., 2013b

6. Retinoic acid Natural Jurkat, HL-60, HCT- 116 & MCF-7 In vitro Carneiro et al., 2012

7. Pentacyclic triterpenediol fromBoswellia serrata Natural HL-60 cells - Bhushan et al., 2012

8. Mitoxantrone, ethotrexate & paclitaxel Synthetic MCF- 7 Intra-venous Zhuang et al., 2012

9. Docetaxel Synthetic A549 cells In vitro Mussi et al.., 2012

10. Curcumin Natural HL-60, A549 & PC3 cells In vitro Vandita et al., 2012

11. Topotecan and vincristine Natural Myriad cells In vitro Zucker et al., 201112. Noscapine Natural Glioblastoma cells In vitro Madan et al., 201213. 3-Chloro-4-[(4-methoxyphenyl) MCF-7 Intra-venous Fang et al., 2012

42JAMIA HAMDARD

chapter - 2 SO£.ID LIPID NANCPARTI^LES: A REVIEW

amino] furo [2,3 -b] quinoline Synthetic

14. Aspirin, curcumin and free sulforaphane

Combination of Natural and

syntheticMIA PaCa-2 & Panc-1 In vitro Sutaria et al., 2012

15. Camptothecin Natural NCI-H460 cells Oral Nekkanti et al., 201116. c-Met siRNA Synthetic Glioblastoma cells Intra-venous Jin et al., 2011

17. 2-Methoxyestradiol Synthetic MCF-7, PC-3, & SK-N- SH In vitro Guo et al., 2012

18. Curcumin Natural HCT-116 cells In vitro Chirio et al., 201119. Benzophenone-3 Synthetic HaCaT cells Topical Marcato et al., 201120. Doxorobucin Synthetic U87MG cells In vitro Kuo & Liang, 2011

21. Topotecan Natural Ovarian and small-cell lung cancer In vitro Souza et al., 2011

22. Tryptanthrin Natural MCF-7 In vitro Fang et al., 201123. Carmustine Synthetic U87MG cells In vitro Kuo & Liang, 201124. Docetaxel Synthetic Ovarian cancer In vitro Zhang et al., 201025. 5-Flurouracil Synthetic Colon cancer In vitro Yassin et al., 201026. Hypericin Natural Hep G2 cells In vitro Youssef et al., 201227. Epirubicin A549 cells In vitro Hu et al., 2010

28. Simvastatin-tocotrienolCombination of

Natural & synthetic

malignant +SA mammary epithelial

cellsIn vitro Ali et al., 2010

29. Doxorobucin Synthetic MCF-7/ADR cells In vitro Kang et al., 201030. Podophyllotoxin Natural 293T & HeLa cells In vitro Zhu et al., 200931. Docetaxel Synthetic BEL7402 cells Intra-venous Xu et al., 200832. 5-Florouracil Synthetic MDA-MB-468 Subcutaneous Jain et al., 200833. Podophyllotoxin Natural Hela cells In vitro Shi et al., 200834. Vinorelbine bitartrate Synthetic MCF-7 cells In vitro Wan et al., 2008

35. Etoposide Natural Dalton's lymphoma ascites bearing mice Intra-venous Reddy et al., 2006

36. Beta-elemene Natural Cancer Intra-venous Wang et al., 200537. Monostearin Natural A549 cells Intra-venous Ding et al., 200438. Cholesteryl butyrate Synthetic HT-29 cells In vitro Serpe et al., 2004

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2.1. solid lipid nanoparticles (slns) -...

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