Solid Lipid Nanoparticles: Tuneable Anti-Cancer Gene/Drug Delivery Systems

With the advent of multifunctional nano delivery systems, simultaneous imaging and therapy aspires to detect and treat tumors at a very early stage with promising out‐ comes. In this context, numerous anti-cancer drug/gene delivery systems have been ex‐ plored with the primary aim to increase the treatment efficacy without compromising safety. Secondary goals include enhancing bioavailability, specific targeting, apart from the enhanced stability of the formulation [1]. The multifaceted applications of nanoparti‐ cles are the direct result of their ability to deliver high pay loads of drugs or biomarkers to the desired sites within the body. Design and development of tumor specific nanopar‐ ticles could significantly amplify the delivering capacity to a specific target of interest, without affecting healthy cells [2]. Technological advances in nanomaterials and nano‐ technology have paved the way for several carriers such as liposomes [3], dendrimers [4], and micelles [5], solid lipid nanoparticles (SLN) [6] and recently nanostructured lipid carriers [1, 7]. Polymeric micelles, or nanosized (~10–100 nm) supramolecular constructs composed of amphiphilic block-copolymers, are emerging as powerful drug delivery ve‐ hicles for hydrophobic drugs. Liposomes are currently the most popular nanosized drug delivery systems, with one or several lipid bilayers enclosing an aqueous core. Liposomeencapsulated formulations of doxorubicin earlier approved for the treatment of Kaposi’s sarcoma, are now used against breast cancer and refractory ovarian cancer. Breast cancer in particular has been the focus of many studies involving liposome-based chemothera‐ peutics, in part due to the clinical success of various drugs such as Doxil, which is a lip‐ osomal formulation currently used to treat recurrent breast cancer [7]. The anthracycline doxorubicin is the active cytotoxic agent and is contained within the internal aqueous core of the liposome. The encapsulation of doxorubicin within liposomes significantly re‐


Introduction
With the advent of multifunctional nano delivery systems, simultaneous imaging and therapy aspires to detect and treat tumors at a very early stage with promising outcomes.In this context, numerous anti-cancer drug/gene delivery systems have been explored with the primary aim to increase the treatment efficacy without compromising safety.Secondary goals include enhancing bioavailability, specific targeting, apart from the enhanced stability of the formulation [1].The multifaceted applications of nanoparticles are the direct result of their ability to deliver high pay loads of drugs or biomarkers to the desired sites within the body.Design and development of tumor specific nanoparticles could significantly amplify the delivering capacity to a specific target of interest, without affecting healthy cells [2].Technological advances in nanomaterials and nanotechnology have paved the way for several carriers such as liposomes [3], dendrimers [4], and micelles [5], solid lipid nanoparticles (SLN) [6] and recently nanostructured lipid carriers [1,7].Polymeric micelles, or nanosized (~10-100 nm) supramolecular constructs composed of amphiphilic block-copolymers, are emerging as powerful drug delivery vehicles for hydrophobic drugs.Liposomes are currently the most popular nanosized drug delivery systems, with one or several lipid bilayers enclosing an aqueous core.Liposomeencapsulated formulations of doxorubicin earlier approved for the treatment of Kaposi's sarcoma, are now used against breast cancer and refractory ovarian cancer.Breast cancer in particular has been the focus of many studies involving liposome-based chemotherapeutics, in part due to the clinical success of various drugs such as Doxil, which is a liposomal formulation currently used to treat recurrent breast cancer [7].The anthracycline doxorubicin is the active cytotoxic agent and is contained within the internal aqueous core of the liposome.The encapsulation of doxorubicin within liposomes significantly re-duces the cardiotoxicity that commonly results from the use of unencapsulated anthracyclines by decreasing the amount of the drug being delivered to the heart [7].As such, patients can receive much higher doses of the chemotherapeutic in the liposomal formulation compared to unencapsulated, thereby allowing tumor tissue to potentially be exposed to a lethal dose of the drug while minimizing deleterious side effects.This inherent advantage associated with the use of liposomes as drug delivery vehicles also serves to minimize the many other toxic side effects associated with doxorubicin including gastrointestinal toxicity and complications arising from myelosuppression.
Each delivery system however, has its advantages and limitations.Advantages afforded for drug delivery include the presence of an inner core for lipophilic drug entrapment, as well as a hydrophilic outer shell that prevents particle aggregation and opsonisation [8].This complexation prevents uptake by the reticuloendothelial system (RES), thereby improving circulation times which, combined with nanoscale sizing, confers preferential accumulation in tumor tissue.In general, nanovectors can be targeted to tumors by passive and active targeting approaches, where a passive strategy takes advantage of a nanvector's small size permitting it to penetrate and accumulate in the tumor.Most solid tumors are sustained by extensive angiogenesis leading to hypervascular tissue with an incomplete vascular architecture.They also have an impaired lymphatic drainage and an increased production of permeability factors resulting in the accumulation and inefficient clearance of nanoparticles leading to the enhanced permeability and retention effect [9].The hyperpermeable nature of tumor vasculature is characterized by a pore cut off size ranging between 380 and 780 nm allowing particles less than 780 nm to extravasate into the tumor interstitium [10].In addition, active targeting to various tissues may be achieved via utilization of ligands on the surface of nanoparticles, reducing the side effects to the normal tissue by limiting drug/gene distribution to the target organ [11].An excellent example is Abraxane, an albumin bound nanoparticle formulation of Paclitaxel (PTX), approved by FDA in January 2005 for the treatment of metastatic breast cancer.Abraxane has been shown to outperform standard PTX in the treatment of breast cancer [12].Utility of this drug was initially limited due to its poor aqueous solubility [13], requiring use of an excipient, Cremophor, which is satisfied by novel engineered nanovectors.A recent Gynecologic Oncology Group Phase II evaluation of albumin-bound paclitaxel nanoparticles to treat recurrent or persistent platinum-resistant ovarian, fallopian tube, or primary peritoneal cancer, concluded that these nanoparticles are as effective and tolerable in their cohort of refractory ovarian cancer patients previously treated with paclitaxel [14].Nanoparticles fabricated with albumin [15], poly(lactic-co-glycolic acid) [16] and poly lactic acid have also been loaded with PTX and used to passively target tumors.Albumin has been shown to be nontoxic, non-immunogenic, biocompatible and biodegradable making it an ideal candidate to fabricate nanoparticles for drug delivery.Site-specific drug delivery allows for the clinical translation of chemotherapeutic agents with safer targeted cell killing, that are otherwise abandoned due to insolubility, toxicity and safety concerns.Moreover, these new delivery devices can preferentially confine treatments to tumors within the nodal space while sparing healthy tissues.

Solid lipid nanoparticles
Solid lipid nanoparticles [17] or lipospheres are rapidly emerging as new class of safer and efficient gene/drug delivery vectors.SLNs are sub-micron colloidal carriers, ranging from 50 nm to 1 μm, that are composed of physiological lipid dispersed in water or in aqueous surfactant solution (Figure 1).SLNs function as an alternative drug carrier system to other novel delivery approaches such as emulsions, liposomes, and polymeric nanoparticles.SLNs offer several advantages conferred by their colloidal dimensions including: i) feasibility of incorporation of lipophilic and hydrophilic drugs; ii) improved physical stability; iii) controlled release; iv) improved biocompatibility; v) potential for site specific drug delivery; vi) improved drug stability; vii) better formulation stability; viii) the ability to freeze dry and reconstitute; ix) high drug payload; x) controllable particle size; xi) the avoidance of carrier toxicity; xii) low production cost; and xiii) easy scale-up and manufacturing [18].In addition, significant toxicity and acidity associated with a number of biodegradable polymeric materials are not observed with SLNs.And, in contrast to emulsions and liposomes, the particle matrix of SLNs is composed of solid lipids.SLNs can be prepared using wide variety of lipids including lipid acids, mono-(glycerol monostearate), di-(glycerol bahenate) or triglycerides (tristearin), glyceride mixtures or waxes (e.g.cetyl palmitate) and stabilized by the biocompatible surfactants(s) of choice (non-ionic or ionic).Lipids most commonly used are triglyceride esters of hydrogenated fatty acids, including hydrogenated cottonseed oil (Lubritab™ or Sterotex™), hydrogenated palm oil (Dynasan™ P60 or Softisan™ 154), hydrogenated castor oil (Cutina™ HR), and hydrogenated soybean oil (Sterotex™ HM, or Lipo™) as typical examples [19].Various emulsifiers and their combination (Pluronic F 68, F 127) have also been added to stabilize the lipid dispersion by more efficiently preventing particle agglomeration.The disavantages associated with SLNs relate mostly to their preparation, which generally involves high pressure and rapid temperature changes that can lead to high pressure-induced drug degradation, lipid crystallization, gelation phenomena and the co-existence of several colloidal species [20].The drug loading capacity of a conventional SLN is limited by the solubility of drug in the lipid melt, the structure of the lipid matrix and the polymeric state of the lipid matrix.If the lipid matrix consists of highly similar molecules (i.e.tristearin or tripalmitin), a perfect crystal with few imperfections is formed.Since incorporated drugs are located between fatty acid chains, between the lipid layers and also in crystal imperfections, a highly ordered crystal lattice cannot accommodate large amounts of drug.This may also lead to the fast release of a large dose of drug initially, generally known as "burst effect", followed by slow and incomplete release of drug.Since high lipid crystallinity is the major cause of burst release of drug from SLNs, this undesirable phenomenon may be minimized by choosing lipids that do not form good crystals, including mono-or di-glycerides, or triglycerides with chains of different lengths.For this reason, in formulation design use of more complex lipids is recommended for higher drug loading.Nanostructured lipid carriers or NLCs were designed to overcome these disadvantages with the main goal to increase drug loading and prevent drug expulsion [21].For NLCs, the highest drug load could be achieved by mixing solid lipids with small amounts of liquid lipids (oils).These types of NLCs are called multiple types NLC, and are analogous to w/o/w emulsions since it is an oil-in-solid lipid-in-water dispersion.

SLN preparation methods
There are two main established SLN synthesis techniques, namely, the high-pressure homogenisation technique described by Müller and Lucks [21], and the microemulsion-based technique described by Gasco [22,23].SLNs are prepared from lipid, emulsifier and water/ solvent using different methods, discussed below.

High Pressure Homogenization (HPH)
High Pressure Homogenization (HPH) is a very reliable technique in the production of SLNs.High pressure homogenizers are employed to push a liquid with high pressure (100-2000 bar) and the fluid accelerates on a very short distance to very high velocity (>1000 Km/h).. Very high shear stress and cavitation forces disrupt the particles down to the submicron range.Generally 5-10% lipid content is used but up to 40% lipid content has also been investigated.Typical SLNs production conditions are 500 bar and two or three homogenisation cycles.Two general approaches of HPH are hot and cold homogenization, both working on the same concept of mixing the drug in bulk of lipid melt.

Hot homogenization
Hot homogenization is carried out at temperatures above the melting point of the lipid and can therefore be regarded as the homogenization of an emulsion (Figure 2).A pre-emulsion of the drug loaded lipid melt and the aqueous emulsifier phase (same temperature) is obtained by high-shear mixing device.The quality of the pre-emulsion affects the quality of the final product to a great extent and it is desirable to obtain droplets in the size range of a few Novel Gene Therapy Approaches micrometers.HPH of the pre-emulsion is carried out at temperatures above the melting point of the lipid.Usually, lower particle sizes are obtained at higher processing temperatures because of lowered viscosity of the lipid phase [24].
Hot homogenisation is the most frequently applied technique in which even temperature sensitive compounds can be processed because of the short exposure time to the elevated temperatures [25].However, high temperatures increase the degradation rate of the drug and the carrier.Increasing the homogenization pressure or the number of cycles often results in an increase of the particle size due to high kinetic energy of the particles.The cold homogenisation technique is therefore recommended for extremely temperature sensitive compounds and hydrophilic compounds, which might partition from the liquid lipid phase to the water phase during the hot homogenisation.

Cold homogenization
During cold homogenization, the drug containing lipid melt is cooled and, after solidification, the lipidic mass is ground to yield lipid microparticles [26].The lipid microparticles are dispersed in cold surfactant solution by stirring, yielding a macro-suspension.This suspension is then passed through a high-pressure homogeniser at or below room temperature, where the microparticles are broken down to solid lipid nanoparticles.However, compared to hot homogenization, larger particle sizes and a broader size distribution are typical of cold homogenized samples.

Ultrasonication or high speed homogenization
SLNs are also developed by high speed stirring or sonication [27].The ultrasonic dispersion may offer an appropriate alternative for laboratory scale productions due to its rapid nature and the relatively low cost of required apparatus.So far, its suitability has only been evaluated for SLN [17,28].The primary disadvantage of this method is the broader particle size distribution that is yielded, ranging into the micrometer range.Potential metal contamination due to ultrasonication is another issue presented by this method.To generate more stable formulations, high speed stirring and ultrasonication may be used in combination at high temperature.

Solvent emulsification/evaporation
In this method, the lipidic material, such as glyceride is dissolved in an organic solvent (e.g.chloroform, cyclohexane) and the solution is emulsified in an aqueous phase [29].After evaporation of the solvent the lipid precipitates to form nanoparticles with a mean diameter of around 30 nm using cholesterol acetate as a model drug and lecithin/sodium glycocholate blend as an emulsifier [30].The solution is emulsified in an aqueous phase by high pressure homogenization and the organic solvent is removed from the emulsion by evaporation under reduced pressure (40-60 mbar).

Supercritical fluid
This platform technology, with several variations for powder and nanoparticle preparation, is a relatively new technique for SLN production and offers the advantage of solvent-less processing [31].SLNs can be prepared by the rapid expansion of supercritical carbon dioxide solutions (RESS) method, where carbon dioxide (99.99%) is a good choice as solvent.

Microemulsion method
This method is based on the dilution of microemulsions that are two-phase systems composed of an inner and outer phase (e.g.o/w microemulsions) [32].They are made by stirring an optically transparent mixture at 65-70°C, which typically composed of a low melting fatty acid (e.g.stearic acid), an emulsifier (e.g.polysorbate 20), co-emulsifiers (e.g.butanol) and water.The hot microemulsion is dispersed in cold water (2-3°C) with stirring.SLN dispersion can be used as granulation fluid for transferring into solid product (tablets, pellets) by granulation process, but in case of low particle content, excess water must first be removed.High-temperature gradients facilitate rapid lipid crystallization and prevent aggregation.Due to the dilution step, achievable lipid contents are considerably lower compared with the HPH based formulations.Novel Gene Therapy Approaches

Spray drying method
Spray drying is an alternative procedure to lyophilization in the transformation of an aqueous SLN dispersion into a solid drug product.This method results in particle aggregation due to high temperature, shear forces and partial melting of the particle.The use of lipid with melting point >70°C for spray drying is recommended [33].Best results are obtained with an SLN concentration of 1% in a solution of trehalose in water or 20% trehalose in ethanol-water mixtures (10/90 v/v).

Double emulsion method
For the preparation of hydrophilic loaded SLN, double emulsion method, a novel approach based on solvent emulsification-evaporation can be employed.Here, 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 [34]

SLNs cellular uptake, pharmacokinetics and bio-distribution
Research on cellular uptake mechanisms has repeatedly demonstrated that endocytosis is the preferred route of internalization of non-viral gene vectors via a number of distinct endocytic processes.The most studied mechanisms include macropinocytosis, circular dorsal ruffles, clathrin-mediated endocytosis and several clathrin-independent endocytic pathways [35].Endocytic uptake mechanisms are highly dependent on cell type and on the nature of gene vectors [36].Clathrin-mediated processes are limited to particles under 200 nm in size, whereas caveolae-dependent uptake prevails for particles between 200 and 500 nm [37].The prevalent pathway for the cell internalization of PEI polyplexes is however, clathrin-dependent [38].
Apart from overcoming cellular barriers of uptake, an anticancer drug must be specifically targeted to the tumor in order to maximize its therapeutic effect, and therefore biodistribution studies are critical to assess the safety of a nanomedicine.However, since most groups work on healthy instead of tumor-bearing animals, it is difficult to confirm whether SLNs can lead to increased tumor drug concentrations by way of enhanced permeability and retention [9].Recently, Zhang et al. (2010) evaluated antitumor efficacy of docetaxel-loaded solid lipid nanoparticles (DSN) in a murine ovarian cancer model [39].In this study, SLN biodistribution from RES more toward the circulation system was observed.Moreover, SLNs in comparison to the free drug demonstrated more potent in vivo anti-ovarian cancer activity with improved pharmacokinetics.In contrast, paclitaxel loaded in pegylated solid lipid nanoparticles were mainly taken up by the RES after intravenous administration in rats, showing 8fold and 3-fold higher levels in liver and spleen, respectively, 8 h after administration compared to paclitaxel in Taxol® [40].Moreover, paclitaxel levels in kidney, heart and lung were indistinguishable between the two formulations.The difference in biodistri-bution of SLNs reported in literature may be due to several factors including variations in size, surface functionalization and composition.
The biodistribution of an anticancer drug delivered by SLN may be further manoeuvred by route of injection to achieve the desired therapeutic goal.Harivardhan Reddy et al. (2005) compared the biodistribution of free 99 m Technetium-labeled etoposide and radio-labeled etoposide loaded SLNs in Dalton's lymphoma tumor-bearing mice [41].They showed that administration via the subcutaneous route resulted in high tumor uptake of etoposide and etoposide loaded tripalmitin nanoparticles and was the preferred route as compared to intravenous or intraperitoneal administration.However, elevated tumor drug concentrations were also found with intravenously administered etoposide loaded SLN in comparison to the free drug, (approximately 67% increase 1 h post-injection, 30% increase 24 h post-injection).In yet another study by Zara et al. (2002) duodenal administration of idarubicin-loaded SLN led to higher bioavailability than intravenously administered SLNs [42].Also, idarubicin and its main metabolite, idarubicinol, were detected in the brain after IDA-SLN administration, indicating that the SLNs were able to pass the blood-brain barrier; an attractive attribute in the tratement of brain tumors.Thus, the route of administration of SLN formulation is a key consideration in the design of animal or clinical anti-cancer drug delivery studies.

SLNs as anti-cancer gene/drug delivery vectors: Challenges and successes
Solid lipid nanoparticles have rapidly established themselves during the past decade as stable, reliable and easy to produce vectors.SLN advantages over other existing transfection vectors include safety, good storage stability, possibility of lyophilization and a high degree of flexibility in design and optimization [25].Cationic SLNs can efficiently bind DNA directly via ionic interaction and mediate gene transfection.However, as with all non-viral vectors, many cellular obstacles have to be overcome to achieve satisfactory levels of transfection activity: i) binding to the cell surface; ii) cellular internalization; iii) escape from the endolysosomal compartment; and iv) translocation through the nuclear envelope.In order to surmount these barriers, cationic SLNs are designed as multifunctional "smart" carriers for efficient gene expression [43].Components such as chitosan [44] and surface functionalization moieties e.g.poly(styrene-4 sodium sulfonate) (PSS) and poly(L-lysine hydrochloride) (PLL) [45], folate-chitosan and cholesterol derivative (CHETA) [46], cetyltrimethyl ammonium bromide (CTAB) [47] and a phyto-ceramide [48] and TAT peptides [49], may each individually assist in overcoming the barriers of efficient transfection.In addition, protamine a cationic small protein rich in arginine exerts both DNA condensation activity and proton sponge effect facilitating endosomal escape as well as assisting nanovectors to enter the nucleus owing to its nuclear localization signal (NLS) [50].Nanovectors offer the potential to both detect and treat cancer at a very early stage, thereby maximizing survival rates.The NCI (National Cancer Institute) Alliance for Nanotechnology in Cancer provides up-to-date information in nano-cancer research and its promise for cancer diagnosis and treatment (http://nano.cancer.gov/).Using siRNA molecules loaded in nanovectors, early proof-of-principle experiments in various tumor cells suggest that RNA silencing may have great potential as a strategy for treating cancer.However, siRNA therapeutics are hindered by poor intracellular uptake, limited blood stability and undesirable non-specific immune stimulation [61].An interesting strategy used to target the vector employs threeamino-acid peptide, arginine-glycine-aspartic acid (known by its amino acid code RGD) that binds to integrins, which in turn are involved in angiogenesis, tumor cell growth, metastasis, and inflammation.Intravenous administration into tumor-bearing mice of nanoparticles combined with a dual strategy of siRNA inhibiting vascular endothelial growth factor receptor-2 and RGD peptide ligand attached at the distal end of the polyethylene glycol [40], conferred selective tumor uptake, and inhibition of both tumor angiogenesis and growth rate, achieving both tissue and gene selectivity [62] .This targeted nanoparticle-based compound called "BIND-014" is currently the first one to enter clinical trial, although with small number of only 17 patients.Patients with advanced or metastatic cancer receive an injection of the nanodrug once every three weeks and are showing signals of efficacy even at relatively low doses.This initial but positive result shows promise and the potential impact of nanomedicines as a paradigm shift in the treatment of cancer.
Very recently, Vighi E et al. (2012) developed a multicomponent cationic SLN as a pDNA delivery vehicle.The formulations were prepared using stearic acid as the main component in the lipid phase, stearylamine, the main component in the aqueous phase, as cationic agent and protamine as transfection promoter along with the phosphatidylcholine (SLN-PC), cholesterol (SLN-Chol) or both (SLN-PC-Chol).Transfection results on various cell lines in this study revealed the best transfection for SLN-PC-Chol on COS-1 cells (African green monkey kidney cell line) [64].However, lower transfection levels than poly [62] were observed on HepG2 cells (human hepatocellular liver carcinoma cell line), regardless of the SLN composition.Using COS-1 monkey kidney fibroblast-like cells, SLNs and liposomes formulated from the same cationic lipids, demonstrated equipotent in vitro transfection efficiencies [65].This study suggests that only the lipid composition in the tested lipid-based formulations affected transfection efficiencies.The intrinsic toxicity that is common in cationic gene delivery vehicles may also be minimized, while maintaining high transfection efficiency, by selecting Novel Gene Therapy Approaches good combinations of two-tailed cationic lipids and matrix lipids.Hence, structural or compositional design changes of nanovectors may influence the outcome in relation with cell physiology, cell internalization pathways and transfection efficiency.The above results support the use of SLNs to serve as nano/microcarriers for anti-cancer gene therapies.
Under optimised conditions SLNs can be designed to incorporate lipophilic or hydrophilic drugs and seem to fulfil the requirements for an optimum particulate carrier system.Stability studies were performed on SLNs loaded with all-trans retinoic acid (ATRA), another compound that is sensitive to light, heat and oxidants, and quickly degrades into less active products such as isotretinoin and all-trans-4-oxo [66].After 3 months of storage at 4 °C, more than 90% of the ATRA drug molecules in SLN remained chemically intact.This can be compared to approximately 50% drug degradation when stored at the same temperature in the form of methanol solution or 1% polysorbate-80 solution for only 1 month.Hence, SLNs are useful for the protection of anticancer compounds that are sensitive to light, and probably heat and oxidants as well.In a study conducted by our group, modulatory effects of encapsulated and free forms of sesamol (anti-oxidant and anti-cancer compound) were evaluated by the topical delivery systems in a skin cancer mice model.Both free sesamol and SLN dispersion were applied as gels (using 1% w/v of Carbopol 934P®) on the skin of mice.Encapsulated or nanosesamol was found to safely exert chemopreventive effects by decreasing the lipid peroxidation levels and increasing the anti-oxidant levels, thereby decreasing the development and promotion of skin tumors.Immunofluorescence studies of pro-and antiapoptotic markers, bcl-2 and bax protein expression revealed higher expression of antiapoptotic protein, bcl-2, in the tissue sections of tumor bearing mice in comparison to their control counterparts and groups which received sesamol treatment, reinforcing the role of bcl-2 in skin carcinogenesis.Higher expression of bax was also observed in sesamol treated animals as compared to the tumor bearing mice.Up-regulation of bax in the control and sesamol treated groups suggests that it follows the intrinsic pathway of apoptosis (unpublished results).
Ongoing work by our group compared neutraceutical curcumin-loaded SLNs to the free form as a chemopreventive topical delivery system in 7,12-dimethylbenz [a]anthracene (DMBA) induced skin cancer model mice.In order to understand the molecular events underlying nanocurcumin-mediated chemoprevention, protein expression of various biomolecules e.g.anti and pro inflammatory cytokines (Il-4 and Il-1β) were analyzed by Western immunoblotting and immunoflourscence.For cancer induction, male Balb/c mice were subcutaneously injected with 30 mg/Kg body weight of DMBA (in olive oil) once a week for three weeks.DMBA skin cancer induced mice were topically applied free and encapsulated curcumin (50mg/Kg b.w) as a chemopreventive agent from one week before DMBA injection to the experiment's end (18 weeks).We found that free and nanocurcumin treatment of DMBA treated mice reduced the levels of malondialdehyde, a by-product of lipid degradation (Figure 3).Antioxidant analysis revealed increased levels of enzymes (SOD, Catalase, Reduced Glutathione, Total Glutathione) in encapsulated nanocurcumin treated group as compared to free curcumin group (Figure 4-7).Immunofluorscence studies and western blot analysis of Il-4 and Il-1β suggest enhanced anti-inflammatory potential of encapsulated curcumin in comparison to mice treated with free curcumin.Mice bearing skin tumors showed increased expression of pro-inflammatory interleukins when compared to the control, which was decreased on treatment with curcumin (Figure 8).Furthermore, the immunoflourscence assay of antiinflammatory interleukin (IL-4) showed a far greater increase in IL-4 expression by topical treatment with encapsulated curcumin as compared to the free curcumin in mice bearing skin tumors (Figure 9).

Conclusion
Solid Lipid Nanoparticles serve as efficient and safe DNA/ drug loaded nanosystems in both the imaging and treatment of cancer.Traditional drug delivery systems are often hindered by their low bioavailabilty, low solubility, toxicity and rapid clearance.In future, clinicians and researchers will be able to "tune and time" the amount of DNA/Drug delivery by controlling the release at specific location thereby minimizing their toxicity and side-effects.

Figure 2 .
Figure 2. Partitioning effects on drug during the hot homogenization technique production of SLNs.Left: Partitioning of drug from the lipid phase to the water phase at increased temperature.Right: Re-partitioning of the drug to the lipid phase during cooling of the produced O/W nanoemulsion.Source: Muller RH et al.Solid lipid nanoparticles (SLN) for controlled drug delivery -a review of the state of the art.European Journal of Pharmaceutics and Biopharmaceutics 50: (2000) 161-177.

Table 1 .
Table 1 lists some of the successful SLN formulations evaluated as anti-cancer agents in various cancers.Solid Lipid Nanoparticles loaded with DNA/ Drug as anti-cancer delivery systems in various cancers.Solid Lipid Nanoparticles: Tuneable Anti-Cancer Gene/Drug Delivery Systems http://dx.doi.org/10.5772/54781 Hrkach et al. (2012)Calando Pharmaceuticals, in Pasadena, Canada, and the National Cancer Institute (NCI) entered into a collaborative development program for a nanoparticle-based siRNA therapeutic aimed at treating neuroblastoma, the most common extracranial solid tumor in children less than five years of age.Previous attempts to develop targeted nanoparticles were unsuccessful due to the inherent difficulties of designing and scaling up a particle capable of targeting, long-circulating via immune-response evasion and controlled drug release.Very recently,Hrkach et al. (2012)reported the preclinical development and clinical translation of a docetaxel nanoparticle with prostate-specific membrane antigen, a clinically validated tumor antigen expressed on prostate cancer cells and on the neovasculature of most non-prostate solid tumors including breast, head, lung, neck, prostate and stomach[63]