IntroductionChronic kidney disease (CKD) is a slowly progressive systemic disease that results in an irreversible long-term loss of kidney function [1, 2]. CKD is a life-threatening disease that is becoming more common, involving about 10% of the global population. According to recent estimates, 800 million of the global population suffer from CKD [3, 4]. Since 1990, this prevalence has risen by around 30% [5]. The three main pathological characteristics of CKD are interstitial inflammation, tubular atrophy, and fibrosis. Functional impairment of the kidney is more strongly connected to tubulointerstitial damage than to glomerular injury, regardless of the underlying etiology [6], which sets off an inflammatory cascade, including T-cell recruitment and macrophage activation. This in turn, produces profibrotic mediators, like transforming growth factor-β (TGF-β), which is a crucial cytokine that has been of increasing concern in recent years [7], as a dysregulated TGF-β signal plays an essential role in contributing to fibrosis via promoting the extracellular matrix deposition [8].The treatment of CKD is a great challenge in healthcare that requires an innovative approach to address its complex nature. RNA nanotechnology has emerged in recent years and continues to develop because of its potential therapeutic applications [9]. The RNA nanotechnology platform is unique compared to many other well-developed nano-delivery technologies, including liposomes, polymers, dendrimers, inorganic, and viral. Adding ligands to polyvalent RNA nanoparticles (RNPs) allows them to be delivered to target cells [10]. 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 [11]. RNA instability is no longer an obstacle that can be managed through various chemical modifications. notably, RNA can be used as a medication, including anti-miRNA, siRNA, aptamer, miRNA, and ribozyme [12, 13].Guo presented the first proof-of-concept study demonstrating the viability of RNA nanotechnology in 1998 [14]. The thermodynamic stability of the bacteriophage phi29 packaging RNA three-way junction (pRNA-3WJ) may be exploited to create multifunctional nanoparticles with therapeutic effects [15]. The phi29 pRNA-3WJ motif offers the perfect framework for creating multifunctional RNPs. The RNPs with regulated topologies, defined sizes, accurate stoichiometries, and polyvalent functionalities can be created using a bottom-up self-assembly method by integrating various functional modules into the branching region of a three-way junction (3WJ) [16]. The three branches of the 3WJ can be expanded to carry flexible functional modules that can be utilized as targeting, imaging, and therapeutic modules without compromising the system’s overall stability and structure. This allows the 3WJ to operate as a platform for target recognition systems. Because of its huge payloads and remarkable enzymatic and thermal stability, pRNA-3WJ is a valuable tool for RNA nanotechnology and the construction of nanomaterials [9].One essential protein that shields the kidneys is Klotho. The deficiency of Klotho is linked to the pathogenesis, development, and progression of CKD as well as extrarenal complications. Its deficiency enhances renal tubular and vascular cell senescence induced by oxidative stress. It also suppresses TGF-β activity, TGF-receptor II, and Wingless-related integration site (WNT) signaling activity promoting renal fibrosis [17, 18]. The kidneys produce sirtuin 1 (SIRT1), which mediates several physiological processes and protects and maintains appropriate kidney cell function. SIRT1 suppresses TNF-dependent transactivation of Nuclear factor kappa B (NF-kB), suppressing the expression of many pro-inflammatory genes and Tumor necrosis factor alpha (TNF-α) -induced cytokine production in fibroblast cells. SIRT1 also interacts with TGF-β signaling to provide an anti-fibrosis impact in CKD [19].Renal fibrosis is the final common pathway of all progressive renal diseases, so it is an interesting target for CKD treatment. The TGF-β as a “core signaling pathway” was found to regulate several microRNAs (miRs), which function as downstream mediators of distinct pro-fibrotic effects. While overexpression of miR-34 induces renal fibrosis, its downregulation has been shown to decrease renal fibrosis [7]. For this reason, the present study aimed to design, prepare, and characterize multifunctioning (antimir-34a and DNA aptamer-kidney targeted) RNA nanoparticle (RNPs) based on bacteriophage phi29 packaging RNA three-way junction (pRNA-3WJ), and then explore their in vivo toxicity and therapeutic potentials in mice model of CKD. Besides the molecular mechanisms involved in their therapeutic effect, the stability, safety, and efficient renal targeting of the prepared anti-miR-34a 3WJ-RNPs were evaluated.Materials and methodsIn silico phaseDesigning the core and therapeutic three-way junction RNA nanoparticles (3WJ-RNPs)The core 3WJ-RNPs (3WJ) were designed using the basic three single-stranded RNA (ssRNA) sequences (3WJ-a, 3WJ-b, and 3WJ-c) [20]. The sequences of these strands are presented in Table 1. Based on the sequences of the basic three ssRNA of the core 3WJ-RNPs we designed four single strands for the construction of the therapeutic 3WJ-RNPs (3WJ-Kapt/anti-miR-34a) which is aptamer-targeted to renal tissues and functionalized with an anti-miR-34a domain as follows: Strand-1: RNA sequence of 3WJ-a plus 3´-extending arm consisting of 25 ribonucleotides, Strand-2: Start from the 5´-end with anti-miR-34a hepta-deoxyribonucleotide (CACTGCC) followed by 25 Deoxyribonucleotides complementary to the extending arm of strand 1, Strand-3: RNA sequence of 3WJ-b plus 3´- extending 43 Deoxyribonucleotides of Kapt [21], Strand-4: RNA sequence of 3WJ-c.Table 1 The sequences of the core three-way junction (3WJ) and the therapeutic three-way junction (3WJ-Kapt/anti-miR-34a) RNA nanoparticle strands.Full size tableThe 3WJ-c strand and strand-4 were labeled with Alexa Fluor 647 to facilitate the detection of the RNPs in vivo, all U and C ribonucleotides were replaced with 2’-Fluoro (2′-F) U and C ribonucleotides and the anti-miR-34a sequence (7- deoxyribonucleotides) used as locked nucleic acid (LNA) to increase the enzymatic and thermal stability of the RNPs.The proper folding of these strands into correct 3WJ RNPs folding was predicted using the VfoldMCPX online tool (http://rna.physics.missouri.edu/vfoldMCPX2) which predicts the 2D structures of multi-strand RNA complexes [22]. The sequences of the strands with modifications were obtained from Viviantis Technologies (Malaysia).In vitro phaseStepwise bottom-up assembly of 3WJsEach strand was prepared in the corresponding volume of Diethylpyrocarbonate water as documented in the datasheet to prepare equimolar concentrations (20 µM). Each type of 3WJs (core or therapeutic) is stepwise prepared.Preparation of core 3WJThe RNPs were constructed by mixing 3WJ-b, and 3WJ-c strands after 10 min at room temperature, then 3WJ-a strand was added (Table 1) at an equal molar concentration in TMS buffer (50 mM Tris pH 8.0, 100 mM NaCl, 10 mM MgCl2), followed by heating to 85 °C for 5 minutes and slowly the particle size (ps), and zeta potential (zp) were then measured based on the dynamic light scattering (DLS) method, using the Malvern Zetasizer Ultra. cooled over 40 minutes to 4 °C on a thermal cycler [20].Preparation of 3WJ-Kapt/anti-miR-34aThe RNPs were constructed by mixing strands (3 and 4) after 10 min at room temperature, then strands (1 and 2) were added (Table 1) at an equal molar concentration in TMS buffer, followed by heating to 85 °C for 5 minutes and slowly cooled over 40 minutes to 4 °C on a thermal cycler [20].Characterization of the prepared RNA nanoparticlesThe prepared RNA nanoparticles were characterized by using agarose gel electrophoresis, thermal stability by thermal cycler (Tm), The 3WJs were dissolved in diethylpyrocarbonate-treated water to a final concentration of 1–2 μg/μl.In vivo phaseThe in vivo experiments were conducted on 97 c57bl/6 mice with an average weight of 20-25 g obtained from the animal house of the Medical Research Institute, Alexandria University, Egypt. The sample size was decided using power calculations and don’t used the randomization and blinding methods. All mice had free access to food and water with a 12:12 hour light/dark cycle and constant environmental conditions before experimentation. The use of experimental animals in the study protocol was carried out by the Ethical Guidelines of The Institutional Animal Care and Use Committee (IACUC) at Alexandria University, Egypt (approval no. AU012237921).In vivo experiments were divided into two parts: part 1, a toxicity study to examine the safety of the prepared 3WJ, and 3WJ-Kapt/anti-miR-34a RNPs on the control mice. Part 2: a therapeutic study to examine the targeting and therapeutic efficiency of the prepared 3WJ-Kapt/anti-miR-34a RNPs on the CKD mice compared with the untreated CKD mice or mice treated with 3WJ RNPs (as a vehicle).Explore in vivo toxicity, safety, and targeting efficiency of the prepared renal cell-targeted multifunctioning 3WJ and 3WJ-Kapt/anti-miR-34a RNPsA toxicity study was conducted to examine the safety of the prepared 3WJ and 3WJ-Kapt/anti-miR-34a RNPs on healthy mice. 19 normal C57BL/6 mice were divided into three groups: untreated healthy control, 3WJ-treated, and 3WJ-Kapt/anti-miR-34a-treated. All mice were injected once intravenously in the tail vein with 200 µL of TMS, 3WJ (50 µg/kg), or 3WJ-Kapt/anti-miR-34a (50 µg/kg), respectively. After 24 hours two mice from 3WJ-Kapt/anti-miR-34areated groups were sacrificed to obtain different organs for the biodistribution assessment using a confocal microscope (LEICA DMI8, Germany). The remaining mice were followed up for 4 weeks then the mice were sacrificed, and blood was obtained for separation of serum and both kidneys were dissected out. The serum was used for the assessment of liver and kidney functions and the tissues were used for assessing inflammatory and oxidative markers.Therapeutic studyThis study involved 80 C57BL/6 mice, 20 normal mice, and 60 mice with CKD. The CKD was induced by oral administration of adenine at a dose of 50 mg/kg body weight (dissolved in 0.5% Carboxymethyl cellulose, CMC) by oral gavage for 28 days [23]. To confirm the establishment of CKD, a serum sample was withdrawn on day 29 after induction for the measurement of urea and creatinine levels. A CKD was considered if the urea level reached more than 100 mg/dL and the creatinine level was higher than 1 mg/dl [24].Experimental designThe mice were divided into two main groups: The control group (20 mice) that received single i.v. injection in the tail vein with 200 µL saline and CKD group (60 mice). The latter was subdivided into three subgroups: Untreated CKD group: CKD mice that received no treatment, CKD + 3WJ group: CKD mice that received single i.v. injection in the tail vein with 200 µL of 3WJ (50 µg/kg) [16], and CKD + 3WJ-Kapt/anti-miR-34a group: CKD mice that received single i.v. injection in the tail vein with 200 µL of 3WJ-Kapt/anti-miR-34a (50 µg/kg) [16].The mice had free access to food and water and were followed up for 4 weeks post-treatment. Every week 5 mice of each group were selected randomly and sacrificed under isoflurane anesthesia to obtain the blood and to dissect kidneys, liver, heart, spleen, and brain.Samples preparationBlood samples were collected in 2 tubes. In a plain tube, the samples were left for 20 min at room temperature and centrifuged at 3000 ×g for 10 minutes to obtain serum for the assessment of urea, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities phosphate, erythropoietin (EPO), Kidney Injury Molecule-1 (KIM-1), and N-acetyl-β-D-glucosaminidase (NAG). The remaining blood was collected into tubes containing EDTA as an anticoagulant to assess the hemoglobin concentration.The left kidneys were quickly dissected out, rinsed with phosphate buffer saline (PBS), and used for fluorescence scanning and histological analysis. The right kidneys were divided into two aliquots: the first aliquot was used for total RNA extraction for subsequent gene expression analysis of miR-34a, Fibroblast Growth Factor 2 (FGF2), Suppressor of Mothers Against Decapentaplegic (SMAD7), TGF-β, β-Klotho, α-Klotho, WNT1, β-catenin, and SIRT1 (the methods and primers used were provided in the supplementary file). The second one was homogenized in RIBA buffer (1:9) and used for the determination of malondialdehyde (MDA) level, then centrifuged at 10,000×g for 10 min at 4 oC, and the supernatant was stored in aliquots for subsequent determinations of total protein level by Lowry method [25], TGF-β, SMAD2, SMAD3, α-Klotho KIM-1, NAG, TNF-α, and IL-6 (the methods and sources of the used kits were provided in the supplementary file). All the other collected tissues (liver, heart, spleen, and brain) were used for fluorescence scanning to detect the biodistribution of the RNPs. Slides for histopathological settings were stained with hematoxylin and eosin (H&E). A detailed methodology for each measurement is provided in the supplementary file.Histopathological examination and lesion scoringFollowing necropsy, tissue specimens from the kidneys were immediately fixed in phosphate-buffered formalin (10%, pH 7.4) for 24 hours, then processed using the conventional paraffin embedding technique [26]. Sections (5 μm thick) were sliced, mounted on slides deparaffinated in xylene, and rehydrated using decreasing ethanol concentrations. Slides were stained with hematoxylin and eosin (H&E) for routine histopathological setting. Stained sections were blindly evaluated using a light microscope (Leica, DM500) and photographed at magnification power 100 using a digital camera (EC3, Leica, Germany).A histological lesion-scoring approach was adopted to show the severity of histopathological lesions. In each animal group, five H&E-stained slides (one slide/rat) were examined blindly to grade the pathological lesions. The severity of pathological lesions (glomerular and tubular injury scores, interstitial inflammation, interstitial fibrosis, and intratubular casts)was evaluated according to the percentage of tissue affected in the entire section as none (0): normal histology with zero involvement of the examined field, mild (1): 5–25% of the tested field was involved, moderate (2): 26–50% of the examined field was involved, severe (3): ˃50% of the examined field was involved. Each animal’s score was evaluated, and the median score was calculated for the various pathological alterations per group. The sums of the various lesion scores were used as the total renal tissue lesion score for each group [27].Statistical analysisData were analyzed using Prism software package version 5 (GraphPad Prism 5.0). The data were expressed as mean ± SD. The Kolmogorov-Smirnov test was used to study the normal distribution of the studied parameters. The analysis of variance (ANOVA) was made and followed by a post hoc (Bonferroni test) to compare the mean values between and within treated groups compared to untreated and control groups. For the histopathology scoring, nonparametric data were represented as median (min-max) and were analyzed by the Kruskal–Wallis Test, followed by Dunn’s post hoc test. Differences were considered statistically significant at p-value