6. Cationic Solid Lipid Nanoparticles Dered Apolipoprotent of Liver Fibrosis

Cationic solid lipid nanoparticles derived from apolipoprotein-free LDLs for target speci ?c systemic treatment of liver ?brosis

Won Ho Kong a ,Kitae Park b ,Min-Young Lee a ,Hwiwon Lee a ,Dong Kyung Sung c ,Sei Kwang Hahn a ,b ,*

a

Department of Materials Science and Engineering,Pohang University of Science and Technology (POSTECH),San 31,Hyoja-dong,Nam-gu,Pohang,Kyungbuk 790-784,Republic of Korea b

School of Interdisciplinary Bioscience and Bioengineering,Pohang University of Science and Technology (POSTECH),San 31,Hyoja-dong,Nam-gu,Pohang,Kyungbuk 790-784,Republic of Korea c

Department of Pediatrics,Samsung Medical Center,Sungkyunkwan University,School of Medicine,Seoul 135-710,Republic of Korea

a r t i c l e i n f o

Article history:

Received 6September 2012Accepted 26September 2012Available online 22October 2012Keywords:

Cationic solid lipid nanoparticles Connective tissue growth factor siRNA

Targeted delivery Liver ?brosis

a b s t r a c t

Low density lipoprotein (LDL)plays an important role in transporting fat molecules including choles-terols in the body.In this work,cationic solid lipid nanoparticles (CSLNs),bioinspired and reconstituted from natural LDLs,were designed and applied to target speci ?c systemic delivery of connective tissue growth factor siRNA (siCTGF)for the treatment of liver ?brosis.They could form a nuclease-resistant stable nano-complex with siRNA,which was ef ?ciently internalized into cells achieving targeted gene silencing in the presence of serum with a remarkably low cytotoxicity.After intravenous injection,CSLN/siCTGF complex was target speci ?cally delivered to the liver and resulted in a signi ?cant reduction in collagen content and pro-?brogenic factors like tumor necrosis factor alpha (TNF-a ),transforming growth factor beta (TGF-b ),interleukin-6(IL-6),and CTGF with the dramatic improvement of patho-physiological symptoms in liver ?brosis model rats.The bio-distribution study by ?uorescence bio-imaging and single-photon emission computed tomography (SPECT)con ?rmed the target speci ?c delivery and accumulation of CSLN/siCTGF complexes to the liver tissues.

ó2012Elsevier Ltd.All rights reserved.

1.Introduction

Chronic liver injury and persistent wound healing in the liver can lead to a ?brogenic process with the imbalance of parenchymal and non-parenchymal cell population,and the activation of hepatic stellate cells (HSCs)[1,2].The increased production and deposition of hepatic extracellular matrix (ECM)components constitute the ?brous scars in the liver reducing its physiological performance [3,4].Hepatitis virus infection is one of the major causes of chronic liver diseases like liver ?brosis and cirrhosis [5,6].The hepatic ?brosis can be reversed by new anti ?brotic therapeutics on the cellular and molecular basis of ?brogenesis [7].Recently,various small interfering RNAs (siRNAs)have shown a therapeutic potential for liver diseases by hepatotropic viruses [8e 10].Several pioneer-ing studies have demonstrated the anti ?brotic effect of siRNA on down-regulating pro-?brogenic growth factors such as connective

tissue growth factor (CTGF)and transforming growth factor beta (TGF-b )in the liver [11,12].Especially,the cooperative interaction of CTGF in TGF-b signaling pathway was reported to be highly related with the HSC activation and hepatic ?brosis in the damaged liver [13e 16].Consequently,much of attention has been devoted to CTGF as an emerging target gene for anti ?brotic therapy [12,17].The clinical progress of siRNA therapeutics has relied on the development of siRNA delivery carriers.In particular,a great effort has been directed toward the development of lipid-based siRNA delivery systems including liposomes [18,19],micelles [20,21],emulsions [22,23],and solid lipid nanoparticles (SLNs)[24,25]due to their high transfection ef ?ciency,improved pharmacokinetic characteristics,and relatively low cytotoxicity [26,27].Among them,lipoprotein-like lipid nanoparticles have emerged as a promising candidate for the delivery of siRNA [28e 31].Lipoproteins are endogenous particles that transport lipids to various cell types in the liver.Since the liver plays a major role in their metabolism,they are recognized and taken up via speci ?c receptors in the liver [32].Their endogenous nature allows them to be biodegradable and non-cytotoxic without triggering the reticuloendothelial system (RES)and the immune responses during the systemic circulation [32].Accordingly,recombinant and reconstituted lipoprotein-like lipid nanoparticles have been developed for targeted siRNA delivery

*Corresponding author.Department of Materials Science and Engineering,Pohang University of Science and Technology (POSTECH),San 31,Hyoja-dong,Nam-gu,Pohang,Kyungbuk 790-784,Republic of Korea.Tel.:t82542792159;fax:t82542792399.

E-mail address:skhanb@postech.ac.kr (S.K.

Hahn).Contents lists available at SciVerse ScienceDirect

Biomaterials

journal h omepage:

https://www.360docs.net/doc/657194119.html,/locate/biomaterials

0142-9612/$e see front matter ó2012Elsevier Ltd.All rights reserved.https://www.360docs.net/doc/657194119.html,/10.1016/j.biomaterials.2012.09.067

Biomaterials 34(2013)542e 551

using commercially available natural and synthetic lipids,and serum derived or recombinant apolipoproteins[30e34].They can be recognized by lipoprotein receptors in the liver like low density lipoprotein(LDL)receptor and remnant receptor,and then taken up by hepatic cells during systemic circulation[32,35].The practical applications revealed that these lipoprotein-like lipid nanoparticles complexed with siRNA had as small a diameter as capable of passing through the fenestrae(100e110nm)in the sinusoidal endothelium of the liver[28e31,36].

In this work,we developed biomimetic cationic solid lipid nanoparticles(CSLNs)by reconstituting the composition of natural apolipoprotein-free LDLs[28,36].The CSLN was designed to have a nanostructure composed of an outer cationic lipid and rarely distributed PEG shell surrounding an inner solid lipid core.CSLN with a positively charged shell layer was expected to condense negatively charged siRNA into a stable nano-sized polyelectrolyte complex.The hydrodynamic size,z-potential,and morphology of CSLNs were investigated by dynamic light scattering(DLS)and atomic force microscopy(AFM).After cytotoxicity tests,CSLN/ siRNA complex was applied to in vitro hepatitis B virus(HBV) treatment.Furthermore,CSLN/siRNA complex was exploited for an anti?brotic therapy in liver?brosis animal models.The anti?brotic effect was assessed by blood chemistry,cytokine analysis,Western blot analysis of CTGF,histological analysis with hematoxylin& eosin(H&E)and Masson’s trichrome stating,and immunohisto-chemical analysis for the expression levels of alpha smooth muscle actin(a-SMA)and CTGF.Their bio-distribution was investigated by confocal microscopy,?uorescence bioimaging,and single-photon emission computed tomography(SPECT).

2.Materials and methods

2.1.Materials

Cholesteryl oleate,glyceryl trioleate(triolein),cholesterol hydrochloride,L-a-dioleoyl phosphatidylethanolamine(DOPE),3b-[N-(N0,N0-dimethylaminoethane) carbamoyl]cholesterol(DC-Chol),and1,2-distearoyl-sn-glycero-3-phosphoetha-nolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-PEG2k)were purchased from Avanti Polar Lipids(Alabaster,AL).N-Nitrosodimethylamine(NDMA), radioisotope-labeled cholesterol(Carbone-13),40,6-diamidino-2-phenylindole (DAPI),and radioimmuno-precipitation assay(RIPA)buffer were purchased from Sigma e Aldrich(St.Louis,MO).Quantum dot(Qdot705)was purchased from Life Technologies(Grand Island,NY),branched polyethyleneimine(PEI,MW?25kDa) was obtained from Polysciences(Warrington,PA),and Lipofectamine2000from Invitrogen(Carlsbad,CA).Connective tissue growth factor(CTGF)siRNA(siCTGF, sense:50-CAA UAC CUU CUG CAG GCU GGA dTdT-30and antisense:50-UCC AGC CUG CAG AAG GUA UUG dTdT-30),scrambled CTGF siRNA(scCTGF),candidate siRNAs to hepatitis B virus(HBV)genes,and siRNA containing a?uorescent FAM labeled sense strand were purchased from Bioneer Co.(Daejeon,Korea).Genedia HBV surface antigen(HBsAg)ELISA3.0was obtained from Green Cross(Yongin,Korea).Anti-CTGF antibody and anti-a-Tubulin antibody were purchased from Abcam(Cam-bridge,MA),and horseradish peroxidase(HRP)conjugated secondary antibody was obtained from DAKO(Carpinteria,CA).Alexa?uor488goat anti-mouse IgG and Alexa?uor568goat anti-rabbit IgG were purchased from Molecular Probes(Eugene, OR).Mouse anti-a-SMA monoclonal antibody and rabbit anti-CTGF polyclonal antibody were purchased from Abcam(Cambridge,MA).EZ-Cytox cell viability kits were obtained from Daeil Lab Service(Seoul,Korea).Tumor necrosis factor alpha (TNF-a),transforming growth factor beta(TGF-b),and interleukin-6(IL-6)ELISA kits were purchased from R&D Diagnostics(Minneapolis,MN).Hepatocellular carcinoma (HepG2cell)and HBV e producing hepatoma(HepG2.2.15cell)were kindly donated by Prof.YH Kim(Suwon University,Korea).Sprague Dawley(SD)rats were obtained from Orient Bio(Seoul,Korea).

2.2.Preparation and characterization of CSLNs

CSLNs were prepared by the modi?ed emulsi?cation and solvent evaporation method[28,36].Brie?y,cholesterol oleate(22.5mg,45.0%w/w),triolein(1.5mg, 3.0%w/w),cholesterol(4.95mg,9.9%w/w),DOPE(7.0mg,14%w/w),DC-Chol

(14.0mg,28%w/w),and DSPE-PEG2k(0.05mg,0.1%w/w)were mixed to give

a lipid suspension with a?xed total lipid concentration of25mg/mL in the co-solvent of chloroform/methanol at a volume ratio of2/1.After10mL of deionized water was added,the mixture was vortexed for2min and subsequently sonicated for5min using a Sonics Vibra-cell soni?er VC750equipped with a micro-tip (Newtown,CT)at amplitude?35%,pulse-on?5.0s,and pulse-off?3.0s.The suspension was transferred to a round-bottom?ask and the solvent was rapidly evaporated at60 C and20mmHg using a rotary evaporator(EYELA,Japan).The resulting CSLNs were puri?ed by extensive dialysis against deionized water(MW cutoff?10kDa).To prepare Qdot or radioisotope-labeled CSLNs,0.5nmol of Qdot or 0.5m mol of isotope-labeled cholesterol was further added to the CSLN formulation. The topological analysis of CSLNs was carried out with a tapping-mode AFM(Veeco NanoScope IV Multimode AFM,Santa Barbara,CA).The average particle size was determined by measuring the diameters of more than30particles on the images.To assess in vitro cytotoxicity of CSLNs,HepG2cells were seeded on a48-well plate at a density of2?104cells per well,and cultivated in MEM medium supplemented with10%(v/v)fetal bovine serum(FBS)and1%antibiotics at37 C for24h.The cells were exposed to CSLN or PEI at varying concentrations up to72m g/mL for10h.The cells were washed with PBS and treated with10m L of EZ-Cytox reagent at37 C for 1h.The relative cell viability was determined by measuring the absorbance at 450nm using a Bio-Rad microplate reader(Hercules,CA)and normalized with respect to the untreated control group.

2.3.Preparation and characterization of CSLN/siRNA complex

To prepare CSLN/siRNA complex,CSLNs were gently mixed with siRNA at various weight ratios of CSLN to siRNA in0.1M PBS(pH7.4)and then incubated at room temperature for15min.The resulting complexes were characterized by agarose gel electrophoresis at100V for5min in TAE buffer solution containing40m M tris-acetate and1m M EDTA at pH8.3.After staining with ethidium bromide,the gel image was taken under UV illumination.The effect of CSLN on siRNA protection was determined by nuclease protection assay.Naked siRNA(5m g)and its complexes with CSLN were prepared just before the experiments.Then,each sample was incubated with50m L of reaction buffer containing10units of RNase ONE ribonuclease at room temperature for1h.The sample was subsequently incubated with10IU of heparin at room temperature for30min to disintegrate siRNA from the complex.Afterward, the remaining siRNA was analyzed by gel electrophoresis and quanti?ed by densi-tometric analysis using Image J software.The hydrodynamic diameter and z-potential value of CSLNs and CSLN/siRNA complexes were measured in PBS(0.1M, pH7.4)at37 C with a Zetasizer Nano ZS(Malvern,UK).Each sample was properly diluted to maintain a count rate above300and measured in triplicate.

2.4.Confocal microscopy and FACS analysis

HepG2.2.15cells were maintained in MEM medium supplemented with10%(v/v) FBS and G418antibiotic solution(200m g/mL)at37 C and5%CO2.Following the cultivation on a cover slide at a density of2?104cells per slide for24h,HepG2.2.15 cells were treated with CSLN/FAM e siRNA complexes(25n M)at37 C for2h.The cells were washed with PBS(pH7.4)three times and then?xed with4%(w/v)formal-dehyde solution in PBS(pH7.4).After DAPI(1.5m g/mL in PBS)staining for10min,the cells were examined with a Radiance2100confocal microscope(Bio-Rad,Hercules, CA).Intracellular uptake of CSLN/siRNA complex with increasing weight(w/w)ratio of CSLN to siRNA was also characterized by FACS analysis.Twenty-four hours prior to the experiments,HepG2.2.15cells were inoculated on a100-mm culture dish at a density of1?106cells per dish and cultivated in MEM medium supplemented with 10%(v/v)FBS and1%antibiotics.The cells were exposed to free FAM e siRNA or CSLN/ FAM e siRNA complex for2h.Then,the cells were collected,washed with PBS(pH7.4) three times,and?xed by adding4%(w/v)formaldehyde solution.As a control experiment,the cells were also treated with Lipofectamine/FAM e siRNA complexes for2h and then harvested by the same procedures.The?uorescence of each cell was analyzed with a?ow cytometer(FACSVantage,Franklin Lakes,NJ).

2.5.In vitro gene silencing for HBV surface antigen knockdown

To assess the feasibility of CSLN as a hepatic siRNA delivery carrier,four kinds of candidate siRNAs to HBV genes were tested for the knockdown of HBsAg. HepG2.2.15cells were inoculated on a48-well plate at a density of4?104cells per well and cultivated as described above for24h.The cells were then exposed to CSLN/ siHBV complexes at siRNA concentrations up to200n M in10%FBS-supplemented complete medium.After incubation for5h,the culture medium was replaced with a fresh culture medium and further cultivated for6h.The medium was aspi-rated to remove endogenously secreted HBsAg and then replaced with a fresh culture medium.After incubation for48h,the concentration of HBsAg released from the cells was measured with a quantitative sandwich ELISA kit.The relative HBsAg levels were calculated based on the HBsAg expression of untreated control cells which was set to100%.

2.6.In vivo gene silencing for hepatic?brosis in SD rats

SD male rats weighing around35e40g at a mean age of three weeks were used for the induction of hepatic?brosis.A total of36animals were divided into four groups:(A)Control group(n?6)which received daily iv injections of normal saline (0.15M NaCl)2h after ip injections of normal saline without NDMA for7days,(B)

W.H.Kong et al./Biomaterials34(2013)542e551543

NDMAtsiCTGF group(n?12)which received daily iv injections of CSLN/siCTGF complexes containing1mg of siCTGF per kg body weight2h after ip injections of NDMA at a dose of10mg/kg body weight for seven days,(C)NDMAtscCTGF group (n?12)which received daily iv injections of CSLN/scCTGF complexes containing 1mg of scCTGF per kg body weight2h after ip administration of NDMA at a dose of 10mg/kg body weight for seven days,and(D)NDMAtCSLN group(n?6)which received daily iv injections of free CSLNs at a dose of30mg/kg body weight2h after ip injections of NDMA at a dose of10mg/kg body weight for seven days.All injec-tions were given without anesthesia.The experimental animals were sacri?ced by cardiac perfusion13days after the initial administration of normal saline,CSLN/ siCTGF complex,CSLN/scCTGF complex,or free CSLN.At the end,all the animals were anaesthetized with1mg/kg xylazine and50mg/kg ketamine hydrochloride to collect blood samples and sacri?ced by cardiac perfusion with saline nitrite?ush, followed by4%paraformaldehyde?xation in0.1M phosphate buffer.After that,the liver tissue samples were quickly removed and one portion was instantly frozen in liquid nitrogen for Western blot analysis.Another portion was simultaneously?xed in10%phosphate-buffered formalin for histopathological analysis.The remaining liver tissue was stored atà80 C for further analysis.The animal protocol was approved by the Committee for the Care and Use of Laboratory Animals,Samsung Biomedical Research Institute(SBRI),Korea.

2.7.Biochemical analysis of serum samples

Blood samples were collected intracardially.After centrifugation at300?g for 15min,the serum samples were separated and stored in a deep freezer before use. The serum levels of alanine transaminase(ALT),aspartate transaminase(AST),total bilirubin(TBIL),and total albumin(ALB)were determined with a Fuji DRI-CHEM 3000automatic chemistry analyzer(Fuji Photo Film,Tokyo,Japan).

2.8.Cytokine analysis and Western blot analysis

The serum concentrations of TNF-a,TGF-b,and IL-6were measured in triplicate with speci?c ELISA kits according to manufacturer’s protocols.For Western blot analysis,the frozen liver tissue samples were minced in3mL of freshly prepared ice-cold RIPA buffer per gram of tissue and homogenized on an ice bath with a Polytron homogenizer(IKA Works,Wilmington,NC).The homogenized lysate was left on ice for30min and centrifuged at13,000?g and4 C for30min.The supernatants were collected and protein concentrations in the supernatants were determined with micro BCA protein assay kits.All protein samples were subsequently boiled in SDS buffer and separated on a10%(w/v)SDS-PAGE.The separated proteins were elec-trophoretically transferred to a nitrocellulose membrane(Millipore,MA). Membrane was blocked with5%skim milk in tris-buffered saline containing0.05% (v/v)Tween-20.Primary and secondary antibodies were diluted as recommended by the manufacturer in blocking buffer,and incubated with the membranes at room temperature for1h.Anti-CTGF antibody,anti-a-Tubulin antibody,and anti-mouse IgG HRP antibody were diluted1/5000,1/1000,and1/1000,respectively.The detection of HRP-conjugated secondary antibodies was performed using Amersham enhanced chemiluminescence reagent.

2.9.Histological analysis of hepatic?brosis

The pathogenesis of NDMA-induced hepatic?brosis and the effect of CLSN mediated delivery of CTGF siRNA on hepatic?brosis were assessed by H&E and Masson’s trichrome staining.The paraf?n-embedded tissues were cut into sections of5mm thickness,deparaf?nized,and hydrated in water.The serial sections were then treated for H&E and Masson’s trichrome staining as per standard protocols.The stained sections were examined and photoimaged with a bright?eld microscope (Eclipse E800,Nikon,Japan).To visualize hepatocellular uptake of CSLN/FAM e siRNA complex in the liver,SD rats were treated by iv injection of CSLN/FAM e siRNA complex containing1.0mg FAM e siRNA per kg body weight.Twelve hours after treatment,animals were anesthetized and perfused intracardially with4%(w/v) formaldehyde in PBS(pH7.4).The liver tissue was excised and post-?xed in form-aldehyde for24h,cryoprotected in30%sucrose in PBS(pH7.4)at4 C for24h,and frozen in tissue freezing medium.After that,serial sections were cut at10m m thickness using the Shandon cryotome(Thermo Electron Co.,Pittsburgh).The slides were rinsed with PBS,counterstained with DAPI,and visualized with a confocal microscope(Bio-Rad,Hercules,CA).

2.10.Immunohistochemistry for a-SMA and CTGF on liver sections

The immunohistochemical analysis for a-SMA and CTGF on deparaf?nized sections was carried out by immuno?uorescence histochemistry.The sections were treated with1%(w/v)bovine serum albumin(BSA)containing0.1%(v/v)Triton X-100 for30min and blocked with3%(v/v)normal goat serum containing0.1%(v/v) Triton X-100in PBS at room temperature for1h.Then,the sections were incubated with1/200diluted mouse anti-a-SMA antibody or1/500diluted rabbit anti-CTGF antibody at4 C overnight,rinsed with PBS,and incubated with1/500diluted Alexa?our568goat anti-mouse IgG or1/200diluted Alexa?our488goat anti-rabbit IgG in a blocking solution for1h.Confocal microscopy was carried out with a Radi-ance2100confocal microscope(Bio-Rad,Hercules,CA).

2.11.Biodistribution of CSLN/siRNA complex

To investigate ex vivo biodistribution,SD rats were treated by iv injection of Qdot-incorporated-CSLN/siRNA complex.The rats were anesthetized with sodium pentobarbital and then four different tissue samples were dissected after cardiac perfusion with PBS.Fluorescence image of each tissue sample was obtained using an in vivo?uorescence imaging system(IVIS;Xenogen,Alameda,CA).Photonic emis-sions were assessed using Living image2.20.1software(Xenogen).In vivo SPECT imaging was carried out to noninvasively visualize the biodistribution of CSLN/ siRNA complex after systemic delivery and to demonstrate the targeted therapeutic potential of the complex in the liver.The SD rats were anesthetized with sodium pentobarbital and treated by iv injection of radioisotope-labeled-CSLN/siRNA complex containing30m g per kg bodyweight of CSLNs.Two days after the treat-ment,animals were scanned on Siemens dual modality SPECT/MicroCAT II scanner (Knoxville,TN).

2.12.Statistical analysis

All experiments were replicated more than three times and the resulting data were statistically analyzed by unpaired t-test using the commercially available software of GraphPad Prism(GraphPad,La Jolla,CA).

3.Results and discussion

3.1.Design,preparation,and characterization of CSLNs

We have designed apolipoprotein free LDL-like solid lipid nanoparticles to prepare highly uniform and stable nano-sized particles with a PEG-protected and positively charged surface monolayer for targeted delivery of siRNA to the liver(Fig.1).Native human plasma LDLs are composed of a hydrophobic inner core and a surrounding surface monolayer of polar phospholipids.The inner core is known to be predominantly45%(w/w)of cholesterol ester and3%(w/w)of triglycerides.The outer-layer is10%(w/w)of cholesterol,22%(w/w)of phospholipids,and20%(w/w)of apoli-poproteins.As a bioinspired CSLN formulation,surface polar lipid constituents in natural lipoproteins were reconstituted with28% (w/w)cationic polar lipid DC-Chol and14%(w/w)fusogenic lipid DOPE to provide a positive surface charge,facilitate endosomal escape,and improve intracellular traf?cking.The resulting nano-particles maintained the same composition of ca.10%(w/w) cholesterol,and nonpolar lipids of45%(w/w)cholesteryl oleate and 5%(w/w)triglyceride in the inner core.Additionally,the surface monolayer of CSLN was further modi?ed with0.1%(w/w)PEGy-lated phospholipid of DSPE-PEG2k to improve hydrodynamic stability and protect their siRNA cargo from nuclease degradation during systemic circulation.As reported elsewhere[28,36],CSLNs were prepared by a modi?ed emulsi?cation and solvent evapora-tion method.All lipid components were initially dissolved in a co-solvent of chloroform/methanol(2/1).Then,the solvent mixture containing all lipid constituents was added to deionized water and ultra-sonicated to form a homogenous oil-in-water emulsion.The hydrophobic core lipid constituents of cholesteryl oleate and triglyceride might be localized inside the oil phase droplets. Subsequent solvent evaporation under a reduced pressure might result in the swift assembly of amphiphilic lipid building blocks around the hydrophobic core of nonpolar lipid constituents.A solid inner core was obtained by concomitant cooling down.The cho-lesteryl oleate has a high melting temperature(T m?52 C)above body temperature.The resulting highly stable CSLNs were thought to be more advantageous than conventional DC-Chol/DOPE lipo-some formulations.The hydrodynamic size distribution,z-poten-tial,and surface topology of CSLNs were analyzed by DLS and AFM. CSLNs had a narrow size distribution with a diameter in the range of106.2?5.4nm showing a positive z-potential of64.3mV.AFM image of CSLNs in Fig.2a also revealed the well-dispersed spherical

W.H.Kong et al./Biomaterials34(2013)542e551 544

CSLN

Component Component Cholesteryl oleate

45.03.045.03.010.020.022.09.928.014.00.1

Triolein S u r f a c e

C o r e

Cholesterol

+

-----

Cholesteryl oleate

Triolein Cholesterol Apolipoprotein Phospholipid

--

Cationic DC-Chol Fusogenic DOPE DSPE-PEG 2k

Portion (%, w/w)Portion (%, w/w)Natural LDL

a

b

Fig.1.(a)A schematic representation of the bioinspired cationic solid lipid nanoparticle (CSLN)and its composition in comparison with natural low density lipoprotein (LDL).(b)A schematic illustration for the formation of CSLN/siRNA complex by electrostatic interaction between positively charged CSLN and negatively charged siRNA.

1201008060402001251007550250

Nuclease -+--+--+

Heparine

siRNA

CSLN/siRNA Complex

CSLN/siRNA ratio (w/w)

siRNA

10

15

20

25

0.0

Average Size = 102 nm

2.2 μm

30

35

20V i a b l e C e l l s (%)

Concentration (μg/mL)

B a n d I n t e n s i t y (%)

406080

CLNPs PEI 25k

a

b

d

c

Fig.2.Characterization of CSLNs.(a)Atomic force microscopy (AFM)of CSLNs.(b)Cytotoxicity of CSLN in comparison with PEI 25k.(c)The agarose gel retardation at various weight ratios of CSLN to siRNA.(d)The effect of CSLN on the protection of siRNA from ribonuclease degradation.The protective effect was quanti ?ed by the densitometric analysis.

W.H.Kong et al./Biomaterials 34(2013)542e 551545

assemblies with an average diameter of 102.0?2.4nm.The size,z -potential,and homogeneity of resulting CSLNs were well main-tained at room temperature for the experimental period.Then,we examined the cytotoxicity of CSLN in comparison with PEI as a control with increasing concentration.CSLN exhibited no severe cytotoxicity up to a concentration of 72m g/mL,whereas PEI signi ?cantly reduced the cell viability to ca.5.1%under the same condition (Fig.2b).The IC 50value of CSLN (333.5m g/mL)was remarkably higher than that of PEI (6.7m g/mL).These results con ?rmed the biocompatibility of CSLNs as a safe delivery vehicle of siRNA.

3.2.Preparation and characterization of CSLN/siRNA complex The complex formation of CSLN with siRNA as schematically shown in Fig.1b was examined by agarose gel electrophoresis.When CSLNs were mixed with siRNA at various weight ratios from 10to 35in 0.1M PBS (pH 7.4),the electrophoretic migration of siRNA revealed a gradual retardation within the gel with increasing weight ratio of the complexes (Fig.2c).The migration of siRNA was completely retarded above a weight ratio of 30.The results indi-cated that positively charged CSLNs effectively interacted with negatively charged siRNA molecules via electrostatic interaction.On the basis of these results,the optimal composition for CSLN/siRNA complexes was determined to be a weight ratio of 30,which was used for the following experiments.According to DLS analysis,the hydrodynamic diameter of CSLN/siRNA complexes (113.0? 3.2nm)was slightly larger than that of CSLN alone (106.2?5.4).In addition,the z -potential value of CSLNs signi ?-cantly decreased from 6

4.3?2.5mV to 32.1?3.3mV upon addi-tion of siRNA re ?ecting the electrostatic complex formation.It was noteworthy that the siRNA binding ability to CSLN was not signif-icantly altered by the incorporation of PEGylated phospholipid DSPE-PEG on the surface of CSLN.Then,the effect of CSLN in CSLN/siRNA complexes was assessed on the protection of siRNA from nuclease attack.As shown in Fig.2d,CSLN/siRNA complex retained 7

5.2?3.3%of siRNA even after treatment with 10units of nuclease.The CSLN/siRNA complex was dissociated in the presence of

100020406080100

020*********

020*********

020*********

020*********

020*********

1011021.0%47.8%

62.2%73.6%87.7%

42.0%w/w = 15FAM-siRNA

Lipopfectamine

CSLN/FAM-siRNA complex

w/w = 20w/w = 25w/w = 30

FAM-siRNA Fluorescene

C e l l N u m b e r

103104100101102103104100101102103104100101102103104100101102103104100101102103104

a

b

Fig.3.(a)Confocal microscopic images of HepG2.2.15cells after incubation with CSLM/FAM e siRNA complex for 2h.(b)Flow cytometric analysis for the intracellular uptake of CSLN/FAM e siRNA complex at different weight ratios.

W.H.Kong et al./Biomaterials 34(2013)542e 551

546

negatively charged heparin molecules at a concentration of 50m g/mL and the siRNA was completely degraded in 60min.From the results,we could con ?rm the formation of stable nano-sized CSLN/siRNA complexes protecting the payload from enzymatic degradation.

3.3.In vitro gene silencing of CSLN/siRNA complexes

Before in vitro gene silencing tests,the intracellular delivery and subcellular localization of CSLN/siRNA complex were examined by confocal microscopy using ?uorescently labeled FAM e siRNA.CSLN/FAM e siRNA complexes were internalized evenly into HepG.2.2.15cells in 10%(v/v)serum containing medium after incubation for 2h (Fig.3a).The internalized complexes accumulated near the nucleus in many cells.The cellular uptake of CSLN/FAM e siRNA complexes was further evaluated quantitatively by ?ow cytometry at various weight ratios from 15to https://www.360docs.net/doc/657194119.html,mercially available Lipofectamine was used as a control.As shown in Fig.3b,the cellular uptake of the complex gradually increased with increasing weight ratio of the complex showing a maximum at a weight ratio of 30.The percentage of FAM-positive cells reached ca.87.7%.In contrast,only a moderate level of cellular uptake was observed for the Lip-ofectamine/siRNA complexes (47.8%).The ef ?cient internalization of CSLN/siRNA complexes to the cells was ascribed to their nano-scale dimension and positive z -potential promoting their initial

interaction with cell membrane through electrostatic interaction.Moreover,the fusogenic lipid components,DOPE and cholesterol,might also facilitate the cellular entry and subsequent endosomal escape of the complexes.The gene silencing ef ?ciency of CSLN/siRNA complexes was assessed using four different candidate siR-NAs speci ?c for overlapping HBV open reading frames (ORF)(Fig.4a).The release of HBsAg was drastically reduced to the statistically signi ?cant levels after treatment with 200n M of CSLN/si257and CSLN/si1826complexes (P <0.0001)in 10%(v/v)serum containing culture condition (Fig.4b and c).The results were thought to re ?ect that CSLN/siRNA complexes were readily inter-nalized to the cells and escaped from the endosome releasing siRNA into cytoplasm for the effective gene silencing.3.4.In vivo anti ?brotic effect of CSLN/siCTGF complex

We carried out the systemic delivery of CSLN/siCTGF complex for the treatment of hepatic ?brosis.CTGF is produced by a variety of different cell types including HSCs and hepatocytes,which promotes ?brogenesis and survival of activated HSCs [37].With the progress of liver ?brosis and cirrhosis,HSCs were reported to be proliferated by 10e 20times and squeezed through the fenestrae [38].According to our previous bioimaging study using Qdots,hyaluronic acid (HA)derivatives were signi ?cantly delivered to the HSCs re ?ecting the easy access to the protruded HSCs [39].

Because

Fig.4.The gene silencing effect of CSLN/siRNA complex on hepatitis B virus surface antigen (HBsAg)expression in HBV producing HepG2.2.15cells.(a)A map of the HBV genome indicating the location of siRNA target sequences and their sequences.The HBsAg expression in HepG2.2.15cells after treatment with (b)increasing concentration of CSLN/si257complex,and (c)200n M of CSLN/si257,CSLN/si1658,CSLN/si1826,and CSLN/si2422complexes.

W.H.Kong et al./Biomaterials 34(2013)542e 551547

of the paracrine stimulation,the gene silencing of CTGF in the liver might result in signi?cant anti?brotic effect.The biochemical analysis for ALT,AST,TBIL,and ALB revealed signi?cant improve-ment of?brotic symptoms in SD rats treated with CSLN/siCTGF complex2h after daily injection of NDMA at a dose of10mg/kg for 7days(Fig.5).Serum cytokine levels of TNF-a,TGF-b,and IL-6were also signi?cantly improved when compared with CSLN treated group(P<0.001)(Fig.6).In addition,quantitative evaluation of CTGF intensity in Western blot analysis also showed a more signi?cant decrease in the production of CTGF in the liver after treatment with CSLN/siCTGF complex than CSLN alone(P<0.001).Fig.7shows the histological and immunohistochemical analyses of dissected?brotic liver tissues after treatment with CSLN/siCTGF complexes.The control group showed a normal lobular architecture and a normal hepatic cell structure with central veins and radiating hepatic cords(Fig.7a).In contrast,?brosis and early cirrhosis with multifocal hepatocyte necrosis and neutrophilic in?ltration were observed in SD rats treated with CSLN after daily injection of NDMA at a dose of10mg/kg for7days.There was no signi?cant alternation or prevention in the livers treated with CSLN/scCTGF complexes after NDMA injection for7days showing extensive bridging,well developed?bers,severe centrilobular congestion,and dilatation

of

Fig.5.The anti?brotic effect of the control,CSLN/siCTGF complex,CSLN/scCTGF complex,and CSLN on the liver function after systemic delivery to?brotic SD rats treated by the daily injection of NDMA at a dose of10mg/kg for7days.(a)ALT,(b)AST,(c)TBIL,and(d)albumin levels in the

serum.

Fig.6.The anti?brotic effect of the control,CSLN/siCTGF complex,CSLN/scCTGF complex,and CSLN on(a)TNF-a,(b)TGF-b,(c)IL-6,and(d)CTGF/a-Tubulin ratio after systemic delivery to?brotic SD rats treated by the daily injection of NDMA at a dose of10mg/kg for7days.

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sinusoids with focal hemorrhage.Interestingly,only mild dilatation of sinusoids and neutrophilic in ?ltrations were observed in animals treated with CSLN/siCTGF complex 2h after the injection of NDMA (Fig.7a).Liver ?brosis is characterized by overproduction and irregular deposition of collagens by liver ?brogenic cells resulting in subsequent loss of the normal function.As shown in Fig.7b,Mas-son ’s trichrome staining showed no histological alteration and collagen deposition in the livers for the control group.In contrast,the massive bridging and deposition of mature collagen ?bers were observed in the livers treated with CSLN or CSLN/scCTGF complex after NDMA injection for 7days.Importantly,only mild collagen disposition was observed in the livers treated with CSLN/siCTGF complex,which was in good agreement with the results in Fig.7a.The immunohistochemistry for CTGF and a -SMA was performed to assess the liver ?brosis.While remarkable staining for CTGF and a -SMA was observed in the livers treated with CSLN or CSLN/scCTGF complex after the NDMA injection for 7days,only focal marginal staining was observed in the livers treated with CSLN/siCTGF complex (Fig.7c).The target speci ?c gene silencing in the livers were also con ?rmed by the confocal microscopy of liver cry-osections showing remarkable cellular uptake of CSLN/FAM e siRNA complexes into the liver parenchyma (Fig.8).From the results,apolipoprotein-free LDL-like CSLN was thought to target-speci ?cally deliver siCTGF to the liver for ef ?cient silencing of

Control a

b

c

NDMA + siCTGF NDMA + scCTGF

NDMA + CSLN

Fig.7.Histological and immunohistochemical analyses of dissected ?brotic liver tissues after systemic treatment with the control,CSLN/siCTGF complex,CSLN/scCTGF complex,and CSLN.(a)H&E staining.(b)Masson ’s trichrome staining.(c)Immunohistochemistry for a -SMA (green),CTGF (red),and nucleus (blue).(For interpretation of the references to color in this ?gure legend,the reader is referred to the web version of this

article.)

Fig.8.Confocal microscopic images of (a)left and (b)right lateral liver robe cryosections 12h after systemic delivery of CSLN/FAM e siRNA complex.

W.H.Kong et al./Biomaterials 34(2013)542e 551549

CTGF gene exhibiting the therapeutic effect in liver ?brosis model rats.

3.5.Biodistribution of CSLN/siRNA complex in the body

To corroborate the targeted delivery of CSLN/siRNA complexes to the liver,their bio-distribution was investigated after iv injec-tion of siRNA complexed with CSLN encapsulating Qdots and radioisotope-labeled CSLN,respectively (Fig.9).According to ex vivo ?uorescence bioimaging,Qdot-CSLN/siRNA complexes containing 20m g of siRNA per 20g of body weight were target-speci ?cally delivered to the liver after single iv injection.A large portion of complexes were also accumulated gradually in the kidney re ?ecting the renal clearance (Fig.9a).In contrast,there was only negligible NIRF signal intensity in spleen and lung during the entire experimental period.Fig.9b and c shows the quanti ?-cation of NIRF signal intensity in each organ recorded as photon per sec (p/s)and photons per sec per square centimeter per steradian (p/sec/cm 2/sr).The biodistribution of CSLN/siRNA complexes was further evaluated by noninvasive whole-body SPECT imaging using the radiolabeled CSLNs.In accordance with the ?uorescence bioimaging,the SPECT imaging revealed that radiolabeled-CSLN/siRNA complexes preferentially accumulated in the liver.The radioactive signal was also observed in the kidney

but not in the spleen and lung.These results strongly supported that CSLN/siRNA complex might be successfully exploited for target speci ?c treatment of various liver diseases as demonstrated above.

4.Conclusions

CSLNs were successfully developed for targeted delivery of siRNA to the liver.The CSLNs were prepared by emulsi ?cation and solvent evaporation of the lipid components reconstituted from natural apolipoprotein-free LDLs.The CSLN was able to form a stable nano-sized complex with CTGF siRNA via electrostatic interaction and protect the payload against enzymatic degradation.The CSLN/siHBV complex showed a signi ?cant in vitro gene silencing with a drastically reduced HBsAg expression.Further-more,after iv injection,CSLN/siCTGF complexes were target-speci ?cally delivered to the liver and resulted in a signi ?cant reduction in collagen content and pro-?brogenic factors like TNF-a and TGF-b ,IL-6,and CTGF showing dramatically improved patho-physiological symptoms in liver ?brosis model rats.The bio-distribution studies by ex vivo ?uorescence bioimaging and SPECT clearly con ?rmed that CSLN/siCTGF complexes were preferentially delivered and accumulated in the liver for the therapeutic gene silencing.

12 h L i v e r T o t a l F l u x (p /s x 107)

R a d i a n c e (p /s /c m 2 x 107)

K i d n e y L u n g

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Fig.9.(a)Ex vivo ?uorescence imaging (left upper panel)of dissected organs and non-invasive SPECT imaging (right upper panel)of whole-body after systemic delivery of Qdot-or radioisotope-labeled CSLN/siRNA complexes.(b)Quanti ?ed total ?ux and (c)average radiance at dissected organs by the ex vivo ?uorescence bioimaging.

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Acknowledgments

This work was?nancially supported by the Converging Research Center Program through the National Research Foundation of Korea (NRF)funded by the Ministry of Education,Science and Technology (2009-0081871).This study was also supported by Mid-career Researcher Program through NRF grant funded by the MEST (No.2012R1A2A2A06045773).

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