INTRODUCTION:

Based on synthetic polymers or natural macromolecules, Particulate drug carriers are include under oil-in-water (O/W) emulsions, liposomes, micro particles and nanoparticles.¹ For parenteral nutrition, O/W emulsions have been introduced successfully to the clinic in the fifties. Based on these drug-containing emulsion formulations have been developed, e.g. containing diazepam and etomidate.² Nanoparticles are composed of synthetic or semi-synthetic polymers. Their size ranges between 10 and 1000 nm. The solid lipid nanoparticles are sub-micron colloidal carriers, and their size range between 50-100 nm, which are composed of physiological lipid, dispersed in water or in aqueous surfactant solution.

SLNs are used as colloidal drug carrier combines the advantage of polymeric nanoparticles fat emulsions and liposomes. The liquid lipid was replaced by solid lipid in order to overcome the disadvantages associated with the liquid state of the oil droplets, which eventually transformed into solid lipid nanoparticles.^{3, 4}

SLNs introduced in 1991 represent an alternative carrier system to colloidal carriers such as emulsions, liposomes and polymeric micro – and nanoparticles.⁵ Nanoparticles made from solid lipids are used as novel colloidal drug carrier for intravenous applications as they have been proposed as an alternative particulate carrier system. SLNs offer unique properties such as large surface area, small size, and high drug loading. They are attractive for their potential to improve performance of pharmaceuticals.^{6, 7}

The reasons for the increasing interest in lipid based system are many, they include:

· Lipids enhance oral bioavailability.

· Reduce plasma profile variability.

· Better characterization of lipoid excipient.

· An improved ability to address the key issues of technology transfer and manufacture scale-up.

2. Advantages of SLN: ^{5, 8-10}

· Improve stability of pharmaceuticals.

· High drug content.

· Easy to sterilize.

· Better control over release of encapsulated compounds.

· Enhanced bioavailability.

· Chemical protection of labile incorporated compounds.

· Much easier to manufacture than bio polymeric nanoparticles.

· No special solvent required.

· Raw materials are easy available and essential the same as in emulsions.

· Very high long-term stability.

· Application versatility.

· Control drug release.

· Target drug release.

· Large scale production is possible.

· High concentration of functional compound can be achieved.

· Lyophilization possible.

3. Disadvantages of SLN: ^{9, 11, 12}

· Growth of Particle.

· High tendency towards gelation.

· Unexpected dynamics of polymeric transitions.

· Drug loading capacity is poor.

· Relatively high water content of the dispersions (70-99.9%).

4. Aims of solid lipid nanoparticles:^{11, 13}

· Possibility of controlled drug release.

· Increased drug stability.

· High drug pay load.

· No bio-toxicity of the carrier.

· Avoidance of organic solvents.

· Incorporation of lipophilic and hydrophilic drugs.

5. Factors to be considered in the manufacturing of solid lipid nanoparticles:

SLNs are particles made from solid lipids (i.e., lipids solid at room temperature and also at body temperature) and stabilized by surfactant(s). Common ingredients used in the preparation of SLNs are emulsifiers, co emulsifiers, lipids, and water.

Excipients used in the formulation of SLNs:

Emulsifiers:

Phosphatidyl choline 95%, Egg lecithin, Poloxamer188, Poloxamer 407, Polysorbate 80.

Co-emulsifiers:

Tyloxapol, Taurocholate sodium, Taurodeoxycholic acid sodium salt, Sodium dodecyl sulphate, Sodium glycholate, Sodium oleate, Cholesteryl hemisuccinate.

Lipid matrices:

Behenic acid, Caprylic acid, cetylpalmitate, Glyceryltrimyristate, Cholesterol, Hardened fat, Glycerylmonostearate, Bees wax, Glyceryltrilaurate, Glyceryltristearate, Glyceryltripalmitate, Glycerylbehenate, Stearic acid, Solid paraffin, Softisan142.

Cryoprotectants:

Trehalose, Glucose, Mannose, Maltose, Lactose, Sorbitol, Mannitol, Glycine, Polyvinyl pyrrolidine, Gelatin.

Charge modifiers:

Stearylamine, Diacetyl phosphate dipalmitoyl phosphatidylcholine (DPPC)

Preservatives: Thiomersal.

Selection of emulsifier:

Emulsifier influences the size of the particle and also their stability. An emulsifier should be compatible with other excipient, capable of producing desired size, with minimum amount used, nontoxic, and also provide adequate stability to the SLNs. Another aspect to be considered in the selection of emulsifier is its in vivo fate. It should be optimum to cover the surface of nanoparticles.

Selection of co-emulsifier:

Phospholipids used in SLNs are neither soluble in continuous phase nor form highly dynamic micelles. The excess phospholipids molecules form small unilamellar vesicles, which causes limited mobility. Low mobility of the phospholipids molecules leads to aggregation and increase in particle size of SLNs, which can be avoided by using co emulsifiers like glycocholate (ionic) as well as Tyloxapol (non-ionic polymer).¹⁴

Selection of lipids:

The important factors to be considered while selecting a lipid are lipophilicity, loading capacity, melting point, crystalline nature and purity of lipid. SLNs are prepared using lipids of less ordered crystal lattices which exhibit successful drug inclusion, compared to those prepared using highly ordered crystal packing lipids. Drug expulsion is caused by the lipids that form highly crystalline particles with a perfect lattice. Lipids with less perfect crystals have many imperfections. As the hydrocarbon chain length increases Lipophilicity of the glycerides increase. Therefore, lipophilic drugs are better soluble in lipid melt (longer fatty acid chain lengths). Production of SLNs of good quality depends on Purity of the lipids. Impurities may affect stability due to alter in zeta potential of formulation. Lipid matrices used for the production of SLNs for I.V administration should be toxicologically accepted, biodegradable and suitable for sterilization by autoclaving. ¹⁵

Drug lipid solubility:

Solubility of the drug in the lipid melt is more than in the solidified lipid and this is the important parameter that decides the entrapment efficiency and loading capacity. Medium chain glycerides possess the optimal characteristic of solubilisation of the drugs as well as formation of micro emulsions. On the other hand, long chain glycerides with higher melting points are necessary for preparation of SLNs. The presence of mono and diglycerides in the lipid as matrix material promotes drug solubilisation.

6. Types of solid lipid nanoparticles:

They are classified depend on the chemical nature of the active ingredient and lipid, the solubility of active ingredients in the melted lipid, nature and concentration of surfactants, type of production and the production temperature. They are 3 types:

Type I - Homogenous matrix model:

They are derived from a solid solution of lipid and active ingredient. A lipid blend can be produced containing the active in a molecularly dispersed form. After solidification of this blend, it is ground in its solid state to avoid or minimize the enrichment of active molecules in different parts of the lipid nanoparticles. A solid solution can be obtained when SLN are produced by the cold homogenation method.

Type II or drug enriched shell model:

They are prepared when SLN are produced by the hot technique, and the active ingredient concentration in the melted lipid is low during the cooling process of the hot o/w nanoemulsion the lipid will precipitate first; leading to increasing concentration of active molecules in the remaining melt, an outer shell will solidify containing both active and lipid. The enrichment of the outer area of the particles causes burst release.

Type III or drug enriched core model:

Core model SLN is achieved when the active ingredient concentration in the lipid melt is high and relatively close to its saturation solubility. Cooling down of the hot oil droplets in most cases reduce the solubility of the active in the melt. When the saturation solubility exceeds, active molecules precipitate leading to the formation of a drug enriched core. Due to the different chemical shifts it is possible to attribute the NMR signals to particular molecules or their segments. Simple 1H spectroscopy permits an easy and rapid detection of super cooled melts. To investigate SLN dispersions ESR spectra requires the addition of paramagnetic spin probes. Micro viscosity and micro polarity information was determined from ESR spectra. Experimental results demonstrate that storage induced crystallization of SLN leads to an expulsion of the probe out of the lipid into the aqueous phase.¹⁶

7. Preparation of solid lipid nanoparticles:

SLNs are made up of emulsifier, co emulsifiers, solid lipid, and water. The lipids used may be triglycerides (tri-Stearic), partial glycerides, fatty acids (Stearic acid, palmitic acid), steroid and waxes (cetyl palmitate). Various emulsifiers and their combination (Pluronic F 68, F 127) have been used to stabilize the lipid dispersion.¹⁷There are different methods of SLNs preparation like:

7.1. High shear homogenization.

7.2. Hot homogenization.

7.3 Cold homogenization.

7.4. Ultrasonication or high speed homogenization.

7.5. Micro emulsion based SLN preparations.

7.6. SLN preparation by using supercritical fluid.

7.7 SLN prepared by solvent emulsification / evaporation.

7.8. Double emulsion method.

7.9. Spray drying method.

7.1. High shears homogenization:

Solid lipid Nanoparticle dispersions were initially produced by High shear homogenization techniques. Both methods are widespread and easy to handle. This method is used to produce SLN by melt emulsification. Olbrich investigated different process parameters, including stirring rate, emulsification time, and cooling condition on the particle size. Lipids used in this preparation are trimyristin, tripalmitin, and mixture of mono, di and triglycerides (Witepsol W35, Witepsol H35).Steric stabilizers like glycerol behenate and poloxamer 188 are used. For Witepsol W35 dispersions the best SLN quality was obtained after stirring for 8 min at 20,000 rpm followed by cooling 10 min and stirring at 5000 rpm at a room temperature. In contrast, the best conditions for Dynasan116 dispersions were a 5 min of cooling at 5,000 rpm in cool water and 10-min emulsification at 25,000 rpm. Higher stirring rates slightly improved the polydispersity index but do not change the particle size. ^{18, 19}

7.2. Hot homogenization:

It is carried out at temperatures above the melting point of the lipid. It is similar to the homogenization of an emulsion. The aqueous emulsifier phase and a pre-emulsion of the drug loaded lipid melt (same temperature) are obtained by high-shear mixing device (like Silverson -type homogenizer). But the problem is with pre-emulsion which affects the quality of the final product to a great extent. High pressure homogenization of the pre-emulsion is done above the lipid melting point. Usually, at higher processing temperatures because of lowered viscosity of the lipid phase lower particle sizes are obtained. This might also accelerate the drug and carrier degradation. Good quality products are obtained after passing several times through the high-pressure homogenizer (HPH), usually 3-5 passes. High pressure processing always increases the temperature of the sample (approximately 10°C at 500bar). In most cases, 3-5 homogenization cycles at 500-1500 bar are sufficient. Increasing the homogenization causes increase in particle size due to particle coalescence, this occurs due to high kinetic energy of the particles. ^20,
21

7.3. Cold homogenization:

The cold homogenization process is done with the help of solid lipid and therefore is similar to milling of a suspension at elevated pressure. Effective temperature regulation is helpful to ensure the solid state of the lipid during homogenization. It has been developed to overcome the following problems of the hot homogenization technique such as:

Loss of drug in aqueous phase during homogenization. Polymorphic transitions of the lipid due to complexity of the crystallization step of the nanoemulsion leading to several modifications. The first preparatory step is the same as in the hot homogenization procedure and includes the dispersion of the drug in the lipid melt. However, the subsequent steps differ from hot homogenization procedure. For homogenous drug distribution in the lipid matrix the drug which is having the melt is cooled rapidly (using dry ice). The drug containing solid lipid is pulverized to microns. The SLNs are dispersed in a chilled emulsifier solution. The dispersion is subjected to high pressure homogenization at room temperature or below, with appropriate temperature control. However, compared to hot homogenization broader size distribution, and larger particle sizes are typical of cold homogenized samples.²²

Advantages:

· Capital cost is Low.

· Demonstrated is done at lab scale.

Disadvantages:

· Energy intensive process.

· Demonstrated at lab scale Bio molecule damage.

· Polydisperse distributions.

· Unproven scalability.

7.4. Ultrasonication or high speed homogenization:^23,24

SLN were also developed by sonication (high speed stirring). The problem of this method is broader particle size distribution ranging into micro meter range. This causes physical instabilities like particle growth in the preparation upon storage. Ultrasonication causes Potential metal contamination which is also a major problem in this method. So for a stable formulation, studies have been performed by various research groups that high speed stirring and Ultrasonication are used combined and performed at high temperature.

Advantages:

· This method Reduce shear stress.

Disadvantages:

· Potential metal contamination is a big problem.

· Physical instability like particle growth upon storage.

7.5. Micro emulsion based SLN preparations:

SLN preparation techniques which are based on the dilution of micro emulsions were developed by Gasco and co-workers. They are prepared by stirring an optically transparent mixture which is composed of a low melting fatty acid (Stearic acid), an emulsifier (polysorbate20, and Polysorbate 60), co-emulsifiers (sodium monooctylphosphate) and water. The hot micro emulsion is dispersed in cold water under stirring. The dilution process is determined by the composition of the micro emulsion. The droplet structure is already present in the micro emulsion state and therefore, no energy is used to achieve submicron particle sizes. The volume ratios of the hot micro emulsion to cold water are in the range of 1:25 to 1:50. The scientist named Fessi, produced polymer particles by dilution of polymer solutions in water. According to De LaTourette particle size is determined by the velocity of the distribution processes. Nanoparticles were produced with the help of solvents which distribute very rapidly into the aqueous phase (acetone), whereas larger particle sizes were obtained with more lipophilic solvents. The hydrophilic co-solvents of the micro emulsion might play a similar role in the formation of lipid nanoparticles as the acetone for the formation of polymer nanoparticles. ^25-30

7.6. SLN preparation by using supercritical fluid:

SLN preparation by using supercritical fluid is a relatively new technique and has the advantage of solvent-less processing. There are several variations in this platform technology for powder and nanoparticles preparation. Rapid expansion of supercritical carbon dioxide solutions (RESS) method is used for preparation of SLN. Carbon dioxide (99.99%) is used as a solvent for this method. ^31-33

Advantages:

· Avoid the use of solvents.

· Particles are obtained as a dry powder.

· Mild pressure and temperature conditions.

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

7.7. SLN prepared by solvent emulsification/ evaporation:

For the production of nanoparticles dispersions by precipitation in o/w emulsions the lipophilic material is dissolved in water-immiscible organic solvent (cyclohexane) that is emulsified in an aqueous phase. Nanoparticles dispersion is formed by precipitation of the lipid in the aqueous medium upon evaporation of the solvent. The mean diameter of the obtained particles was 25 nm. Here cholesterol acetate is used as model drug and lecithin/sodium glycocholate blend as emulsifier. Siekmann and Westesen produced the cholesterol acetate nanoparticles of mean size 29 nm.^{34, 35}

7.8. Spray drying method:

In order to transform an aqueous SLN dispersion into a drug product spray drying method is used which is an alternative procedure to lyophilization. It is less in cost compared to lyophilization. In this method due to high temperature, shear forces and partial melting of the particle, particle aggregation is observed. Freitas and Mullera recommends the use of lipid with melting point >700 for spray drying. The best result was obtained with SLN concentration of 1% in a solution of trehalose in water or 20%trehalose in ethanol-water mixtures (10/90 v/v).³⁶

7.9. Double emulsion method:

For the preparation of hydrophilic loaded SLN, a novel method based on solvent emulsification-evaporation has been used. Here to prevent drug partitioning to external water phase during solvent evaporation in the external water phase of w/o/w double emulsion the drug is encapsulated with a stabilizer. ³⁷

8. Applications:

Solid lipid Nanoparticles has a better stability and ease of upgradability to production scale as compared to liposomes. This property plays an important role in many modes of targeting. They form the basis of colloidal drug delivery systems, which are biodegradable. They can be stored for at least one year. Important in drug deliver to the liver in vivo and in vitro to cells which are actively phagocytic.³⁸ There are many potential applications of SLNs some of which are given below:

8.1. SLNS as gene vector carrier:

It has an application in gene vector formulation, used in carrying genetic/peptide materials such as DNA, plasmid DNA and other nucleic acid. They (lipid nucleic acid nanoparticles) are prepared from a water miscible organic solvent where both lipid and DNA are dissolved separately by removing the organic solvent. Lipid-nucleic acid nanoparticles of 70-100 nm size were formed. They are stable and their size is homogeneous. They are known as genospheres. It is targeted specific by insertion of an antibody-lipo polymer conjugated in the particle. The gene transfer was exploited by incorporation of a diametric HIV-1 HAT peptide into SLN gene vector.^39-41

8.2. SLNS as cosmeceuticals:

They are used in the preparation of sunscreens, used as an active carrier agent for molecular sunscreens and UV blockers. Better localization has been achieved for vitamin A in upper layers of skin with glyceryl behenate SLNs compared to conventional formulations. The in vivo study showed that skin hydration will be increased by 31% after 4 weeks by addition of 4% SLN to a conventional cream. SLN and NLCs have application in controlled release innovative occlusive topicals.^42-44

8.3. SLNS for potential agriculture application: ^{45, 46}

Artemisia arboreseens L when incorporated in SLN Essential oil were extracted, and were able to reduce the rapid evaporation and they have been used in agriculture as a suitable carrier of ecologically safe pesticides. Compritol 888 ATO as lipid and Miranol Ultra C32 as surfactant were used in preparation of SLN.

8.4. SLNS as a targeted carrier for anticancer drug to solid tumors:

SLNs have been used as drug carriers for the treatment of neoplasm’s. Tamoxifen, which is used as an anticancer drug incorporated in SLN to prolong release of drug after i.v administration in breastcancer and to enhance the permeability and retention effect. SLNs which are loaded with drugs like methotrexate and camptothecin are used in Tumour targeting. ^47-49

8.5. SLNS in breast cancer and lymph node metastases:

Mitoxantrone - loaded SLN which are local injections were prepared to reduce the toxicity and improve the bioavailability and safety of drug. Doxorubicin (Dox) Efficacy has been reported to be increased by incorporation in SLNs. Dox-loaded solid lipid nanoparticles were prepared by combining Dox with soybean-oil-based anionic polymer and dispersed together with a lipid in water. The system has reduced breast cancer cells by increasing its efficacy. ^{50, 51}

8.6. Oral SLNS in antitubercular chemotherapy:

An antitubercular drug such as rifampicin, isonizide, pyrazinamide-loaded SLN systems, decreases the dosing frequency and increase patient compliance. By using the emulsion solvent diffusion technique this anti tubercular drug loaded solid lipid nanoparticles are prepared. The nebulization in animal is done by incorporating the above drug in SLN which causes improving the bioavailability of the drug.

8.7. Stealth nanoparticles:

These provide a novel and unique drug-delivery system. They evade quick clearance by the immune system. Theoretically, such type of nanoparticles can target specific cells. They have been successfully tested in animal models with the use of marker molecules and drugs. Studies with antibody labelled stealth lipobodies have shown increased delivery to the target tissue. ^{52, 53}

8.8. SLNS as potential new adjuvant for vaccines:

To enhance the immune response adjutants are used in vaccination. The safer new subunit vaccines are less effective in immunization and therefore effective adjutants are required. Increase the amount of antigen delivered is not a solution because this also increases the costs. The side effects of Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) are too strong to be employed. But Freund's complete adjuvant is still considered as a “gold standard” when developing new adjutants. The adjuvant frequently used for many years consist of aluminium hydroxide particles, however they can also exhibit side effects.⁵⁴ They are oil-in-water emulsions that degrade rapidly in the body. Being in the solid state, the lipid components of SLN will be degraded more slowly providing a longer lasting exposure to the immune system. Degradation can be slowed down even more when using stabilizing surfactants.^55-57In a first study SLN have been tested as adjuvant in comparison to FIA in sheep. The two unoptimized SLN formulations exhibited 43 and 73% of the immune response (antibody titer) of FIA investigated as standard. These data are promising and currently the SLN are being optimized regarding their surface properties to give a maximum immune response. Advantages compared to traditional adjutants are the biodegradation of SLN and their good tolerability by the body.⁵⁴

9. Administration route ^58-64

The in vivo fate of the SLN will depend mainly on the route of administration and distribution process in the body. SLN are made of physiological lipids or waxes. The pathways which are present in the body for transportation and metabolism contribute to a large extent to the in vivo fate of the carrier. For the degradation process of SLN particles, Lipase is the most important enzymes. Which are present in various organs and tissues? Lipases are helpful in splitting the ester linkage and form free fatty acids and partial glycerides. Most oil/water interface is required for activation of most lipases. In vitro experiment indicates that SLN show different degradation velocities by the lipolytic enzyme.

9.1. Parenteral administration:

For parenteral administration Peptide and proteins drugs are usually used, as their oral administration is not possible due to enzymatic degradation in GI tract. They are very suitable for drug targeting. They reduce the possible side effects of drug incorporated with the increased bioavailability.⁶⁵

9.2. Oral administration:

Self-emulsifying formulations, self-emulsifying solid dispersion formulations and single-component lipid solutions are the different types of oral lipid based formulation. The most commonly used excipients for preparing oral lipid-based formulations were lipid soluble solvents (propylene glycol, glycerine, ethanol), dietary oils composed of medium (coconut or palm seed oil) or long-chain triglycerides (corn, olive, peanut, soybean oils, including hydrogenated soybean oils), and various pharmaceutically acceptable surfactants (Polysorbate 20 or 80; D-α-tocopherol polyethylene glycol 1000 succinate (TPGS®); Span 20; various Labrafils®, Labrasol®, and Gelucires®). These formulations, which are in the form of either bulk oral solutions or liquid-filled hard / soft gelatine capsules, were used in instances where conventional approaches (solid wet or dry granulation) did not provide sufficient bioavailability.

Oral lipid-based formulations can provide some benefits which included:

· Reduction or elimination of positive food effect.

· Causes increase and decrease in GI absorption of poorly water-soluble and lipophilic drugs.

· Possible reduction in, or elimination of, a number of development and processing steps (Detection of a stable crystalline form of the drug, coating, taste masking, and reduced clean-up requirements and need for containment during manufacture of highly-potent drug products).

· Readily manufacture using available equipment.

The amount of drug present in a unit-dose capsule product ranges from 0.25 μg to 500 mg. And for oral solution products ranges from 1 μg/ml to 100 mg/ml. The total amount of lipid excipient administered in a single dose of a capsule formulation ranges from 0.5 to 5 g. Some of these products tolerate room temperature storage for only brief periods of time and require long-term storage at 2-8 ° due to chemical / physical stability issues.⁶⁶

9.3. Rectal administration:

Rectal administration is preferred, in some circumstances when rapid pharmacological effect is seen. This route is used for paediatric patients due to easy application.

9.4. Nasal administration:

Nasal route is preferred due to its fast absorption and rapid onset of drug action also avoiding degradation of labile drugs in the GIT and insufficient transport across epithelial cell layers.

9.5. Respiratory delivery:

Nebulisation of SLN carrying anti-asthmatic drugs, anti-tubercular drugs, and anti-cancer was observed to be useful in improving drug bioavailability and reducing the dosing frequency which is for better management of pulmonary action.

9.6. Ocular administration:

Muco-adhesive and Biocompatibility properties of SLN improve their interaction with ocular mucosa. They also prolong corneal residence time of the drug, with the aim of ocular drug targeting.

9.7. Topical application:

SLN are considered as the next generation of delivery system after liposomes. They are composed of well-tolerated excipients. Due to their small particle size they possess similar adhesive properties leading to film formation on the skin. Distinct advantages of SLN are ability to protect chemically labile ingredients against chemical decomposition, their solid state of the particle matrix, and the possibility to modulate drug release. Many cream bases do not exhibit a melting peak below 100^OC that means the content of SLN in a cream can be quantified by their melting peak determined by DSC. Just by looking at the change in melting enthalpy the stability of SLN during storage can easily be monitored. Analysis is even possible in cases where a cream contains a fraction which melts below 100^OC. If there is an overlapping, one can determine the total melting energy as a function of time. This special property of SLN opens new markets for topical products containing colloidal carriers for active ingredients.

Adhesiveness is a general property of nanoparticles of different kinds. A better example from practical life is iced sugar which sticks much better to bakery products than crystalline sugar. The SLN are forming adhesive films onto the skin Similar to liposomes. Previously it was assumed that SLN would be forming films of densely packed spheres, recent results suggest that under the pressure of application the spheres form a coherent film. Such a lipid film formation will be able to restore damaged protective lipid film on the skin. In addition such a film can have an occlusive effect.^67-69SLN were incorporated into a commercial cosmetic O/W cream and tested in the Franz cell regarding their effect on drug penetration and occlusiveness. SLN containing cream base were applied to the skin and analysis was performed after 24 h of incubation. The occlusive effects were assessed by staining vertical skin slices by eosin. Untreated skin showed a compact stratum corneum with corneocyte layers closely conjuncted. A different result was obtained for the SLN cream, the stratum corneum appeared swollen and overall thickness had increased.

The in vivo occlusive effect of SLN is somewhat controversial. It is partially attributed to the differences in the formulations tested. From the results obtained we can concluded that SLN added to a formulation do not have an additional occlusive effect when the formulation itself is already highly occlusive. The only parameter to assess the ability of a delivery system is its effect on drug/active ingredient penetration into skin. And its therapeutic affects in cosmetic applications the effect on skin appearance. A range of cosmetic ingredients like coenzyme Q10, vitamin E and its derivatives and retinol have been incorporated into SLN. The skin caring properties of a commercial retinol cream have been compared to the same cream containing retinol-loaded SLN, reference was untreated skin. Parameters assessed were skin elasticity, and skin roughness as standard read out parameters. The moisture level of the SLN-containing formulation was raised by 33% (SLN-free base 23%) after a 1-week period of treatment compared to untreated skin. Besides this the cream containing retinol-loaded SLN improved the skin smoothness by 10.3%, the SLN-free cream achieved only 4.1%.^70-75

A completely new, recently discovered area of application of SLN is in sun-protective creams. Due to the reduction of the protective ozone layer there is a steep increase in skin cancer, especially in countries like Australia Side effects of molecular sunscreens (UV-blockers) are penetration into the skin and consequently irritation. Particulate sunscreens like titanium dioxide were also found to possibly penetrate into the skin. This can be avoided or minimized by entrapping molecular and particulate sunscreens into the SLN matrix. It was found that the SLN themselves have also a sun-protective effect. Due to their particulate character they are protective due to scattering of UV light (similar to titanium dioxide).Molecular sunscreens are much more effective after incorporation into SLN and at the same time side effects are reduced.⁷⁶

10. Sterilization of SLN:

Sterilization of SLN is an issue in the case of pulmonary or parenteral administration. The SLN melt during autoclaving and recrystallize during cooling down. However, autoclaving is not possible when a certain structure has been given to the SLN. This special structure - leading to the desired modulated release profile - would be lost when the particles melt again during the autoclaving and recrystallize in a non - controlled way. Autoclaving at 121^oC cannot be performed by using sterile stabilizing polymers, e.g. poloxamer series. The autoclaving temperature seems to be too close to the critical occulation temperature (CFT) of the polymers, at least the polymer adsorption layer seems partially to collapse leading to insufficient stabilization and particle aggregation. This can be avoided by reducing the autoclaving temperature (e.g. 121 to 110^oC).

The physical stability during autoclaving cannot be stated in general manner, it depends on the composition of the SLN formulation. SLN dispersions can also be sterilized by filtration. It is highly important to filter them in the liquid state; this allows even particles with a size larger than the pores in the filter to be filtered. This technology is well known from parenteral emulsions and easy to apply to SLN. Alternatively, the SLN can be produced aseptically. To sum up, SLN dispersions can be sterilized or prepared aseptically using already established techniques in the pharmaceutical industry.^77-80

11. Characterization of SLNs:

Characterization of SLN is an important aspect due to the colloidal size of the particles and the dynamic nature of the delivery system. The important parameters which are to be evaluated for the SLNs are degree of crystallinity, particle size, size distribution kinetics, and lipid modification (polymorphism), coexistence of additional colloidal structures (micelles, liposome, super cooled, melts), drug content, in vitro drug release and surface morphology. The particle size may be studied by using photon correlation spectroscopy (PCS), scanning tunnelling microscopy (STM), or freeze fracture electron microscopy (FFEM), transmission electron microscopy (TEM), scanning electron microscopy (SEM) atomic force microscopy (AFM).

11.1. Measurement of particle size and zeta potential:

The most powerful techniques used for measurements of particle size are Photon correlation spectroscopy (PCS) and laser diffraction (LD). The Coulter method is rarely used due to difficulties in the assessment of small nanoparticles. PCS used in measurement of the fluctuation of the intensity of the scattered light (caused by the particle movement).PCS is not able to detect larger micro particles but is a good tool to characterize nanoparticles. They can be detected with the help of LD measurements which is based on the diffraction angle on the particle radius. Compared to the larger ones smaller size particles cause more intense scattering at high angles. An advantage of LD it includes a broad size which ranges from the nanometre to the lower millimetre. The development of polarization intensity differential scattering (PIDS) technology increases the sensitivity of LD to smaller particles. However, it is highly recommended to use PCS and LD simultaneously. It should be known that both methods do not ‘measure’ particle size, but they detect light scattering effects which are used to calculate particle size. For example, uncertainties may result from non–spherical particle shapes. Further, for the samples which contain different size particles difficulties may arise in measurements for both in PCS and LD. Electron microscopy provides, in contrast to PCS and LD, direct information on the particle shape. However, the investigator should pay special attention to possible artifacts which may be developed by the sample preparation. For example, solvent removal may lead to modifications which will influence the particle shape. Zeta potential is an important product characteristic because of its high value which is expected to lead to deaggregation of particles in the absence of other complicating factors such as steric stabilizers or hydrophilic surface appendages. It is usually measured by zeta meter. ⁸¹

11.2. Dynamic light scattering (DLS):

DLS, also known as PCS records the variation in the intensity of scattered light on the microsecond time scale. This variation under the influence of Brownian motion results from interference of light scattered by individual particles, and is quantified by compilation of an autocorrelation function. This function is fit to an exponential, or some combination or modification thereof, with the corresponding decay constant(s) being related to the diffusion coefficient(s). Using standard assumptions of spherical size, low concentration, and known viscosity of the suspending medium, particle size is calculated from this coefficient.

The advantages of the method are:

· The speed of analysis,

· Lack of required calibration,

· Sensitivity to sub micro meter particles ^{81, 82}

11.3. Static light scattering/Fraunhofer diffraction:

The method is fast and rugged. Main reason is it requires more cleanliness than DLS, and advance knowledge of the particles' optical qualities Static light scattering (SLS) is an ensemble method in which the pattern of light scattered from a solution of particles is collected and fit to fundamental electromagnetic equations in which size is the main variable.

11.4. Electron microscopy:

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) provide a way to directly observe nanoparticles and physical characterization of nanoparticles. TEM has a smaller size limit of detection, is a good validation for other methods and one must be cognizant of the statistically small sample size and the effect that vacuum can have on the particles.

11.5. Nuclear magnetic resonance (NMR):

NMR is used to determine both nature and size of nanoparticles. The selectivity afforded by chemical shift complements the sensitivity to molecular mobility to provide information on the physicochemical status of components within the nanoparticles.

11.6. Atomic force microscopy (AFM):

In this technique, a probe tip with atomic scale sharpness is kept across a sample to produce a topological map based on the forces at play between the tip and the surface. The probe can be dragged across the sample (contact mode) or allowed to hover just above (noncontact mode), with the exact nature of the particular force employed serving to distinguish among the sub techniques. ^{81, 83}

11.7. Acoustic methods:

It measures the attenuation of sound waves as a means of determining size through the fitting of physically relevant equations. In addition, by the movement of charged particles the oscillating electric field generated under the influence of acoustic energy can be detected to provide information on surface charge.

11.8. X-ray diffraction (powder X-ray diffraction) and differential scanning calorimetry (DSC):

The geometric scattering of radiation from crystal planes within a solid allow the presence or absence of the former to be determined thus permitting the degree of crystallinity to be assessed. Another method that is a little different from its implementation with bulk materials, DSC can be used to determine the nature and speciation of crystallinity within nanoparticles through the measurement of glass and melting point temperatures and their associated enthalpies.⁸⁴

12. Pharmacological performance of SLN systems:

Drug delivery to the brain is an exciting area of possible application for lipid nanoparticles technology. Lipid nanoparticles can be administered intravenously due to their nanoscale size. Without proper surface modification the cells of the reticuloendothelial system, particularly the liver and spleen, rapidly clear colloidal particles. Incorporation of polyoxyethylene and polypropylene block copolymers has increased lipid nanoparticles tumour accumulation, antibacterial activity of antifungal drugs, and extravasations of the blood brain barrier (BBB) of anticancer drugs normally incapable of crossing the BBB. Lipid nanoparticles drug formulations have shown to produce improved pharmacokinetic profiles compared to traditional drug formulations. Doxorubicin plasma concentrations increased 3-5 times when formulated in lipid nanoparticles, showed a bi-exponential curve with high Area under the curve (AUC), exhibited longer circulation half-lives, decreased the volume of distribution, and toxic side effects in rats. As noted earlier removal of hydrophobic colloidal particles, a role of Buffer cells in the liver. If liver targeting is desirable, then in the case of non-stealth nanoparticles passive targeting can be accomplished. Similarly, macrophages throughout the circulation offer a passive targeting opportunity. However, targeting of the liver and macrophages is avoided through the use of stealth technology. By the leaky vasculature associated with cancer Passive targeting of cancer tumour is made possible. The leaky vasculature generated during cancer driven angiogenesis (to feed the tumour cells) allows extravasations of colloidal particulates. Interestingly, stealth lipid nanoparticles have demonstrated a strong propensity to accumulate in the brain.BBB penetration is extremely difficult and is one of the critical challenges facing pharmaceutical therapeutics. Lipid nanoparticles accumulation in the brain may be blood protein mediated. Adsorption of blood proteins such as lipoproteins on lipid nanoparticles surfaces may lead to interactions with endothelial cells that facilitate crossing the BBB. Diminazen aceturate because of its hydrophilicity it alone does not cross the BBB. Lipid nanoparticles enhanced BBB transport also has been demonstrated for tobramycin, doxorubicin, and idarubicin.

13. Use of SLN in various cancer therapies:

Introduction:

The main importance of the chemotherapy and radiation is to kill the tumour cells as they are more susceptible to the actions of these drugs, this is due to their growth at a much faster rate than healthy cells, at least in adults. Current research include new therapeutic targets such as blood vessels fueling tumour growth, development of carriers to allow alternative dosing routes and targeted therapeutics that are more specific in their activity. Clinical trials have shown that patients are open to new therapeutic options and the goal of these is to increase survival time and the quality of life for cancer patients. In all cases, the effectiveness of the treatment is directly related to the treatment’s ability to target and to kill the cancer cells while affecting as few healthy cells as possible. The degree of change in the patient’s quality of life and eventual life expectancy is directly related to this targeting ability of the treatment. Most current cancer patients’ only selectivity in their treatment is related to the inherent nature of the chemotherapeutic drugs to work on a particular type of cancer cell more intensely than on healthy cells. Unfortunately, not all treatments, even if carried through to the oncologists specifications, are effective in killing the cancer before the cancer kills the patient. The advances in treatment of cancer are progressing quickly both in terms of new agents against cancer and new ways of delivering both old and new agents. Hopefully this progress can move us away from near-toxic doses of non-specific agents. This review will primarily address new methods for delivering therapies, both old and new, with a focus on nanoparticles formulations and ones that specifically target tumours.⁸⁵

13.1. Liver cancer:

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, which is a primary malignancy of the liver. The mortality rate from HCC is the third highest worldwide for any cancer-related diseases. And since the 1990s, HCC has been the cause of the second highest mortality rate due to cancer in China.⁸⁶Liver is the most common organ where tumor metastases occur, In addition to primary tumors. For the delivery of antisense oligonucleotide (AS-ODN) to liver endothelial cells (in-vitro and in-vivo), Bartsch and co-workers (2004) proposed stabilized lipid coated lipoplexes. ⁸⁷

13.2. Breast Cancer:

It is one of the most frequently occurring cancers in women and the second leading cause of cancer deaths in women. However, since 1989, due to improvements in breast cancer prevention as well as treatment the breast cancer mortality rate has decreased 1.8% per year.⁸⁸An important phenomenon termed multidrug resistance (MDR) which is the main clinical obstacle in cancer therapy which indicates the development of resistance to a multitude of chemotherapeutic agents. Generally Chemo resistance result from either by physically impairing delivery to the tumor (e.g., increased metabolism/excretion, poor absorption, and/or poor diffusion of drugs into the tumor mass); or by intracellular mechanisms that raise the threshold for cell death.^89-93 Nanoparticles are known beneficial tumor targeting vehicles due to their passive targeting properties by the enhanced permeability and retention (EPR) effect ⁹⁴

13.3. Colorectal Cancer:

It is the most common cancer in Western countries and is the second leading cause of cancer-related deaths in the United States, accounting for nearly 60,000 deaths each year.⁹⁵ Hyaluronic acid–coupled chitosan nanoparticles bearing oxaliplatin (L-OHP) encapsulated in Eudragit S100–coated pellets were developed for effective delivery to colon tumors.⁹⁶ SLN have been proposed as new approach of drug carriers.⁹⁷ SLN carrying cholesteryl butyrate (chol-but), doxorubicin and paclitaxel had been developed previously. However, doxorubicin is not so active against colorectal cancer.⁹⁸ Depending on the preparation method of SLN they are in the colloidal size range and can be loaded with hydrophilic and lipophilic drugs. ^{99, 100} The composition of the warm micro emulsions from which SLN are prepared is flexible, and can be varied to suit the type of drug and administration route.¹⁰¹

13.4. Lung Cancer:

It is one of the leading causes of death worldwide.¹⁰² Adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma, are referred to as ‘‘non-small cell lung cancers’’ (NSCLCs) which together make up the majority of lung cancers. Patients with early stage NSCLC are typically treated with surgery. 5-year survival rates range from 25% to 80%, depending on the stage of the disease.¹⁰³ Current treatments for lung cancer have shown little success because they cannot cure disseminated tumors with an acceptable level of toxicity. Generally lung cancers are caused by mutations in p53 gene.^104-107 which leads to loss of tumor-suppressor function, loss of mutational repair, increase of drug resistance, increase of tumor angiogenesis, proliferation of cells, and inhibition of apoptosis.¹⁰⁸Gene therapy is one of the alternative strategies that have shown promise in the treatment of lung cancer. Viral and non-viral vectors are two main groups of vectors used in gene delivery. Toxicity and immunogenicity are associated with viral vectors have led to an active interest in non-viral systems for gene delivery.¹⁰⁹Among non-viral vectors, biodegradable nanoparticles have shown advantage over other carriers by their increased stability and their controlled-release ability.^110,
111 Nanoparticles are divided into two systems, cationic and anionic nanoparticles in gene delivery systems. Cationic nanoparticles are associated with the ionic interaction between the cationic polymers and the anionic plasmid DNA, forming stable polymer.^112,113 Cationic lipid formulations, solid lipid nanoparticles (SLNs) have gained increasing attention as promising colloidal carrier systems.¹¹⁴ It was reported that p53 gene/cationic lipid complexes was used for the treatment of early endobronchial cancer.¹¹⁵ In spite of being low potent compared to those of viral vectors, cationic lipids may present advantages in the context of long-term administration to multiple tumor sites. Most nonviral gene delivery systems that are being considered show no immunogenicity.^116-118

13.5. Brain Tumor:

SLN are Lipid-based nanoscale compounds which are developed for brain tumor drug delivery. They are prepared by high-pressure homogenization or by micro-emulsion of solid physiologic lipids.¹¹⁹ But the exact mechanism by which SLN’s cross the BBB and BTB is unknown. The process of endocytosis occurs to the SLN surface by the adsorption of circulating plasma proteins.¹²⁰ The lipid matrix of SLN is useful in loading of drugs which are protected from degradation. The unloading of drugs can be maintained depending on the surface coating of the SLN and its constituent lipids.¹²¹ Nanoparticles which are coated with the polysorbate (Tween) are useful in transport of drugs across the BBB.¹²² SLN are useful in both preoperative and intraoperative brain tumor detection. The incidence of primary brain tumors in the United States has been estimated at approximately 43,800 per year.^123-125 There has been a rapid expansion of the application of nanodevices to the diagnosis and treatment of brain tumors, since the application of nanotechnology was proposed.¹²⁶ A wide variety of nanoparticle targeting options have been reported including peptides, cytokines, drugs, antibodies and ferromagnetic agents. Nanoparticles when administered systemically they are cleared swiftly by the reticuloendothelial system. This process involves phagocytosis by macrophages, opsonisation of nanoparticles, and uptake in the liver and spleen.¹²⁷ Clearance of nanoparticles can be partially blocked by the attachment of hydrophilic molecules to their surface (reticuloendothelial system).¹²⁸ However, common agents which are preferred to achieve hydrophilic coatings are polyethylene glycol or pluronic can be immunogenic.¹²⁹ Passage of the BBB on cerebral endothelial cells was possible by the toxic effect of nanoparticles (about 200 nm),¹³⁰ although for similar nanoparticles (about 300 nm) this was contradicted in another study.¹³¹ In addition this effect was not found for a different type of nanoparticles.¹³² Physical contact of the drug to the nanoparticles was necessary for drug delivery into the brain.¹³¹ Also other SLN like manganese oxide by the olfactory route was shown to translocate to the brain,¹³³ based on measurements of manganese in different parts of the brain.

13.6. Gastro-intestinal Cancer:

In gastrointestinal cancers, drugs are administered by oral route in the form of SLP. They were introduced as a novel drug carrier system for oral delivery.¹³⁴The adhesive properties of nanoparticles cause reduction or minimize erratic absorptionand increase bioavailability.¹³⁵ Several mechanisms cause absorption of nanoparticles through mucosa of the intestine namely through the Peyer’s patches, by intracellular uptake or by the paracellular pathway. Pinto and Muller in 1999investigated SLN release for oral administration and incorporated SLN into spherical pellets.¹³⁶ SLN powders can be put into capsules, incorporated into pellets or compressed into tablets. The conversion of the liquid dispersion into a dry product is necessary for some of these applications by spray-drying or lyophilization.^{136, 137}The stability of colloidal carriers in GI fluids is essential in order to predict their suitability for oral administration. Critical parameters have been widely overlooked in the design of new and efficient colloidal drug carrier systems for oral use: firstly, their stability upon contact with GI fluids since they are composed of biodegredable materials and particle size in nanorange maximizes the surface area for enzymatic degradation.¹³⁸ secondly, particle aggregation due to environmental conditions of the GI tract causing decrease in the interaction capability of particles.¹³⁹

13.7. Nanoparticle and quantum dot for cancer treatment:

Nanoparticles in the field of cancer research with the help of nanotubes, liposomes dendrimers and polymers have recently improved diagnosis, targeting and drug delivery.^140-142 other nanoparticles, such as quantum dots, possess excellent photo physical properties.¹⁴³ They are one of the most rapidly evolving products of nanotechnology, with great potential as a tool for biomedical and bio analytical imaging. Their physical properties and sometimes multifunctional surfaces are suitable for applications in various biological models.^144,145 Semiconductor quantum dots and nanoparticles composed of metals, lipids or polymers have emerged with applications for early detection and therapy of cancer. Quantum dots are commonly made of cadmium contained semiconductors. This is toxic to living cells, and humans, potentially hazardous, and is not yet systematically investigated. Therefore, search for less toxic materials with similar targeting and optical properties are done. Cancer treatment requires high accuracy in delivering ionizing radiation to reduce toxicity to surrounding tissues. For production of radicals upon absorption of visible light in developing photosensitizing quantum dots recently some research has been focused. This approach is suitable to treat only superficial tumours, as visible light is safe. ^146,147

FUTURE TRENDS:

SLN constitute an attractive colloidal drug carrier system due to successful incorporation of active compounds and their related benefits. Although most of the technologies have focused on the delivery of single chemotherapeutic agents to the tumors, it is increasingly becoming clear that an integrative approach may work better than a reductionist approach. Nanotechnology platforms can provide the unique niche within this space by enabling multimodal delivery with a single application. Although SLN’s may be used for drug targeting, when reaching the intended diseased site in the body the drug carried needs to be released. So, for drug delivery biodegradable nanoparticle formulations are needed as it is the intention to transport and release the drug in order to be effective. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. Nevertheless, we believe that the next few years are likely to see an increasing number of nanotechnology-based therapeutics and diagnostics reaching the clinic.

CONCLUSION:

Solid lipid nanoparticle, although in its nascent stage, has a great potential to cure the cancer, with least side effects. It is the technology that will grow in years to come, and probably, the human race will have a 100% cure to cancer. Anticancer drugs are more diverse class of drugs in terms of physicochemical properties and molecular structure. SLN are new class of drug carrier systems having ability to incorporate both lipophilic and hydrophilic anticancer drugs. A number of anticancer drugs loaded SLN have been formulated and tested for cytotoxic studies, pharmacokinetic parameters, bio distribution studies and compared them with conventional drug formulations. Results showed that their superiority over conventional formulations. In future various modified forms of SLN include stealth SLN, NCL, PLN, targeted SLN, LDL deliveries designed and developed perfectly to improve the therapeutic efficacy, selective targeting and to reduce side effects of various anticancer drugs for chemotherapy.

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Received on 19.04.2013 Modified on 10.05.2013

Research J. Pharm. and Tech. 6(8): August 2013; Page 825-837