2.1. solid lipid nanoparticles (slns) -...
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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).
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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
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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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• 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
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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
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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
• Toxicity of solvent in final product
• 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
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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
• Toxicity of solvent in final product
• 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
• Suitable for thermo-labile drugs
• Small and uniform particle size distribution
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• Avoidance of any thermal stress
• Avoids temperature induced drug degradation
Disadvantages
• Extra step of filtration
• Toxicity of solvent in final product
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
• Toxicity of solvent in final product
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
• Toxicity of solvent in final product
• Bigger particle size
• Not suitable for thermo-labile drugs
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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
• Not suitable for thermo-labile drugs
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
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(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
• Suitable for thermo-labile drugs
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
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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).
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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
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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.
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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
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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.
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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
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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.
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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):
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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).
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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.
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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.
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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).
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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
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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
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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).
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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.
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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
CHAPTE R - 2 SOL.ID LI^ID NANOP^ R ^ LES: A RE VIEW
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.
CHAPTE R - 2 SOL.ID LI^ID NANOP^ RTI^LES: A REVIEW
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
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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
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21. Topotecan Natural Ovarian and small-cell lung cancer In vitro Souza et al., 2011
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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
JAMIA HAMDARD43