SiRNA showntremendous interest in siRNA technology in cancer

SiRNA anti-cancer drugs utilize endogenous RNAi mechanisms to silence oncogene expression, which promotescancer remission. RNAi might constitute a novel therapeutic approach for cancer treatment because researchers caneasily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumorinitiation and progression. However, the limited cellular uptake, low biological stability, and unfavorablepharmacokinetics of siRNA have limited their application in the clinic. Indeed, blood nucleases easily degradenaked siRNAs, and the kidney rapidly eliminates these molecules. Furthermore, at the level of target cells, thenegative charge and hydrophilicitty of siRNAs strongly impair their cellular internalization. Therefore, thetranslation of siRNA to the clinical setting is highly dependent on the development of an appropriate deliverysystems, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, nanoparticles(NPs) and their formulations provide immense opportunities to substantially alter the treatment regimens. Theadvantages of using NPs for siRNA delivery, particularly at a systemic level, include i) the prolonged half-life ofsiRNA in blood, ii) improved pharmacokinetics, and iii) preferential targeting of tumor tissues by an EnhancedPermeation and Retention (EPR) effect. The purpose of this review is to explore current developments in shortinterfering RNA (siRNA) delivery systems in nanooncology, in particular NPs that encapsulate siRNA for targetedtreatment of cancer. siRNA has a high specificity towards the oncogenic mRNA in cacer cells, while application ofNPs can improve stable delivery and enhance efficacy.In 1998, Fire et al. 1 discovered the ability of double-stranded RNA (dsRNA) to silence gene expression inCaenorhabditis elegans. In 2001, Elbashir et al. 2 demonstrated that synthetic small interfering RNA (siRNA)could knock down a gene in a sequence specific manner. Further, he in vivo therapeutic potential of siRNA wasdemonstrated in transgenic mice with a sequence from hepatitis C virus 3. Several others 4,5 have showntremendous interest in siRNA technology in cancer therapy in particular. RNAi is one of the fastest advancing fieldsin biology. In 2004, the technology entered a Phase I clinical trials in humans for wet agerelated maculardegeneration 6,7. In 2010, the first-in-human Phase I clinical trial was started 8, against solid tumors using atargeted NP delivery system.Although siRNA is thought to be more effective in treating disease compared to other methods 9, severalchallenges are associated with delivering small interfering RNAs (siRNAs) to diseased sites for gene therapy 10.Two main approaches for the delivery of RNAi molecules have been developed: viral and non-viral vectors.However, NPs have recently received attention for use in siRNA. The expansion of novel NPs for drug delivery isan exciting and challenging research filed, in particular for the delivery of emerging cancer therapies, includingsmall interference RNA (siRNA) and microRNA (miRNAs)-based molecules. The paradigm shift to the use of NPsfor RNAi molecules delivery is attributed to unique benefits provided by NPs in comparison to other carriers.Nanoparticles as carrier for siRNALimitations to clinical application of naked siRNA drugs in oncology exist because of their physicochemicalproperties 11. Consequently, encapsulation of siRNA with NPs will help shield the siRNA from plasmaticnucleases and immune responses thus assisting in endocytosis and successful siRNA delivery. Furthermore ligandbound NPs increase selectivity of NP delivery of siRNA to tumor cells 12. The constituents of nanocarriers appliedto siRNA can be fold into three categories. Such as,Delivery of siRNA-Nanoparticles:Recently, NPs have received considerable attention as vectors for gene delivery 13. NPs are particulatedispersions or solid particles with particle sizes in the range of 10–1000 nm. One advantage of using NPs fordelivery is the enhanced permeability and retention (EPR) effect, which enables nanocarriers to accumulate intumors at much higher concentrations than in normal tissues 14. Nanocarriers can protect RNAi moleculesfrom enzymatic degradation and immune recognition, have much higher transportation efficiency across the cellmembrane compared to other carriers 15, and can prevent excretion if the carrier size and surface coating areappropriate 16.With the avalanche of research using NPs, exciting possibilities and workable outcomes may resolve most of thecurrent challenges in drug delivery. This is also likely to remove the hurdles to discovering the “magic bullet” forcancer, by changing unprofitable treatment regimens. With the advent of nanotechnology, a number of NPtherapeutics has been made commercially available. Here are some examples,Achieving Target and Releasing siRNAGrowing tumors induce angiogenesis. Due to rapid angiogenesis, tumor blood vessels are often irregular, leaky andhave poorly organized endothelial cells with large fenestrations which can be as larger than 260 nm. Tumor tissue iseasily infiltrated by macromolecules, NPs and liposomes. Macromolecule retention within tumors is favored due tothe poor venous return in tumor tissue and limited lymphatic drainage. This is the Enhanced Permeability andRetention (EPR) effect leading to the development of passive targeting strategy 17. Delivery systems that promotespecific tumoural uptake would lead to increased therapeutic efficacy and reduction of side effects. Hence, siRNANPscan easily enter into target tumor cells. The extensive and leaky vasculature of tumor capillaries can be exploited to allow for a natural and selective accumulation of long circulating NPs impregnated with anticanceragents within tumor tissues.Understanding the Fate of Delivered siRNAMany fluorescence based techniques have been developed recently. One such method is based on fluorescenceresonance energy transfer (FRET) technique 18. In a different approach FRET-based imaging was used by Jarve etal. in tracking and understanding the siRNA integrity in intracellular environment 19. To study the organ specificdelivery and to monitor the gene silencing efficiency of payload siRNA, a fluorescent dye that have both imagingand targeting capability was conjugated to polymer NPs by Press et al 20. In a different strategy Liu et al.encapsulated red fluorescent protein (RFP) in chitosan NPs that carry siRNA 21. Alabi et al. utilized a FRET-basedapproach in studying the formation, stability and disassembly of NP-siRNA complex 22. Altogether, developmentof many such tools and assays has been very helpful in assessing the transfection efficiency of RNAi, and indetection, quantification and analysis of extracellular and intracellular siRNA. Preclinical Studies:The nanoparticle-based delivery of siRNA can produce multiple advantages. Many studies, as given in the table 2,have identified the efficient applications of RNAi delivered by diverse nanocarriers.1. In Vitro StudiessiRNA delivery agents would be rejected as therapeutic agents if they caused an unacceptable level of toxicity oneither a cellular or systemic level. Cytotoxicity can be measured via a colorimetric assay in which cell viability iscompared between untreated versus treated samples reflecting the level of toxicity to cells 26. Biodegradability isa key feature that will allow the clearance of larger molecular mass materials. The usage of biodegradable, highmolecular mass polymers containing linkages that can undergo intracellular cleavage and degradation can helpreduce cytotoxicity 26. Endocytosis is an evolutionarily conserved process in eukaryotes where extracellularsubstances are internalized by cells through invagination of plasma membrane to form vesicles 27.Macropinocytosis involve plasma membrane extensions called lamellipodia which forms large endocytic vesicles of non -standard size and shape 27. Additionally other endocytic routes have also been identified: phagocytosisand caveolae independent endocytosis. These various endocytic routes vary in coat composition, size of endosomes,and fate of endocytosed NPs 27. Regulation of uptake pathways is possible through adjusting NP size. This isbecause each endocytic pathway is specific to individual NP sizes 27.2. In Vivo StudiesToxicity is a crucial parameter for evaluation of a therapeutic agent prior to clinical use. According to a micemodel, siRNA formulated in the GC4-targeted nanoparticles did not induce IL-6, IL-12, and IFN-? significantlyadditionally aspartate aminotransferase and alanine aminotransferase levels also remained similar to untreatedanimals 28. To determine the therapeutic outcomes, lung metastasis–bearing mice were treated with differentformulations with two consecutive intravenous administrations. The results indicate that the combined siRNAsdelivered by GC4 targeted NPs could inhibit the growth of lung metastasis 28. Another in vivo experimentwas performed which indicates that surviving, an inhibitor of apoptosis protein (IAP) family, which isfrequently expressed in most human cancers and is an attractive therapeutic target 29. Intravenous delivery oforganic NPs complexed with 10 mg of siRNA to mice resulted in approximately 92% size reduction of theprostate tumor 30. Intravenous injections targeting bcl-2 family members were performed in mice with siRNAutilizing both polyethylene glycol (PEG) coated or PEGylated cationic liposomes and atelocollagen complexeslimiting tumor growth by 40–65% 30. Most promisingly, oral administration of AuNP-siRNA-glycol chitosantaurocholicacid nanoparticle to specific delivery of Akt2 siRNA was successfully developed against colorectalliver metastasis where AuNP-siRNA was firstly formed then; biofunctional glycol chitosan-taurocholic acidconjugates enclosed AuNP-siRNA to protect Akt2 siRNA from GI degradation. Hence, Active transportation hasbeen take place via enterocytes that increased selective accumulation in cancerous cells. After oraladministration (animal model), low production of Akt2 had been appeared by means of initiation of apoptosis incancerous cells 34.Limitations Associated with siRNA-NPsEven though, due to advancement of NPs based siRNA delivery, cancer treatment has become more specific,targeted and easy, NP-based drug delivery still has some challenges. First, the vehicles must cross the biologicalbarriers in the mucosa as well as the cellular and humoral components of the immune system, in order to reach thetarget cells. The particles are also sequestered by negatively charged blood serum proteins. Moreover, they are liableto opsonization by immune cells. Even though nanostructures generally undergo rapid clearance, thereticuloendothelial system is able to trap and degrade intravenously injected particles >100 nm in diameter, byactivated monocytes and macrophages. On the other hand, particles <4 nm in diameter usually are able to undergorapid renal clearance. Research indicates that NPs have systemic toxicity, mostly in the liver 32. Again, theheterogeneity of tumors could also induce the development of inherent resistance to some RNAi, owing to factorssuch as somatic mutations, and germline single nucleotide polymorphisms 33.ConclusionsiRNA technology has remarkably progressed from mere academic discovery to a potential class of treatmentmodality against human diseases. Despite siRNA being a relatively new technique, the list of diseases for whichsiRNA is being tested as a therapeutic agent is extensive. Although much has been accomplished, obstacles remainthat will hamper their clinical application. To overcome poor results obtained using single siRNA delivery, possibleconcomitant strategies can be used by combining several siRNA to simultaneously disrupt various pathways orutilizing single siRNA in conjunction with other therapeutic agents. On the other hand,The advent of nanomedicinehas represented a milestone, contributing strongly towards a solution to problems often encountered in the clinicalsetting. However, their delivery remains one of the daunting challenges to the use of siRNA therapeutics in theclinical setting. Even though tremendous progress has been made in the use of siRNA-based nps in cancertherapeutics, the ability to overcome the challenges is still the key indicators of the success of siRNA-based therapy.On the whole, it is expected that siRNA-based nanotherapeutics will be the forerunner in the pool of strategies fordrug development for cancer therapy.