Cancer arises from a single cell that is unable to repair their mutations produced in DNA which leads to abnormal cell division, invasion and metastasis 1. Normal cells can transform into tumor cells due to various reasons, such as genetic factors, radiation, chemical carcinogens such as asbestos, tobacco smoke, BPA and biological carcinogens infections from viruses, bacteria, and parasites. Among cancer’s cervical is the second most common cancer and predominant gynaecological cancer in women, causing most cancer related deaths world over 64 2. There are several factors which contribute to high incidence of this disease are early age of marriage, multiple sexual partners, multiple pregnancies, poor genital hygiene, smoking and use of oral contraceptives. Cervical growth is a malignancy emerging from the cervix 3. It is because of the irregular development of cells that can attack or spread to different parts of the body. Initial stage of development there was no indications are seen. Later indications may incorporate unusual vaginal bleeding and pelvic pain. While bleeding after sex may not be serious, it may also indicate the presence of cervical cancer 4.
Cervical cancer is the commonest cancer cause of death among women in developing countries. Mortality due to cervical cancer is also an indicator of health inequities, 2 as 86% of all deaths due to cervical cancer are in developing countries 5. Every year in India, 122,844 women are diagnosed with cervical cancer and 67,477 die from the disease. India has a population of 432.2 million women aged 15 years and older who are at risk of developing cancer 6. It is the second most common cancer in women aged 15–44 years. India also has the highest age standardized incidence of cervical cancer in South Asia at 22, compared to 19.2 in Bangladesh, 13 in Sri Lanka, and 2.8 in Iran. Therefore, it is vital to understand the epidemiology of cervical cancer in India 6.
Figure 1.1. Structure of the female reproductive system
1.1.1. Signs and symptoms
The early phases of cervical malignancy might be totally free of side effects. Vaginal bleeding, contact bleeding or a vaginal mass may show the nearness of danger. Similarly the, direct agony amid sex and vaginal release are side effects of cervical growth. In cutting edge ailment, metastases might be available in the midriff, lungs, or somewhere else 4. The major Side effects of cervical disease such as : loss of voracity, weight reduction, pelvic torment ,weakness, leg torment, back torment, overwhelming vaginal dying ,swollen legs, bone cracks, spillage of pee or dung from the vagina. Seeping subsequent to douching or after a pelvic exam is a typical side effect of cervical malignancy 4.
Infection with HPV is the most serious danger element for cervical tumor, which is induced by smoking 7. Similarly HIV disease mediated a danger also variable. The majority of the reasons for cervical malignancy are not known, a few may be an unknown cause 8.
1.2. Human Papillomavirus
Human papillomavirus sorts are the major reason for 75% of cervical malignancy cases, while 25% are the another reasons 109Ladies who have numerous sexual accomplices have a more serious danger of the HPV infection. Genital warts, are a particular type of amiable tumor of epithelial cells, which are additionally brought by different strains of HPV 4. Some of the HPV serotypes are generally not identified with cervical malignancy. It is very rare to have different strains in the mean time, including those that can bring about cervical growth along side those that cause warts. Disease with HPV was move suscetable get cervical tumor 10.
Cigarette smoking, both dynamic and uninvolved, leads to the danger of cervical malignancy in women. Among HPV-contaminatedwomen, present and previous smokers have showed approximately a few times higher rate of obtrusive growth 11. In active smoking connected with expanded danger, however to a lesser degree. Smoking has connected with the development of cervical disease. Smoking can build the danger in ladies a couple of various ways, which can be immediate or backhanded strategies 12. An immediate method for getting this tumor is a smoker has a higher shot of CIN3 happening which has the capability of shaping cervical malignancy 10. At the point when CIN3 sores lead to disease. Therefore the majority of can get HPV infection, yet that is not generally the situation, which is the reason it can be viewed as an immediate connection to cervical malignancy. Overwhelming smoking and long term smoking appear to get a more danger of getting the CIN3 sores than the lighter smoking. Albeit smoking has been also connected to cervical tumor, it helps in the advancement of HPV which is the main source of this sort of malignancy. Likewise, not just does it help in the advancement of HPV, additionally if the women is now HPV-positive; she has the prominent probability of getting cervical malignancy 13.
1.4. Oral contraceptives
Long term utilization of oral contraceptives can leads to expanded danger of cervical cancer development. Ladies who have utilized oral contraceptives for 5 to 9 years have higher frequency of obtrusive growth, and the individuals who utilized them for a long time can have around more high danger of getting cervical cancer 4.
Having numerous of pregnancies is connected with an expanded danger of cervical tumor 14. Among HPV-contaminated ladies, the individuals who have had seven or all the more full-term pregnancies have the higher danger of cervical cancer growth .Any pregnancies, and a few times the danger of ladies who have had maybe a couple full-term pregnancies 14.
The Pap smear can be utilized as a screening test, however there was a more change of getting false negative to half of 15. Aconfirmation analysis of cervical malignancy or precancer requires a biopsy of the cervix 16. This is frequently done through colposcopy, an amplified visual assessment of the cervix supported by utilizing weaken acidic corrosive 17. Therapeutic gadgets utilized for biopsy of the cervix incorporate with puncher using forceps, SpiraBrushCX, SoftBiopsy, or Soft-ECC etc . Colposcopy impression, the appraisal of malady seriousness in light of the visual review, frames part of the conclusion. Supplementary indicative and treatment strategies are loop electrical excision procedure and conization, which the internal coating of the cervix is expelled near be inspected pathologically. These are finished the biopsy affirms serious cervical intra epithelial neoplasia. This enormous squamous carcinoma (base of picture) has wrecked the cervix and attacked the lower uterine sector 4. The uterus similarly has a round leiomyoma up higher. Regularly before biopsy, the specialist requests healing imaging to discount different reasons for lady’s side effects. Imaging modalities such as ultrasound, CT output and MRI has been utilized to investigate for exchanging malady, spread of tumor and impact on nearby structures. Regularly, they show up the heterogeneous mass in the cervix 18.
1.5.2. Cancer Subtypes
Histologic subtypes of obtrusive cervical carcinoma integrate the additional: Though squamous cell carcinoma is the cervical growth with the most frequency, the rate of adenocarcinoma of the cervix has been expanding in late decades 19.
• squamous cell carcinoma (around 80-85% (citation needed))
• adenocarcinoma (around 15% of cervical tumors in the UK 38)
• adenosquamous carcinoma
• small cell carcinoma
• glassy cell carcinoma
• villoglandular adenocarcinoma
Noncarcinoma malignancies which can once in the while occur in the cervix integrate melanoma and lymphoma.The FIGO stage does not incorporate lymph hub involvement as different to the TNM arranging for most different growths. For cases treated surgically, data acquired from the pathologist to be utilized as a part of relegating a different pathologic stage however is not to supplant the first clinical stage. Stage 0: The carcinoma is confidential to the surface layer (cells coating) of the cervix, Similarly called carcinoma in situ (CIS).4
Checking the cervix by the Papanicolaou test, or Pap smear, for cervical disease has been credited with drastically decreasing the quantity of instances of and mortality from cervical tumor in created nations 20. Pap smear screening at regular intervals with fitting follow-up can decrease cervical disease rate up to 80% 21. Unusual results may propose the nearness of precancerous changes, permitting examination and conceivable preventive treatment. The treatment of second rate injuries may antagonistically influence ensuing fruitfulness and pregnancy. Individual solicitations urging ladies to get screened are compelling at improving the probability they will do as such. Instructive materials likewise improve the probability ladies will go for screening, however they are not as successful as solicitations 4.
As indicated by the 2010 European rules, the age at which to begin screening ranges somewhere around 20 and 30 years old, “yet specially not before age 25 or 30 years”, and relies on upon weight of the illness in the populace and the accessible assets In the United States, screening is prescribed to start at age 21, paying little heed to age at which a lady started having intercourse or other danger variables. Pap tests ought to be done like clockwork between the ages of 21 and 654 . In ladies beyond 65 years old, screening might be stopped if no unusual screening results were seen inside the past 10 years and no history of CIN 2 or higher exists 22. HPV inoculation status does not change screening rates. Screening can happen like clockwork between ages 30 and 65 when a mix of cervical cytology screening and HPV testing is utilized and this is preferred 23. However, it is adequate to screen this age bunch with Pap spread alone at regular intervals. Screening is not helpful before age 25 as the rate of illness is low 4. Screening is not gainful in ladies more seasoned than 60 years on the off chance that they have a background marked by negative results 4.
Fluid based cytology is another potential screening technique. In spite of the fact that it was presumably proposed to improve the exactness of the Pap test, its fundamental favorable position has been to decrease the quantity of lacking smears from around 9% to around 1% 24. This lessens the need to review women for a further spread. The United States anticipatory Services Task Force bolsters screening at regular intervals in the individuals who are somewhere around 30 and 65 years when cytology is utilized as a part of mix with HPV testing 4. Pap smears has not been as viable in creating nations. This is a imperfect extent in light of the fact that a number of these nations has a bankrupted social insurance framework, excessively few prepared and talented experts, making it impossible to get and interpret Pap smears, naive ladies who motivate lost to postliminary, and a protracted pivot time to get results 4. These substances have brought about the examination of cervical screening approaches that utilization less assets and offer fast results, for example, visual assessment with acidic corrosive or HPV DNA testing 25.
1.6.2. Barrier protection
Obstruction security and/or spermicidal gel use amid sex diminishes malignancy hazard. Condoms offer insurance against cervical growth 26. Proof on whether condoms secure against HPV disease is blended, yet they may ensure against genital warts and the forerunners to cervical growth.. They additionally give assurance against different STIs, for example, HIV and Chlamydia, which are connected with more serious dangers of creating cervical growth Condoms may likewise be helpful in treating conceivably precancerous changes in the cervix 4. Presentation to semen seems to expand the danger of precancerous changes (CIN 3), and utilization of condoms causes these progressions to relapse and clears HPV 27. One study proposes that prostaglandin in semen may fuel the development of cervical and uterine tumours and that influenced ladies may profit by the utilization of condoms. Forbearance likewise forestalls HPV contamination 4.
Two HPV antibodies (Gardasil and Cervarix) diminish the danger of malevolent or precancerous changes of the cervix and perineum by around 93% and 62%, individually3 .The antibodies are somewhere around 92% and 100% influential against HPV 16 and 18 up to less than 8 years. HPV antibodies are commonly given to age 9 to 26 as the immunization is just powerful if given before contamination happens. The immunizations has been appeared to be powerful for less than 4 to 6 years, and they are conventional to be convincing for more; be that as may, the term of adequacy and whether a supporter will be required is incomprehensible 4. The high cost of this antibody have been a reason for anxiety. A few nations have considered (or are thinking about) projects to subsidize HPV inoculation.
Since 2010, young ladies in Japan have been qualified to get the cervical disease immunization for free. In June 2013, the Japanese Ministry of Health, Labor and Welfare mandated that, before overseeing the antibody, therapeutic foundations must educate ladies that the Ministry does not prescribe it. In any case, the immunization is still accessible at no expense to Japanese ladies who acknowledge the inoculation 4.
Vitamin An is connected with a lower hazard as are vitamin B12, vitamin C, vitamin E, and beta-carotene 28.
The treatment of cervical disease differs around the world, to a great extent because of access to specialists gifted in radical pelvic surgery, and the rise of “fertility-sparing therapy “in created countries. Since cervical malignancies are radiosensitive, radiation might be utilized as a part of all phases where surgical alternatives don’t exist 4.
Microinvasive malignancy (stage IA) might be dealt with by hysterectomy (expulsion of the entire uterus including part of the vagina). For stage IA2, the lymph hubs are evacuated, too. Options incorporate nearby surgical methods, for example, a circle electrical extraction strategy or cone biopsy. For 1A1 infection, a cone biopsy (cervical conization) is viewed as therapeutic 29. In the event that a cone biopsy does not deliver clear edges (discoveries on biopsy demonstrating that the tumor is encompassed by malignancy free tissue, recommending the majority of the tumor is expelled), one more conceivable treatment choice for ladies who need to protect their ripeness is a trachelectomy. This endeavors to surgically expel the disease while safeguarding the ovaries and uterus, accommodating a more moderate operation than a hysterectomy . It is a practical alternative for those in stage I cervical tumor which has not spread; be that as it may, it is not yet considered a standard of care, as few specialists are talented in this system. Indeed, even the most experienced specialist can’t guarantee that a trachelectomy can be performed until after surgical infinitesimal examination, as the degree of the spread of tumor is obscure. On the off chance that the specialist is not ready to infinitesimally affirm clear edges of cervical tissue once the lady is under general anesthesia in the working room, a hysterectomy may in any case be required 4. This must be done amid the same operation if the lady has given earlier assent. Because of the conceivable danger of growth spread to the lymph hubs in stage 1b diseases and some stage 1a tumours, the specialist may likewise need to expel some lymph hubs from around the uterus for pathologic assessment 4. A radical trachelectomy can be performed abdominally or vaginally and conclusions are clashing as to which is better. A radical stomach trachelectomy with lymphadenectomy typically just requires an a few day healing center stay, and most ladies recuperate rapidly (around six weeks) 30. Difficulties are exceptional, in spite of the fact that ladies who can imagine after surgery are defenseless to preterm work and conceivable late unnatural birth cycle. Hold up no less than one year is by and large prescribed before endeavoring to end up pregnant after surgery. Repeat in the leftover cervix is extremely uncommon if the tumor has been cleared with the trachelectomy. However, ladies are prescribed to hone watchful anticipation and subsequent consideration including Pap screenings/colposcopy, with biopsies of the rest of the lower uterine section as required (like clockwork for no less than 5 years) to screen for any repeat notwithstanding minimizing any new exposures to HPV through safe sex hones until one is effectively attempting to imagine 4 .
Early stages (IB1 and IIA under 4 cm) can be treated with radical hysterectomy with evacuation of the lymph hubs or radiation treatment. Radiation treatment is given as outer shaft radiotherapy to the pelvis and brachytherapy (inner radiation). Ladies treated with surgery who have high-chance components found on pathologic examination are given radiation treatment with or without chemotherapy to lessen the danger of backslide 31.
Bigger early-arrange tumors (IB2 and IIA more than 4 cm) might be treated with radiation treatment and cisplatin-based chemotherapy, hysterectomy (which then as a rule requires adjuvant radiation treatment), or cisplatin chemotherapy took after by hysterectomy. At the point when cisplatin is available, it is thought to be the most dynamic single specialist in occasional sicknesses 32. Propelled stage tumors (IIB-IVA) are treated with radiation treatment and cisplatin-based chemotherapy. On June 15, 2006, the US Food and Drug Administration affirmed the utilization of a mix of two chemotherapy drugs, hycamtin and cisplatin, for ladies with late-organize (IVB) cervical disease treatment. Blend treatment has noteworthy danger of neutropenia, frailty, and thrombocytopenia symptoms. For surgery to be therapeutic, the whole growth must be expelled with no tumor found at the edges of the evacuated tissue on examination under a magnifying instrument. This methodology is known as exenteration 4.
Forecast relies on upon the phase of the malignancy. The possibility of a survival rate around 100% is high for ladies with minuscule types of cervical disease .With treatment, the five-year relative survival rate for the soonest phase of obtrusive cervical tumor is 92%, and the generally speaking (all stages consolidated) five-year survival rate is around 72%. These insights might be enhanced when connected to ladies recently analyzed, remembering that these results might be incompletely in light of the condition of treatment five years back when the ladies concentrated on were initially analysed 4.With treatment, 80 to 90% of ladies with stage I disease and 60 to 75% of those with stage II tumor are alive 5 years after conclusion. Survival rates reduction to 30 to 40% for ladies with stage III tumor and 15% or less of those with stage IV growth 5 years after analysis 4. As per the International Federation of Gynecology and Obstetrics, survival enhances when radiotherapy is joined with cisplatin-based chemotherapy 33.
As the tumor metastasizes to different parts of the body, visualization drops significantly on the grounds that treatment of nearby injuries is for the most part more powerful than entire body medicines, for example, chemotherapy 34. Interim assessment of the lady after treatment is basic. Intermittent cervical tumor recognized at its most punctual stages may be effectively treated with surgery, radiation, chemotherapy, or a mix of the three. Around 35% of ladies with obtrusive cervical malignancy have tenacious or repetitive sickness after treatment 4. Normal years of potential life lost from cervical malignancy are 25.3. Around 4,600 ladies were anticipated to pass on in 2001 in the US of cervical tumor, and the yearly rate was 13,000 in 2002 in the US, as figured by SEER. In this manner, the proportion of passings to frequency is around 35.4% . Consistent screening has implied that precancerous changes and early-organize cervical malignancies have been distinguished and treated early. Figures recommend that cervical screening is sparing 5,000 lives every year in the UK by averting cervical tumor 96. Around 1,000 ladies for every incredible cervical tumor in the UK. The greater part of the Nordic nations has cervical malignancy screening programs set up. Pap smear was coordinated into clinical practice in the Nordic nations in the 1960s 4.
In our study Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase enzyme that maintains telomere ends by the addition of telomeric repeats to the ends of chromosomal DNA and that may generates immortal cancer cells. Hence, the activity of telomerase is raised in cancer cells including cervical cancer. Unique siRNAs targeting hTERT mRNA sequences were designed to knockdown the expression of hTERT. Methods The siRNA is easily degraded in the cytoplasm of many cells and also has low sustainability. To overcome this problem, ThesiRNA loaded chitosan coated Poly lactic-co-glycolic acid (PLGA) nanoparticle was synthesised by double emulsion solvent diffusion method (DESE). Characterisation of nano formulation were measured to determine efficient siRNA delivery. MTT, RT-PCR and Western blot were performed to evaluate silencing efficiency of Nano-formulation.
2. REVIEW OF LITERATURE
Nanoparticles (NPs) are key components of nanomedicine. Currently, there are varies types nanoparticle of cure exist 35. Therefore, terms such as engineered nanomaterials, nonbiological complex drugs (NBCDs), nanomedicals/nanomedicines, etc. are commonly used. Many nanomaterials can replicate some functions of globular biological macromolecules. 36 Many of these nanoscale materials have unique size- and shape dependent optical, electronic, and magnetic properties, and these properties are dependent upon methods to synthesize, to purify, and to characterize them. Many researchers stated that small changes in size and shape can significantly affect the properties of the NPs. Precision syntheses are therefore necessary to produce samples with tightly focused distributions in order to achieve the targeted functions specifically and to correlate observed functions with specific NP characteristics.37 .The characterization of NP samples that are used in a medical application is also critical because one must know and understand what is being injected into the body. In addition to using nanomedicine to diagnose and to treat diseases, it is also important to establish NPs’ efficacy and safety in biological systems. 36 If they do not get cleared, the long-term fate of the NPs is not clear. These particles may degrade and get cleared renally because they are small enough to transport through the kidney’s filtration slits,35?37 or they may accumulate in different organs and interact with off-target cells. The in vivo fate of NPs can potentially be a dynamic process, and thus, there is a need to understand nanobiokinetics (nanopharmacokineticsand pharmacodynamics), which may relate to unique and interesting toxicological responses of NPs. Many of these concepts are still at the early stages of development, but some have already reached clinical practice.38
Magnetic nanoparticles have been widely used as a magnetic resonance imaging (MRI) contrast agents for medical diagnoses of the liver, spleen, and bone marrow.81 In addition, recent studies have attempted to apply magnetic nanoparticle-based gene delivery systems for simultaneous diagnostic and therapeutic functions in vivo.39 For effective gene delivery, it is necessary to modify the surface of magnetic nanoparticles for the stable attachment of therapeutic molecules. This surface functionalization process can allow the effective loading of siRNA onto magnetic nanoparticles. Nanoparticles are physical entities with a characteristic length of 1–100 nm. 40 They can be considered as a unit in term of physicochemical properties. In nanotechnology, the synthesis and characterization of these objects is a broad field in constant development, heading towards “smart” structures with superior capabilities in various scientific and technological contexts, including catalysis, energy conversion, sensors, and actuators. The most promising and rapidly growing applications of nanoparticles are directly linked to biomedical issues, with cellular trafficking and accurate in vitro and in vivo tracking of biological systems being two examples of the new frontiers opened up for biological study by the application of luminescent inorganic nanoparticles.Nanoparticles can be categorized as organic or inorganic according to the bulk constituent materials in their structure. Classifications based on size, shape, surface, structure, and chemical behavior can be devised. Overall, what is particularly attractive about nanostructures is their surface to volume ratio. Nanosized entities enable a very limited volume to provide an enormous surface area for transport, chemical reactions, and interaction with biological systems 41.These features, combined with enhanced permeability to biological barriers, make nanoparticles the basis of nanomedicine and a powerful and promising tool in the design of new diagnostic and therapeutic devices. Interest in nanoparticles has consistently grown in every branch of medicine.42 It has been driven by the potential of nanoparticles to achieve a high therapeutic index and corresponding clinical success with improved patient compliance and reduced side effects. Nanoparticle-based drug delivery systems play an important role in the development and future applications of new pharmaceutical formulations.43. They may be capable of improving bioavailability and reducing the frequency and dosage needed for currently available drugs. With this novel type of drug formulation, in which nanoparticles act as drug carriers, it may be possible to: improve drug stability and carrier capacity; target drug molecules to a specific diseased cell or tissue, avoiding toxicity to normal cells; confine release of the drug to the site of interest; allow efficient permeation of epithelial and endothelial barriers; and combine therapies with codelivery of two or more drugs.44 . Nanoparticles suitable for multistage drug delivery systems are hybrid nanostructures (for instance, with a core-shell structure) designed to reach injured tissues in a selective manner. Each part of the hybrid nanoparticle responds differently in the biological environment.42
Liposomes are among the most advanced nanoparticle drug delivery systems successfully applied in the clinic.Nanoparticles can be engineered in terms of size, surface functionalization, and core structures to overcome these barriers. Nanoparticles have often been reported to have systemic toxicity, mostly in the liver. Hemolysis, thrombogenicity, and complement activation can also occur as a consequence of certain physical or chemical features of nanoparticles. It is important that the nanomaterial carrier is able to be eliminated harmlessly from the body in a reasonable period of time after releasing its cargo, and, in case of theranostic nanoparticles, accomplishing a desired diagnostic function.45 The drug could be adsorbed, dissolved, or dispersed throughout the matrix of the nanoparticles, or, alternatively, it can be covalently attached to the surface or inside the matrix. In the body, the drug loaded in nanoparticles is usually released from the matrix by diffusion, swelling, erosion, or degradation. There are two kinds of targeting strategies are possible, ie, passive targeting and active targeting. Passive targeting does not require surface modification of the nanoparticles, and is achieved using the unique pathophysiology of diseased cells and controlling particle mobility through human tissues, according to particle size and route of administration. Thus, the optimal diameter of nanoparticles for intratumoral delivery, avoidance of the reticuloendothelial system, and renal clearance should be in the range of 5–100 nm 46 . The higher interstitial fluid pressure found in solid tumor tissue could prevent diffusion of nanoparticles. Strategies adopted to overcome this issue could include normalizing the tumor vasculature (eg, with antiangiogenic therapy) and using nanoparticles with a diameter larger than that of normal vessel fenestrations. The molecules must also face the extracellular matrix, and often the desmoplastic process in cancer creates difficulties in this respect 47. Nanoparticulate systems have emerged in last few years as an alternative material for advanced diagnostic and therapeutic applications in medicine. Compared to molecular medicine, nanoparticles offer many advantages that overcome a range of challenges and barriers summarized in the previous section, particularly, bioavailability and biodistribution of therapeutic agents 48.The first remarkable property of nanoparticles is their superior in vivo retention by decreasing enzymatic degradation and sequestration by phagocytes of the reticulo-endothelial systems. This is mostly attributed to their immunochemically inert surfaces in contact with the biological environment. Increased deposition to the diseased sites viacompromised vasculatures in the phenomenon called enhanced permeability and retention effect also contributes to their improved deposit to diseased sites and efficacy 49. There are various types of polymers today, such as chitosan, polyethylenimine (PEI), dendrimers, cyclodextrin (CD), and metallic core nanoparticles. In this sense, nanoparticles are designed to be part of a delivery system and it is necessary to consider the overall behavior of such systems, ie, carrier, payload, and host system properties, as well as mutual interactions 50. In a gene delivery context, a key feature of the carrier is the ability to protect the gene payload until it reaches the target site, with no modification in molecular structure of the gene or its biochemical activity. The pursuit of gene delivery has been undertaken using viral-based and nonviral-based vectors 41. These topics have already been extensively reviewed, so are not discussed in depth in this paper.Some tumor characteristics can be exploited for drug delivery. The enhanced permeability and retention effect takes advantage of the abnormal neovascularization typical of many cancers. This effect is often found in tumors as well as at sites of inflammation and body tissue with other types of disease. The blood vessels are either disrupted or not fully formed, allowing molecules of a certain size to be retained to a greater extent in abnormal tissues than in healthy ones. 51. It has been shown that therapeutic nanoparticles can accumulate in specific target tissues as a result of this effect, potentially allowing for drug release over a longer period of time than for the same drug administered as a conventional preparation.The development of nanoparticles to aid in the delivery of a drug to the brain via inhalation holds considerable promise for the treatment of neurological disorders such as Parkinson disease, Alzheimer disease, and multiple sclerosis 46. Indirect techniques use X-rays or neutron beams and obtain their information by mathematically analyzing the radiation scattered or diffracted by the nanoparticles. The techniques of greatest relevance to nanoscience are small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), along with their surface-specific analoguesGISAXS and GISANS, where GI is “grazing incidence,” and X-ray or neutron reflectometry (XR/NR). The advantage of those techniques is that they are able to simultaneously sample and average very large numbers of nanoparticles and often do not require any particular sample preparation. Indirect techniques have many applications. For example, in studies of nanoparticles in raw sewage, scientists used SANS measurements, in which neutrons readily penetrated the turbid sewage and scattered strongly from the nanoparticles, to follow the aggregation behaviour of the particles over time 52.
The isolation of nanoparticles from colloidal and larger matter involves specialized techniques, such as ultra centrifugation and field-flow fractionation. Such laboratory-based techniques are normally coupled to standard spectroscopic instrumentation to enable particular types of chemical characterization. These features, combined with conventional targeting against upregulated cell surface antigens, allow precise spatiotemporal drug delivery in selected areas of the host system. Furthermore, in the design of nanocarriers, it is possible to achieve stimuli-responsive behavior using pH-sensitive and/or temperature-sensitive materials 53.These carriers hold promise for realizing effective spatiotemporal delivery of drugs. Silica and silicon-based delivery systems have emerged in drug delivery applications due to their controllability in nanopore formation and surface modifications. This enabled a multi-component and multi-functional bio-delivery systems that can possibly perform a sequence of functions at various stages of the delivery route. Several spherical mesoporous silica nanoparticles (MSNPs) with core size range from 50 to 200 nm and modified with polyethyleneimine 54.
In 1998, Fire and Mello et al. discovered that potent and specific RNA interference can be induced by double-stranded RNA (dsRNA) in Caenorhabditis elegans . Further investigations confirmed that similar dsRNA-triggered phenomena also exist in many other species such as plants , Drosophila , and mammalian cells . The past decade has witnessed an explosion of research on small regulatory RNAs that has yielded a basic understanding of many types of small RNAs in diverse eukaryotic species and the functions of key protein factors amidst the RNA silencing pathways.55 RNA silencing is recognized as a widespread mechanism of gene regulation in eukaryotes. The key machinery of RNAi pathway is that dsRNA molecules, experimentally or naturally occurring, can be recognized and cleaved into 21-23 nucleotide duplex termed small interfering RNA by Dicer homologues that have dsRNA binding domain and sRNaseIII-like enzyme activity, The siRNAs are incorporated into the multi-subunit effector complex called RNA-induced silencing complex (RISC), therefore activate the helicase activity leading to cleavage of the sense strand of siRNA 56 The remaining antisense strand recognizes the homologue region with base-pairing and degrading the target messenger RNA (mRNA) mediated by the Argonaute (Ago) family proteins with endonuclease activity, which is the catalytic core of active RISC, resulting in inhibition of gene expression . RNAi technology has become a routine laboratory research tool for gene functional study and is making its way as a revolutionary class of therapeutics for treatment of cancers and different viral infections. The RNAi was considered as a highly conserved natural mechanism that controls the gene expression triggered by a long dsRNAin the cytoplasm.6Cancer is a disease that is often characterized by mutations in multiple signal transduction pathways, leading to uncontrolled cell proliferation. It is thus difficult to identify the key genes governing cell proliferation or survival which should be targeted to induce cell death 57. RNAi technology can be useful for cancer therapy because of its high efficacy and specificity in downregulating gene expression, and it is clearly important to understand the role of altered genes in the development of cancer. It must be stressed that blockage of a single gene is not sufficient to cure or control most cancers. One of the major likely benefits of the RNAi technique in the treatment of cancer is the feasibility of developing combination therapeutic RNAi approaches to inhibit multiple oncogenes or genes of proteins involved in tumorigenesis 58. A unique approach could involve hitting multiple pathways simultaneously and/ or combining gene silencing with other therapies. Many in vitro and in vivo studies have evaluated the silencing of genes involved in pathways that drive cancer, such as oncogenesis, apoptosis, cell cycle regulation, cell senescence, tumor-host interaction, and resistance to conventional therapies. Moreover, because miRNAs may work as both tumor suppressors and oncogenes, these molecules could also be investigated as therapeutic targets, in that they could be knocked down when expressed in excess or replaced in cells when they have been pathologically reduced or lost 59. Deregulated miRNA could also serve as a helpful biomarker for diagnosis and monitoring of disease. Biomarkers are easily and objectively measurable biological characteristics which can be used as indicators of normal and pathological processes. Remarkable, miRNAs can be detected in body fluids, such as blood and cerebrospinal fluid, enabling noninvasive and early detection of disease. A further example of the potential applications of miRNA is expression profiling in order to distinguish cancer from normal tissue and to classify, eg, cancer subtypes or the origin of metastatic cancer tissue 60. The clinical utility of RNAi is still a matter of debate. There has been increasing interest in harnessing this versatile and multifaceted mechanism as a novel pharmacological approach to the treatment of human disease. As has already happened in other developing human therapeutic fields, including gene and antibody therapy, there has been a cooling off after initial enthusiasm. This was driven by a realistic understanding of the various complex milestones that needed to be reached for efficient RNAi-based therapy, before eventual approval for use in human therapy. This trend is now reversing as a result of the excitement generated by the advent of nanomedicine as a potential way of overcoming the challenges in this field. In recent years, there has been an increase in the numbers of preclinical and clinical RNAi-based trials being undertaken. Early proof-of-principle studies in animal models have strengthened the usefulness of RNAi as specific and powerful inhibitors of gene expression without significant toxicity. Translation of animal studies has in some cases progressed to early human trials 42.
In particular, the research interest is strongest when the aim is targeting genes with single nucleotide polymorphism mutations, as found in dominant-negative disorders, genes specific for pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases. In these cases, RNAi could represent the only entities providing an opportunity for a potent and specific approach 61.
The relevant clinical trials have addressed many kinds of disease, including retinal degeneration, dominantly inherited brain and skin diseases, viral infections, respiratory disorders, cancer, and metabolic disease). However, large-scale Phase III clinical trials and regulatory approvals remain distant. Use of RNAi for respiratory tract and neurological disorders, metabolic disease (such as hypercholesterolemia), and hepatic cancer has been widely revised in recent times. Most of the more advanced clinical trials focus on the treatment of age-related macular degeneration, which is a leading cause of blindness arising from excessive growth of blood vessels and their rupture within the cornea. . However, effective and well controlled in vivo delivery is challenging, and presently limits the use of RNAi in the clinic .In fact, these molecules, without any other modifications (“naked”), encounter several obstacles that reduce their therapeutic efficacy. To be efficacious, they need to reach the cytoplasm of the cell in sufficient amounts to enable sustained target inhibition 41.
RNAi is a fundamental regulatory pathway for most eukaryotic cells. It consists of complex enzymatic machinery able to control post-transcriptional gene expression through homology-dependent degradation of target mRNA by siRNA, ie, nontranslated dsRNA molecules. siRNA molecules are 21–23 nucleotides in length with a characteristic and highly specific structure (2–3 nucleotide 3? overhangs and 5? phosphate and 3? hydroxyl groups) to prevent erroneous gene silencing. They also contain a sense (passenger) strand and an antisense (guide) strand. On the other hand, the potential advantage of DNA-based RNAi is its stable introduction into cells as gene therapy, bypassing transient effects, so avoiding repeated or continuous administration in the clinical setting 62. Intact RNAi machinery is clearly essential for mammalian cells, as suggested by the early embryonic lethality of Dicer knockouts. The advantage of siRNA as therapeutics is that siRNA can target many undruggable genes. Other than antibody-based therapeutics that mainly targets receptors present on the cell surface, only a very small number of targets, mostly kinases, have been validated for traditional small molecule drugs. In addition, it is found that diseases such as cancer, genes are often deregulated by high-level amplifications. Such genes are particularly interesting as therapeutic targets for treatment of patients that are refractory to existing therapies. The properties that have been validated in chemotherapeutics are also exploited for siRNA packaging and delivery. The effort began with stable association of siRNA molecules with the nanoparticles and their retention in circulation. Methods of conjugating siRNAs with other inert and biocompatible molecules, such as cholesterol and long-chain fatty acids have been reported. 11Unlike long dsRNA with over 100 bp, synthetic siRNA has 21 ~ 23 bp with a 2-nucleotide overhang at both of the 3′ ends. In addition, siRNA is incorporated into RISC without a Dicer processing step. Synthetic siRNA has attracted much attention because it can easily be designed and customized for any gene. Although many hindrances remain for applying these technologies in the treatment of cancer, early clinical results are exciting, and suggest that we are moving forward quickly and also highlight how far we have already come 63.
Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases 64. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure 65. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA.
An example of the clinical feasibility of siRNA application for cancer therapy is represented by chronic myeloid leukemia. In more than 90% of cases, this disease is caused by a chromosomal aberration, ie, the so-called Philadelphia chromosome. This abnormality is due to fusion between the Abelson (Abl) tyrosine kinase gene at chromosome 9 and the breakpoint cluster (Bcr) gene at chromosome 22, resulting in the chimeric oncogene Bcr-Abl and a constitutively active Bcr-Abl tyrosine kinase implicated in the pathogenesis of chronic myeloid leukemia. The findings of this study imply that the clinical application of synthetic siRNA is feasible and safe, and has real potential for a genetic-based therapy using a synthetic nonviral carrier 64. In this small Phase I clinical trial, nanoparticles packed with siRNA were administered systemically on days 1, 3, 8, and 10 every 21 days via a 30-minute intravenous infusion to patients with melanoma refractory to standard of care. This new “drug” was able to deliver its cargo to melanoma cells and to silence the targeted gene.Terminal modification (5? or 3?) of siRNA molecules using cholesterol is a useful strategy for increasing their stability and cellular uptake. In particular, it increases binding to serum albumin, with consequent improved biodistribution in certain target tissues, eg, the liver. The improvement in cellular uptake is mediated by in vivo interaction and incorporation into low-density and high-density lipoproteins. Cholesterol-modified siRNA are capable of silencing apolipoprotein B targets in the mouse liver and jejunum, and of ultimately reducing total cholesterol levels.65. However, although this type of chemical modification has improved siRNA delivery to tissues, it is often associated with impaired biological activity and increased toxicity. In addition, siRNA can be conjugated with other lipid-like molecules, such as long-chain or medium-chain fatty acids and bile salt derivatives. These interact with high-density and low-density lipoprotein receptors, enhancing delivery into the liver and gene silencing in vivo. Moreover, albumin serves as a primary carrier for siRNA conjugated to medium-chain fatty acids. . Among the successful in vivo demonstrations, are antibody-protamine fusions that bind siRNA 63. Along with antibodies, aptamers99 have been used for site-specific delivery of siRNA because of their high affinity and specificity for targets, with the advantages of a much smaller size (