Target specific tumor treatmentby
Target speci ?c tumor treatment by VEGF siRNA complexed with reducible polyethyleneimine e hyaluronic acid conjugate
Kitae Park a ,Min-Young Lee b ,Ki Su Kim b ,Sei Kwang Hahn a ,b ,*
a
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 b
Department of Materials Science and Engineering,POSTECH,San 31,Hyoja-dong,Nam-gu,Pohang,Kyungbuk 790-784,Republic of Korea
a r t i c l e i n f o
Article history:
Received 27January 2010Accepted 9March 2010
Available online 7April 2010Keywords:
Hyaluronic acid Polyethyleneimine siRNA
Target delivery Gene silencing
a b s t r a c t
Target speci ?c delivery of small interfering RNA (siRNA)has been regarded as one of the most important technologies for the development of siRNA therapeutics.In this work,non-toxic low molecular weight (MW)polyethyleneimine (PEI,2000Da)was cross-linked with cystamine bisacrylamide (CBA)to prepare reducible PEI-SS in the body.Then,PEI-SS was conjugated with hyaluronic acid (HA)in the form of block-copolymer to enhance serum stability and facilitate target speci ?c cellular uptake of siRNA by HA receptor mediated endocytosis.The cytotoxicity of (PEI-SS)-b-HA conjugate appeared to be negligible likely due to the degradation of PEI-SS to low MW PEI in the cytosol.Flow cytometric and confocal microscopic analyses con ?rmed the HA receptor mediated endocytosis of siRNA/(PEI-SS)-b-HA complex.The siRNA/(PEI-SS)-b-HA complex demonstrated an excellent in vitro gene silencing ef ?ciency in the range of 50e 80%reducing the mRNA expression level in the absence and presence of 50vol%serum.Moreover,intra-tumoral injection of vascular endothelial growth factor (VEGF)siRNA/(PEI-SS)-b-HA complex resulted in dramati-cally inhibited tumor growth with reduced VEGF mRNA and VEGF levels in the tumors.
ó2010Elsevier Ltd.All rights reserved.
1.Introduction
A small interfering RNA (siRNA)is a short double-stranded RNA which has been regarded as novel potential therapeutics for the treatment of various diseases by speci ?c gene silencing of the complementary mRNA [1,2].However,the ef ?ciency of gene silencing by siRNA is very low in the body because of its rapid degradation by nuclease and the renal clearance [3,4].Accordingly,siRNA delivery has been emerged as a key issue for the development of siRNA therapeutics.Various natural polymers like chitosan [5],and synthetic polymers such as polyethyleneimine (PEI)[6]and cationic dendrimers [7]have been investigated for intracellular delivery of siRNA.Cationic peptides,such as poly-L -lysine,poly-histidine [8],and cell penetrating peptides (CPP)[9],have been also used for intracel-lular delivery of siRNA with improved biocompatibility.In addition,cationic lipids were developed as non-viral vectors for siRNA delivery due to their positive surface charge and endosomal escape capability [10,11].Recently,high-throughput screening approach was applied to
facilitate rapid preparation of various lipid-like siRNA carriers with high intracellular delivery and gene silencing ef ?ciencies [12].In order for targeted delivery to speci ?c tissues such as liver [13]and lung [14],the siRNA carriers were further modi ?ed with targeting ligands.
Among various candidates,however,branched PEI (bPEI)with a high molecular weight (MW)of 25,000Da has been regarded as one of the most effective non-viral vehicles for in vitro gene silencing [15].The high positive charge of bPEI is advantageous to form a tight complex with siRNA by electrostatic interaction and its strong proton sponge effect plays essential roles in the endosomal escape of siRNA [16].Paradoxically,the strong positive surface charge of bPEI causes severe cytotoxicity and non-speci ?c binding with serum proteins making dif ?cult in vivo gene silencing applications [17,18].Amine groups of cationic polymers interact with complements and induce innate immune responses [19].Until now,siRNA therapeutics have been applied to the treatment of various diseases,such as cancer [20,21],AIDS [2,22],liver and pulmonary diseases [13,23],and so on.Especially,siRNA therapeutics have been extensively investigated for the treatment of cancer.Angiogenesis for tumor growth was reported to be suppressed ef ?ciently by down-regulating the gene expression of vascular endothelial growth factor (VEGF)[24].There have been many reports on therapeutic application of VEGF siRNA (siVEGF)for the treatment of cancer [25,26].
*Corresponding author.School of Interdisciplinary Bioscience and Bioengi-neering,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 ScienceDirect
Biomaterials
journal h omepage:
https://www.360docs.net/doc/00829291.html,/locate/biomaterials
0142-9612/$e see front matter ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.biomaterials.2010.03.018
Biomaterials 31(2010)5258e 5265
We previously reported the tumor treatment with siVEGF complexed with PEI grafted hyaluronic acid(HA)[27,28].HA is a natural anionic polysaccharide which can be ef?ciently up-taken to the cells by HA receptor mediated endocytosis in the body.The HA in the outer-layer of PEI-g-HA conjugate reduced the cytotox-icity of PEI and made possible the target speci?c delivery to the tissues with various HA receptors,such as liver,lymphatic vessels, and tumor tissues[28].However,the inherent cytotoxicity of high MW PEI should be alleviated for further development.In this work, reducible bPEI was synthesized by the crosslinking of low MW PEI with cystamine bisacrylamide(CBA).PEI with a MW of2,000Da is known to have relatively negligible cytotoxicity and low siRNA delivery ef?ciency[29,30].The cross-linked PEI containing disul?de bonds(PEI-SS)was expected to show comparable siRNA delivery capability to high MW bPEI with remarkably reduced cytotoxicity likely due to the degradation by glutathione in the cytosol[31e33]. The amine groups of PEI-SS were further conjugated to the carboxyl groups of HA in the form of block-copolymer by reductive amida-tion.The resulting(PEI-SS)-b-HA conjugate was exploited to make a complex with siRNA by the electrostatic interaction between negatively charged siRNA and positively charged PEI in(PEI-SS)-b-HA conjugate.The siRNA/(PEI-SS)-b-HA complex was characterized and applied to in vitro and in vivo gene silencing for target speci?c treatment of tumor.
2.Materials and methods
2.1.Materials
HA with a MW of6,700Da was purchased from Lifecore Co.(Chaska,MN).Cy3-siRNA,siLuc,and siVEGF were purchased from Bioneer Co.(Daejeon,Korea).Cy3-siRNA has the sequences of5’-CCUACGCCAAUUUCGUdTdT-3’(sense)and5’-ACGAAAUUG-GUGGCGUAGGdTdT(antisense).The siRNA was labeled with Cy3at the5’-end of its sense strand.The sequences of siLuc were5’-UUGUUUUGGAGCGAAAdTdT-3’(sense) and5’-UUUCCUUCCAAAACAAdTdT-3’(antisense).The sequences of siVEGF were5’-AUGUGAAUGCAGACCAAAGAATT-3’(sense)and3’-TTUAACACUUACGUCUGGUUU-CUU-5’(antisense).PEI with the MW of2000and25,000,sodium cyanoborohydride, sodium borate decahydrate,boric acid,and paraformaldehyde were purchased from Sigma-Aldrich(St.Louis,MO).N,N’-Cystamine bisacrylamide(CBA)was purchased from Polyscience(Warrington,PA)and dimethyl sulfoxide(DMSO)from Junsei Chemical Co.(Tokyo,Japan).B16F1cells of murine melanoma were obtained from Korean Cell Line Bank(Seoul,Korea).Dulbecco’s modi?ed Eagle’s medium(DMEM), fetal bovine serum(FBS),antibiotics,phosphate buffered saline(PBS),SuperScript III First-Strand Synthesis System,and Dulbecco’s phosphate buffered saline(DPBS)were purchased from Invitrogen(Carlsbad,CA).MTT assay kit,pVMC-Luc(pVMC Luciferase) plasmid,lysis solution,and luciferase assay reagents were obtained from Promega (Madison,WI).TRI-reagent was purchased from Molecular Research Center Inc. (Cincinnati,OH).SYBR green was purchased from Takara-Bio Inc.(Otsu,Shiga,Japan). Mouse VEGF ELISA kit was purchased from R&D systems(Minneapolis,MN).All reagents were used without further puri?cation.
2.2.Synthesis of HA-b-(PEI-SS)
The synthesis of PEI-SS was carried out as described elsewhere[34].Brie?y,2g of PEI with a MW of2000Da and various ratios of CBA were dissolved in10% aqueous methanol at55 C.After reaction for24h,excess amount of PEI(1g)was added to quench the remaining acryloyl groups of CBA.The solution was mixed for 6h and the resulting product was puri?ed by exhaustive dialysis.The obtained PEI-SS was analyzed by1H NMR and then conjugated to the reductive end of HA molecules by reductive amidation as described elsewhere[35].In detail,97.45mg of PEI-SS and62.36mg of HA(6.7kDa)were dissolved in0.1M sodium borate buffer (pH8.5)containing1M NaCl.After that,NaBH3CN was added to be a?nal concentration of0.3M and reacted at37 C for3days.The reaction solution was poured into dialysis tube(MWCO7000)and dialyzed against a large excess amount of1M NaCl solution for2days and pure water for additional2days.The recovered (PEI-SS)-b-HA conjugate was also analyzed by1H NMR[36].
2.3.Preparation and characterization of siRNA/(PEI-SS)-b-HA complex
The siRNA/(PEI-SS)-b-HA complex was prepared by mixing1m L of Luc siRNA (100m M)with the speci?ed amount of(PEI-SS)-b-HA solution(10mg/mL)followed by incubation at room temperature for15min.The weight ratio of(PEI-SS)-b-HA to siRNA varied from4to38.The complex formation was con?rmed by gel electro-phoresis.The siRNA and siRNA/(PEI-SS)-b-HA complexes were loaded in the wells of 1.0wt%agarose gel containing ethidium bromide at a concentration of0.1m g/mL, which was applied to100V electrodes in50mM borate buffer(pH8.98)for20min. The siRNA was visualized by bromide staining and the gel image was taken under UV.After dilution of the complex solution with1mL of water,the hydrodynamic volume and surface charge of siRNA/(PEI-SS)-b-HA complex were measured at25 C using a particle analyzer(Zetasizer Nano,Malvern Instrument Co.,UK).
2.4.Cell culture and cytotoxicity test
B16F1cells were cultured in5%CO2incubator at37 C.DMEM was supple-mented with10vol%FBS and10IU/mL of antibiotics(penicillin).The cytotoxicity of (PEI-SS)-b-HA was evaluated by MTT assay.B16F1cells at a population of5?103 were dispensed in each well of96well plate.After a day,fresh medium containing various concentrations of PEI25kDa,PEI-SS,and(PEI-SS)-b-HA were added and incubated for24h.Then,20m L of2mg/mL MTT solution in DMEM was added to each well and incubated at37 C for2h.After removal of the medium containing MTT,100m L of DMSO was added to dissolve the formazan crystal formed by live cells.The optical density was measured at540nm with an absorbance microplate reader(EMaxòmicroplate reader,Bucher Biotec AG,Basel,Switzerland).Cell viability(%)was calculated by the following equation:Cell viability(%)?[OD540 (sample)/OD540(control)]?100,where OD540(sample)represents the optical density from
the wells treated with samples and OD540(control)represents that from the wells treated with PBS.
2.5.Intracellular delivery of Cy3-siRNA/(PEI-SS)-b-HA complex
The cellular uptake of Cy3-siRNA/(PEI-SS)-b-HA complex was investigated by ?uorescence microscopic and?ow cytometric analyses.For the cytometric analysis, B16F1cells were seeded on6well plate at a density of2?106cells/well and incubated for24h.Then,culture medium was exchanged with serum free DMEM containing Cy3-siRNA/(PEI-SS)-b-HA complex at an siRNA concentration of1m g/mL. After incubation for2h,the cells were detached with trypsin and washed with DPBS three times.Cells were?xed with1%paraformaldehyde and analyzed using?ow cytometry(BD FACSCalibur?,BD Bioscience,San Hose,CA).For the microscopic analysis,B16F1cells were placed on culture slides(Bedford,MA)at a density of 1.0?104cells/well and incubated for24h.Then,the culture medium was replaced with200m L of serum free DMEM containing Cy3-siRNA/(PEI-SS)-b-HA complex at a weight ratio of1/38.The culture slide was incubated at37 C for24h.The cells were washed with cold DPBS three times and?xed with1wt%of paraformaldehyde. After additional washing three times,the cells were observed with a confocal laser scanning microscope(LSM510,Carl-Zeiss Inc.,Thornwood,NY).For competitive binding test,Cy3-siRNA/(PEI-SS)-b-HA was incubated in the DMEM with and without20m g/mL of free HA.
2.6.In vitro gene silencing of siRNA/(PEI-SS)-b-HA complex
B16F1cells were dispensed into24-well plate at a population of5?105cells/well and incubated at37 C for the transfection of pVMC-Luc plasmid DNA.The siLuc/ (PEI-SS)-b-HA complex was co-transfected as described elsewhere[19].Brie?y,cells were pre-transfected with0.5mL of DMEM containing1m g of pVMC-Luc vector using a jetPEI?reagent following the product instruction.After incubation for3h and the subsequent PBS washing,various weight ratios of siLuc/(PEI-SS)-b-HA complexes were transfected for the gene silencing in DMEM containing10vol%FBS.After incubation for24h,the transfected cells were lysed with a lysis buffer of1wt%Triton X-100and5m L of the lysed solution was mixed with25m L of luciferase assay solution. The luciferase activity was measured with a luminescence microplate reader(Lumi-noskan Ascent,Lab systems,Germany).In order to evaluate the serum stability,the gene silencing by siLuc/(PEI-SS)-b-HA complex was carried out in the absence and presence of50vol%FBS.Furthermore,after treatment of B16F1cells with siVEGF/PEI, siVEGF/PEI-SS,and siVEGF/(PEI-SS)-b-HA complexes in the presence of50vol%serum for24h,mRNA was extracted using TRI-reagent and reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System for RT-PCR kit as per manufac-turer’s protocol.Then,the RT-PCR(CFX96Real-Time PCR Detection System,Bio-Rad, CA)was performed using SYBRòpremix according to the manufacturer’s protocol with VEGF primer and GAPDH primer as a normalizing control.
2.7.In vivo tumor treatment with siVEGF/(PEI-SS)-b-HA complex
In order to generate tumors,50m L of1?106CT-26single-cell suspension in5wt% glucose was injected subcutaneously to the middle of the right?ank in6-to 7-week-old female balb/c mice(in total24heads).Tumor size was measured using a vernier caliper across its longest(a)and shortest(b)diameters and its volume was calculated using the formula of V?0.5a?b2.Tumor treatment with four kinds of samples started after8days when the tumor size became approximately70mm3: 5wt%glucose solution as a control,non-speci?c siRNA of siLuc/(PEI-SS)-b-HA complex,siVEGF/PEI-SS complex,and siVEGF/(PEI-SS)-b-HA complex in5wt% glucose solution,respectively.Each50m L of5wt%glucose solution and three complex solutions at a dose of4.5m g siRNA/head was injected intra-tumorally to balb/c mice three times once every3days.The treated mice were examined for
K.Park et al./Biomaterials31(2010)5258e52655259
Fig.1.Schematic representations for (A)the synthesis of reducible polyethyleneimine-block-hyaluronic acid [(PEI-SS)-b-HA]conjugate by the reductive amidation of HA with low molecular weight PEI cross-linked with cystamine bisacrylamide (CBA)and (B)the complex formation of siRNA with (PEI-SS)-b-HA by electrostatic
interaction.
Fig.2.1H nuclear magnetic resonance (NMR)spectra of reducible polyethyleneimine-block-hyaluronic acid [(PEI-SS)-b-HA]conjugate.The characteristic peak of PEI shifted to 2.5e 3.0ppm after conjugation with HA.
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appearance,necrosis growth,and physical activity.The tumor volumes were measured every3days.Three mice from each group were sacri?ced1day after last injection of siRNA complexes,and the excised tumors were homogenized for RNA extraction using TRI-reagent.The VEGF mRNA level was analyzed by RT-PCR,and normalized with GAPDH mRNA level.In addition,to measure the VEGF level in tumor tissues after20days,the excised tumors from three mice were homogenized in PBS with protease inhibitor.After centrifugation,the amount of VEGF in each supernatant was measured by ELISA.
2.8.Statistical analysis
The data are expressed as means?S.D.from several separate experiments. Statistical analysis was carried out via the two-way analysis of variance(ANOVA)tests using the software of SigmaPlot9.0and a value for*P<0.05was considered statis-tically signi?cant.
3.Results and discussion
3.1.Synthesis and characterization of(PEI-SS)-b-HA conjugate
(PEI-SS)-b-HA conjugate was successfully developed as a target speci?c delivery carrier of siRNA.The siRNA delivery has been regarded as one of the most important technologies for the devel-opment of siRNA therapeutics.As shown in Fig.1,to alleviate the inherent cytotoxicity and low serum stability of high MW PEI,rela-tively non-toxic low MW PEI of2000Da was cross-linked with CBA to prepare reducible PEI-SS in the body,which was conjugated with polyanion of HA.The PEI-SS was synthesized by Michael addition between acryloyl group of CBA and amine group of branched PEI. After reaction at55 C for24h,excess PEI was added to quench the unreacted acryloyl groups.1H NMR analysis con?rmed the successful synthesis of PEI-SS.The peaks of PEI-SS between d?2.5ppm and d?3.0ppm correspond to the speci?c chemical shift of PEI by the conjugation.The acryloyl peaks of CBA between d?5.8and
d?6.5ppm disappeared con?rming that CBA was completely reac-ted with PEI.Although siRNA/PEI-SS complex showed comparable gene silencing ef?ciency and low toxicity in vitro,highly positive surface charge of siRNA/PEI-SS complex was known to cause non-
speci?c interaction with serum proteins in the body[17e19].
Accordingly,the PEI-SS was conjugated with HA in the form of(PEI-
SS)-b-HA by the reductive amidation between primary amine groups
of PEI-SS and reducing end groups of HA(Fig.1).Previously,we
reported the grafting of PEI to carboxyl groups of HA using1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide(EDC)for the preparation of
siRNA/PEI-g-HA complex[27,28].However,(PEI-SS)-b-HA conjugate
would be better to make more precise core-shell structure with
siRNA containing unmodi?ed carboxyl groups of HA on the surface
than PEI-g-HA conjugate(Fig.1).The MW of HA was optimized to be
6,700Da,since the polyanion of HA appeared to interfere with the
electrostatic interaction between negatively charged siRNA and
positively charged PEI in(PEI-SS)-b-HA conjugate.1H NMR analysis
con?rmed the successful synthesis of(PEI-SS)-b-HA conjugate
(Fig.2).There was a peak shift of PEI-SS between d?2.5ppm and d?3.0ppm re?ecting that protons in the amine groups of PEI-SS reacted for the conjugation[36].The PEI-SS was degraded by the
treatment with DTT and/or glutathione.
3.2.Preparation and characterization of siRNA/(PEI-SS)-b-HA
complex
The formation of siRNA/(PEI-SS)-b-HA complex by the electro-
static interaction between negatively charged siRNA and positively
charged PEI in(PEI-SS)-b-HA conjugate was assessed by agarose gel
electrophoresis(Fig.3A).The siRNA/(PEI-SS)-b-HA complex
appeared to be formed when the weight ratio of(PEI-SS)-b-HA
conjugate to siRNA was higher than15.With increasing weight ratio,
the surface charge of siRNA/(PEI-SS)-b-HA complex decreased proportionally.Zeta-particle size analysis revealed that mono-disperse complex was formed with a weight ratio of(PEI-SS)-b-HA to siRNA higher than23.A compact complex with a particle size of ca. 110nm was formed at a weight ratio of38.When the weight ratio of (PEI-SS)-b-HA to siRNA varied from15,23,to38,the surface charge of the complex decreased fromt12.1,t6.8,tot1.8mV(Fig.3B).
The Fig.3.Characterization of siRNA/reducible polyethyleneimine-block-hyaluronic acid [(PEI-SS)-b-HA]complex:(A)Agarose gel electrophoresis of siRNA/(PEI-SS)-b-HA complex with various ratios of(PEI-SS)-b-HA conjugate to siRNA.(B)Surface charge characteristics of siRNA/(PEI-SS)-b-HA complex with increasing weight
ratios.
Fig.4.B16F1cell viability(%)with increasing concentration of polyethyleneimine (PEI2kDa),reducible PEI(PEI-SS)prepared with PEI2kDa,(PEI-SS)-block-hyaluronic acid[(PEI-SS)-b-HA]conjugate,and PEI25kDa(n?3),respectively.
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negatively charged HA contributed to the decrease of the complex surface charge preventing the non-speci ?c binding of siRNA/(PEI-SS)-b-HA complex to the serum proteins and/or cell membranes [17e 19].To investigate the cytotoxicity of (PEI-SS)-b-HA,MTT assay was performed on B16F1cells (Fig.4).The cytotoxicity of PEI-SS and (PEI-SS)-b-HA was as ignorable as that of low molecular weight PEI,whereas PEI 25kDa caused severe cytotoxicity with increasing concentration up to 100m g/mL.To optimize the weight ratio of (PEI-SS)-b-HA to siRNA,the gene silencing ef ?ciency of siRNA/(PEI-SS)-b-HA complex with various weight ratios was measured on B16F1cells using luciferase gene co-transfection method.In the range of the weight ratio from 4to 38,the higher weight ratio resulted in the better gene silencing ef ?ciency around 80%,which was compa-rable to that of PEI 25kDa (N/P ratio ?8:1).The siRNA/(PEI-SS)-b-HA complex at the weight ratio of 38was used for the following experiments.
3.3.Intracellular uptake of Cy3-siRNA/(PEI-SS)-b-HA complex The effect of HA conjugation with PEI-SS on cellular uptake of Cy3-siRNA/(PEI-SS)-b-HA complex was assessed by ?ow cytometry and confocal microscopic analysis,as shown in Fig.5.Cy3-siRNA/(PEI-SS)-b-HA complex was up-taken into B16F1cells more ef ?ciently than Cy3-siRNA/PEI-SS complex.The results might be ascribed to the receptor mediated endocytosis of siRNA/(PEI-SS)-b-HA complex to B16F1cells with HA receptors.There are various kinds of HA recep-tors,such as LYVE-1,CD44,HARE,and so on [37,38].To con ?rm the HA receptor mediated endocytosis,we carried out competitive binding tests with free HA molecules using ?uorescence microscopic analysis (Fig.5A).As expected,the ?uorescence intensity of Cy3from the cells decreased drastically for the case of the medium containing free HA.It might be explained by the fact that high concentration of free HA molecules pre-dominated the HA receptors like CD44and LYVE-1on the B16F1cell surface and reduced the receptor mediated endocy-tosis of Cy3-siRNA/(PEI-SS)-b-HA complex.Cy3-siRNA/(PEI-SS)-b-HA distributed evenly in the cells 24h after transfection as shown in Fig.5.The results were well matched with the ?ow cytometric analysis results in Fig.5B.Considering all these results,HA in the outer surface of siRNA/(PEI-SS)-b-HA complex was thought to signi ?cantly contribute to the improved cellular uptake by
HA
Fig.5.(A)Confocal microscopic images of B16F1cells after treatment with (A-1)the control,(A-2)Cy3-siRNA/reducible polyethyleneimine-block-hyaluronic acid [(PEI-SS)-b-HA]complex,and (A-3)free HA and Cy3-siRNA/(PEI-SS)-b-HA complex.(A-4)Magni ?ed image of B16F1cells treated with Cy3-siRNA/(PEI-SS)-b-HA complex.The scale bars represent 20m m .(B)Flow cytometric analysis for the intracellular uptake of Cy3-siRNA/PEI-SS and Cy3-siRNA/(PEI-SS)-b-HA
complexes.
Fig.6.(A)Luciferase gene silencing by siRNA/polyethyleneimine (PEI 25k),siRNA/reducible PEI (PEI-SS),and siRNA/(PEI-SS)-block-hyaluronic acid [(PEI-SS)-b-HA]complexes in B16F1cells in the absence and presence of 50vol%serum.The results represent mean ?SD (n ?3).*P <0.05versus PEI and **P <0.01versus PEI-SS.(B)Semi-quantitative RT-PCR analysis for VEGF mRNA level using a normalized graph with housekeeping gene expression of GAPDH.The results represent mean ?SD (n ?2).*P <0.05versus PEI and PEI-SS.
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receptor mediated endocytosis making possible target speci?c delivery to the tissues with HA receptors.
3.4.Gene silencing of siRNA/(PEI-SS)-b-HA complex
On the basis of cellular uptake test results,(PEI-SS)-b-HA conju-gate was assessed as a target speci?c gene silencing carrier.In the absence of serum,the gene silencing ef?ciency was ca.80%for siRNA/PEI25k,siRNA/PEI-SS,and siRNA/(PEI-SS)-b-HA complexes (Fig.6A).Prior to in vivo applications,we tested the serum effect on gene silencing.In the presence of50vol%FBS,the gene silencing ef?ciency of siRNA/(PEI-SS)-b-HA complex was maintained around 50%at the weight ratio of38,while the gene silencing ef?ciency of PEI25kDa and PEI-SS decreased drastically to the range of only 20e30%(Fig.6A).The neutral surface charge characteristics of siRNA/(PEI-SS)-b-HA complex was thought to contribute to the enhanced serum stability and the effective gene silencing.The gene silencing by(PEI-SS)-b-HA conjugates in the presence of serum was also assessed by the quantitative analysis of VEGF mRNA level using RT-PCR after treatment with siVEGF/PEI25k,siVEGF/PEI-SS,and siVEGF/(PEI-SS)-b-HA complexes.As shown in Fig.6B,siVEGF/(PEI-SS)-b-HA complex resulted in statistically more signi?cant decrease of VEGF mRNA expression level than siVEGF/PEI and siVEGF/PEI-SS complexes in50vol%serum condition.Approximately2-fold reduction of VEGF mRNA level was observed after treatment with siVEGF/(PEI-SS)-b-HA complex in the presence of50vol%serum.3.5.In vivo tumor treatment
Encouraged by in vitro gene silencing test results,we applied the siRNA/(PEI-SS)-b-HA complex to the treatment of tumor.There are several factors related with angiogenesis for tumor growth.Among them,VEGF is known to have a critical role in the tumor angio-genesis.Accordingly,we decided to apply siVEGF/(PEI-SS)-b-HA complex to target speci?c treatment of tumor by inhibiting the VEGF production of tumor cells.CT-26cells at a population of 1?106was inoculated to balb/c mice for the preparation of tumor models.CT-26tumor cell line is known to express the HA receptors such as CD44,RHAMM,LYVE-1,and so on[39].After8days,the average of tumor volume increased to approximately70mm2,and siVEGF/(PEI-SS)-b-HA complex was injected intra-tumorally three times once every3days.As control groups,5%glucose solution, non-speci?c siLuc/(PEI-SS)-b-HA,and siVEGF/PEI-SS complexes were also injected in the same manner.Fig.7A showed that siVEGF/ (PEI-SS)-b-HA complex signi?cantly suppressed the tumor growth compared with other control groups(P<0.01).Tumor tissues recovered after20days con?rmed the gene silencing ef?ciency of siVEGF/(PEI-SS)-b-HA complex(Fig.7B).To con?rm the gene silencing by siVEGF/(PEI-SS)-b-HA complex more clearly,the VEGF mRNA expression level was also investigated after sacri?ce of three mice in each group at24h after last sample injection.The siVEGF/ (PEI-SS)-b-HA down-regulated the VEGF mRNA level effectively compared with other control groups(Fig.7C).Fig.7D shows
the
Fig.7.Anti-tumoral therapeutic effect of VEGF siRNA(siVEGF)/(PEI-SS)-b-HA complex in female balb/c mice where CT-26colon cancer cells were injected for tumor inoculation and growth.(A)Tumor volume change with increasing time after intra-tumoral injection of a control of5%glucose solution,siVEGF/PEI-SS,non-speci?c Luc siRNA(siLuc)/(PEI-SS)-b-HA, and siVEGF/(PEI-SS)-b-HA complexes.The treatments were performed three times after8,11,and14days.The results represent mean?SD(n?3).*P<0.05versus the control.
(B)Photo-images of dissected tumor tissues after20days.(C)VEGF mRNA levels in tumor tissues one day after last treatment with the samples(after15days).The data were normalized with mouse GAPDH mRNA level.*P<0.05versus siVEGF/PEI-SS.(D)Comparison of VEGF levels in tumor tissues after20days.*P<0.05versus siVEGF/PEI-SS.
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relative VEGF level in tumor tissues,which also con?rmed the statistically signi?cant inhibition of VEGF production by siVEGF/ (PEI-SS)-b-HA complex.As shown in Fig.7B-D,siRNA/PEI-SS complex was also slightly effective for the inhibition of tumor growth.The tumor tissues after treatment with siRNA/PEI-SS complex were recovered in the form of several small pieces,which was the reason for the overestimation of tumor volumes in Fig.7A. Considering all these results,we could conclude that(PEI-SS)-b-HA conjugate ef?ciently delivered siRNA to target tissues with HA receptors,and contributed to VEGF gene silencing and the following therapeutic effect for the treatment of tumor.The siVEGF/ (PEI-SS)-b-HA complex would be successfully developed as anti-angiogenic therapeutics for the treatment of various diseases in the tissues with HA receptors.
4.Conclusions
(PEI-SS)-b-HA conjugate was successfully developed as a target speci?c and non-toxic delivery system of siRNA therapeutics.PEI-SS prepared by the crosslinking of non-toxic low MW PEI(2000Da) with CBA was further conjugated with HA by reductive amidation in the form of(PEI-SS)-b-HA conjugate.The cytotoxicity of(PEI-SS)-b-HA appeared to be negligible likely due to the degradation of PEI-SS to low MW PEI in the cytosol.The effective cellular uptake of siRNA/(PEI-SS)-b-HA complex by HA receptor mediated endocy-tosis was con?rmed by?ow cytometric and confocal microscopic analyses.In vitro gene silencing ef?ciency of siRNA/(PEI-SS)-b-HA complex was in the range of50e80%in the absence and presence of 50vol%serum.HA in the outer surface of siRNA/(PEI-SS)-b-HA complex contributed to not only effective cellular up-take by HA receptor mediated endocytosis but also enhanced serum stability alleviating the non-speci?c binding with serum proteins.Further-more,siVEGF/(PEI-SS)-b-HA complex was successfully applied to the treatment of tumor dramatically retarding the tumor growth. Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science and Technology(2009-0072986).This research was also supported by the Converging Research Center Program through the National Research Founda-tion of Korea(NRF)funded by the Ministry of Education,Science and Technology(2009-0081871).
Appendix
Most of the?gures in this article have parts that are dif?cult to interpret in black and white.The full color images can be found in the on-line version,at doi:10.1016/j.biomaterials.2010.03.018. References
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