Engineered nanoplex mediated targeted miRNA delivery to rescue dying podocytes in diabetic nephropathy
Nidhi Raval, Piyush Gondaliya, Vishakha Tambe, Kiran Kalia, Rakesh K. Tekade *
National Institute of Pharmaceutical Education and Research-Ahmedabad (NIPER-A), An Institute of National Importance, Government of India, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Palaj, Opp. Air Force Station, Gandhinagar 382355, Gujarat, India
A R T I C L E I N F O
Keywords: microRNAs delivery Nanoplexes
Gene therapy Podocyte targeting Diabetic model
A B S T R A C T
MicroRNAs (miRNA) is vital for gene expression regulation and normal kidney function. Mainly, miRNA-30a is responsible for the homeostasis of podocytes. In the diabetic nephropathic condition, miRNA-30a is directly and primarily suppressed by hyperglycemic kidney induced Notch signaling pathway leads to podocyte damage and apoptosis. Thus, transferring the exogenous miRNA-30a to podocytes might improve albuminuria as well as podocytes injury. The deprived stability, poor targetability, and low specificity in vivo are critical limitations to attain this objective. This investigation reports the specific and efficient delivery of miRNA-30a mimic via cyclo (RGDfC)-gated polymeric-nanoplexes with dendrimer templates to alleviate podocyte conditions. The nanoplexes able to protect RNase enzyme and to exhibit greater cellular uptake viaαvβ3 receptor selective binding in HG treated podocytes. The nanoplexes up-regulated the expression level of miRNA-30a and repress the elevated Notch-1 signaling in HG exposed podocytes.
The critical results of in vivo experimentation attribute marked suppression of Notch-1 in streptozotocin (STZ) induced diabetic C57BL/6 mice and reduced glomerular expansion and fibrosis in the glomerular area. Developed nanoplexes represents an efficient platform for the targeted delivery of exogenous miRNA to podocytes. The approach developed herein could be extrapolated to other gene therapeutics and other kidney-related diseases.
1. Introduction
A group of small non-coding RNA as microRNA (miRNA), classically 19–25 nucleotides, which regulate the expression of genes (post-tran- scriptional level) via target mRNA degradation or translation suppres- sion (Treiber et al., 2018). miRNA is associated with regulating numerous downstream effects of diverse biological processes, e.g., cell survival, cell cycle regulation, differentiation, and apoptosis (Gebert and MacRae, 2018). The gene expression is regulated via aiming the 3′-un- translated regions. The mammalian genome comprises more than a thousand diverse miRNAs wherein a single miRNA targets several hundred genes (Hausser and Zavolan, 2014; Khalid et al., 2019).
miRNA played an essential role in podocytes, tubular epithelial cells, and mesangial cells. In the experimental diabetic nephropathy (DN) model, miRNA-192 induces fibrosis in mesangial cells and tubule- interstitial fibrosis employing TGF-b/Smad3 pathway activation (Chung et al., 2010). Moreover, miR-1 92, miR-216, and miR-217 associated with the E-box repressors Zeb1/2 and target PTEN (phos- phatase and tensin homologue), an inhibitor of Akt activation, led toglomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-β and which is primarily associated with mesangial cells rather than podocytes (Trionfini and Benigni, 2017; Long et al., 2010).
Besides, during an early stage of organ development, such as kidney organogenesis, miR 30 played a crucial role, and miR-30a is mainly expressed in human podocytes and in addition to protecting them against apoptosis by directly targeting Notch1 and p53, they promote podocyte actin fibre stability through controlling calcium/calcineurin signaling via the inhibition of several components of this pathway (TRPC6, PP3CA, PP3CB, PPP3R1, and NFATC3). Dysregulation of cal- cium/calcineurin signaling leads to podocyte cytoskeletal damage, a key feature of various glomerular diseases, such as diabetic nephropathy and Focal Segmental glomerulosclerosis characterized by the early onset of podocyte injury (Trionfini and Benigni, 2017). Therefore, it is reported that miR-30a downregulation is able to induce disruption of actin fiber and podocyte loss, signifying that miR-30 s are involved in podocyte actin fiber and cytoskeletal stability via an undetermined mechanism (Wu et al., 2015). Hence, reports suggested that miRNA played a vital
* Corresponding author.
E-mail address: [email protected] (R.K. Tekade).
https://doi.org/10.1016/j.ijpharm.2021.120842
Received 19 January 2021; Received in revised form 13 June 2021; Accepted 14 June 2021
Available online 1 July 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.
role in podocytes apoptosis. In contrast, the miRNA-30 family is pro- fusely presented in kidney podocytes concerning glomerular cells in mice and played a vital role in podocytes’ physiology (Wu et al., 2014). Reports suggested that miRNA-30a is employed mainly in main- taining the homeostasis of podocytes. Moreover, the serum concentra- tion of miRNA-30a-5p increases and down-regulated its expression in the kidney in type-2 diabetes, nephrotic syndrome, focal segmental glomerulosclerosis, and acute pyelonephritis (Petrillo et al., 2017; Tri- onfini et al., 2015). In kidney disease conditions, miRNA-30a specif- ically down-regulated in podocytes via the Notch-1 pathway and TGF-β up-regulation. This podocytopathy-related gene miRNA 30a is directly linked to podocytes apoptosis. In comparison, glucocorticoids suppress Notch-1 and p53 signaling and regulate the miRNA-30 level but not specifically regulate the level of miRNA-30a (Wu et al., 2014). There- fore, transporting exogenous miRNA-30a to podocytes improves albu-
minuria, Notch signaling as well as podocytes injury.
The cell’s natural gene silencing process is RNA interference (RNAi viz. siRNA, miRNA), found most propitious and rapidly growing frontier in biology and drug development currently. Its discovery has been recognized with the 2006 Nobel Prize in Physiology or Medicine (Fire and Mello, 2006). Despite extensive research and the potential of miRNA, its clinical translation was made ineffective. The central limiting stage that hampers clinical applicability is rapid renal clear- ance, reduced half-life, off-target effect, and degradation by endonu- cleases enzyme of serum, and degradation in the endo-lysosomal compartment (Kauffman et al., 2016). Therefore, to successfully deliver miRNA-30a mimics as RNAi therapeutic in diabetic kidney disease treatment, it must be delivered to protect miRNA from enzymatic degradation, enhance cellular uptake, and escape the endosomal compartment.
However, viral vectors hold tremendous promise for miRNA de-
livery, but activation of host immune reaction limits its applicability. Several marketed lipid-based delivery systems are available, such as Lipofectamine™, Oligofectamine™, TransGene™, RNAifect™, X-trem- eGENE™, Xfect RNA™ have been utilized for RNAi (e.g. siRNA, miRNA) delivery (Yu et al., 2016). But therapeutic effect of miRNA loaded commercially available lipid based delivery system and cell penetration peptide is compromised via cellular toxicity, reduced bioavailability, in vivo degradation, and by passive targeting (Andre et al., 2016; Saar et al., 2005). The cationic polymers are also having more significant potential for delivery of miRNA intracellularly. But the cationic polymer such as polyethyleneimine, ethylenediamine, poly(propylene imine), poly (lysine) showed cellular toxicity owing to extreme positive charge (Jones et al., 2013). Polymeric nanoconstruct embraces greater poten- tials in this regard; numerous nanoconstruct has been chased for effec- tive gene silencing and found safe and effectively deliver miRNA pre- clinically as well (Devulapally et al., 2015).
As a natural biopolymer, albumin (USFDA approved carrier) is
intrinsic, biocompatible, non-immunogenic, easily modified, and biodegradable (Parodi et al., 2019). Albumin-based nanoconstruct comprising Paclitaxel anticancer drug has already present in the market as Abraxane™ of Celgene Corporation, USA (Houghton et al., 2015). The anionic architecture of albumin dose not capable of effectively loading and delivering negatively charged miRNA via anionic interac- tion resulted in less entrapment and premature release of miRNA in blood circulation. Anionic side chain (–COOH) limits of albumin impede entrapment of the anionic miRNA. Moreover, albumin is not enough for complete endosomal escape, leading to miRNA degradation in the harsh endo/lysosomal environment reported in our publications (Raval et al., 2020; Tekade et al., 2015).
Thus, the presented investigation’s primary goal is to develop a novel albumin-based delivery tactic with greater miRNA entrapment effi- ciency, pH dependable endosomal escape, RNase stability, and silencing of Notch-1. The hypothesis was proved on the HG-treated podocytes model and diabetes-induced mice. As exogenous miRNA, miRNA-30a mimics arebeing utilized in this investigation to reduce podocytes
apoptosis and podocytes damage. This study can be extended to other types of RNAi therapeutics and a combination of RNAi therapeutics. It is reported that more than 10 nm nanoparticles underwent rapid renal clearance, whereas less than 200 nm size nanoparticle evade uptake in the spleen. For selective targeting of nanoplexes to glomerular podo- cytes, the selected size was 70 nm (Kamaly et al., 2016). It is docu- mented that 10 nm nanoparticles undergo renal clearance and are excreted from the kidney (Tan and Ho, 2018). In normal conditions, the glomerular filtration barrier includes glomerular endothelium com- prises ≈150 nm pores. The glomerular basement membrane (GBM) al- lows passing 3–10 nm size molecules according to size and charge (Choi et al., 2011; Jefferson et al., 2008; Zuckerman et al., 2012).
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Moreover, the interdigitating foot process of podocytes adjacent to GBM encompasses filtration slits. The podocytes slit diaphragm abides a connection with the size of 30–40 nm under normal physiology (Choi et al., 2011; Zuckerman et al., 2012). In DN, the thickening of the GBM creates the irregular fenestration 10–80 nm size (Du et al., 2018; Ota et al., 1995). While this GBM is adjacent with the podocytes attached integrin (αvβ3 integrin predominantly). This αvβ3 integrin of podocytes integrin enhances selective cellular uptake of nanoparticles (Jefferson et al., 2008).
For selective targeting towards kidney podocytes, we judiciously selected cyclo-(Arg-Gly-Asp-D-Phe-Cys) (cyclo(RGDfC)) as an αvβ3 integrin receptor ligand.
It is majorly expressed in glomerular capsular epithelial cells, podocytes, and mesangial cells (Hayek et al., 2017). In diabetic kidney condition enhanced expression of VEGF, bFGF, TNFα, and other cytokines, it enhances the expression of αvβ3 integrin on podocytes’ surface (Hafdi et al., 2000). It is also reported that in pro- teinuric kidney diseases comprising diabetic nephropathy, the soluble urokinase receptor stimulates the αvβ3 receptor, leading to the efface- ment of podocytes (Koh et al., 2019). Here, the albumin architect has altered m-maleimidobenzoyl N-hydroxysuccinimide (MBS) crosslinker. Further, poly amidoamine dendrimer (PAMAM, 2.0G) with sixteen terminal primary amino groups was utilized to enhance the entrapment of miRNA via dendrimer:miRNA ratio, to avoid premature release, protection from RNase, dendrimeric template approach has been incorporated to load miRNA inside albumin nanoplexes (Raval et al., 2020). pH-sensitive dendrimer was undergoing protonation owing to the free primary amino group at the periphery.
Thus, tactically adopted dendrimer templated approach to facilitate endo/lysosome escape after receptor-mediated cellular uptake of cRGD gated nanoplexes as reported in our study (Indian patent at Indian Patent Office (IPO), Mumbai, India; Application No.: 201921019898; Date of Application: 18/05/2019) (Raval et al., 2019a). The prototype formulation process and its
composition for loading, stability, and delivery of siRNA comprised al- bumin nanoplexes were evaluated for the in vitro and in vivo DN model reported in our previously reported by our group in Scientific Reports (Raval et al., 2019a). Its follow-up study, where the author reported developing a fit-to-purpose cRGD targeted HDAC4 siRNA comprised nanoplexes specifically to the kidney in DN (Raval et al., 2020).
A further extrapolation of the investigation of studies is reported here for miRNA-30a comprised nanoplexes. The targeted nanoplexes were prepared, characterized, and evaluated for RNase degradation. The receptor-based cellular uptake of model FITC loaded nanoplexes, and gene silencing efficiency of miRNA-30a mimics loaded nanoplexes were also evaluated on HG-treated podocytes. The receptor-based targeting was also confirmed through competition assay. Lastly, the gene silencing effect was also demonstrated in the streptozotocin (STZ)-induced C57BL/6 diabetes mice model. Renal histological evaluation of casted cRGD gated nanoplexes is performed on diabetes-induced mice kidney sections.
2. Experimental section
2.1. Material and reagents
Bovine Serum Albumin-Fraction V was procured from HiMedia Laboratories Pvt Ltd (Mumbai, India). PAMAM dendrimer (cystamine core), m-maleimidobenzoyl N-hydroxysuccinimide (MBS), was bought from Sigma-Aldrich (Missouri, USA). DEPC treated RNase free water was procured from Invitrogen (Thermo scientific, Massachusetts, USA). The Cyclo(RGDfC) peptide utilized to modify albumin was procured from Peptide synthetics (Fareham, UK). Other reagents included sodium chloride, sodium acetate, and sodium phosphate, were procured from Sigma-Aldrich (Missouri, USA). The bicinchoninic acid protein assay (BCA) kit and (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) reagent was acquired from Thermo Fisher Scientific (Massachusetts, USA).
Agarose, polyacrylamide-bisacrylamide were acquired from Sigma
Aldrich (Missouri, USA). Media for cell culture experiments such as fetal bovine serum (FBS), RPMI 1640 media, penicillin–streptomycin, trypsin-EDTA, 1x ITS (Insulin-Transferrin-Selenium), as well as Hoechst 33,342 dye was bought from Invitrogen (California, USA) and Opti- MEM® was purchased from Gibco® (Life Technologies, Grand Island, USA). mirVana® miRNA-30a mimic and, mirVana™ miRNA mimic Negative Control were procured from Ambion® (Thermo Fisher Scien- tific, MA, USA). TaqMan™ Universal Master Mix II, no UNG purchased from Applied Biosystem (California, USA). Hsa-miR-30a-5p TaqMan™ assay probe and U6snRNA TaqMan™ assay probe purchased from Applied Biosystem (California, USA). Unless otherwise specified, all the other solvents and reagents were of analytical grade.
2.2. Preparation of albumin miRNA-30a mimic nanoplexes (miAnp-30a mm) and cRGD targeted albumin miRNA-30a mimic nanoplexes (miAnp- cRGD-30a mm)
Preparation of albumin-cRGD (A-cRGD) conjugation was done as reported by our previous publication (Raval et al., 2019b, 2020). The miAnp-cRGD-30a mm was formulated according to the protocol earlier reported by our group with suitable changes to attain 70 nm particle size (Raval et al., 2019b, 2020; Tekade and Chougule, 2013; Tekade et al., 2015; Wang et al., 2017). For the preparation of miAnp-30a mm, firstly, albumin solution (4 %w/v in DEPC treated nuclease-free water) was prepared, and albumin solution with miRNA-30a mimic was incu- bated (200 pmol; 3–4 µg/µL siRNA) with stirring at 300 rpm for 1 h. Under optimized stirring condition (2 h; 700 rpm) (IKA Magnetic Stirrer, Model RT5, Staufen, Germany) ethanol (4 mL) (desolavating agent) was added dropwise into albumin:miRNA 30a mm solution at a rate of 50 µL/min. Nanoplexes formed were cross-linked with genipin (20 µL; 0.1% w/v). This leads to the stabilization of the primary amino group. Free miRNA was removed by centrifugation at 14,000 g for 25 min. The pellet was redispersed in DEPC-treated RNase free water. Further, its particle size, polydispersity index (PDI), and zeta potential was determined.
For the preparation of miAnp-cRGD-30a mm, albumin: A-cRGD (w:
w::90:10) was incubated with miRNA-30a mimic (200 pmol; 3–4 µg/µL siRNA) for 1 h under stirring condition at 300 rpm). Then, ethanol was added at 50 µL/min under stirring at 700 rpm for 2 h. Lastly, nanoplexes were crosslinked via genipin. As described previously, the un-entrapped miRNA-30a mimic was removed via the centrifugation method. The obtained nanoplexes were analyzed for their particle size, PDI, and zeta potential.
2.3. Preparation of Dendrimer-Templated Albumin miRNA-30a mimic nanoplexes (DTmiAnp-30a mm) and cRGD targeted Dendrimer-Templated Albumin miRNA-30a mimic nanoplexes (DTmiAnp-cRGD-30a mm)
DTmiAnp-30a mm were prepared by incubating optimized d:miR complex (200 pmol; n/p ratio of 0.5; 3–4 µg/µL (Raval et al., 2019b,
2020) with albumin (4 %w/v) for 1 h under moderate stirring. After that, ethanol was added dropwise at a rate of 500 µL/min under stirring conditions (700 rpm for 2 h). They were cross-linked with the addition of genipin (0.1% w/v). The free miRNA-30a mimic was removed by centrifugation. Hydrodynamic particle size, PDI, and surface zeta po- tential of nanoplexes was determined.
Similarly, the DTmiAnp-cRGD-30a mm were developed through d: miR complex incubation (200 pmol; n/p ratio: 0.5; 3 to 4 µg/µL (Raval et al., 2019b, 2020) under moderate stirring condition 300 rpm with albumin:A-cRGD (w:w::90:10) for 1 h. Then, ethanol was added for desolvation at 500 µL/min under stirring at 700 rpm for 2 h. The DTmiAnp-cRGD-30a mm were stabilized by genipin, and the free miRNA-30a mimic was removed by centrifugation at 14,000 g for 25 min. Trehalose (3% w/v) was used to lyophilize the resultant nanoplexes (Wang et al., 2018). For further experimentations, depending upon the study requirement, the lyophilized nanoplexes was reconstituted in DEPC treated nuclease-free water or PBS (1X). For the cellular uptake study, nanoplexes were prepared with DTAnp-cRGD-FITC instead of miRNA-30a mimic.
2.4. Characterization of miRNA-30a mimic nanoplexes
The particle size, PDI, and surface zeta potential were measured at
25 2 ◦C by Zetasizer (Nano-ZS90, Malvern Instruments, Cambridge, UK). Nanoplexes were used after ten-fold dilution and characterized at 633 nm and an angle of 173◦. All the samples were measured in tripli- cate. Results were represented as mean SD (Pandey et al., 2019).
Additional characterization for morphology and particle size of nanoplexes was determined by transmission electron microscope (TEM; Philips, Tecnai 20, Holland). The acceleration voltage was 200 kV. Samples were negatively stained with phosphotungstic acid (1% v/v) and placed on a carbon-coated copper grid. After drying samples, they were focused and visualized at a magnification of 40,000 (Raval et al., 2018). Morphology was also determined with Atomic force microscopy (AFM; Bruker Multimode 8, Bruker, USA). It was completed under constant tapping force 0.02–0.77 N/m with a fixed height of 10–15 nm. The scan area was covered by 90 90 (µm), and using a charge-coupled device monitor, the cantilever and samples were positioned. Images were analyzed using NanoScope 8.15 Software (Lu et al., 2015).
2.5. Entrapment of miRNA-30a mimic complex
After preparation of miAnp-cRGD-30a mm and DTmiAnp-cRGD-30a mm, miRNA-30a mimic entrapment efficiency was determined by the gel retardation assay. Nanoplexes were mixed with 6X loading dye (5 µL), and the sample volume was preserved via DEPC treated nuclease- free water. Then, the sample (15 µL; 0.3 µg miRNA-30a mimic) was loaded onto agarose gel (2 %w/v) comprising ethidium bromide dye (2 μg/mL) for miRNA-30a mimic identification. The gel was run in 1X Tris/ Borate/EDTA (TBE) buffer at 80 V for 1 h. The UV transilluminator (Bio- Rad Laboratories, California, USA) was used to determine the retention of miRNA-30a mimic within the nanoplexes (Sarett et al., 2016).
2.6. RNase protection assay for miRNA-30a mimic
RNase protection assays of miRNA-30a mimic from miAnp-30a mm, DTmiAnp-30a mm, miAnp-cRGD-30a mm, and DTmiAnp-cRGD-30a mm was performed by gel electrophoresis with appropriate modification (Yuan et al., 2010). Briefly, 0.3 μL (0.25 %v/v) RNase was added into eight μL of nanoplexes (0.2 μg miRNA-30a mimic) and incubated at 37
± 2 ◦C for 1 h with shaking. All the samples’ aliquots were treated with
EDTA (4 μL; 0.25 M) for 10 min to stop the reaction or inactivate RNase. After that, samples were mixed with sodium dodecyl sulfate (SDS in 1 M NaOH, 1.0 %w/v (pH 7.3–7.6), to make the final volume (20 μL). Then, the samples were again incubated for 30 min. Then, gel electrophoresis was performed (2 %w/v agarose gel) via TBE buffer for 30 min at 80 V.Free miRNA-30a mimics treated cells were considered as the positive control and nanoplexes treated (RNase –ve or without RNase treated cells) cells were considered as negative controls for superior evaluation of the RNase protection.
2.7. Stability study
RNAi therapeutics are not stable at room temperature and remained stable only at the refrigeration condition (4 2 ◦C) (Kundu et al., 2012). To study the stability profile of the miRNA-loaded nanoplexes over the period, the lyophilized nanoplexes were intended to store under refrigeration conditions at 4 2 ◦C. For the stability assay, DTmiAnp- cRGD-30a mm was stored for 45 days. Briefly, the lyophilized nano- plexes powder was reconstituted in DEPC treated RNase free water and stability was measured in terms of particles size, PDI, zeta potential as well as presence of the entrapped miRNA in nanoplexes. The particle size and PDI of DTmiAnp-cRGD-30a mm was measured at different time points by dynamic laser light scattering method at room temperature by using Malvern Zetasizer ZS90. Each measurement was performed three times (n 3). Analysis of the charge density of DTmiAnp-cRGD-30a mm at various time points was performed by examining their zeta potential using Malvern Zetasizer ZS90 (Raval et al., 2019a). HG-treated Human podocytes model generation.
±
Human podocytes cells (received from Dr. Jeffrey Kopp Lab, Na- tional Institute of Health (NIH), Maryland, USA) were cultured as per the protocol, as explained earlier (Kopp and Heymann, 2019; Saleem et al., 2002). Herein, Type-1 collagen-coated tissue culture flask with RPMI 1640 containing 10 %v/v FBS, 1X ITS (Insulin-Transferrin-Selenium), and antibiotics, namely penicillin (100 IU/mL) and streptomycin sulfate (100 μg/mL) were used. They were incubated at 33 ± 0.5 ◦C with 5 % CO2 (growth permissive stage). After sufficient confluency (70–80%), cells were shifted to 37 0.5 ◦C with 5 %CO2 for 10–14 days to differentiate the podocytes. Following this, the HG-treated podocytes model was developed on differentiated podocytes seeded at a density
105 cells per well in 6 well plates using 30 mM (20–40 mM) glucosein FBS compromised RPMI 1640 media for 48 h (Liu et al., 2013). The developed HG-treated podocytes model was used in further experi- ments, as discussed below (Imasawa et al., 2016).
2.8. Cell viability assay
To evaluate the cellular viability of formulated nanoplexes on podocyte cells, an MTT assay was performed (Pandey et al., 2019). Briefly, the HG-treated podocytes model was generated on differentiated podocyte cells, as discussed. After that, Free miRNA-30a mimic, miRNA- 30a mimic from miAnp-30a mm, DTmiAnp-30a mm, miAnp-cRGD-30a mm, and DTmiAnp-cRGD-30a mm (10 pmol; miRNA-30a mimic /well) were treated on podocytes. After 24 h, the cells were treated for 4 h with MTT reagent (5 mg/mL; 20 µL per well; Sigma-Aldrich, Missouri, USA). After that, DMSO (100 µL) was added to each well to solubilize the formazan crystals. Further, absorbance was recorded at 575 nm by a UV microplate reader (Multiscan GO, Thermo Scientific, USA) at 37 0.5 ◦C (Zhang et al., 2016).
2.9. Cellular uptake assay of nanoplexes
Cellular uptake efficiency of DTAnp-cRGD-FITC and DTAnp-FITC was demonstrated on the HG-treated podocytes model. As described
above, differentiated podocytes cells were seeded onto a glass coverslip in a 6-well plate (2×105 cells per well) for 24 h. Then, the HG-treated
podocytes model was developed as described previously. Further, media was changed with an Opti-MEM® (Minimal Essential Medium (MEM) with reduced fetal bovine serum (FBS) supplement by at least
50% and comprises insulin, transferrin, hypoxanthine, thymidine, and trace elements for better transfection) comprising FITC solution, DTAnp- cRGD-FITC, and DTAnp-FITC. After that 8 h, the nucleus of the cells was stained with Hoechst 33342. The cells were then washed with PBS (1X) and fixed in paraformaldehyde (4 %v/v). It was again washed with PBS (1X). The coverslip was removed and mounted on a glass slide (Borosil, Mumbai, India). Fluorescence signal was determined using via confocal laser scanning microscope (Emission max: 520 nm; Excitation max: 495 nm; Leica TCS SP5 AOBS Confocal microscopy system (Leica, Germany) and mean fluorescent intensity was evaluated by ImageJ (Liu et al., 2015).
2.10. Competitive receptor binding assay for receptor-mediated internalization
As per the research αvβ3 receptor, integrin is the primary integrin expressed in podocytes and having an important role in homeostasis. While, during diabetic nephropathy, due to hyperglycemic condition of kidney the overexpressed αvβ3 receptor activates urokinase receptor (uPAR) at podocytes level and further leads to proteinuria via podocytes effacement and loss of podocytes filtration barrier. Further, the expression of αvβ3 receptor in HG DN model of human podocytes was also confirmed via RT-PCR with reference to 18 s (Lennon et al., 2014; Maezawa et al., 2013; Maile et al., 2014; Reiser et al., 2010).
The competitive receptor binding assay was examined by FITC
loaded nanoplexes through flow cytometry. For that, differentiated podocytes were seeded at a density of 2×105 cells per well in 6-well
plates, and the HG-treated podocytes model was developed as previ- ously discussed. The media was changed with FBS compromised media containing free cRGD (100 nM). Post 1 h, free cRGD treated cells were incubated with DTAnp-cRGD-FITC for 6 h. After that, cells were tryp- sinized with trypsin-EDTA (1X), collected, and then centrifugated at 2000 g for 7 min to form a cell pellet. It was resuspended in PBS (1X; 500 µL) and repeated in the centrifugation step. Finally, the pellet was sus- pended in PBS (300 µL; 1X). The fluorescence intensity of FITC present in the prepared cell suspension cells was determined via Beckman Coulter flow cytometer (S3e™ Cell Sorter, Bio-Rad Laboratories, Cali- fornia, USA). At least ten thousand gated events were attained from each sample (Kong et al., 2016).
2.11. Delivery effect of miRNA-30a mimic loaded nanoplexes to HG- treated podocytes
HG-treated podocytes were treated with free miRNA-30a mimic, DTmiAnp-cRGD-scramble (scramble miRNA control loaded nanoplexes; scramble sequence is randomly rearranged nucleotide sequence or non- targeting sequence), DTmiAnp-30a mm, and DTmiAnp-cRGD-30a mm for 48 h. After that, cells were washed with PBS (1X) and then harvested. Further, targeted gene up-regulation was evaluated using quantitative real-time PCR (qRT-PCR) at the mRNA level (Oe et al., 2014). Cells were harvested for total RNA extraction using the RNeasy mini kit (Qiagen, Hilden, Germany) as stated by the manufacturer’s protocol. Total RNA extracted was quantified using Nanodrop® 2000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). For up-regulation of miRNA-30a, TaqMan® microRNA Assays were accomplished based on the manufacturer’s instruction (Applied Biosystems, California, USA). Briefly, total RNA (1 µg) was taken for the reverse transcription using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, California, USA). miRNA-30a specific probe has-miR-30a and U6 snRNA as endogenous control (Applied Biosystems, California, USA) were used. RT-PCR was performed via Taqman Assay. The experiment’s final vol- ume was 20 μL using Taqman Universal PCR Master Mix (Applied Bio- systems, California, USA), and three experimental sets were performed. PCR was performed on StepOne™ systems (Applied Biosystems, Cali- fornia, USA). The results were analyzed by the comparative CT method
(ΔΔCT Method).
2.12. Western blot analysis: In vitro
The expression of the downstream target gene Notch-1 of miRNA- 30a was evaluated in HG-treated podocytes through western blot anal- ysis. HG-treated podocytes incubated with free miRNA-30a mimic, DTmiAnp-cRGD-scramble, DTmiAnp-30a mm, and DTmiAnp-cRGD-30a mm equivalent to 30 pmol per well for 48 h. Further, cell lysate was prepared in radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing a protease inhibitor cocktail (1 µL). The concentration of protein was evaluated by Bicinchoninic acid assay (BCA) reagent, and protein (25 µg) was loaded on an acrylamide gel and transferred over polyvinylidene difluoride (PVDF) membrane using RTA Trans Turbo kit (Bio-Rad Laboratories, California, USA). Protein was detected via pri- mary antibody incubation (Notch-1 (1:2000) in 1% w/v Casein blocker; Abcam, Cambridge, UK) and β-actin (1:5000) dilution in 1% w/v Casein), Santacruz, USA) at 4 2 ◦C overnight. Following overnight incubation, TBST (0.1% w/v) was utilized for membrane washing and then incubated with secondary antibody (HRP-conjugated Goat anti- mouse IgG-HRP 1:20000, Santacruz and Goat anti-rabbit IgG-HRP 1:20,000, Abcam, Cambridge, UK) at room temperature for 2 h. The protein bands were detected using chemiluminescence substrate (Bio- Rad Laboratories, California, USA) and quantified using ImageJ soft- ware (NIH, Bethesda, Maryland) (Zhou et al., 2017).
2.13. Apoptosis assay in HG-treated podocytes model
HG-induced expression of Notch-1 in podocytes via downregulation of protective miRNA-30a which leads to inflammation, apoptosis, and injury to the podocytes. After treatment with miRNA-30a mimic nano- plexes the consequence of podocytes apoptosis exhibited was measured using flow cytometry. Differentiated podocytes were exposed to HG for 48 h to generate HG-treated podocytes model induction (Pandey et al., 2019). Further, cells were treated with free miRNA-30a mimic, DTmiAnp-30a mm, and DTmiAnp-cRGD-30a mm (30 pmol/well) 24 h. After treatment, the cells were trypsinized with trypsin-EDTA, collected, and centrifuged at 2000 g for 7 min. The pellet was suspended in binding buffer (1X; 200 μL) comprising propidium iodide (2 μL) and annexin V (5 μL) and kept for 15 min in dark conditions in room temperature. The apoptotic cell population was evaluated by flow cytometry with Annexin V FITC Apoptosis Kit (Thermo Fisher Scientific, Massachusetts, USA) in Beckman Coulter flow cytometer (Bio-Rad Laboratories, California, USA) by following the manufacturer’s protocol (Bio-Rad Laboratories, California, USA). At least ten thousand gated events were obtained from each sample. Results are presented as the total percentage of apoptotic cells (Annexin V positive and PI) in the gated cell population (Reddy et al., 2016).
2.14. In vivo diabetic model in C57BL/6 male mice
All animal experiments were performed following the National Institute of pharmaceutical education and research (NIPER-A) Guide- lines for Care and Use of Laboratory Animals. The animal study pro- tocols were approved by the Institutional Animal Ethics Committee at NIPER-Ahmedabad, Gujarat, India (Approval number: NIPER-A/IAEC/ 2017/034; Date: 17/07/2017).
For the examination, streptozotocin (STZ) at a dose (50 mg/kg) in citrate buffer (0.1 M) was administered via intraperitoneal injection for five consecutive days to C57BL/6 male mice (average weight: 21 gm) (Zydus Research Centre, Ahmedabad, India) to avoid acute toxicity of STZ. Plain vehicle as citrate buffer (0.1 M) was administered to control animals. The diabetes model (STZ-DN) generation in mice was confirmed by measuring blood glucose levels AccuChek-Active gluc- ometer (Accucheck active, Roche, Indiana, USA) (Pandey et al., 2019; Raval et al., 2020). After the blood glucose levels of mice were increased
(fasting glucose > 12 mmol), mice were randomized and divided into
five experimental groups with six mice/groups, namely STZ-DN mice,
free miRNA-30a mimic, DTmiAnp-cRGD-scramble, DTmiAnp-30a mm, and DTmiAnp-cRGD-30a mm. They were administered with a dose (1 mg/kg) of equivalent miRNA twice a week intravenously until 4 weeks. After this, mice were kept in a metabolic cage to assess metabolic pa- rameters comprising blood urea nitrogen level (BUN), blood glucose, creatinine clearance, serum creatinine through a creatinine test kit (identity, Jeev diagnostic Pvt. Ltd, Tamil Nadu, India). According to the manufacturer’s protocol of albumin mouse ELISA kit (Abcam, Cam- bridge, UK), urinary albumin was evaluated as urinary albumin excre- tion rate (UAER).Further, as per the manufacturers’ protocol N-acetyl glucosamine activity (NAG; Abcam, USA) was also determined (Jha et al., 2016). After treatment, mice were anesthetized with ketamine/xylazine and sacrificed, and kidneys were excised immediately. One portion was fixed by 4 %w/v paraformaldehyde for histological evaluation, and the other portion was homogenized in the lysis buffer used for western blot analysis (Gai et al., 2014).
2.15. Western blot analysis: In vivo
In vivo gene silencing efficacy in kidneys removed from treatment sets, including (i) normal healthy mice, (ii) STZ-DN mice, and (iii) miRNA-30a mm nanoplexes treated STZ-DN mice, was determined by western blot analysis. The miRNA-30a mm were treated at 1 mg/kg miRNA-30a mm given twice a week to STZ-DN mice. Post-treatment, the mice were sacrificed, and kidneys were removed. Its cortex portion or glomerular area was homogenized by tissue lyser (TissueLyser LT, Qiagen, Germany) and centrifuged for 5 min at 12 K. The collected su- pernatant was utilized for protein content was measured using the BCA method. Western blotting was performed by resolving (10 %v/v) and stacking (5 %v/v) SDA-PAGE gel. A detectable amount of protein (50 µg/mL) was loaded into the gel. Protein was overnight incubated with Notch-1 primary antibody against (Abcam, Cambridge, UK) and (β-actin, Santa Cruz, Texas, USA) at 4 2 ◦C. Afterward, the secondary antibody was incubated for 2 h (HRP-conjugated; Goat anti-mouse IgG- HRP, Santa Cruz, Texas, USA and Goat anti-rabbit IgG, Abcam, Cam- bridge, UK) at room temperature. Chemiluminescence substrate (Bio- Rad Laboratories, California, USA) was used to detect the specific pro- tein bands, which were quantified via ImageJ software (NIH, Bethesda, Maryland) (Zeng et al., 2019).
2.16. Renal histological evaluation
The removed kidneys were immersed in 4 %w/v paraformaldehyde for fixing and inserted in paraffin using (Sawaguchi et al., 2018) to make paraffin block. A microtome (Leica RM2235, Germany) was used to cut three-micrometer-thick kidney sections. They were stained as stated by the manufacturer’s protocol with hematoxylin and eosin staining (H&E) as well as modified Masson’s trichrome staining (Abcam, Cambridge, UK)) (Shi et al., 2018). H&E staining was used to measure the glomer- ular area from fifteen random cortex region images per mouse. The fibrotic area was analyzed through Masson’s trichrome-stained sections with ImageJ software as defined (Grange et al., 2019a).
2.17. Statistical analysis
One-way analysis of variance (ANOVA) was used to determine sta- tistical changes among the group. This was followed by Bonferroni’s
post-test using GraphPad Prism 6.01™ software (GraphPad Software Inc., San Diego, California). A level of probability as p > 0.05 greater than that would be considered as non-significant. A level of probability as p < 0.05, p < 0.01, and p < 0.001 was considered to be significant and highly significant well. All the results are represented as mean ± SD or mean ± SEM.
3. Results
3.1. Characterization of miRNA-30a mimic nanoplexes
The DLS was performed for the evaluation of miRNA-30a mimic nanoplexes. The hydrodynamic particle size for DTmiAnp-cRGD-30a mm was found 73.51 ± 1.43 nm (PDI; 0.268 ± 0.049), and zeta po- tential was –20.32 ± 1.03 mV. Likewise, the particle size for DTmiAnp- 30a mm and miAnp-30a mm was found 68.19 ± 0.52 (PDI: 0.254 ± 0.052) and 67.57 ± 1.21 nm (PDI: 0.136 ± 0.064), respectively. The obtained zeta potential was –19.62 1.62 and –26.9 0.22 mV, respectively, as shown in Fig. 1A and B. Further, the morphology of DTmiAnp-cRGD-30a mm was confirmed using TEM and AFM. The TEM images were showed an average particle size of approximately around 60–80 nm with narrow size distribution; it was found in good agreement with the result of DLS (Fig. 1C). Likewise, AFM was also showed spherical morphology with a smooth surface of DTmiAnp-cRGD-30a mm (Fig. 1D).
3.2. Entrapment of miRNA-30a mimic in nanoplexes
Here, the gel shift assay was performed to investigate the entrapment
of miRNA-30a mimic inside nanoplexes. As shown in Fig. 1E, the miRNA migration was found from the miAnp-cRGD-30a mm and DTmiAnp-30a mm. The intensity of free miRNA in DTmiAnp-30a mm was less compared to miAnp-cRGD-30a mm. However, no migration of free miRNA was found in DTmiAnp-cRGD-30a mm than miAnp-cRGD-30a mm and DTmiAnp-30a mm.
3.3. RNase protection assay for miRNA-30a mimic
The ability of making protection of miRNA from RNase enzyme was confirmed via gel assay. The miRNA loaded nanoplexes and intact free miRNA were incubated with the RNase enzyme, loaded onto an agarose gel. The results of the gel assay depicted in Fig. 1F, free miRNA-30a mimic was entirely degraded by RNase treatment before performing gel electrophoresis compared to without RNase treated free miRNA-30a mimic (Lane 1, Lane 2). Because the RNase enzyme easily came in contact with the free miRNA and degrades immediately after coming in contact. While in the case of miAnp-cRGD-30a mm, miRNA-30a mimic was not showing any band of miRNA compared to non-RNase enzyme- treated group. Therefore, it suggested that in after RNase treatment of miAnp-cRGD-30a mm, miRNA was degraded compared to non-RNase treated miAnp-cRGD-30a mm (Lane 3, Lane 4). However, in. (A) Hydrodynamic particle size (B) zeta potential (C) TEM image (D) AFM image of DTmiAnp-cRGD-30a mm (E) gel electrophoresis for evaluation of entrapment of miRNA-30a mimic in nanoplexes. Lane specified as (i) free miRNA-30a mimic (ii) DTmiAnp-cRGD-30a mm (iii) miAnp-cRGD-30a mm and (iv) DTmiAnp —30a mm (F) RNase protection assay, Lane:1 and 2, free miRNA-30a mimic; lane: 3 and 4, miAnp-cRGD-30a mm; lane: 5 and 6, DTmiAnp-cRGD-30a mm.
DTmiAnp-cRGD-30a mm, miRNA was found intact after exposure with RNase with reference to non-RNase treated DTmiAnp-cRGD-30a mm. It depicted tightly complexed miRNA with dendrimer remains inside the well (Lane 5, Lane 6). Thus, from the results it can be said that DTmiAnp- cRGD-30a mm was able to entrapped miRNA-30a mimic compactly and protect from unwanted RNase degradation effectively.
3.4. Stability study
The stability study was investigated for 45 days to determine the stability profile of the ANp, DTANp, DTmiAnp, and DTmiAnp-cRGD-30a mm at 4 ± 2 ◦C. The data given in Fig. 2 shows the effect on particle size, PDI, ζ potential, and stability of entrapped RNAi concerning time. In thecase of ANp and DTANp, the particle size, PDI, and ζ potential were relatively stable at 4 2 ◦C for 45 days, and non-significant (p > 0.05) change was observed compared to 0 days. While miRNA-loaded nano- plexes (DTmiAnp, and DTmiAnp-cRGD-30a mm) were found much better and stable during the entire investigation time (45 days) at 42 ◦C after reconstitution (p > 0.05). This ascribed the stabilization effect of cryoprotectant as help in avoiding the aggregation of nanoplexes.Overall the formulations were found to be more stable at 4 2 ◦C. The presented results of the stability of nanoplexes are found in good agreement with the reported literature by Kundu et al. (Kundu et al., 2012)
3.5. Cell viability assay
The free miRNA-30a mimic (95.6 3.49%), free dendrimer (91.04
10.01%), DTmiAnp-30a mm (94.25 1.55%), and DTmiAnp-cRGD- 30a mm (94.31 2.37%), treated on an HG-treated podocytes model for 24 h. The cellular viability assay result suggested no such notable cytotoxicity proceedings were observed in all the treatment groups
(Fig. 3C p > 0.05, ns). Therefore, it concluded that nanoplexes were found safe as well as biocompatible towards HG-treated podocytes.
3.6. Cellular uptake assay of nanoplexes
FITC loaded DTAnp-cRGD-FITC (71.23 0.49 (PDI: 0.28 0.022)),
and DTAnp-FITC (69.31 1.68 (PDI: 0.243 0.062) nanoplexes were incubated with HG-treated podocytes. Then nuclei were stained with Hoechst 33342. The appeared green fluorescence of FITC in the cyto- plasm of podocytes via confocal laser scanning microscopy was due to cellular uptake, and delivery of FITC loaded nanoplexes inside the cells (Fig. 3A). It was demonstrated using mean fluorescent intensity and found that greater cellular uptake (67.79 0.79% and 76.55 1.22%) was observed in DTAnp-cRGD-FITC compared to DTAnp-FITC and free
FITC (Fig. 3B; p < 0.001). The result directed that the DTAnp-cRGD-FITC
nanoplexes could internalize significantly higher than the non-targeted counterpart, DTAnp-FITC. A minimal signal was obtained in the control group (without treatment of nanoplexes).
3.7. Competitive receptor binding assay for receptor-mediated internalization
The obtained results for the expression of αvβ3 receptor suggest the overexpression of αvβ3 receptor at podocytes level in hyperglycemic condition of DN (Fig. S1). Following, the cellular uptake profile (flow cytometry) of DTAnp-cRGD-FITC was demonstrated in the existence of excess free cRGD, which able to bind selectively to the αvβ3 receptors to confirm the binding efficiency of the αvβ3 receptor. Initially, podocytes were treated with free cRGD, then after cells were treated with DTAnp- cRGD-FITC. The DTAnp-cRGD-FITC treated cells showed mean fluores- cence intensity (MFI) was found 1685.2 ± 31.54, and free cRGD treated cells exhibited MFI was 475.2 ± 39.35. Therefore, from the result it can
(A) Confocal scanning microscopy of podocytes after treatment of prepared nanoplexes (B) mean fluorescent intensity (MFI) of FITC. Nuclei were stained with Hoechst 33,342 (Scale bar: 20×) **p < 0.01 (C) cellular viability of podocytes.be said that DTAnp-cRGD-FITC (free cRGD ve) started performing similar to DTAnp-FITC. The experiment proved that cRGD gated nano- plexes enhanced cellular uptake (Fig. 4A and B; p < 0.001).
3.8. Delivery effect of miRNA-30a mimic loaded nanoplexes to HG- treated podocytes
Next, we tested the definite up-regulation of miRNA-30a in HG- treated podocytes, known to suppress homeostatic gene miRNA-30a specifically in podocytes. For maintaining the expression level of miRNA-30a in podocytes, a miRNA-30a mimic was utilized, and a scramble sequence of miRNA was used as a negative control. Here, for the assay, free miRNA-30a mimic, DTmiAnp-30a mm, DTmiAnp-cRGD- scramble, and DTmiAnp-cRGD-30a mm incubated with HG-treatedpodocytes. As a result (Fig. 4C), up-regulated expression of miRNA- 30a was observed in DTmiAnp-30a mm by 3.38 ± 1.79 fold (p > 0.05) compared to free miRNA-30a mimic (0.62 ± 0.10 fold; p > 0.05).
Whereas DTmiAnp-cRGD-30a mm treated podocytes were exhibited 2117.96 11.21 fold (p < 0.001) up-regulation of miRNA-30a compared to control podocytes. DTmiAnp-30a mm and free miRNA showed 2.38 1.18 fold and 0.37 0.08 fold (p > 0.05) miRNA upregulation with reference to control podocytes. Likewise, DTmiAnp-cRGD-30a mm enhance expression of miRNA-30a up to 99.97 5.25% (p < 0.001) and 99.84 0.95% (p < 0.001), respectively compared to DTmiAnp-30a mm and free miRNA-30a mimic. Therefore, results suggested that successful delivery and upregulation of miRNA- 30a, mimic in podocytes by DTmiAnp-cRGD-30a mm.
It was further established via western blot analysis at the protein level. Oniszczuk et al. created nanoparticle templates composed of chitosan (polycation) and nucleic acid (polyanion) for podocytes damage and able to suppress c- mip expression ~80% at podocytes cell level compared to control group (Oniszczuk et al., 2021). Effect of miRNA-30a mimic loaded nanoplexes on the expression of Notch-1 of HG-treated podocytes model.
The Notch-1 signaling is activated via suppression of miRNA-30a
(A) Flow cytometry examination of HG-treated podocytes after treatment with free cRGD following DTAnp-cRGD-FITC (+) and DTAnp-cRGD-FITC (-) (B) quantitative evaluation of cell uptake by means of MFI p < 0.001 vs. cRGD DTAnp-cRGD-FITC (C) qRT-PCR data represents relative up regulation of miRNA-30a after treatment of miRNA-30a mimic loaded nanoplexes, here U6snRNA was considered as an endogenous control (healthy podocytes) ***p < 0.001; DTmiAnp-cRGD- 30a mm vs. DTmiAnp-30a mm and ***p < 0.001; DTmiAnp-cRGD-30a mm vs. free miRNA-30a mimic (D) western blot analysis for Notch-1 expression after incubation of free miRNA-30a mimic, DTmiAnp-30a mm-scramble, DTmiAnp-30a mm and DTmiAnp-cRGD-30a mm. β-actin was taken as endogenous control. **p < 0.01; DTmiAnp-cRGD-30a mm vs. DTmiAnp-30a mm and ***p < 0.001; DTmiAnp-cRGD-30a mm vs. free miRNA-30a mimic.homeostatic gene in podocytes due to proteinuria in diabetic kidney disease. After treatment with miRNA-30a mimic loaded nanoplexes to HG-treated podocytes, the relative expression of Notch-1 was found 8.3
3.9. Effect of miRNA-30a mimic loaded nanoplexes on apoptosis of HG- treated podocytes
The total apoptotic cell ratio was found significantly increased (p < 0.001) in HG treated podocytes cells (15.26 0.82) due to hypergly- cemic condition compared to healthy control podocytes (2.95 0.83).After exposure with HG, podocytes were incubated with free miRNA-30a mimic, DTAnp-30a mm, and DTAnp-cRGD-30a mm to understand the effect of apoptosis on podocytes. After treatment with free miRNA-30a mm, significant suppression of cell apoptosis was not detected. Be- sides, enhanced total cellular apoptosis was observed in HG exposed, and free miRNA-30a mimic treated cells up to 15.26 0.82% (Fig. 5). After the treatment of DTmiAnp-30a mm, total apoptosis was lessened
(A) Flow cytometric assay showing apoptosis of HG-treated podocytes after treatment; Q1: live cells, Q2: early apoptotic cells, Q3: late apoptotic cells; Q4: dead cells, total apoptotic ratio is total cell from early and late apoptotic phase (B) representing total apoptotic cell of HG-treated podocytes after treatment, *p < 0.05; DTAnp-cRGD-30a mm vs. DTAnp-30a mm and ***p < 0.001; DTAnp-cRGD-30a mm vs. Free miRNA-30a mimic. (C) Western blot analysis at protein level showing expression of Notch-1 after the treatment of free miRNA-30a mimic, DTmiAnp-cRGD-scramble, DTmiAnp-30a mm, and DTmiAnp-cRGD-30a mm, (B)
Relative quantification of Notch-1 concerning β-actin. The analysis was done by ImageJ (NIH, Bethesda, MD). ***p < 0.001 vs. Free miRNA-30a mimic; *p < 0.05 vs. DTmiAnp-30a mm treated STZ-DN mice group. Results are represented as mean ± SEM (n = 3). 0.25% (p < 0.05). Whereas, DTmiAnp-cRGD-30a mm treated cell significantly able to suppress apoptosis (3.34 0.67%; p < 0.001) compared to free miRNA-30a mimic and DTmiAnp-30a mm.
3.10. Evaluation of metabolic parameter: In vivo diabetic model (STZ- DN) in C57BL/6 male mice
Next, the therapeutic competence of miRNA-30a mimic nanoplexes in STZ-DN mice was evaluated, as shown in Fig. 6. After inducing dia- betes to C57BL/6 mice, the STZ-DN mice displayed a significant eleva- tion in glucose level by 24.15 ± 3.07 mmol/L; p < 0.001 compared to the Metabolic parameters after treatment of miRNA-30a mimic loaded nanoplexes in STZ-DN induced mice. Metabolic parameters were determined in control as well as in STZ-DN mice after the treatment of free miRNA-30a mimic, DTmiAnp-cRGD-scramble, DTmiAnp-30a mm, and DTmiAnp-cRGD-30a mm at a dose of 1 mg/kg equivalent to miRNA. The blood glucose level was evaluated utilizing AccuChek-Active glucometer (AccuCheck active, Roche, Indiana, USA). The serum creatinine and urine creatinine levels were evaluated via the creatinine test kit (identifi, Jeev diagnostic Pvt. Ltd, Tamil Nadu, India). Serum, as well as urine urea measurement, was performed via urea kinetic test kit (identifi, Jeev diagnostic Pvt. Ltd, Tamil Nadu, India). Furthermore, UAER for Albumin was measured by mouse ELISA kit (Abcam, Cambridge, UK) as per manu- facturer’s protocol. NAG activity was measured using the NAG activity kit according to the manufacturer’s protocol (NAG; Abcam, USA). Results are represented as mean ±
SD (n = 6). Here, **p < 0.01 and ***p < 0.001 vs STZ-DN mice. BUN: blood urea nitrogen, NAG: N-acetyl glucosamine activity, UAER: urinary albumin excretion rate.
Histological analysis of hematoxylin-eosin staining via ImageJ analysis of STZ-DN treated mice kidney (A) control (B) STZ-DN mice (C) DTmiAnp-cRGD- scramble (D) free miRNA-30a mimic (E) DTmiAnp-30a mm (F) DTmiAnp-cRGD-30a mm (G) represented glomerular area after the treatment of miRNA nano-
plexes, here, p < 0.001 vs. STZ-DN mice. Modified Masson trichrome staining (H) control (I) STZ-DN mice (J) DTmiAnp-cRGD-scramble (K) free miRNA-30a mimic(L) DTmiAnp-30a mm (M) DTmiAnp-cRGD-30a mm (N) indicating percentage fibrosis, calculated from ten random glomeruli, here, ***p < 0.001 vs. STZ-DN mice; *p< 0.05 vs. free miRNA-30a mimic.
DTmiAnp-30a mm. Thus, miRNA-30a mimics the loaded nanoplexes treated STZ-DN mice group significantly suppressed the expanded glomerular area or bowman’s space. The results of the glomerular area were found in good agreement with the previously published reports by Grange et al. (2019b).
Collagen is a crucial marker for the determination of renal fibrosis. Extracellular matrix deposition or fibrosis was further characterized by Masson Trichrome stained paraffin sections. The percentage of fibrosis
4. Discussion
Ever since the finding of RNAi significantly enhances gene regula- tion, it has also been offered as the potential for treatment for diabetic kidney diseases due to its excellent potency and selectivity for a particular gene. As homeostatic gene miRNA-30a was found down- regulated in the podocytes in kidney diseases viz. diabetes, focal segmental glomerulosclerosis, acute nephritis, nephrotic syndrome, etc. They were moreover, deprived of a proficient and safe delivery system for RNAi therapeutics such as miRNA acted poorly in vivo. Lesser size of RNAi ( 5 nm) exhibited a shorter half-life and degradation via endo- nucleases enzyme of serum. This limitation hindered the knockdown of the targeted gene as well as the clinical translation. Therefore, several tactics have been developed to generate a potent miRNA delivery system with a high safety level, miRNA protection capability in vivo, site- specific delivery potential, and commercial translatability. In context, positively charged polymeric systems have been copiously utilized, whereas cellular-based toxicity hindered significant utilization for effi- cient delivery of miRNA. This investigation reports simple tactics for loading, stabilizing, and effectively delivering miRNA using albumin as a biopolymeric and USFDA permitted construct. Plain albumin embraced miRNA nanoconstruct was incapable and deficient in miRNA entrapment and released cargo in the cytoplasm. This is endorsed by anionically charged albumin that cannot extra negatively chargedmiRNA and prematurely releases miRNA payload in blood circulation.
Besides, the abridged efficacy of albumin nanoconstruct was evinced by lacking endosomal escape propensity in the cytosolic compartment of the cell.
For effective entrapment of miRNA in albumin biopolymer, the dendrimer templated (PAMAM dendrimer 2.0G) concept was utilized owing to the principle of electrostatic interaction. Our earlier reports suggested that 0.5n/p ratios for dendrimer to RNAi (siRNA or miRNA) complex were found efficient to entrap negatively charged RNAi in positively charged dendrimer (Indian patent at Indian Patent Office (IPO), Mumbai, India; Application No.: 201921019898; Date of Appli- cation: 18/05/2019). As well as it was also reported and evaluated that cRGD truncated albumin nanoplexes were significantly up taken via HG exposed podocytes and also able to escape endo/lysosomal compart- ment that lead to efficient gene silencing (Raval et al., 2020).
Therefore, cRGD gated and dendrimer templated nanoplexes were
prepared with an average hydrodynamic particle size of ≤70 nm owing to glomerular fenestration (70–100 nm). The suggested sized nanoplexes could not clear via kidney and specifically bind with the αvβ3 receptor and selectively localized in kidney podocytes. The formed DTmiAnp- cRGD-30a mm was able to entrap miRNA significantly compared to
miAnp-cRGD-30a mm due to dendrimeric template. Whereas the lesser amount of miRNA was entrapped in DTmiAnp-30a mm evinced by the presence of miRNA band.
It suggests the effective interaction of posi- tively charged dendrimer:miRNA complex with the anionic charged albumin/A-cRGD conjugate.
The stability of the entrapped miRNA is another encounter for sys- temic delivery of miRNA owing to RNase degradation. The results of the RNase protection experiment suggested that casted DTmiAnp-cRGD-30a mm (Fig. 1F) enhanced RNase protection of miRNA compared to miAnp- cRGD-30a mm. The reason behind this finding might be the availability of polycationic architect of dendrimeric template in DTmiAnp-cRGD- 30a mm, which may not permit the unnecessary transfer of miRNA with other environmental ions of blood that resulted in the efficient delivery of miRNA. Hence, miRNA nanoplexes were found compatible and non-cytotoxic to HG-treated podocytes. In contrast, cellular uptake and cytoplasmic release of loaded miRNA payload are necessary for efficient gene silencing.
Here is a model fluorescent substance FITC that was loaded inside the nanoplexes. The cellular uptake assay showed that significant cellular uptake of DTAnp-cRGD-FITC was found due to receptor-mediated endocytosis via overexpressed αvβ3 receptors in HG treated podocytes as subsists in diabetic kidney condition. The prepared dendrimer templated nanoplexes better the capability to escape the endosomal compartment due to the proton sponge effect of the den- drimer template at low pH (Raval et al., 2019a; Raval et al., 2020).
The selective binding effect of DTAnp-cRGD-FITC with αvβ3 re- ceptors of podocytes was also performed via competition binding assay. The results (Fig. 4A-B) suggested that the free cRGD pre-treated group could not uptake more DTAnp-cRGD-FITC due to the unavailability of free αvβ3 receptors to bind with nanoplexes and start behaving like DTAnp-FITC. DTAnp-cRGD-FITC (without free cRGD pre-treated) significantly enhanced the cellular uptake due to the availability of αvβ3 receptors for binding with DTAnp-cRGD-FITC.
In a healthy condition, miRNA-30a selectively expressed and regu- lated the glomerular podocytes functioning. Enhanced expression of Notch signaling induced podocytes apoptosis, which leads to suppres- sion of the miRNA-30a. The upregulation of miRNA-30a via exogenously delivered miRNA-30a mimic loaded nanoplexes was the ultimate goal for achieving the significant therapeutic effect and suppressing apoptosis of HG-treated podocytes. The DTmiAnp-cRGD-30a mm treated cells significantly up-regulate the level of miRNA-30a in HG- treated podocytes (Fig. 4C). The protein level of Notch-1 further confirmed it. In line with the qRT-PCR results, the level of miRNA-30a as up-regulated by DTmiAnp-cRGD-30a mm treatment could suppress Notch-1 more efficiently than free miRNA-30a mimic and DTmiAnp-30a mm. Therefore, the DTmiAnp-cRGD-30a mm significantly suppressed the apoptosis induced via HG-treated podocytes compared to free miRNA-30a mimic DTmiAnp-30a mm (Fig. 5B).
Moreover, miRNA-30a loaded nanoplexes were administered to the
STZ-DN C57BL/6 mice model and found significantly lowered elevated serum BUN and enhanced creatinine clearance level through DTmiAnp- cRGD-30a mm treatment (Fig. 7). Whereas elevated serum creatinine level in STZ-DN mice known to reduce glomerular filtration rate prominently. The serum creatinine level was noticeably reduced via DTmiAnp-cRGD-30a mm compared to DTmiAnp-30a mm and free miRNA-30a mimic. Proteinuria is a vital marker for diabetic kidney disease progression, also found reduced by DTmiAnp-cRGD-30a mm proficiently in comparisons to DTmiAnp-30a mm and free miRNA-30a mimic. Thus, DTmiAnp-cRGD-30a mm alleviates the functionality of the kidney and the metabolic parameter in STZ-DN mice. It is reported that suppression of homeostatic miRNA-30a gene due to overexpression of Notch-1 leads to podocytes apoptosis and inflammation. Therefore, after the treatment of DTmiAnp-cRGD-30a mm, effective suppression of Notch-1 was showed due to selective targeting of DTmiAnp-cRGD-30a mm towards podocytes.
The pathological differences were detected employing hematoxylin-
eosin staining and modified Masson trichrome staining. The glomerularbasement membrane expansion and thickening of the glomerular base- ment membrane are the main pathological changes observed in the diabetic kidney. The histopathological analysis results suggested that the DTmiAnp-cRGD-30a mm significantly reduced the expanded glomerular area and bowman space (Fig. 7). The nominal amount of collagen is expressed in healthy kidney conditions when collageninterests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The research was carried out at the National Institute of Pharmaenhancement and extracellular matrix accumulation led to renal
ceutical Education and Research-Ahmedabad with financial supportfibrosis. The finding indicated that in vivo delivered DTmiAnp-cRGD-30a mm could suppress fibrosis and kidney injury by reducing the accu- mulation of extracellular matrix in the glomerular area. Therefore, the administered nanoplexes passing through larger pore of glomerular endothelial (~150 nm) can reach GBM from the blood circulation. The pathophysiology of the DN mice modified the GBM morphology and resulted in enlarged GBM pores approx. 10–80 nm due to GBM thick- ening (Raval et al., 2020). This phenomenon allows the nanoplexes to reach towards podocytes through GBM attached αvβ3 integrin utilizing cRGD. It gives receptor-mediated uptake of nanoplexes towards the podocytes in DN. This might be a possible mechanism for disseminating and delivering the cRGD targeted nanoplexes to the DN kidney.
The finding acquired from this investigation directed towards a po- tential and straightforward approach for selective targeting and delivery of miRNA-30a mimic to podocytes; it can be stretched for other diseases. The presented work may provide a possible solution to glitches related to the bench-to-bed translation of miRNA as a targeted therapeutic system to treat kidney-related ailments. Owing to the easy preparation, flexible and efficient delivery system, in forthcoming would take note- worthy paces to develop clinically applicable nanoplexes to treat the podocytes-related disorders.
5. Conclusions
The depleted miRNA-30a and elevated Notch-1 signaling levels have been identified as a primary cause for depleting the podocyte in diabetic nephropathy patients. This investigation reports the development and placement of engineered nanoplexes for targeted silencing of miRNA- 30a expression to rescue dying podocytes in Diabetic Nephropathy. The engineered nanoplexes were resistant to RNase and could protect the RNAi-loaded therapeutics from degradation by RNAse enzyme. The nanoplexes showed binding affinity towards the αvβ3 receptors that get over-expressed over podocytes of the diabetic patient. The presence of αvβ3 peptide component resulted in high cellular uptake of nanoplexes in diabetic podocytes. The treatment of nanoplexes successfully up- regulated the expression levels of miRNA-30a and represses the elevated Notch-1 signaling in HG exposed podocytes cell model. Anal-
ogous to in vitro results, the in vivo assays also marked the suppression of
from the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India. RKT would like to acknowledge the Science and Engineering Research Board (Statutory Body Established Through an Act of Parliament: SERB Act 2008), Department of Science and Technology, Government of India for a grant (Grant #ECR/2016/ 001964) and N-PDF funding (PDF/2016/003329) for work in Dr. Tekade’s Laboaratory. The authors would also like to acknowledge Dr. Jeffrey Kopp, National in the statute of Health (NIH), Maryland, the USA, for the gift sample of the immortalized human podocyte cell line.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijpharm.2021.120842.
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¨
administration also reduced the glomerular expansion and fibrosis in the glomerular area of the kidney. The developed nanoplexes represent a potential approach for the targeted delivery of exogenous miRNA to podocytes. The engineered nanoplexes treated group exhibited abridged albuminuria and might aid in the alleviation of abnormal kidney func- tion. The approach developed with this investigation could be extrap- olated to other gene therapeutics and other kidney-related diseases.
Author contribution
Nidhi Raval: Investigation, Methodology, Formal analysis, Valida- tion, Writing – original draft. Piyush Gondaliya: Investigation, Meth- odology. Vishakha Tambe: Formal analysis. Kiran Kalia: Resources, Methodology. Rakesh K. Tekade: Conceptualization, Visualization, Project administration, Supervision, Resources, Reviewing and Writing – original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
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