Research Article Delivery Efficiency of miR-21i -CPP-SWCNT...

Research ArticleDelivery Efficiency of miR-21i-CPP-SWCNT and Its InhibitoryEffect on Fibrosis of the Renal Mesangial Cells

Hong Liu12 Guobao Wang3 Yihong Yang1 Lei Yu1 Leyu Wang1 Zhengda Wen1

Xiaofang Hu4 Hequn Zou12 and Xiaozhong Qiu1

1Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering Southern Medical UniversityGuangdong Guangzhou 510515 China

2Department of Nephrology Third Affiliated Hospital Southern Medical University Guangzhou Guangdong 510603 China3Division of Nephrology Nanfang Hospital Southern Medical University Key Lab for Organ Failure ResearchMinistry of Education Guangzhou Guangdong 510515 China

4Department of Anatomy Zhuhai School Campus of Zunyi Medical College Zhuhai Guangdong 519041 China

Correspondence should be addressed to Hequn Zou hequnzouhotmailcom and Xiaozhong Qiu qqiuxzh163com

Received 13 September 2016 Accepted 30 October 2016

Academic Editor Xiaolian Sun

Copyright copy 2016 Hong Liu et alThis is an open access article distributed under theCreativeCommons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

MicroRNA 21 (miR-21) was proved to cause renal fibrosis and the inhibition of miR-21 would improve the poor prognosis inrenal cell carcinoma diseases The complementary oligonucleotide of mature miR-21 was considered to be an effective intracellularmiR-21 inhibitor (miR-21i) The directly effective delivery of miR-21i into fibrotic cell is a facile method for treatment of renalfibrosis Herein the miR-21i-CPP-SWCNT delivery system synthesized via single-walled carbon nanotube (SWCNT) and cell-penetrating peptide (CPP) was taken as a novel fibrosis-targeting therapeutic carrier The miR-21i and CPP firstly bind togethervia electrostatic forces and subsequently miR-21i-CPP binds to the surface of SWCNTs via hydrophobic forces CPP could endowthe delivery system with targeting property while SWCNT would enhance its penetrating ability The exogenous miR-21i releasedfrom the designed miR-21i-CPP-SWCNTs had successfully inhibited the expression of fibrosis-related proteins in renal mesangialcells (RMCs) We found that the expression of TGF-1205731 proteins was more sensitive to miR-21i-CPP-SWCNT than the expressionof 120572-SMA proteins

1 Introduction

High level of microRNA 21 (miR-21) was reported to causerenal fibrosis [1] and led to poor prognosis in renal cellcarcinoma patients [2]The elevated level of circulating miR-21-5p of renal transplant recipients is associated with theseverity of fibrosis [3] The anti-miR-21 may be an effectivetherapeutic tool in renal fibrosis Effective delivery of miR-21 inhibitor (miR-21i) into cells would be a novel therapeuticmethod to treat kidney injury and fibrosis [4] For require-ment of biological safety the nonviral vectors have beengradually taken as a clinical alternative to viral vectors forthe appropriate expression and delivery of therapeutic genes[5] Locked nucleic acids (LNAs) are the class of syntheticnucleotides which are often taken as oligonucleotide-based

therapeutic tools such as anti-miR-21s [6] However theLNA-anti-miRNA heteroduplexes still suffer some limita-tions including instabilities and the complex locking-to-unlocking steps [7] Moreover multistep chemical reactionsare not an easy route to miR-21i delivery and release whenthey were introduced into the cytosol to silence the targetmRNA

Our previous studies indicated that single-walled carbonnanotube (SWCNT) was an excellent nucleic acid deliv-ery vector due to its high surface-volume ratio adjustablehydrophobicity unparalleled electric and uniquely mechan-ical properties [8 9] SWCNT-RNAhybrid could also protectthe RNA away from enzyme digestion Nucleic acid couldstably bind SWCNT through 120587-120587 conjugation on SWCNTsand improved the poor nanotube dispersity SWCNT-RNA

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 3451685 8 pageshttpdxdoiorg10115520163451685

2 Journal of Nanomaterials

+

FAMmiR-21i

CPPSWCNT

miR-21i

CPP

aaaaaaaaUCAACAU AUCGAAUAGUCUGACYGRKKRRQRRRGGGLGASWHRPDKGKKKKKK

Scheme 1 Schematic illustration of the facile synthesis of miR-21i-CPP-SWCNT hybrid and the influence of miR-21i-CPP-SWCNT hybridson renal mesangial cells (RMCs) The miR-21i and CPP firstly bind together via electrostatic forces subsequently miR-21i-CPP binds to thesurface of SWCNT via hydrophobic forcesThemiR-21is released from themiR-21i-CPP-SWCNThybrids would inhibit the fibrosis of RMCsFAM 5-carboxyfluorescein

hybrid shows better self-delivery capability and intracellularstability compared to free RNA [8] However RNA exhibitsmore rearrangement on the SWCNT than DNA while DNAhas much greater conformational stability on SWCNT thanRNA [10] The stable nucleic acid structure on nanotubes isimportant to regulate the properties of the hybrids As forthe short-length single strand RNAs they are more difficultto form stable RNA-SWCNT hybrids than those long lengthRNA fragments double strand siRNAs and DNAs

microRNAs (miRNAs) are small endogenous noncodingRNA of approximately 21 bp and make it difficult to designan efficient short-length miRNA inhibitor delivery vector[11] The single complementary strand of mature miR-21-5poligonucleotide (51015840-UCAACAUCAGUCUGAUAAGCUA-31015840) is an effective intracellular miR-21i [12] However thenaked miR-21i has negative charge which means it is difficultto cross cell membrane Moreover the naked miR-21i is alsoeasily degraded by RNase before its transportation into thecell Cell-penetrating peptide (CPP)was usually employed fordirect delivery of siRNA and miRNA [8 13] The synthesizedCPP (CRGDKGPDC) fragment was reported to assemblemiR-34a delivery systems [14] We previously developed anew artificial CPP with three segments for the delivery ofdouble strand negative siRNAs [8] Hereby we try to developa new effective SWCNT-based miR-21i delivery system The

CPP (YGRKKRRQRRRGGGLGASWHRPDKGKKKKKK)was designed as two hydrophilic segments linked with ahydrophobic segment (GGGLGA) The positively chargedCPP segment and the negatively charged miR-21i couldform miR-21i-CPP complex via electrostatic adsorptionThe SWCNT was wrapped with miR-21i-CPP complexthrough ultrasonic treatment and hydrophobic interactionbetween the hydrophobic segments of CPP (GGGLGA) andthe SWCNT surface After ultrasonic treatment the twohydrophilic end segments of CPP could form hydrogen bondto increase the stability of the miR-21i-CPP-SWCNTdeliverysystem Here a single complementary strand of mature miR-21-5p oligonucleotide (51015840-aaaaaaaaUCAACAUCAGUCU-GAUAAGCUA-31015840) was designed as an anti-miR-21 inhibitor(miR-21i) The developed miR-21i-CPP-SWCNT hybrid wasused to deliver miR-21i into the rat renal mesangial cells(RMCs) (see Scheme 1)

Carbon nanotubes (CNTs) are often used widely in tissueengineering and biomedical fields due to their particularphysicochemical properties Owing to high lengthdiameterratio CNTs could easily and efficiently penetrate biologicalmembranes and accumulate into cells The RNAs bindingto CNTs would help to overcome many problems of ther-apeutic or diagnostic molecules including insolubility easydegradability and inability to cross cellular barriers 15 Our

Journal of Nanomaterials 3

previous study implied that CPP-siRNA-SWCNT can silencethe target gene through endosome escape pathway 8 Theeffective delivery of miR-21i into fibrotic cell via miR-21i-CPP-SWCNThybrid is a facilemethod for inhibition of renalfibrosis through directly binding to miR-21

2 Materials and Methods

21 Cell Culture Rat renal mesangial cell lines (RMCs) werecultured in DMEM medium (GIBCO Grand Island NYUSA) supplemented with 10 FBS (GIBCO Grand IslandNY) and 1 penicillin streptomycin at 37∘C under a humidi-fied 95 air 5 CO

2atm The cell cultures were maintained

under the same controlled conditions The mediums werereplaced every other day

22 Assembly of miR-21i-CPP-SWCNT Hybrids The de-signed miR-21 inhibitor was synthesized by Sangon BiotechCo (Shanghai China) according to the following sequence51015840-aaaaaaaaUCAACAUCAGUCUGAUAAGCUA-31015840 Thesequence of CPP was synthesized as following NH

2-YGR-

KKRRQRRRGGGLGASWHRPDKGKKKKKK-COOHTheCPP was synthesized by Qiangyao Biotech Co (GuangzhouChina) The COOH-end polylysine was designed to increaseits cell adhesion ability The miR-21i adhere to the positivelycharged segments of CPP

The miR-21i-CPP-SWCNT hybrids were preparedaccording to our previous methods [8 10] Briefly 8 nmolCPPs were incubated with 2 nmol miR-21i in 1mL ofDEPC-treated water for 30min at room temperature toprepare the miR-21i-CPP complex and then 05120583g SWCNTs(purity 90 purchased from Carbon Nanotechnologies)were added into the miR-21i-CPP complex and mixed inaqueous solution successively followed by 60min sonicationat a power level of 40W As for the preparing the miR-21i-SWCNT complex the pure 2120583M miR-21i and 05120583gSWCNTs were mixed in aqueous solution successivelyfollowed by 60min sonication at a power level of 40W All ofthe mixtures were then centrifuged at 12000 rmin for 30minto discard the pellet (uncoated SWCNT) The other threegroups were prepared as follows PBS (the control group)pure miR-21i group and miR-21i-CPP group All miR-21iswere labeled with 5-carboxyfluorescein (FAM) at 51015840-terminalfor confocal laser scanning microscopy (CLSM) detection

23 Characterization of the miR-21i-CPP-SWCNT HybridsThe sizes and surface charges of miR-21i-SWCNT andmiR-21i-CPP-SWCNT hybrids were measured on MalvernZetasizer apparatus After centrifugation the hybrids onsupernatant were collected and deposited on mica underatomic force microscopy (AFM) using Nanoscope III AtomicForce Microscope (Digital Instruments Inc) in air at roomtemperature in contact mode with a resonance frequency of242 kHz

24 Confocal Laser ScanningMicroscopicObservation RMCswere seeded on glass covers lips and placed in a 24-well plateRenal mesangial cells (RMCs) were cultured in low glucose

DMEM medium (GIBCO) The medium was supplementedwith 10 FBS (GIBCO) and 1 penicillin streptomycin Cellswere incubated at 37∘C in a humidified 95 air and 5CO2atm The medium was replaced every other day The

FAM-labeled miR-21i and their hybrids were added whenthe culturing cells reached confluence of 80 The cells weredivided into five groups for different treatment as follows PBS(the control group)miR-21is miR-21i-CPPhybrids miR-21i-SWCNThybrids andmiR-21i-CPP-SWCNThybrids respec-tively After being treated for 6 hrs or 12 hrs the coverslipswere washed thrice with PBS Subsequently the coverslipswere incubated with 200 nM red fluorescence mitochondriontracker for 30min and then stained with DAPI and fixed with4 paraformaldehyde overnight The FAM-miR-21i (green)mito-tracker (red) and DAPI (blue) were excited at 488568 and 358nm separately under confocal laser scanningmicroscopy (CLSM) (FV-1000 Japan)

25 Immunofluorescence Staining Assay Immunofluores-cence staining assay was performed to explore the cellularexpression of 120572-smooth muscle actin (120572-SMA) transform-ing growth factor-1205731 (TGF-1205731) and mothers against DPPhomolog 3 (Smad3) in RMCs treated by different reagentsThe cells seeded on the coverslips were incubated with PBSmiR-21i miR-21i-CPP hybrids miR-21i-SWCNT hybridsand miR-21i-CPP-SWCNT hybrids separately for differenttreatment time Then the samples were washed by PBS forthree times and fixed by 4 paraformaldehyde overnight at4∘C The fixed samples were then rinsed with PBS for threetimes and permeabilized with 02 Triton X-100 in PBS atroom temperature for 10mins After being blocked with 1BSA for 1 h the samples were incubated with mouse anti-120572-SMA antibody (1 100 Sigma A2547) rabbit anti-TGF-1205731antibody (1 200 Abcam ab170874) and rabbit anti-Smad3antibody (1 200 CST 9523) overnight at 4∘C respectivelyThen the samples were washed by PBS three times andincubated inAlexa Fluor 488 donkey anti-mouse IgG (1 500)or Alexa Fluor568 donkey anti-rabbit IgG (1 500) for 1 hAfter being rinsed with PBS the samples were stained withDAPI for 1 h and then imaged under a confocal microscope

26 Western Blotting Analysis The total proteins were ex-tracted by RIPA buffer (50mmolL Tris-HCl pH 80150mmolLNaCl 01 SDS 1NP-40 025 sodiumdeoxy-cholate and 1mmolL EDTA) The protein concentrationswere measured using BCA Protein Assay Kit (KeyGEN)10120583glane proteins were loaded on 10 SDS-PAGE sepa-rating gels for electrophoresis then transferred to PVDFmembranes The transferred membranes were blocked in 5(wv) skimmilk for 2 h at room temperature and then washedthree times using TBS-T buffer (10mmolL TrissdotHCl pH 75500mmolL NaCl 005Tween 20) and then incubated withdifferent antibodies including antibodies against 120572-SMA(1 1000 Sigma A2547) against TGF-1205731 (1 1000sim1 5000Abcam ab170874) against Smad3 (1 1000 CST 9523) andagainst GAPDH (1 5000 Tianjin Sungene) respectively at4∘C overnight Then the membranes were washed threetimes with TBS-T buffer and incubated with the appropriate


10 100 10000

2

4

6

8

10

12

14

16

18

Num

ber (

)

Size distribution

Size (dmiddotnm)

Mean with +minus1 standard deviation error bar

50nM

(a1)

(a)

10 100 1000

30

20

10

0

Num

ber (

)

Size distribution

Size (dmiddotnm)

100nM


(b1)

(b)

500000

400000

300000

200000

100000

0minus200 0

Zeta potential distributionminus171

Tota

l cou

nts

Zeta potential (mV)

(c)

minus200 0

120000

100000

80000

60000

40000

20000

0

Zeta potential distribution247

Tota

l cou

nts

Zeta potential (mV)

(d)

Figure 1 Characterization ofmiR-21i-SWCNT andmiR-21i-CPP-SWCNThybrids (a)The size distribution of miR-21i-SWCNT and its AFMimage (a1) (b) The size distribution of miR-21i-CPP-SWCNT and its AFM image (b1) (c) The surface charges of miR-21i-SWCNT hybrids(d) The surface charges of miR-21-CPP-SWCNT hybrids

HRP-linked secondary antibodies for 1 h at room tempera-ture After being washed for three times the chemilumines-cence signalswere used to assess protein content using chemi-luminescent substrate (Invitrogen) according to themanufac-turerrsquos instructions and captured by Tanon 5500 Chemilumi-nescent substrate (Yuwei Biotech Co Guangzhou China)

27 Statistical Analysis All data were presented as the meanplusmn SE from at least three independent experiments Statisticalsignificance (119901 lt 005) of differences among means wasdetermined by analysis of variance (ANOVA)

3 Results and Discussion

31 Characterization of the miR-21i-CPP-SWCNT DeliverySystem The primary structure of polynucleotide affects the

stability of DNARNA-SWCNT complex It was reportedthat a nearly 2500-fold difference in fluorescence emissionof the most fluorescently stable DNA-SWCNT complex thanthat of the least fluorescently stable complex [10] As afunctional microRNA inhibitor the primary structure ofmiR-21 inhibitor is relatively unchangeable A short lengthprimary anti-miR-21 oligonucleotide could not form stableRNA-SWCNT complex Here a polyA

8on the 51015840-terminal

of anti-miR-21 oligonucleotide was used to add the lengthof miR-21i and increase its efficient dispersion ability toSWCNT On the other hand the positively charged segmentof CPP was designed to interact with the negatively chargedmiR-21 inhibitors After ultrasonic treatment the hydropho-bic segments of CPP (GGGLGA) could be combined withSWCNT via Van der Waalsrsquo force Dynamic light scattering(DLS) and atomic force microscopy (AFM) were used tocharacterize the delivery system (Figures 1(a) and 1(b))


(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

Figure 2 Delivery efficiency of miR-21is and their different hybrids (miR-21i-CPP miR-21i-SWCNT and miR-21i-CPP-SWCNT) on RMCsdetected by CLSM (blue) Cell nuclei marked by DAPI (red) mitochondria marked by mito-tracker and (green) FAM-labeled miR-21ifragments The RMCs were treated with different hybrids for 6 hrs (andashe) and 12 hrs (fndashj) (a and f) Double distilled PBS (b and g) miR-21ialone (c and h) miR-21i-CPP hybrids (d and i) miR-21i-SWCNT hybrids (e and j) miR-21i-CPP-SWCNT Scale bar = 10120583m

Dynamic light scattering data showed that the majority ofmiR-21i-SWCNT had average length of about 1503 nm asshown in Figure 1(a) while the majority of miR-21i-CPP-SWCNT (987) had average length of about 5034 nm asshown in Figure 1(b) AFM images of miR-21i-SWCNT areshown in Figure 1(a)(a1) Compared with the diameter ofmiR-21i-SWCNT the diameter of miR-21i-CPP-SWCNT islarger Figure 1(b)(b1) This implied that two hydrophilicend segments of CPP may form hydrogen bond to increasethe stability of miR-21i-CPP-SWCNT delivery system Thezeta potential of miR-21i-SWCNT hybrid was about minus171mV(Figure 1(c)) while that of miR-21i-CPP-SWCNT hybrid wasabout 247mV (Figure 1(d)) The CPP-binding miR-21i viaelectrostatic interactions endowed the positive-charge prop-erties of the miR-21i-CPP-SWCNT hybrids The positivelycharged miR-21i-CPP-SWCNT hybrids were easily binded tothe natural negative cell surfaces

32 The miR-21i-CPP-SWCNTDelivered miR-21i into CytosolDirectly To verify the efficiency of miR-21i-carried vectorsgreen fluorescent FAM (carboxyfluorescein) was labeledon the 51015840 end of the miR-21i and Confocal Laser Scan-ning Microscopy (CLSM) was used to track FAM-labeledmiR-21i in RMCs Before CLSM studies the RMCs wereincubated with PBS (the negative control) FAM-miR-21iFAM-miR-21i-SWCNT FAM-miR-21i-CPP and FAM-miR-21i-CPP-SWCNT for 6 hrs and 12 hrs respectively In thetreatment groups the final concentration of miR-21i was100 nM

CPPs are usually employed for direct delivery of miRNAsinto cells because of its TAT segment [8 13] Significantlyhigh level of G protein-coupled chemokine (C-X-C motif)receptor 4 (CXCR4)was detected in kidney after renal fibrosis[15] The DV3 peptide (LGASWHRPDK) was proved to be abinding domain of CXCR4 [16] In this study the designedCPP peptide (TAT-DV3-polylysine) consisted of three seg-ments the TAT segment (YGRKKRRQRRR)was synthesizedto penetrate the cytomembrane the DV3 was designed as

a target-binding segment for fibrotic renal mesangial cellsand the polylysine (KKKKKK) segment could enhance itsadhesive ability to the cells Owing to the fact that the pureCPPs penetrate the cells mainly depending on endocytosismost of the CPPs-loaded RNAs would be degraded in theearly endosomes [17] Therefore pure CPP could not beserved as a fine RNA-loaded vector As our results the merelyCPP-loaded miR-21is even could not enter into the renalmesangial cells (Figure 2(c))

Although the totally complementary short RNA fragmentofmiR-21was an effective inhibitor few complementary shortRNA fragment was taken as miRNA inhibitor since exoticRNA fragment was not stable and could be digested in cellsOur previous study found that the SWCNT-loaded CPPs canprotect siRNA from RNase degradation in vitro SWCNTcould also help theCPP-loadedRNAs to penetrate the cellularmembrane without being trapped in the endosomelysosomesystem in vivo [8] After the RMCswere treated with differenthybrids for 6 hrs only the miR-21i-CPP-SWCNTs couldpenetrate into cells (Figures 2(a)ndash2(e)) After the RMCs weretreated with different hybrids for 12 hrs both of the miR-21i-SWCNTs and miR-21i-CPP-SWCNTs could penetrate intocells (Figures 2(f)ndash2(j)) Compared with CPP the SWCNTmay be a more suitable cell-penetrating carrier candidate forintroducing miR-21i into RMCs (Figures 2(h) and 2(i)) How-ever the CPP could improve the quantity of carrier-shuttlingmiR-21i in the SWCNT delivery system (Figures 2(d) 2(e)2(i) and 2(j)) and contribute to stable miR-21i-CPP-SWCNTdelivery system through the formation of hydrogen bondbetween its two hydrophilic end segments The cellular miR-21i uptake reflected by fluorescence intensity increased afterbeing treated for 12 hrs either in the miR-21i-SWCNT groupor in the miR-21i-CPP-SWCNT group (Figures 2(d) 2(i)2(e) and 2(j)) The pure SWCNTs could protect RNA awayfrom degradation by endosome just as in our previousreport [8] The miR-21i-CPP-SWCNTs exhibited excellentcell-penetrating ability (Figures 2(e) and 2(j)) This indicated


1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH

Lane 1 the control groupLane 2 miR-21i aloneLane 3 miR-21Ｃ + ００

Lane 4 miR-21Ｃ + ３７４

Lane 5 miR-21Ｃ + ００ + ３７４

(a)

1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(b)

Figure 3 Western blotting analysis of fibrosis-related proteins in RMCs after being treated with miR-21is and their different hybrids (miR-21i-CPP miR-21i-SWCNT and miR-21i-CPP-SWCNT resp) for 6 hrs (a) or 12 hrs (b)

that our designed miR-21i-CPP-SWCNT could be served as afibrotic target-binding delivery system

33 Inhibition of the Expression of TGF-1205731 by miR-21i-CPP-SWCNT Was an Early Event The high level of miR-21was regarded to associate with renal fibrosis [4 18] TGF-120573Smad3 pathway is regarded to play an important role inthe miRNA 21-induced renal fibrosis [19ndash21] TGF-120573 wasidentified as a mediator in renal fibrosis through Smad3-dependent pathway and Smad3-induced high expression ofmiR-21 promoted renal fibrosis [21 22] Here we found thatinhibition of miR-21 by miR-21i-CPP-SWCNT could firstdownregulate the expression of TGF-1205731 in RMCs (Figures3(a) and 3(b)) After the RMCswere challenged with differenthybrids for 6 hrs the high expression of TGF-1205731 proteins wasstill detected in the miR-21i-CPP and the miR-21i-SWCNTtreatment groups using western blot assay (Figure 3(a)) Afterthe RMCs were challenged with different hybrids for 12 hrslow expression of TGF-1205731 proteins was detected in the miR-21i-SWCNTand themiR-21i-CPP-SWCNT treatment groups(Figure 3(b)) There appeared no inhibition of the 120572-SMAexpression in miR-21i-CPP-SWCNT treatment group afterthe RMCs being treated for 6 hrs and 12 hrs The expressionof TGF-1205731 proteins was more sensitive to the inhibition ofmiR-21i-CPP-SWCNT than that of 120572-SMA proteins Down-regulation of TGF-1205731 expression may be an early event in theintracellular knockdown of miR-21

34 The Expression of 120572-SMA Was Inhibited after the RMCsWere Treated with miR-21i-CPP-SWCNT for 24 hrs 120572-SMAacts as a double-edged sword in RMCs because of its pro-motion of cell proliferation and cell fibrosis [23] In orderto verify the relationship between the intracellular miR-21iand cellular fibrosis the FAM-labeled miR-21is were used tosynthesize the FAM-miR-21i FAM-miR-21i-CPP FAM-miR-21i-SWCNT and FAM-miR-21i-CPP-SWCNT hybrids Afterbeing coincubated with RMCs for 24 hrs lots of FAM-miR-21i-CPP-SWCNTs could be detected to penetrate into cytosol

and inhibit the expression of TGF-1205731 (Figures 4(a)ndash4(e)) and120572-SMA (Figures 4(f)ndash4(j)) using immunofluorescence assayAfter 24 hrs of treatment more free miR-21is were releasedfrom the hybrids and inhibited the expression of 120572-SMACompared with 120572-SMA protein TGF-1205731 protein may be amore accurate molecular marker of early fibrosis in renalmesangial cells

The miR-21i-SWCNT also exhibited cellular penetrationand the inhibition of TGF-1205731 (Figure 4(d)) and 120572-SMAexpression (Figure 4(i)) This revealed that pure SWCNT-loaded miR-21i could also penetrate into cytosol and inhibitcell fibrosis while SWCNT endowed miR-21i-CPP-SWCNTwith high penetrating ability CPP alone was not an effectivemiR-21i carrier but CPP can increase the penetrating abilityof SWCNT Furthermore the DV3 segment of CPP canendow miR-21i-CPP-SWCNT with targeting property of thefibrosis cells We indeed found that lots of FAM-labeled miR-21i aggregations gathered in the dividing RMCs after beingtreated with the miR-21i-CPP-SWCNTs for 24 hrs (Figures4(e) and 4(j))

4 Conclusion

Our study designed a very simple and effective approachto make a stable single strand short-length RNA deliverysystem The miR-21i was complexed with CPP via elec-trostatic interactions and the miR-21i-CPP adsorbed ontothe surface of pristine SWCNT via hydrophobic interactionHere our designed miR-21i-CPP-SWCNTs were successfullydelivered into the cytosol of RMCs through cell barriersThe DV3 segment of CPP could endow the carrier withtargeting property for the fibrosis cell The miR-21i-CPP-SWCNT hybrids could inhibit the expression of TGF-1205731in RMCs after being treated for 6 hrs and 12 hrs They caninhibit the expressions of 120572-SMA after being treated for24 hrs Combining the data of western blot assay we provedthat the inhibition of TGF-1205731 was the early event duringthe miR-21i-CPP-SWCNT treatment of kidney fibrosis while


(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

Figure 4 The CLSM analysis of TGF-1205731 and 120572-SMA proteins in RMCs after being treated with miR-21is and their different hybrids (miR-21i-CPP miR-21i-SWCNT and miR-21i-CPP-SWCNT) for 24 hrs (andashe) The CLSM analysis of TGF-1205731 proteins in different groups (blue)Cell nuclei marked by DAPI (red) TGF-1205731 positive expression and (green) FAM-labeled miR-21i fragments (fndashj) The CLSM analysis of120572-SMA proteins in different groups (blue) Cell nuclei marked by DAPI (red) 120572-SMA positive expression and (green) FAM-labeledmiR-21ifragments (a and f) double distilled PBS (b and g) miR-21i alone (c and h) miR-21i-CPP hybrids (d and i) miR-21i-SWCNT hybrids (e andj) miR-21i-CPP-SWCNT Scale bar = 10 120583m

the inhibition of 120572-SMA proteins via miR-21i-CPP-SWCNTwas a late-onset event The anti-miR-21 treatment can betaken as a fibrosis selective treatment targeting for TGF-120573120572-SMA pathway in kidney fibrosis disease The miR-21i-CPP-SWCNT may be an effective drug-delivery system fortreatment of kidney fibrosis

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Hong Liu Guobao Wang and Yihong Yang contributedequally

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (31371000 31572343 51428301 and81670669) and Science and Technology Fund of GuizhouProvince (Qian Ke He J[2014]2179)

References

[1] A D McClelland M Herman-Edelstein R Komers et alldquomiR-21 promotes renal fibrosis in diabetic nephropathy bytargeting PTEN and SMAD7rdquo Clinical Science vol 129 no 12pp 1237ndash1249 2015

[2] L Y Gu H Z Li L Y Chen et al ldquoMicroRNAs as prognosticmolecular signatures in renal cell carcinoma a systematicreview andmeta-analysisrdquoOncotarget vol 6 no 32 pp 32545ndash32560 2015

[3] F Glowacki G Savary V Gnemmi et al ldquoIncreased circulatingmiR-21 levels are associated with kidney fibrosisrdquo PLoS ONEvol 8 no 2 Article ID e58014 2013

[4] I G Gomez N Nakagawa and J S Duffield ldquoMicroRNAs asnovel therapeutic targets to treat kidney injury and fibrosisrdquoAmerican Journal of Physiology Renal Physiology vol 310 no10 pp P391ndashP944 2016

[5] J Pahle and W Walther ldquoVectors and strategies for nonviralcancer gene therapyrdquo Expert Opinion on Biological Therapy vol16 no 4 pp 443ndash461 2016

[6] R Nedaeinia M Sharifi A Avan et al ldquoLocked nucleic acidanti-miR-21 inhibits cell growth and invasive behaviors of acolorectal adenocarcinoma cell line LNA-anti-miR as a novelapproachrdquo Cancer Gene Therapy vol 23 pp 246ndash253 2016

[7] P Jolly P Estrela and M Ladomery ldquoOligonucleotide-basedsystems DNA microRNAs DNARNA aptamersrdquo Essays inBiochemistry vol 60 no 1 pp 27ndash35 2016

[8] X Jiang GWang R Liu et al ldquoRNase non-sensitive and endo-cytosis independent siRNA delivery system delivery of siRNAinto tumor cells and high efficiency induction of apoptosisrdquoNanoscale vol 5 no 16 pp 7256ndash7264 2013

[9] G Wang T Zhao L Wang et al ldquoStudying different bindingand intracellular delivery efficiency of ssDNA single-walledcarbon nanotubes and their effects on LC3-related autophagy inrenalmesangial cells via miRNA-382rdquoACS AppliedMaterials ampInterfaces vol 7 pp 25733ndash25740 2015

[10] M P Landry L Vukovic S Kruss et al ldquoComparative dynamicsand sequence dependence of DNA and RNA binding to singlewalled carbon nanotubesrdquoThe Journal of Physical Chemistry CNanomaterials and Interfaces vol 119 no 18 pp 10048ndash100582015

[11] J Giacomotto S Rinkwitz and T S Becker ldquoEffective heritablegene knockdown in zebrafish using synthetic microRNAsrdquoNature Communications vol 6 article 7378 2015


[12] Y-J Tao Y-J Li W Zheng et al ldquoAntisense oligonucleotidesagainst microRNA-21 reduced the proliferation and migrationof human colon carcinoma cellsrdquoCancer Cell International vol15 article 77 2015

[13] J S Suh J Y Lee Y SChoi PCChong andY J Park ldquoPeptide-mediated intracellular delivery of miRNA-29b for osteogenicstem cell differentiationrdquo Biomaterials vol 34 no 17 pp 4347ndash4359 2013

[14] Q L Hu Q Y Jiang X Jin et al ldquoCationic microRNA-delivering nanovectors with bifunctional peptides for efficienttreatment of PANC-1 xenograft modelrdquo Biomaterials vol 34no 9 pp 2265ndash2276 2013

[15] A Yuan Y Lee U Choi G Moeckel and A KarihalooldquoChemokine receptor Cxcr4 contributes to kidney fibrosisvia multiple effectorsrdquo American Journal of PhysiologymdashRenalPhysiology vol 308 no 5 pp F459ndashF472 2015

[16] Y Liu Y Li HWang et al ldquoBH3-based fusion artificial peptideinduces apoptosis and targets human colon cancerrdquoMolecularTherapy vol 17 no 9 pp 1509ndash1516 2009

[17] Z Qian A Martyna R L Hard et al ldquoDiscovery andmechanism of highly efficient cyclic cell-penetrating peptidesrdquoBiochemistry vol 55 no 18 pp 2601ndash2612 2016

[18] R Bijkerk R G de Bruin C van Solingen et al ldquoSilencing ofmicroRNA-132 reduces renal fibrosis by selectively inhibitingmyofibroblast proliferationrdquoKidney International vol 89 no 6pp 1268ndash1280 2016

[19] A C K Chung Y Dong W Yang X Zhong R Li and H YLan ldquoSmad7 suppresses renal fibrosis via altering expression ofTGF-120573Smad3-regulated microRNAsrdquo Molecular Therapy vol21 no 2 pp 388ndash398 2013

[20] H Y Lan ldquoTransforming growth factor-120573Smad signalling indiabetic nephropathyrdquoClinical and Experimental Pharmacologyamp Physiology vol 39 no 8 pp 731ndash738 2012

[21] X Zhong A C K Chung H-Y Chen X-M Meng and H YLan ldquoSmad3-mediated upregulation of miR-21 promotes renalfibrosisrdquo Journal of the American Society of Nephrology vol 22no 9 pp 1668ndash1681 2011

[22] X-M Meng P M-K Tang J Li and H Y Lan ldquoTGF-120573Smadsignaling in renal fibrosisrdquo Frontiers in Physiology vol 6 article82 2015

[23] R J Johnson H Iida C E Alpers et al ldquoExpression of smoothmuscle cell phenotype by rat mesangial cells in immune com-plex nephritis 120572-Smooth muscle actin is a marker of mesangialcell proliferationrdquo The Journal of Clinical Investigation vol 87no 3 pp 847ndash858 1991

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ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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+

FAMmiR-21i

CPPSWCNT

miR-21i

CPP

aaaaaaaaUCAACAU AUCGAAUAGUCUGACYGRKKRRQRRRGGGLGASWHRPDKGKKKKKK

Scheme 1 Schematic illustration of the facile synthesis of miR-21i-CPP-SWCNT hybrid and the influence of miR-21i-CPP-SWCNT hybridson renal mesangial cells (RMCs) The miR-21i and CPP firstly bind together via electrostatic forces subsequently miR-21i-CPP binds to thesurface of SWCNT via hydrophobic forcesThemiR-21is released from themiR-21i-CPP-SWCNThybrids would inhibit the fibrosis of RMCsFAM 5-carboxyfluorescein

hybrid shows better self-delivery capability and intracellularstability compared to free RNA [8] However RNA exhibitsmore rearrangement on the SWCNT than DNA while DNAhas much greater conformational stability on SWCNT thanRNA [10] The stable nucleic acid structure on nanotubes isimportant to regulate the properties of the hybrids As forthe short-length single strand RNAs they are more difficultto form stable RNA-SWCNT hybrids than those long lengthRNA fragments double strand siRNAs and DNAs

microRNAs (miRNAs) are small endogenous noncodingRNA of approximately 21 bp and make it difficult to designan efficient short-length miRNA inhibitor delivery vector[11] The single complementary strand of mature miR-21-5poligonucleotide (51015840-UCAACAUCAGUCUGAUAAGCUA-31015840) is an effective intracellular miR-21i [12] However thenaked miR-21i has negative charge which means it is difficultto cross cell membrane Moreover the naked miR-21i is alsoeasily degraded by RNase before its transportation into thecell Cell-penetrating peptide (CPP)was usually employed fordirect delivery of siRNA and miRNA [8 13] The synthesizedCPP (CRGDKGPDC) fragment was reported to assemblemiR-34a delivery systems [14] We previously developed anew artificial CPP with three segments for the delivery ofdouble strand negative siRNAs [8] Hereby we try to developa new effective SWCNT-based miR-21i delivery system The

CPP (YGRKKRRQRRRGGGLGASWHRPDKGKKKKKK)was designed as two hydrophilic segments linked with ahydrophobic segment (GGGLGA) The positively chargedCPP segment and the negatively charged miR-21i couldform miR-21i-CPP complex via electrostatic adsorptionThe SWCNT was wrapped with miR-21i-CPP complexthrough ultrasonic treatment and hydrophobic interactionbetween the hydrophobic segments of CPP (GGGLGA) andthe SWCNT surface After ultrasonic treatment the twohydrophilic end segments of CPP could form hydrogen bondto increase the stability of the miR-21i-CPP-SWCNTdeliverysystem Here a single complementary strand of mature miR-21-5p oligonucleotide (51015840-aaaaaaaaUCAACAUCAGUCU-GAUAAGCUA-31015840) was designed as an anti-miR-21 inhibitor(miR-21i) The developed miR-21i-CPP-SWCNT hybrid wasused to deliver miR-21i into the rat renal mesangial cells(RMCs) (see Scheme 1)

Carbon nanotubes (CNTs) are often used widely in tissueengineering and biomedical fields due to their particularphysicochemical properties Owing to high lengthdiameterratio CNTs could easily and efficiently penetrate biologicalmembranes and accumulate into cells The RNAs bindingto CNTs would help to overcome many problems of ther-apeutic or diagnostic molecules including insolubility easydegradability and inability to cross cellular barriers 15 Our








2-YGR-










10 100 10000

2

4

6

8

10

12

14

16

18

Num

ber (

)

Size distribution

Size (dmiddotnm)


50nM

(a1)

(a)

10 100 1000

30

20

10

0

Num

ber (

)

Size distribution

Size (dmiddotnm)

100nM


(b1)

(b)

500000

400000

300000

200000

100000

0minus200 0


Tota

l cou

nts

Zeta potential (mV)

(c)

minus200 0

120000

100000

80000

60000

40000

20000

0


Tota

l cou

nts

Zeta potential (mV)

(d)










(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)








1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(a)

1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(b)







4 Conclusion



(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)



Competing Interests




Acknowledgments


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










2-YGR-










10 100 10000

2

4

6

8

10

12

14

16

18

Num

ber (

)

Size distribution

Size (dmiddotnm)


50nM

(a1)

(a)

10 100 1000

30

20

10

0

Num

ber (

)

Size distribution

Size (dmiddotnm)

100nM


(b1)

(b)

500000

400000

300000

200000

100000

0minus200 0


Tota

l cou

nts

Zeta potential (mV)

(c)

minus200 0

120000

100000

80000

60000

40000

20000

0


Tota

l cou

nts

Zeta potential (mV)

(d)










(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)








1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(a)

1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(b)







4 Conclusion



(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)



Competing Interests




Acknowledgments


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




10 100 10000

2

4

6

8

10

12

14

16

18

Num

ber (

)

Size distribution

Size (dmiddotnm)


50nM

(a1)

(a)

10 100 1000

30

20

10

0

Num

ber (

)

Size distribution

Size (dmiddotnm)

100nM


(b1)

(b)

500000

400000

300000

200000

100000

0minus200 0


Tota

l cou

nts

Zeta potential (mV)

(c)

minus200 0

120000

100000

80000

60000

40000

20000

0


Tota

l cou

nts

Zeta potential (mV)

(d)










(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)








1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(a)

1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(b)







4 Conclusion



(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)



Competing Interests




Acknowledgments


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)








1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(a)

1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(b)







4 Conclusion



(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)



Competing Interests




Acknowledgments


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(a)

1 2 3 4 5

TGF-

-SMA

Smad3

GAPDH



Lane 5 miR-21Ｃ + ００ + ３７４

(b)







4 Conclusion



(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)



Competing Interests




Acknowledgments


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)



Competing Interests




Acknowledgments


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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Research Article Delivery Efficiency of miR-21i -CPP-SWCNT...

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