الفهرس 
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Abstract
The treatment of chronic diseases like chronic kidney diseases (CKD) is a great challenge in healthcare that requires an innovative approach to address the complex nature of these diseases. The field of RNA nanotechnology continues to develop, and researchers are becoming more interested in it because of its potential therapeutic applications. The RNA nanotechnology platform is unique compared to many other well-developed nano-delivery technologies, including liposomes, polymers, dendrimers, inorganic, viral, and so on. The addition of ligands to polyvalent RNA nanoparticles (RNPs) allows them to be delivered to target cells. Because of their uniform nanoscale size, polyvalent nature, exact stoichiometry, low toxicity, low immunogenicity, and target specificity, 3WJ-RNPs have the potential to be used in clinical applications as a targeted therapeutic delivery system to treat a variety of disorders.
Chronic kidney disease (CKD) is a systemic disease that results in a long-term loss of kidney function. CKD progresses slowly and causes irreversible damage that is associated with end-stage renal disease, and/or premature death leading to adverse clinical and economic outcomes. Recent findings indicate that between 700 million and one billion people worldwide have CKD. There is an urgent need to find novel therapeutic approaches that stop or reverse the progression of the disease because existing treatments for CKD are ineffective and only delay disease progression. Renal fibrosis is the final common pathway of all progressive renal disease, so it is considered an interesting target for CKD treatment.
The present study aimed to design, prepare, and characterize multifunctioning (antimir-34a and DNA aptamer-kidney targeted) RNPs. Also, the study explored the specific targeting to renal tissue and the safety of the constructed RNPs in healthy mice, then were used to explore their therapeutic potentials against CKD in mice. The last aim of our study was to evaluate the molecular mechanisms involved in their therapeutic effects.
Using the basic sequences of the three strands of pRNA-3WJ we designed two types of RNPs: core 3WJ (3WJ-C) and therapeutic 3WJ (3WJ-T). The proper folding of the designed RNPs was confirmed using VfoldMCPX online tool. Then the designed RNPs were constructed using 2′-F modified ribonucleotides and LNA. The prepared were characterized.
The size and hydrodynamic diameter of the prepared RNPs were measured by gel electrophoresis and DLS respectively, zeta potential was also assessed by DLS and the results of all confer the correct preparation of these RNPs. The prepared 3WJ-C and 3WJ-T have high Tm that confirm increased thermostability upon using 2′-F, LNA-modified RNA strands.
The study was conducted on 97 male C57BL/6 mice. After confirming the safety and specific targeting prepared core 3WJ (3WJ-C) and therapeutic 3WJ (3WJ-T) RNPs to renal tissue using healthy mice, the CKD was induced in mice using adenine. The mice were classified into two main groups; group I: 20 normal mice. group II: 60 CKD mice that were subdivided into 3 subgroups (20 mice each); as untreated, treated intravenously with single i.v. injection in the tail vein with 200 µL (50µg/Kg) of 3WJ-C, and 3WJ-T, then followed for 4 weeks post-treatment. Every week: 5 mice from each group were selected randomly and sacrificed we obtained blood and both kidneys, liver, heart, spleen, and brain. The obtained serum was used for assessment of ALT and AST activities, urea, creatinine, phosphate, erythropoietin, KIM-1, and NAG levels. The remaining blood was used for the assessment of hemoglobin concentration. The left kidneys were used for florescent scanning and histological analysis, and the right kidneys were divided into two parts: for total RNA extraction for subsequent gene expression. The second part was homogenized and used for the determination of MDA level, and then the supernatant was used for subsequent determinations of total protein level, KIM-1, NAG, TNF-α, and IL-6. All the other collected tissues (liver, heart, spleen, and brain) were used for fluorescence scanning to detect the biodistribution of the RNPs.
CKD mice have significant disturbances in kidney function that resemble humans. Including significant kidney dysfunction throughout the experimental period, markedly elevated levels of serum KIM-1, NAG, and phosphate, besides increased liver enzyme activities AST, and ALT, and significantly declined levels of haemoglobin and erythropoietin that confer impaired kidney function. Also, the CKD mice showed significantly elevated levels of oxidative and inflammatory markers such as MDA, TNF-α, and IL-6. CKD mice suffered from renal fibrosis due to suppressed expression of anti-fibrotic genes such as Klotho, SIRT1, and SMAD7, and enhanced expression of fibrotic genes such as miR-34a, TGF-β, FGF2, WNT1, and βCatenin.
The 3WJ-T nanoparticles completely normalized the elevated urea and creatinine levels and significantly ameliorated renal anemia, liver function, and inflammatory markers throughout the 4-week experimental period. At the histological level, the treatment of CKD mice with the multi-function 3WJ-T significantly ameliorated the pathological lesions observed in untreated CKD mice. The treated mice rapid downregulation of the expression level of miR-34a (complete normalization from the 1st week of treatment). This suppression results in the induction of the antifibrotic factors including α and β klotho, SIRT1, and SMAD7, suppression of profibrotic factors including TGF-β1, FGF2, WNT1, and β-catenin, and inhibition of renal inflammatory mediators (TNFα, and IL-6).
To our knowledge, this is the first study that reported a promising effect of the targeted 3WJ-T as an anti-fibrotic effect and anti-inflammatory effect in the CKD-mice model.
from the present study, we can conclude that:
1. Designing and construction of multifunctional renal targeted 3WJ-RNPs containing anti-miR-34a is a feasible approach.
2. The renal targeting of used RNPs using DNA aptamer is successful.
3. The prepared RNPs have very low or no toxicities on the main organs.
4. The present study provides preliminary and pioneer evidence for the promising treatment of renal fibrosis and CKD through targeting miR-34a in the renal tissue using properly designed and constructed multifunctional antimir-34a pRNA-3WJ stabilized by 2′-F ribonucleotides and LNA and targeted to kidney using DNA aptamer.
5. The mechanism of action of 3WJ-T could be mediated through suppressing the expression of fibrotic genes; TGF-β, FGF2, WNT1, and β-catenin, and inducing the expression of anti-fibrotic genes; Klotho, SMAD7, SIRT1.
6. Future studies are required to explore the molecular mechanism(s), pharmacodynamics, and pharmacokinetics of the used RNPs. The effect of repeated injection of the lower doses of RNPs needs to be explored. The application of multifunctioning RNPs targeting more than one molecular target using a 3WJ or 4WJ platform will be of great importance.