Research ArticleSelf-Assembling Multifunctional PeptideDimers for Gene Delivery Systems
Kitae Ryu1 Gyeong Jin Lee1 Ji-yeong Choi1 Taewan Kim1 and Tae-il Kim12
1Department of BiosystemsampBiomaterials Science andEngineering College of Agriculture andLife Sciences SeoulNationalUniversity1 Gwanak-ro Gwanak-gu Seoul 151-921 Republic of Korea2Research Institute of Agriculture and Life Sciences Seoul National University 1 Gwanak-ro Gwanak-guSeoul 151-921 Republic of Korea
Correspondence should be addressed to Tae-il Kim seal1004snuackr
Received 10 May 2015 Accepted 6 September 2015
Academic Editor Michele Iafisco
Copyright copy 2015 Kitae Ryu et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Self-assembling multifunctional peptide was designed for gene delivery systems The multifunctional peptide (MP) consists ofcellular penetrating peptide moiety (R
8) matrix metalloproteinase-2 (MMP-2) specific sequence (GPLGV) pH-responsive moiety
(H5) and hydrophobic moiety (palmitic acid) (CR
8GPLGVH
5-Pal) MP was oxidized to form multifunctional peptide dimer
(MPD) by DMSO oxidation of thiols in terminal cysteine residues MPD could condense pDNA successfully at a weight ratio of 5MPD itself could self-assemble into submicronmicelle particles via hydrophobic interaction of which criticalmicelle concentrationis about 001mMMPDshowed concentration-dependent but low cytotoxicity in comparisonwith PEI25kMPDpolyplexes showedlow transfection efficiency in HEK293 cells expressing low level of MMP-2 but high transfection efficiency in A549 and C2C12cells expressing high level of MMP-2 meaning the enhanced transfection efficiency probably due to MMP-induced structuralchange of polyplexes Bafilomycin A1-treated transfection results suggest that the transfection of MPD is mediated via endosomalescape by endosome buffering ability These results show the potential of MPD for MMP-2 targeted gene delivery systems due toits multifunctionality
1 Introduction
Lots of peptides have been developed and used for genedelivery systems due to their advantages [1ndash3] Buildingblocks of peptides amino acids are nontoxic and almostnonimmunogenic endogenous molecules possessing vari-ous functional side chains with the diversity for designinggene delivery carriers Peptides can be synthesized by well-established solid-phase chemistry in sequence-controlledmanner yielding almost monodispersed products Peptidesalso can interact with cellular molecules or componentswhich induce biologically important events such as recogni-tion of RGD peptide by integrin receptors [4]
Particularly cellular penetrating peptides such as pene-tratin Tat sequence or oligoarginine have been extensivelyexamined in gene delivery field because they can formnanosized complexes with nucleic acids due to their cationicproperty and possess high cellular uptake efficiency [5ndash9]
Besides oligopeptide forms bioreducible cellular penetratingpeptides which are linked via disulfide bonds also have beendeveloped for gene delivery systems [10ndash13] They can bedegraded in reductive environment such as cytosol showingcontrolled release of nucleic acids and low cytotoxicity [14]
In this work we designed a self-assembling multifunc-tional peptide (MP CR
8GPLGVH
5-Pal) in order to combine
advantages of several functional peptides for gene deliverysystems It is expected that R
8moiety would condense
pDNA into polyplex particles and facilitate the cellular uptakeof the polyplexes [15] GPLGV is a substrate sequence ofmatrix metalloproteinase-2 (MMP-2) which is one of thecollagenases overexpressed from several tumor cells andplays important roles in tumor progression and metastasis[16] It was introduced to cause structural change of thepolyplexes for efficient cellular uptake responding toMMP-2of tumor cells Many studies reported the potential of MMP-cleavable druggene delivery carriers for targeting tumor cells
Hindawi Publishing CorporationAdvances in Materials Science and EngineeringVolume 2015 Article ID 852584 9 pageshttpdxdoiorg1011552015852584
2 Advances in Materials Science and Engineering
[17 18] H5moiety was employed for endosome buffering
ability of imidazole groups to escape from endosomes aftercellular uptake [19 20] Palmitic acid at C-terminal wouldinduce micelle formation via self-assembly of the peptide byhydrophobic interaction H
5moiety also would contribute to
the micelle formation because of hydrophobicity at neutralpH In addition we cross-linked this peptide to dimer(MPD) for improvement ofmicelle formation and performedcharacterizations to examine the potential of MPD for genedelivery systems
2 Materials and Methods
21 Materials Multifunctional peptide (CR8GPLGVH
5-Pal)
was purchased from GL Biochem (Shanghai China) Thia-zolyl Blue Tetrazolium Bromide (MTT reagent) agarose and551015840-dithiobis(2-nitrobenzoic acid) (Ellmanrsquos reagent) werepurchased from Sigma-Aldrich (St Louis MO) Dimethylsulfoxide (DMSO) was purchased from Merck (Germany)Luciferase assay systemwas purchased from Promega (Madi-son WI) BCA protein assay kit was purchased fromPierce (Rockford IL) Fetal bovine serum (FBS) 025trypsin-EDTADulbeccorsquos phosphate buffered saline (DPBS)and Dulbeccorsquos modified Eaglersquos medium (DMEM) werepurchased from Invitrogen (Carlsbad CA) The luciferasereporter gene-encoding plasmidDNA pCN-Luci was ampli-fied in Escherichia coli DH5120572 and isolated by NucleobondXtra Midi kit (Macherey-Nagel Germany) MMP-2 enzymewas purchased from Calbiochem (San Diego CA) All otherchemicals were purchased and used without any furtherpurification
22 Synthesis of Peptide Dimers Multifunctional peptidedimer (MPD)was synthesized by usingDMSO autooxidationreaction [21] Multifunctional peptide (MP) was dissolved inDMSO (05 wt) and the solution was stirred under nitrogengas at room temperature for 24 h After oxidation reactionsynthesized MPD was purified by diethyl ether precipitationand dried under vacuum at room temperature overnightFinally sticky and transparent product was obtained
23 Ellmanrsquos Assay Ellmanrsquos assay was carried out to con-firm the synthesis of MPD by measuring residual thi-ols after dimerization of MPM MPD was dissolved inDMSO (02mM) 50 120583L of Ellmanrsquos reagent solution (DMSO4mgmL) was added to 250 120583L of sample After 15min ofincubation the absorbance at 499 nm was measured by amicroplate reader (Synergy H1 BioTek USA) Thiol amountof MPD was calculated based on calibration curve
24 Agarose Gel Electrophoresis DNA condensation abilityof MPD was identified by agarose gel electrophoresis assayAgarose gel (07 wt) containing ethidium bromide wasprepared in Tris-Acetate-EDTA (TAE) bufferTheMPDpoly-plexes (05 120583g pDNA) having various weight ratios (05 1 25 and 10) were prepared After 30min of incubation at roomtemperature electrophoresis was carried out for 15min at
100V (Mupid-2plus Takara Bio Inc Japan)The locations ofpDNA bands were observed by UV illuminator (ChemiDocXRS+ gel documentation system Bio-Rad Hercules CA)
25 Critical Micelle Concentration (CMC) Measurements Inorder to characterize CMC of MPD pyrene was selectedas a model drug as previously reported [22] 02mM ofpyrene solution was prepared by dilution of pyreneethanolsolution 3 120583L of pyrene solutions was added to vials anddried under vacuum overnight in dark condition Afterethanol evaporation 1mL of MPD solution with variousconcentrations was added to the vials giving the final pyreneconcentration of 6 times 10minus7M The MPDpyrene solution wassonicated for 10min and incubated with gentle mixing atroom temperature for 24 h Absorbance was measured byUVVis spectrometer (Optizen POP BIO Mecasys Korea)using quartz cuvettes in the range of 200 to 400 nmThe totalabsorbance was calculated by sum of four absorbance points(242 272 320 and 336 nm) CMC of theMPD sample was setto the center of the sigmoid
26 Average Particle Size and Zeta-Potential MeasurementsAverage sizes and Zeta-potential values of MPD and MPDpolyplexes were measured by Zetasizer Nano ZS (MalvernInstruments UK) with He-Ne laser beam (633 nm) at 25∘Crespectively 1mL of MPD (001 and 01mM) and MPDpolyplex solutions (5120583g pDNA weight ratios 2 10 and 20)was prepared and used for analysis Sizes of MPD polyplexesafter active MMP-2 cleavage were also examined MPDpolyplexes (1 120583g pDNA weight ratio 20) were prepared asdescribed above The polyplex solution was added to activeMMP-2 containing PBS buffer (pH 74 10 120583gmL MMP-2)and themixture was incubated at 37∘C for 4 hours with gentleshaking After treatment the sizes of the polyplexes samplewere analyzed by Zetasizer Nano ZS Average sizes and Zeta-potential values ofMPD andMPDpolyplexes weremeasured3 times
27 Atomic Force Microscopy (AFM) The morphologies ofMPD particle structures were observed by using atomicforce microscopy (Park Systems Korea) 100 120583L of MPDaqueous solution samples was prepared (001 and 01mM)10 120583L sample aliquots were dropped to fresh mica surfaceby pipette carefully and then excess of sample was removedby absorption of filter papers from the corner of the micaThe mica samples were dried at room temperature for 24 hand used for AFM observation The imaging was set tononcontact tapping mode and the scanning rate was 1Hz
28 Cell Culture Human lung adenocarcinoma epithelialcells (A549) human transformed primary embryonal kidneyepithelial cells (HEK293) and mouse myoblast cells (C2C12)were grown in DMEM supplemented with 10 FBS and1 penicillinstreptomycin (PS) in humidified atmospherecontaining 5 CO
2at 37∘C
Advances in Materials Science and Engineering 3
29 Cytotoxicity Cytotoxicity of MPD was characterized byMTT assay Cells were seeded into 96-well cell culture platesin 100120583L of DMEM (10 FBS 1 PS) at a density of1 times 104 cellswell After 24 h the cells were treated with100 120583L of peptide solutions for 4 h PEI25k was used as acontrol Then the fresh media were exchanged and furtherincubated for 24 h After incubation 25120583L of MTT stocksolution (2mgmL in DPBS) was added to each well and themediawere carefully removed after 2 hThe formazan crystalsformed by proliferating cells were dissolved with 150120583L ofDMSO and the absorbance was measured at 570 nm using amicroplate reader (Synergy H1 BioTek USA) Results werepresented as relative cell viabilities (RCV percentage valuesrelative to value of untreated control cells) All experimentswere performed in quadruplicate
210 Transfection Experiments The transfection efficiencyof MPD was examined by measuring luciferase transgeneexpression Cells were seeded into 24-well cell culture platesin 500120583L of DMEM (10 FBS 1 PS) at a density of 5 times 104cellswell After 24 h the media were exchanged to serum-free media and MPD polyplex solutions (weight ratios 510 20 and 30) were added to cells PEI25k polyplex (weightratio 1) was used as a control After 4 h of incubation thetransfection medium was replaced with fresh DMEM with10 FBS The cells were further incubated for 2 days andrinsed with 240120583L of DPBS twice The cells were lysedwith 120120583L of lysis buffer and shaken for 30min at roomtemperature The cell lysates were centrifuged for 30min at15000 g and 20 120583L of supernatants was used for luciferaseassay Luciferase activities were measured using luciferaseassay reagent on a microplate reader (Synergy H1 BioTekUSA)The total cellular proteins in cell lysateswere quantifiedby BCA protein assay kit The final transfection efficiencyresults were presented in terms of RLUmg cellular proteinAll experiments were performed in triplicate
211 Transfection Experiments with Bafilomycin A1 In orderto characterize the endosome buffering ability and trans-fection mechanism of MPD transfection experiments withBafilomycin A1 treatment were carried out A549 cellswere seeded as explained above 200 nM of BafilomycinA1 solutions was pretreated for 10min before transfectionMPD polyplexes were prepared with weight ratio of 10 20and 30 PEI25K was used as a positive control Polyplexsolutions were treated to the cells for 4 h and the media wereexchanged with fresh DMEM with 10 FBS After 48 h offurther incubation transfection efficiency was examined asdescribed above Final results were presented in terms ofrelative transfection efficiency (transfection efficiency withBafilomycin A1transfection efficiency without BafilomycinA1)
3 Results and Discussion
31 Characterization of MPM and MPD MP consists ofcellular penetrating peptide moiety (R
8) matrix meta-
lloproteinase-2 (MMP-2) specific sequence (GPLGV)
05 1 2 5 10C
Figure 1 Agarose gel electrophoresis result of MPD polyplexes CpDNA only Numbers mean weight ratios of the polyplexes
pH-responsive moiety (H5) and hydrophobic moiety
(palmitic acid) (CR8GPLGVH
5-Pal) Cellular penetrating
moiety was introduced for condensation of pDNA byelectrostatic interaction of cationic arginine residues andfor enhancement of cellular uptake pH-responsive moiety(H5) was introduced for facilitation of endosomal escape
of polyplexes after cellular uptake by proton sponge effectHydrophobic palmitic acid was introduced to C-terminalof MP in order to induce the formation of micelle structureby hydrophobic interaction R
8and other functional blocks
were linked with MMP-2 specific sequence which maylead to structural change of polyplexes by cleavage of thesequence with exposure to MMP-2
The structure of MP was confirmed by the manufacturerMP was dimerized to synthesize MPD by DMSO oxidationvia disulfide bond formation between cysteine thiols at N-terminal MP was insoluble in water but the synthesizedMPD after oxidation was soluble in water probably due tothe facilitated formation of micelle structure by strengthenedhydrophobic interaction of MPD It is thought that disulfidebond formation between MP could cause the proximityof hydrophobic moieties (histidine moieties and palmiticacid) and ease of micelle structure formation by structuralrestriction
The synthesis of MPD was examined by quantification ofremaining thiols of MPD product After DMSO oxidation itwas found that only 03 mole of thiol was detected in MPDproduct which means that almost MP could be dimerized tosynthesize MPD
32 Agarose Gel Electrophoresis pDNA condensing abilityof MPD was investigated by agarose gel electrophoresisAs shown in Figure 1 weak migration of pDNA was stillobserved at a weight ratio of 2 However MPD could retardpDNA completely at a weight ratio of 5 which meansthat pDNA was well condensed by MPD Therefore it wasconfirmed that MPD could condense pDNA due to itscationic R
8moiety
33 CMC Measurement CMC of MPD was measured inorder to confirm the formation of micelle structure by MPDMPD solutions with various concentrations were incubatedwith pyrene and absorbances from specific peaks frompyrene were summed It was reported that absorbance valuessum from 4 specific peaks of pyrene would be increasedunder hydrophobic environment such as core part of micelle
4 Advances in Materials Science and Engineering
Concentration (mM)
Abs
valu
es su
m
00
01
02
03
04
05
1e minus 5 1e minus 4 1e minus 3 1e minus 2 1e minus 1
Figure 2 CMC measurement result of MPD micelle
structures [22] In Figure 2 absorbance values sum of pyrenewas abruptly increased from 001mM of MPD This resultshows that MPD could form micelle structure as expected inaqueous medium and CMC of MPD is about 001mM
34 Characterization of MPD Micelles and MPD PolyplexesAverage sizes and Zeta-potential values of MPDmicelles andMPD polyplexes were measured by Zetasizer (Figure 3) Itwas found that MPD micelles showed submicron averagesize at 001mM and the size was decreased to about 470 nmat 01mM which means the formation of compact andstable micelle structure due to increase of MPD molecules(Figure 3(a)) Zeta-potential values of MPD micelles werealso increased from 106mV to 292mV probably due to theadditional incorporation of cationic MPDmolecules into themicelle structures
The morphology of MPD micelles was also observedby AFM They showed heterogeneous structures contain-ing small sphere or large cylinder structures at 001mMwhich explains large variation of average size measurementresults (Figure 4(a)) At 01mM MPD micelles displayedmore homogenous morphologies of spherical structures(Figure 4(b)) Greater height of particles was found at 01mM(30ndash60 nm) than at 001mM (5ndash8 nm) probably due to theincreased assembly ofMPDmolecules intomicelle structuresat a high concentration
In the case ofMPDpolyplexes average size was 230 nm ata weight ratio of 2 and increased to about 2-3 120583m over weightratios of 20 (Figure 3(b)) However Zeta-potential values ofMPD polyplexes were found to be negative (minus301mV) at aweight ratio of 2 and to be increased to almost zero overweight ratios of 20 Considering Zeta-potential values it isthought that MPD polyplexes could form large aggregatesvia hydrophobic interaction at high weight ratios due to theneutralization of surface charges by interaction of cationicMPD micelles with anionic pDNA It is also elucidatedthat negatively charged and small MPD polyplex could bepartially formed via surface coating of anionic pDNA whichis consistent with agarose gel electrophoresis result
35 Cytotoxicity Measurement The cytotoxicity of MPD wasexamined by MTT assay in various cell lines (A549 C2C12and HEK293) As shown in Figure 5 control agent PEI25kexhibited significant cytotoxicity The viability of PEI25k-treated cells was less than 20 even at a concentration of20120583gmL However the viability of MPD-treated cells wasmore than 80 at the same concentration in all three celllines This result means that cytotoxicity of MPD was low atthis condition although it was found to be concentration-dependent
36 Transfection Experiments of MPD Polyplexes Trans-fection efficiency of MPD polyplexes was investigated bymeasurement of luciferase transgene expression in A549C2C12 and HEK293 cell lines (Figure 6) It is known thatA549 and C2C12 cells express high levels of MMP-2 butHEK293 does not [23ndash25] MMP-2 is a family of collagenaseswhich is important for proliferation and migration of cancercells Therefore we performed transfection experiment invarious cell lines in order to examine the effect of MMP-2 expression on transfection of MPD containing MMP-2specific sequence Interestingly MPD showed high transfec-tion efficiency comparable to PEI25k in A549 and C2C12cells expressing high level of MMP-2 On the other handtransfection efficiency of MPD was found to be low (dozensto hundreds lower than that of PEI25k) in HEK293 cellseven though HEK293 cells are known to be well-transfectedcells and transfection efficiency of PEI25k in HEK293 cellsis about 10-fold higher than that in other cells This resultmeans that MPD possess the potential for gene deliverysystems especially targeting MMP-2 expressing cancer cellsIn addition the average sizes of the polyplexes weremeasuredin the presence of MMP-2 in order to examine the effectof MPD polyplexes structural change on the transfectionefficiency (Figure 6(d)) The size of the polyplexes was foundto be largely decreased in the presence of MMP-2 Thisresult shows that the large polyplex aggregates could beconverted to smaller polyplex particles by MMP-2 cleavageprobably leading to enhanced cellular uptake and trans-fection efficiency Although further study will be requiredfor revealing the detailed mechanism one explanation isthat the structural change of MPD polyplexes by cleavageof MMP-2 such as exposure of cellular penetrating moiety(R8) may facilitate the cellular uptake of MPD polyplexes
for efficient transfection (Scheme 1) On the contrary inHEK293 cells MPD polyplexes would not undergo structuralchange maintaining large aggregate structures with zeroZeta-potential values which are not proper for transfection
37 Transfection Experiments with Bafilomycin A1 Transfec-tion experiments were also performed in the presence ofBafilomycin A1 in order to examine the endosome bufferingability of MPD and the transfection mechanism BafilomycinA1 is an inhibitor of vacuolar type ATPase which cansuppress the proton influx into endosome and disturb theproton pump effect of endosome buffering moiety finallyleading to the decrease of transfection efficiency [26 27]
Advances in Materials Science and Engineering 5
Concentration (mM)
Aver
age s
ize (
nm)
0
200
400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
Size
10minus2 10minus1
Zeta
-pot
entia
l (m
V)
Zeta-potential
(a)
minus40
minus30
minus20
minus10
Size
Aver
age s
ize (
nm)
0
500
2000
30000
10
202 10
Weight ratio
Zeta-potential
Zeta
-pot
entia
l (m
V)
(b)
Figure 3 Average sizes and Zeta-potential values measurement results of MPD micelles (a) and MPD polyplexes (b)
75
5
25
0
(nm
)(n
m)
6
6
4
4
2
2
0
0
64206420
(120583m) (120583m)
(120583m)
(a)
6420
64206420
30
20
10
0
60
40
20
0
(nm
)(n
m)
(120583m)
(120583m)(120583m)
(b)
Figure 4 AFM images and height information of MPD micelles at 001mM (a) and 01mM (b)
6 Advances in Materials Science and Engineering
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(a)Re
lativ
e cel
l via
bilit
y (
)
PEI25KMPD
0
20
40
60
80
100
0 5 10 15 20 25 30
Concentration (120583gmL)
(b)
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(c)
Figure 5 MTT assay results of MPD in A549 (a) C2C12 (b) and HEK293 (c) cells
It is thought that MPD may possess the endosome bufferingability due to imidazole groups of histidine residues ofwhich pKa value is about 6 As shown in Figure 7 thetransfection efficiency of PEI25k in Bafilomycin A1 conditionwas decreased to about 180 times lower value than normalcondition Similar to PEI25kMPD also showed 20ndash170 timeslower transfection efficiency in Bafilomycin A1 conditionthan normal condition This result means that the transfec-tion of MPD is mediated by endosomal escape by endosomebuffering ability after cellular uptake
4 Conclusion
Self-assembling multifunctional peptide was designed anddimerized for gene delivery systems This MPD couldself-assemble to micelle structures and condense pDNAby electrostatic interaction The cytotoxicity of MPDwas concentration-dependent but low in the examinedconcentration range High transfection efficiency ofMPD polyplexes in cancer cells expressing high level ofMMP-2 showed the potential of MPD for targeted gene
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
2 Advances in Materials Science and Engineering
[17 18] H5moiety was employed for endosome buffering
ability of imidazole groups to escape from endosomes aftercellular uptake [19 20] Palmitic acid at C-terminal wouldinduce micelle formation via self-assembly of the peptide byhydrophobic interaction H
5moiety also would contribute to
the micelle formation because of hydrophobicity at neutralpH In addition we cross-linked this peptide to dimer(MPD) for improvement ofmicelle formation and performedcharacterizations to examine the potential of MPD for genedelivery systems
2 Materials and Methods
21 Materials Multifunctional peptide (CR8GPLGVH
5-Pal)
was purchased from GL Biochem (Shanghai China) Thia-zolyl Blue Tetrazolium Bromide (MTT reagent) agarose and551015840-dithiobis(2-nitrobenzoic acid) (Ellmanrsquos reagent) werepurchased from Sigma-Aldrich (St Louis MO) Dimethylsulfoxide (DMSO) was purchased from Merck (Germany)Luciferase assay systemwas purchased from Promega (Madi-son WI) BCA protein assay kit was purchased fromPierce (Rockford IL) Fetal bovine serum (FBS) 025trypsin-EDTADulbeccorsquos phosphate buffered saline (DPBS)and Dulbeccorsquos modified Eaglersquos medium (DMEM) werepurchased from Invitrogen (Carlsbad CA) The luciferasereporter gene-encoding plasmidDNA pCN-Luci was ampli-fied in Escherichia coli DH5120572 and isolated by NucleobondXtra Midi kit (Macherey-Nagel Germany) MMP-2 enzymewas purchased from Calbiochem (San Diego CA) All otherchemicals were purchased and used without any furtherpurification
22 Synthesis of Peptide Dimers Multifunctional peptidedimer (MPD)was synthesized by usingDMSO autooxidationreaction [21] Multifunctional peptide (MP) was dissolved inDMSO (05 wt) and the solution was stirred under nitrogengas at room temperature for 24 h After oxidation reactionsynthesized MPD was purified by diethyl ether precipitationand dried under vacuum at room temperature overnightFinally sticky and transparent product was obtained
23 Ellmanrsquos Assay Ellmanrsquos assay was carried out to con-firm the synthesis of MPD by measuring residual thi-ols after dimerization of MPM MPD was dissolved inDMSO (02mM) 50 120583L of Ellmanrsquos reagent solution (DMSO4mgmL) was added to 250 120583L of sample After 15min ofincubation the absorbance at 499 nm was measured by amicroplate reader (Synergy H1 BioTek USA) Thiol amountof MPD was calculated based on calibration curve
24 Agarose Gel Electrophoresis DNA condensation abilityof MPD was identified by agarose gel electrophoresis assayAgarose gel (07 wt) containing ethidium bromide wasprepared in Tris-Acetate-EDTA (TAE) bufferTheMPDpoly-plexes (05 120583g pDNA) having various weight ratios (05 1 25 and 10) were prepared After 30min of incubation at roomtemperature electrophoresis was carried out for 15min at
100V (Mupid-2plus Takara Bio Inc Japan)The locations ofpDNA bands were observed by UV illuminator (ChemiDocXRS+ gel documentation system Bio-Rad Hercules CA)
25 Critical Micelle Concentration (CMC) Measurements Inorder to characterize CMC of MPD pyrene was selectedas a model drug as previously reported [22] 02mM ofpyrene solution was prepared by dilution of pyreneethanolsolution 3 120583L of pyrene solutions was added to vials anddried under vacuum overnight in dark condition Afterethanol evaporation 1mL of MPD solution with variousconcentrations was added to the vials giving the final pyreneconcentration of 6 times 10minus7M The MPDpyrene solution wassonicated for 10min and incubated with gentle mixing atroom temperature for 24 h Absorbance was measured byUVVis spectrometer (Optizen POP BIO Mecasys Korea)using quartz cuvettes in the range of 200 to 400 nmThe totalabsorbance was calculated by sum of four absorbance points(242 272 320 and 336 nm) CMC of theMPD sample was setto the center of the sigmoid
26 Average Particle Size and Zeta-Potential MeasurementsAverage sizes and Zeta-potential values of MPD and MPDpolyplexes were measured by Zetasizer Nano ZS (MalvernInstruments UK) with He-Ne laser beam (633 nm) at 25∘Crespectively 1mL of MPD (001 and 01mM) and MPDpolyplex solutions (5120583g pDNA weight ratios 2 10 and 20)was prepared and used for analysis Sizes of MPD polyplexesafter active MMP-2 cleavage were also examined MPDpolyplexes (1 120583g pDNA weight ratio 20) were prepared asdescribed above The polyplex solution was added to activeMMP-2 containing PBS buffer (pH 74 10 120583gmL MMP-2)and themixture was incubated at 37∘C for 4 hours with gentleshaking After treatment the sizes of the polyplexes samplewere analyzed by Zetasizer Nano ZS Average sizes and Zeta-potential values ofMPD andMPDpolyplexes weremeasured3 times
27 Atomic Force Microscopy (AFM) The morphologies ofMPD particle structures were observed by using atomicforce microscopy (Park Systems Korea) 100 120583L of MPDaqueous solution samples was prepared (001 and 01mM)10 120583L sample aliquots were dropped to fresh mica surfaceby pipette carefully and then excess of sample was removedby absorption of filter papers from the corner of the micaThe mica samples were dried at room temperature for 24 hand used for AFM observation The imaging was set tononcontact tapping mode and the scanning rate was 1Hz
28 Cell Culture Human lung adenocarcinoma epithelialcells (A549) human transformed primary embryonal kidneyepithelial cells (HEK293) and mouse myoblast cells (C2C12)were grown in DMEM supplemented with 10 FBS and1 penicillinstreptomycin (PS) in humidified atmospherecontaining 5 CO
2at 37∘C
Advances in Materials Science and Engineering 3
29 Cytotoxicity Cytotoxicity of MPD was characterized byMTT assay Cells were seeded into 96-well cell culture platesin 100120583L of DMEM (10 FBS 1 PS) at a density of1 times 104 cellswell After 24 h the cells were treated with100 120583L of peptide solutions for 4 h PEI25k was used as acontrol Then the fresh media were exchanged and furtherincubated for 24 h After incubation 25120583L of MTT stocksolution (2mgmL in DPBS) was added to each well and themediawere carefully removed after 2 hThe formazan crystalsformed by proliferating cells were dissolved with 150120583L ofDMSO and the absorbance was measured at 570 nm using amicroplate reader (Synergy H1 BioTek USA) Results werepresented as relative cell viabilities (RCV percentage valuesrelative to value of untreated control cells) All experimentswere performed in quadruplicate
210 Transfection Experiments The transfection efficiencyof MPD was examined by measuring luciferase transgeneexpression Cells were seeded into 24-well cell culture platesin 500120583L of DMEM (10 FBS 1 PS) at a density of 5 times 104cellswell After 24 h the media were exchanged to serum-free media and MPD polyplex solutions (weight ratios 510 20 and 30) were added to cells PEI25k polyplex (weightratio 1) was used as a control After 4 h of incubation thetransfection medium was replaced with fresh DMEM with10 FBS The cells were further incubated for 2 days andrinsed with 240120583L of DPBS twice The cells were lysedwith 120120583L of lysis buffer and shaken for 30min at roomtemperature The cell lysates were centrifuged for 30min at15000 g and 20 120583L of supernatants was used for luciferaseassay Luciferase activities were measured using luciferaseassay reagent on a microplate reader (Synergy H1 BioTekUSA)The total cellular proteins in cell lysateswere quantifiedby BCA protein assay kit The final transfection efficiencyresults were presented in terms of RLUmg cellular proteinAll experiments were performed in triplicate
211 Transfection Experiments with Bafilomycin A1 In orderto characterize the endosome buffering ability and trans-fection mechanism of MPD transfection experiments withBafilomycin A1 treatment were carried out A549 cellswere seeded as explained above 200 nM of BafilomycinA1 solutions was pretreated for 10min before transfectionMPD polyplexes were prepared with weight ratio of 10 20and 30 PEI25K was used as a positive control Polyplexsolutions were treated to the cells for 4 h and the media wereexchanged with fresh DMEM with 10 FBS After 48 h offurther incubation transfection efficiency was examined asdescribed above Final results were presented in terms ofrelative transfection efficiency (transfection efficiency withBafilomycin A1transfection efficiency without BafilomycinA1)
3 Results and Discussion
31 Characterization of MPM and MPD MP consists ofcellular penetrating peptide moiety (R
8) matrix meta-
lloproteinase-2 (MMP-2) specific sequence (GPLGV)
05 1 2 5 10C
Figure 1 Agarose gel electrophoresis result of MPD polyplexes CpDNA only Numbers mean weight ratios of the polyplexes
pH-responsive moiety (H5) and hydrophobic moiety
(palmitic acid) (CR8GPLGVH
5-Pal) Cellular penetrating
moiety was introduced for condensation of pDNA byelectrostatic interaction of cationic arginine residues andfor enhancement of cellular uptake pH-responsive moiety(H5) was introduced for facilitation of endosomal escape
of polyplexes after cellular uptake by proton sponge effectHydrophobic palmitic acid was introduced to C-terminalof MP in order to induce the formation of micelle structureby hydrophobic interaction R
8and other functional blocks
were linked with MMP-2 specific sequence which maylead to structural change of polyplexes by cleavage of thesequence with exposure to MMP-2
The structure of MP was confirmed by the manufacturerMP was dimerized to synthesize MPD by DMSO oxidationvia disulfide bond formation between cysteine thiols at N-terminal MP was insoluble in water but the synthesizedMPD after oxidation was soluble in water probably due tothe facilitated formation of micelle structure by strengthenedhydrophobic interaction of MPD It is thought that disulfidebond formation between MP could cause the proximityof hydrophobic moieties (histidine moieties and palmiticacid) and ease of micelle structure formation by structuralrestriction
The synthesis of MPD was examined by quantification ofremaining thiols of MPD product After DMSO oxidation itwas found that only 03 mole of thiol was detected in MPDproduct which means that almost MP could be dimerized tosynthesize MPD
32 Agarose Gel Electrophoresis pDNA condensing abilityof MPD was investigated by agarose gel electrophoresisAs shown in Figure 1 weak migration of pDNA was stillobserved at a weight ratio of 2 However MPD could retardpDNA completely at a weight ratio of 5 which meansthat pDNA was well condensed by MPD Therefore it wasconfirmed that MPD could condense pDNA due to itscationic R
8moiety
33 CMC Measurement CMC of MPD was measured inorder to confirm the formation of micelle structure by MPDMPD solutions with various concentrations were incubatedwith pyrene and absorbances from specific peaks frompyrene were summed It was reported that absorbance valuessum from 4 specific peaks of pyrene would be increasedunder hydrophobic environment such as core part of micelle
4 Advances in Materials Science and Engineering
Concentration (mM)
Abs
valu
es su
m
00
01
02
03
04
05
1e minus 5 1e minus 4 1e minus 3 1e minus 2 1e minus 1
Figure 2 CMC measurement result of MPD micelle
structures [22] In Figure 2 absorbance values sum of pyrenewas abruptly increased from 001mM of MPD This resultshows that MPD could form micelle structure as expected inaqueous medium and CMC of MPD is about 001mM
34 Characterization of MPD Micelles and MPD PolyplexesAverage sizes and Zeta-potential values of MPDmicelles andMPD polyplexes were measured by Zetasizer (Figure 3) Itwas found that MPD micelles showed submicron averagesize at 001mM and the size was decreased to about 470 nmat 01mM which means the formation of compact andstable micelle structure due to increase of MPD molecules(Figure 3(a)) Zeta-potential values of MPD micelles werealso increased from 106mV to 292mV probably due to theadditional incorporation of cationic MPDmolecules into themicelle structures
The morphology of MPD micelles was also observedby AFM They showed heterogeneous structures contain-ing small sphere or large cylinder structures at 001mMwhich explains large variation of average size measurementresults (Figure 4(a)) At 01mM MPD micelles displayedmore homogenous morphologies of spherical structures(Figure 4(b)) Greater height of particles was found at 01mM(30ndash60 nm) than at 001mM (5ndash8 nm) probably due to theincreased assembly ofMPDmolecules intomicelle structuresat a high concentration
In the case ofMPDpolyplexes average size was 230 nm ata weight ratio of 2 and increased to about 2-3 120583m over weightratios of 20 (Figure 3(b)) However Zeta-potential values ofMPD polyplexes were found to be negative (minus301mV) at aweight ratio of 2 and to be increased to almost zero overweight ratios of 20 Considering Zeta-potential values it isthought that MPD polyplexes could form large aggregatesvia hydrophobic interaction at high weight ratios due to theneutralization of surface charges by interaction of cationicMPD micelles with anionic pDNA It is also elucidatedthat negatively charged and small MPD polyplex could bepartially formed via surface coating of anionic pDNA whichis consistent with agarose gel electrophoresis result
35 Cytotoxicity Measurement The cytotoxicity of MPD wasexamined by MTT assay in various cell lines (A549 C2C12and HEK293) As shown in Figure 5 control agent PEI25kexhibited significant cytotoxicity The viability of PEI25k-treated cells was less than 20 even at a concentration of20120583gmL However the viability of MPD-treated cells wasmore than 80 at the same concentration in all three celllines This result means that cytotoxicity of MPD was low atthis condition although it was found to be concentration-dependent
36 Transfection Experiments of MPD Polyplexes Trans-fection efficiency of MPD polyplexes was investigated bymeasurement of luciferase transgene expression in A549C2C12 and HEK293 cell lines (Figure 6) It is known thatA549 and C2C12 cells express high levels of MMP-2 butHEK293 does not [23ndash25] MMP-2 is a family of collagenaseswhich is important for proliferation and migration of cancercells Therefore we performed transfection experiment invarious cell lines in order to examine the effect of MMP-2 expression on transfection of MPD containing MMP-2specific sequence Interestingly MPD showed high transfec-tion efficiency comparable to PEI25k in A549 and C2C12cells expressing high level of MMP-2 On the other handtransfection efficiency of MPD was found to be low (dozensto hundreds lower than that of PEI25k) in HEK293 cellseven though HEK293 cells are known to be well-transfectedcells and transfection efficiency of PEI25k in HEK293 cellsis about 10-fold higher than that in other cells This resultmeans that MPD possess the potential for gene deliverysystems especially targeting MMP-2 expressing cancer cellsIn addition the average sizes of the polyplexes weremeasuredin the presence of MMP-2 in order to examine the effectof MPD polyplexes structural change on the transfectionefficiency (Figure 6(d)) The size of the polyplexes was foundto be largely decreased in the presence of MMP-2 Thisresult shows that the large polyplex aggregates could beconverted to smaller polyplex particles by MMP-2 cleavageprobably leading to enhanced cellular uptake and trans-fection efficiency Although further study will be requiredfor revealing the detailed mechanism one explanation isthat the structural change of MPD polyplexes by cleavageof MMP-2 such as exposure of cellular penetrating moiety(R8) may facilitate the cellular uptake of MPD polyplexes
for efficient transfection (Scheme 1) On the contrary inHEK293 cells MPD polyplexes would not undergo structuralchange maintaining large aggregate structures with zeroZeta-potential values which are not proper for transfection
37 Transfection Experiments with Bafilomycin A1 Transfec-tion experiments were also performed in the presence ofBafilomycin A1 in order to examine the endosome bufferingability of MPD and the transfection mechanism BafilomycinA1 is an inhibitor of vacuolar type ATPase which cansuppress the proton influx into endosome and disturb theproton pump effect of endosome buffering moiety finallyleading to the decrease of transfection efficiency [26 27]
Advances in Materials Science and Engineering 5
Concentration (mM)
Aver
age s
ize (
nm)
0
200
400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
Size
10minus2 10minus1
Zeta
-pot
entia
l (m
V)
Zeta-potential
(a)
minus40
minus30
minus20
minus10
Size
Aver
age s
ize (
nm)
0
500
2000
30000
10
202 10
Weight ratio
Zeta-potential
Zeta
-pot
entia
l (m
V)
(b)
Figure 3 Average sizes and Zeta-potential values measurement results of MPD micelles (a) and MPD polyplexes (b)
75
5
25
0
(nm
)(n
m)
6
6
4
4
2
2
0
0
64206420
(120583m) (120583m)
(120583m)
(a)
6420
64206420
30
20
10
0
60
40
20
0
(nm
)(n
m)
(120583m)
(120583m)(120583m)
(b)
Figure 4 AFM images and height information of MPD micelles at 001mM (a) and 01mM (b)
6 Advances in Materials Science and Engineering
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(a)Re
lativ
e cel
l via
bilit
y (
)
PEI25KMPD
0
20
40
60
80
100
0 5 10 15 20 25 30
Concentration (120583gmL)
(b)
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(c)
Figure 5 MTT assay results of MPD in A549 (a) C2C12 (b) and HEK293 (c) cells
It is thought that MPD may possess the endosome bufferingability due to imidazole groups of histidine residues ofwhich pKa value is about 6 As shown in Figure 7 thetransfection efficiency of PEI25k in Bafilomycin A1 conditionwas decreased to about 180 times lower value than normalcondition Similar to PEI25kMPD also showed 20ndash170 timeslower transfection efficiency in Bafilomycin A1 conditionthan normal condition This result means that the transfec-tion of MPD is mediated by endosomal escape by endosomebuffering ability after cellular uptake
4 Conclusion
Self-assembling multifunctional peptide was designed anddimerized for gene delivery systems This MPD couldself-assemble to micelle structures and condense pDNAby electrostatic interaction The cytotoxicity of MPDwas concentration-dependent but low in the examinedconcentration range High transfection efficiency ofMPD polyplexes in cancer cells expressing high level ofMMP-2 showed the potential of MPD for targeted gene
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
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CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
Advances in Materials Science and Engineering 3
29 Cytotoxicity Cytotoxicity of MPD was characterized byMTT assay Cells were seeded into 96-well cell culture platesin 100120583L of DMEM (10 FBS 1 PS) at a density of1 times 104 cellswell After 24 h the cells were treated with100 120583L of peptide solutions for 4 h PEI25k was used as acontrol Then the fresh media were exchanged and furtherincubated for 24 h After incubation 25120583L of MTT stocksolution (2mgmL in DPBS) was added to each well and themediawere carefully removed after 2 hThe formazan crystalsformed by proliferating cells were dissolved with 150120583L ofDMSO and the absorbance was measured at 570 nm using amicroplate reader (Synergy H1 BioTek USA) Results werepresented as relative cell viabilities (RCV percentage valuesrelative to value of untreated control cells) All experimentswere performed in quadruplicate
210 Transfection Experiments The transfection efficiencyof MPD was examined by measuring luciferase transgeneexpression Cells were seeded into 24-well cell culture platesin 500120583L of DMEM (10 FBS 1 PS) at a density of 5 times 104cellswell After 24 h the media were exchanged to serum-free media and MPD polyplex solutions (weight ratios 510 20 and 30) were added to cells PEI25k polyplex (weightratio 1) was used as a control After 4 h of incubation thetransfection medium was replaced with fresh DMEM with10 FBS The cells were further incubated for 2 days andrinsed with 240120583L of DPBS twice The cells were lysedwith 120120583L of lysis buffer and shaken for 30min at roomtemperature The cell lysates were centrifuged for 30min at15000 g and 20 120583L of supernatants was used for luciferaseassay Luciferase activities were measured using luciferaseassay reagent on a microplate reader (Synergy H1 BioTekUSA)The total cellular proteins in cell lysateswere quantifiedby BCA protein assay kit The final transfection efficiencyresults were presented in terms of RLUmg cellular proteinAll experiments were performed in triplicate
211 Transfection Experiments with Bafilomycin A1 In orderto characterize the endosome buffering ability and trans-fection mechanism of MPD transfection experiments withBafilomycin A1 treatment were carried out A549 cellswere seeded as explained above 200 nM of BafilomycinA1 solutions was pretreated for 10min before transfectionMPD polyplexes were prepared with weight ratio of 10 20and 30 PEI25K was used as a positive control Polyplexsolutions were treated to the cells for 4 h and the media wereexchanged with fresh DMEM with 10 FBS After 48 h offurther incubation transfection efficiency was examined asdescribed above Final results were presented in terms ofrelative transfection efficiency (transfection efficiency withBafilomycin A1transfection efficiency without BafilomycinA1)
3 Results and Discussion
31 Characterization of MPM and MPD MP consists ofcellular penetrating peptide moiety (R
8) matrix meta-
lloproteinase-2 (MMP-2) specific sequence (GPLGV)
05 1 2 5 10C
Figure 1 Agarose gel electrophoresis result of MPD polyplexes CpDNA only Numbers mean weight ratios of the polyplexes
pH-responsive moiety (H5) and hydrophobic moiety
(palmitic acid) (CR8GPLGVH
5-Pal) Cellular penetrating
moiety was introduced for condensation of pDNA byelectrostatic interaction of cationic arginine residues andfor enhancement of cellular uptake pH-responsive moiety(H5) was introduced for facilitation of endosomal escape
of polyplexes after cellular uptake by proton sponge effectHydrophobic palmitic acid was introduced to C-terminalof MP in order to induce the formation of micelle structureby hydrophobic interaction R
8and other functional blocks
were linked with MMP-2 specific sequence which maylead to structural change of polyplexes by cleavage of thesequence with exposure to MMP-2
The structure of MP was confirmed by the manufacturerMP was dimerized to synthesize MPD by DMSO oxidationvia disulfide bond formation between cysteine thiols at N-terminal MP was insoluble in water but the synthesizedMPD after oxidation was soluble in water probably due tothe facilitated formation of micelle structure by strengthenedhydrophobic interaction of MPD It is thought that disulfidebond formation between MP could cause the proximityof hydrophobic moieties (histidine moieties and palmiticacid) and ease of micelle structure formation by structuralrestriction
The synthesis of MPD was examined by quantification ofremaining thiols of MPD product After DMSO oxidation itwas found that only 03 mole of thiol was detected in MPDproduct which means that almost MP could be dimerized tosynthesize MPD
32 Agarose Gel Electrophoresis pDNA condensing abilityof MPD was investigated by agarose gel electrophoresisAs shown in Figure 1 weak migration of pDNA was stillobserved at a weight ratio of 2 However MPD could retardpDNA completely at a weight ratio of 5 which meansthat pDNA was well condensed by MPD Therefore it wasconfirmed that MPD could condense pDNA due to itscationic R
8moiety
33 CMC Measurement CMC of MPD was measured inorder to confirm the formation of micelle structure by MPDMPD solutions with various concentrations were incubatedwith pyrene and absorbances from specific peaks frompyrene were summed It was reported that absorbance valuessum from 4 specific peaks of pyrene would be increasedunder hydrophobic environment such as core part of micelle
4 Advances in Materials Science and Engineering
Concentration (mM)
Abs
valu
es su
m
00
01
02
03
04
05
1e minus 5 1e minus 4 1e minus 3 1e minus 2 1e minus 1
Figure 2 CMC measurement result of MPD micelle
structures [22] In Figure 2 absorbance values sum of pyrenewas abruptly increased from 001mM of MPD This resultshows that MPD could form micelle structure as expected inaqueous medium and CMC of MPD is about 001mM
34 Characterization of MPD Micelles and MPD PolyplexesAverage sizes and Zeta-potential values of MPDmicelles andMPD polyplexes were measured by Zetasizer (Figure 3) Itwas found that MPD micelles showed submicron averagesize at 001mM and the size was decreased to about 470 nmat 01mM which means the formation of compact andstable micelle structure due to increase of MPD molecules(Figure 3(a)) Zeta-potential values of MPD micelles werealso increased from 106mV to 292mV probably due to theadditional incorporation of cationic MPDmolecules into themicelle structures
The morphology of MPD micelles was also observedby AFM They showed heterogeneous structures contain-ing small sphere or large cylinder structures at 001mMwhich explains large variation of average size measurementresults (Figure 4(a)) At 01mM MPD micelles displayedmore homogenous morphologies of spherical structures(Figure 4(b)) Greater height of particles was found at 01mM(30ndash60 nm) than at 001mM (5ndash8 nm) probably due to theincreased assembly ofMPDmolecules intomicelle structuresat a high concentration
In the case ofMPDpolyplexes average size was 230 nm ata weight ratio of 2 and increased to about 2-3 120583m over weightratios of 20 (Figure 3(b)) However Zeta-potential values ofMPD polyplexes were found to be negative (minus301mV) at aweight ratio of 2 and to be increased to almost zero overweight ratios of 20 Considering Zeta-potential values it isthought that MPD polyplexes could form large aggregatesvia hydrophobic interaction at high weight ratios due to theneutralization of surface charges by interaction of cationicMPD micelles with anionic pDNA It is also elucidatedthat negatively charged and small MPD polyplex could bepartially formed via surface coating of anionic pDNA whichis consistent with agarose gel electrophoresis result
35 Cytotoxicity Measurement The cytotoxicity of MPD wasexamined by MTT assay in various cell lines (A549 C2C12and HEK293) As shown in Figure 5 control agent PEI25kexhibited significant cytotoxicity The viability of PEI25k-treated cells was less than 20 even at a concentration of20120583gmL However the viability of MPD-treated cells wasmore than 80 at the same concentration in all three celllines This result means that cytotoxicity of MPD was low atthis condition although it was found to be concentration-dependent
36 Transfection Experiments of MPD Polyplexes Trans-fection efficiency of MPD polyplexes was investigated bymeasurement of luciferase transgene expression in A549C2C12 and HEK293 cell lines (Figure 6) It is known thatA549 and C2C12 cells express high levels of MMP-2 butHEK293 does not [23ndash25] MMP-2 is a family of collagenaseswhich is important for proliferation and migration of cancercells Therefore we performed transfection experiment invarious cell lines in order to examine the effect of MMP-2 expression on transfection of MPD containing MMP-2specific sequence Interestingly MPD showed high transfec-tion efficiency comparable to PEI25k in A549 and C2C12cells expressing high level of MMP-2 On the other handtransfection efficiency of MPD was found to be low (dozensto hundreds lower than that of PEI25k) in HEK293 cellseven though HEK293 cells are known to be well-transfectedcells and transfection efficiency of PEI25k in HEK293 cellsis about 10-fold higher than that in other cells This resultmeans that MPD possess the potential for gene deliverysystems especially targeting MMP-2 expressing cancer cellsIn addition the average sizes of the polyplexes weremeasuredin the presence of MMP-2 in order to examine the effectof MPD polyplexes structural change on the transfectionefficiency (Figure 6(d)) The size of the polyplexes was foundto be largely decreased in the presence of MMP-2 Thisresult shows that the large polyplex aggregates could beconverted to smaller polyplex particles by MMP-2 cleavageprobably leading to enhanced cellular uptake and trans-fection efficiency Although further study will be requiredfor revealing the detailed mechanism one explanation isthat the structural change of MPD polyplexes by cleavageof MMP-2 such as exposure of cellular penetrating moiety(R8) may facilitate the cellular uptake of MPD polyplexes
for efficient transfection (Scheme 1) On the contrary inHEK293 cells MPD polyplexes would not undergo structuralchange maintaining large aggregate structures with zeroZeta-potential values which are not proper for transfection
37 Transfection Experiments with Bafilomycin A1 Transfec-tion experiments were also performed in the presence ofBafilomycin A1 in order to examine the endosome bufferingability of MPD and the transfection mechanism BafilomycinA1 is an inhibitor of vacuolar type ATPase which cansuppress the proton influx into endosome and disturb theproton pump effect of endosome buffering moiety finallyleading to the decrease of transfection efficiency [26 27]
Advances in Materials Science and Engineering 5
Concentration (mM)
Aver
age s
ize (
nm)
0
200
400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
Size
10minus2 10minus1
Zeta
-pot
entia
l (m
V)
Zeta-potential
(a)
minus40
minus30
minus20
minus10
Size
Aver
age s
ize (
nm)
0
500
2000
30000
10
202 10
Weight ratio
Zeta-potential
Zeta
-pot
entia
l (m
V)
(b)
Figure 3 Average sizes and Zeta-potential values measurement results of MPD micelles (a) and MPD polyplexes (b)
75
5
25
0
(nm
)(n
m)
6
6
4
4
2
2
0
0
64206420
(120583m) (120583m)
(120583m)
(a)
6420
64206420
30
20
10
0
60
40
20
0
(nm
)(n
m)
(120583m)
(120583m)(120583m)
(b)
Figure 4 AFM images and height information of MPD micelles at 001mM (a) and 01mM (b)
6 Advances in Materials Science and Engineering
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(a)Re
lativ
e cel
l via
bilit
y (
)
PEI25KMPD
0
20
40
60
80
100
0 5 10 15 20 25 30
Concentration (120583gmL)
(b)
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(c)
Figure 5 MTT assay results of MPD in A549 (a) C2C12 (b) and HEK293 (c) cells
It is thought that MPD may possess the endosome bufferingability due to imidazole groups of histidine residues ofwhich pKa value is about 6 As shown in Figure 7 thetransfection efficiency of PEI25k in Bafilomycin A1 conditionwas decreased to about 180 times lower value than normalcondition Similar to PEI25kMPD also showed 20ndash170 timeslower transfection efficiency in Bafilomycin A1 conditionthan normal condition This result means that the transfec-tion of MPD is mediated by endosomal escape by endosomebuffering ability after cellular uptake
4 Conclusion
Self-assembling multifunctional peptide was designed anddimerized for gene delivery systems This MPD couldself-assemble to micelle structures and condense pDNAby electrostatic interaction The cytotoxicity of MPDwas concentration-dependent but low in the examinedconcentration range High transfection efficiency ofMPD polyplexes in cancer cells expressing high level ofMMP-2 showed the potential of MPD for targeted gene
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
4 Advances in Materials Science and Engineering
Concentration (mM)
Abs
valu
es su
m
00
01
02
03
04
05
1e minus 5 1e minus 4 1e minus 3 1e minus 2 1e minus 1
Figure 2 CMC measurement result of MPD micelle
structures [22] In Figure 2 absorbance values sum of pyrenewas abruptly increased from 001mM of MPD This resultshows that MPD could form micelle structure as expected inaqueous medium and CMC of MPD is about 001mM
34 Characterization of MPD Micelles and MPD PolyplexesAverage sizes and Zeta-potential values of MPDmicelles andMPD polyplexes were measured by Zetasizer (Figure 3) Itwas found that MPD micelles showed submicron averagesize at 001mM and the size was decreased to about 470 nmat 01mM which means the formation of compact andstable micelle structure due to increase of MPD molecules(Figure 3(a)) Zeta-potential values of MPD micelles werealso increased from 106mV to 292mV probably due to theadditional incorporation of cationic MPDmolecules into themicelle structures
The morphology of MPD micelles was also observedby AFM They showed heterogeneous structures contain-ing small sphere or large cylinder structures at 001mMwhich explains large variation of average size measurementresults (Figure 4(a)) At 01mM MPD micelles displayedmore homogenous morphologies of spherical structures(Figure 4(b)) Greater height of particles was found at 01mM(30ndash60 nm) than at 001mM (5ndash8 nm) probably due to theincreased assembly ofMPDmolecules intomicelle structuresat a high concentration
In the case ofMPDpolyplexes average size was 230 nm ata weight ratio of 2 and increased to about 2-3 120583m over weightratios of 20 (Figure 3(b)) However Zeta-potential values ofMPD polyplexes were found to be negative (minus301mV) at aweight ratio of 2 and to be increased to almost zero overweight ratios of 20 Considering Zeta-potential values it isthought that MPD polyplexes could form large aggregatesvia hydrophobic interaction at high weight ratios due to theneutralization of surface charges by interaction of cationicMPD micelles with anionic pDNA It is also elucidatedthat negatively charged and small MPD polyplex could bepartially formed via surface coating of anionic pDNA whichis consistent with agarose gel electrophoresis result
35 Cytotoxicity Measurement The cytotoxicity of MPD wasexamined by MTT assay in various cell lines (A549 C2C12and HEK293) As shown in Figure 5 control agent PEI25kexhibited significant cytotoxicity The viability of PEI25k-treated cells was less than 20 even at a concentration of20120583gmL However the viability of MPD-treated cells wasmore than 80 at the same concentration in all three celllines This result means that cytotoxicity of MPD was low atthis condition although it was found to be concentration-dependent
36 Transfection Experiments of MPD Polyplexes Trans-fection efficiency of MPD polyplexes was investigated bymeasurement of luciferase transgene expression in A549C2C12 and HEK293 cell lines (Figure 6) It is known thatA549 and C2C12 cells express high levels of MMP-2 butHEK293 does not [23ndash25] MMP-2 is a family of collagenaseswhich is important for proliferation and migration of cancercells Therefore we performed transfection experiment invarious cell lines in order to examine the effect of MMP-2 expression on transfection of MPD containing MMP-2specific sequence Interestingly MPD showed high transfec-tion efficiency comparable to PEI25k in A549 and C2C12cells expressing high level of MMP-2 On the other handtransfection efficiency of MPD was found to be low (dozensto hundreds lower than that of PEI25k) in HEK293 cellseven though HEK293 cells are known to be well-transfectedcells and transfection efficiency of PEI25k in HEK293 cellsis about 10-fold higher than that in other cells This resultmeans that MPD possess the potential for gene deliverysystems especially targeting MMP-2 expressing cancer cellsIn addition the average sizes of the polyplexes weremeasuredin the presence of MMP-2 in order to examine the effectof MPD polyplexes structural change on the transfectionefficiency (Figure 6(d)) The size of the polyplexes was foundto be largely decreased in the presence of MMP-2 Thisresult shows that the large polyplex aggregates could beconverted to smaller polyplex particles by MMP-2 cleavageprobably leading to enhanced cellular uptake and trans-fection efficiency Although further study will be requiredfor revealing the detailed mechanism one explanation isthat the structural change of MPD polyplexes by cleavageof MMP-2 such as exposure of cellular penetrating moiety(R8) may facilitate the cellular uptake of MPD polyplexes
for efficient transfection (Scheme 1) On the contrary inHEK293 cells MPD polyplexes would not undergo structuralchange maintaining large aggregate structures with zeroZeta-potential values which are not proper for transfection
37 Transfection Experiments with Bafilomycin A1 Transfec-tion experiments were also performed in the presence ofBafilomycin A1 in order to examine the endosome bufferingability of MPD and the transfection mechanism BafilomycinA1 is an inhibitor of vacuolar type ATPase which cansuppress the proton influx into endosome and disturb theproton pump effect of endosome buffering moiety finallyleading to the decrease of transfection efficiency [26 27]
Advances in Materials Science and Engineering 5
Concentration (mM)
Aver
age s
ize (
nm)
0
200
400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
Size
10minus2 10minus1
Zeta
-pot
entia
l (m
V)
Zeta-potential
(a)
minus40
minus30
minus20
minus10
Size
Aver
age s
ize (
nm)
0
500
2000
30000
10
202 10
Weight ratio
Zeta-potential
Zeta
-pot
entia
l (m
V)
(b)
Figure 3 Average sizes and Zeta-potential values measurement results of MPD micelles (a) and MPD polyplexes (b)
75
5
25
0
(nm
)(n
m)
6
6
4
4
2
2
0
0
64206420
(120583m) (120583m)
(120583m)
(a)
6420
64206420
30
20
10
0
60
40
20
0
(nm
)(n
m)
(120583m)
(120583m)(120583m)
(b)
Figure 4 AFM images and height information of MPD micelles at 001mM (a) and 01mM (b)
6 Advances in Materials Science and Engineering
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(a)Re
lativ
e cel
l via
bilit
y (
)
PEI25KMPD
0
20
40
60
80
100
0 5 10 15 20 25 30
Concentration (120583gmL)
(b)
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(c)
Figure 5 MTT assay results of MPD in A549 (a) C2C12 (b) and HEK293 (c) cells
It is thought that MPD may possess the endosome bufferingability due to imidazole groups of histidine residues ofwhich pKa value is about 6 As shown in Figure 7 thetransfection efficiency of PEI25k in Bafilomycin A1 conditionwas decreased to about 180 times lower value than normalcondition Similar to PEI25kMPD also showed 20ndash170 timeslower transfection efficiency in Bafilomycin A1 conditionthan normal condition This result means that the transfec-tion of MPD is mediated by endosomal escape by endosomebuffering ability after cellular uptake
4 Conclusion
Self-assembling multifunctional peptide was designed anddimerized for gene delivery systems This MPD couldself-assemble to micelle structures and condense pDNAby electrostatic interaction The cytotoxicity of MPDwas concentration-dependent but low in the examinedconcentration range High transfection efficiency ofMPD polyplexes in cancer cells expressing high level ofMMP-2 showed the potential of MPD for targeted gene
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
Advances in Materials Science and Engineering 5
Concentration (mM)
Aver
age s
ize (
nm)
0
200
400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
Size
10minus2 10minus1
Zeta
-pot
entia
l (m
V)
Zeta-potential
(a)
minus40
minus30
minus20
minus10
Size
Aver
age s
ize (
nm)
0
500
2000
30000
10
202 10
Weight ratio
Zeta-potential
Zeta
-pot
entia
l (m
V)
(b)
Figure 3 Average sizes and Zeta-potential values measurement results of MPD micelles (a) and MPD polyplexes (b)
75
5
25
0
(nm
)(n
m)
6
6
4
4
2
2
0
0
64206420
(120583m) (120583m)
(120583m)
(a)
6420
64206420
30
20
10
0
60
40
20
0
(nm
)(n
m)
(120583m)
(120583m)(120583m)
(b)
Figure 4 AFM images and height information of MPD micelles at 001mM (a) and 01mM (b)
6 Advances in Materials Science and Engineering
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(a)Re
lativ
e cel
l via
bilit
y (
)
PEI25KMPD
0
20
40
60
80
100
0 5 10 15 20 25 30
Concentration (120583gmL)
(b)
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(c)
Figure 5 MTT assay results of MPD in A549 (a) C2C12 (b) and HEK293 (c) cells
It is thought that MPD may possess the endosome bufferingability due to imidazole groups of histidine residues ofwhich pKa value is about 6 As shown in Figure 7 thetransfection efficiency of PEI25k in Bafilomycin A1 conditionwas decreased to about 180 times lower value than normalcondition Similar to PEI25kMPD also showed 20ndash170 timeslower transfection efficiency in Bafilomycin A1 conditionthan normal condition This result means that the transfec-tion of MPD is mediated by endosomal escape by endosomebuffering ability after cellular uptake
4 Conclusion
Self-assembling multifunctional peptide was designed anddimerized for gene delivery systems This MPD couldself-assemble to micelle structures and condense pDNAby electrostatic interaction The cytotoxicity of MPDwas concentration-dependent but low in the examinedconcentration range High transfection efficiency ofMPD polyplexes in cancer cells expressing high level ofMMP-2 showed the potential of MPD for targeted gene
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
6 Advances in Materials Science and Engineering
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(a)Re
lativ
e cel
l via
bilit
y (
)
PEI25KMPD
0
20
40
60
80
100
0 5 10 15 20 25 30
Concentration (120583gmL)
(b)
Relat
ive c
ell v
iabi
lity
()
0
20
40
60
80
100
PEI25KMPD
0 5 10 15 20 25 30
Concentration (120583gmL)
(c)
Figure 5 MTT assay results of MPD in A549 (a) C2C12 (b) and HEK293 (c) cells
It is thought that MPD may possess the endosome bufferingability due to imidazole groups of histidine residues ofwhich pKa value is about 6 As shown in Figure 7 thetransfection efficiency of PEI25k in Bafilomycin A1 conditionwas decreased to about 180 times lower value than normalcondition Similar to PEI25kMPD also showed 20ndash170 timeslower transfection efficiency in Bafilomycin A1 conditionthan normal condition This result means that the transfec-tion of MPD is mediated by endosomal escape by endosomebuffering ability after cellular uptake
4 Conclusion
Self-assembling multifunctional peptide was designed anddimerized for gene delivery systems This MPD couldself-assemble to micelle structures and condense pDNAby electrostatic interaction The cytotoxicity of MPDwas concentration-dependent but low in the examinedconcentration range High transfection efficiency ofMPD polyplexes in cancer cells expressing high level ofMMP-2 showed the potential of MPD for targeted gene
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
Advances in Materials Science and Engineering 7
PEI25K
RLU
mg
prot
ein
510
2030
MPD
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
(a)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
MPD
(b)
510
2030
PEI25K
RLU
mg
prot
ein
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3MPD
(c)
No MMP
Aver
age s
ize (
nm)
0
1000
2000
3000
4000
5000
MMP
(d)
Figure 6 Transfection experiment results of MPD polyplexes in A549 (a) C2C12 (b) and HEK293 (c) cells Numbers in boxes mean weightratios of MPD polyplexes PEI25k polyplexes were prepared at a weight ratio of 1 (d) Average sizes measurement result of MPD polyplexes(weight ratio 20) in the absence or presence of MMP-2
delivery In addition it was revealed that the transfectionof MPD is mediated via endosomal escape by endosomebuffering ability In the further study encapsulation ofhydrophobic drug molecules in MPD micelles would betried for synergistic effect of druggene codelivery forcancer therapy and the detailed mechanism for structuralchange of MPD polyplex by MMP cleavage also would berevealed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
AcknowledgmentsThis work was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
8 Advances in Materials Science and Engineering
Large polyplex aggregates
MMP-2 cleavage
Small polyplex particles
Cellular penetrating peptide moiety
pH-responsive moiety endosome buffering effect in endosomal pHHydrophobic moiety
S
S-S
S-S
S-S S-SS-S
S-S
S-S
S-S
S-S
S-S
S-S
RRRRRRRR
GPLGV
HHHHH
Palmitic acid
MMP-2 specific sequence structural change by MMP-2 cleavage
S
Scheme 1 A scheme for the proposed mechanism of structuralchange of MPD polyplex in the presence of MMP-2
PEI25K
Relat
ive e
ffici
ency
0001
001
01
1
10
Without Bafilomycin AWith Bafilomycin A
WR 30WR 20WR 10
Figure 7 Transfection experiment results withwithout Bafilo-mycin A1 in A549 cells WR means the weight ratios of MPD poly-plexes Relative efficiency is the ratio of transfection efficiency withBafilomycin A1 to the transfection efficiency without BafilomycinA1
(NRF) funded by the Ministry of Education Science andTechnology (2011-0015045) and by the Ministry of ScienceICT and Future Planning (NRF-2014R1A1A1037692) Theauthors also acknowledge Professor Yan Lee for permissionto take Zetasizer measurements
References
[1] H O Mccarthy J McCaffrey C M Mccrudden et al ldquoDevel-opment and characterization of self-assembling nanoparticlesusing a bio-inspired amphipathic peptide for gene deliveryrdquoJournal of Controlled Release vol 189 pp 141ndash149 2014
[2] S Sundar Y Chen and Y W Tong ldquoDelivery of therapeuticsand molecules using self-assembled peptidesrdquo Current Medici-nal Chemistry vol 21 no 22 pp 2469ndash2479 2014
[3] D M Copolovici K Langel E Eriste and U Langel ldquoCell-penetrating peptides design synthesis and applicationsrdquo ACSNano vol 8 no 3 pp 1972ndash1994 2014
[4] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996
[5] D Derossi A H Joliot G Chassaing and A Prochiantz ldquoThethird helix of the Antennapedia homeodomain translocatesthrough biologicalmembranesrdquoThe Journal of Biological Chem-istry vol 269 no 14 pp 10444ndash10450 1994
[6] H Brooks B Lebleu and E Vives ldquoTat peptide-mediatedcellular delivery back to basicsrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 559ndash577 2005
[7] M Zhao and R Weissleder ldquoIntracellular cargo delivery usingTat peptide and derivativesrdquo Medicinal Research Reviews vol24 no 1 pp 1ndash12 2004
[8] M Zorko and U Langel ldquoCell-penetrating peptides mecha-nism and kinetics of cargo deliveryrdquo Advanced Drug DeliveryReviews vol 57 no 4 pp 529ndash545 2005
[9] Y-W Won P P Adhikary K S Lim H J Kim J K Kim andY-H Kim ldquoOligopeptide complex for targeted non-viral genedelivery to adipocytesrdquo Nature Materials vol 13 pp 1157ndash11642014
[10] A Kiselev A Egorova A Laukkanen V Baranov and A UrttildquoCharacterization of reducible peptide oligomers as carriers forgene deliveryrdquo International Journal of Pharmaceutics vol 441no 1-2 pp 736ndash747 2013
[11] Y-W Won H A Kim M Lee and Y-H Kim ldquoReduciblepoly(oligo-d-arginine) for enhanced gene expression in mouselung by intratracheal injectionrdquo Molecular Therapy vol 18 no4 pp 734ndash742 2010
[12] D Soundara Manickam H S Bisht L Wan G Mao andD Oupicky ldquoInfluence of TAT-peptide polymerization onproperties and transfection activity of TATDNA polyplexesrdquoJournal of Controlled Release vol 102 no 1 pp 293ndash306 2005
[13] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008
[14] T-I Kim and S W Kim ldquoBioreducible polymers for genedeliveryrdquo Reactive and Functional Polymers vol 71 no 3 pp344ndash349 2011
[15] HMargus K Padari andM Pooga ldquoCell-penetrating peptidesas versatile vehicles for oligonucleotide deliveryrdquo MolecularTherapy vol 20 no 3 pp 525ndash533 2012
[16] M E Stearns and M Wang ldquoType IV collagenase (Mr 72000)expression in human prostate benign and malignant tissuerdquoCancer Research vol 53 no 4 pp 878ndash883 1993
[17] G Y Lee K Park S Y Kim and Y Byun ldquoMMPs-specificPEGylated peptide-DOX conjugate micelles that can containfree doxorubicinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 67 no 3 pp 646ndash654 2007
[18] G Gu H Xia Q Hu et al ldquoPEG-co-PCL nanoparticles modi-fied withMMP-29 activatable lowmolecular weight protaminefor enhanced targeted glioblastoma therapyrdquo Biomaterials vol34 no 1 pp 196ndash208 2013
[19] M L Read S Singh Z Ahmed et al ldquoA versatile reduciblepolycation-based system for efficient delivery of a broad rangeof nucleic acidsrdquo Nucleic Acids Research vol 33 no 9 articlee86 2005
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
Advances in Materials Science and Engineering 9
[20] T-I Kim T Rothmund T Kissel and S W Kim ldquoBiore-ducible polymers with cell penetrating and endosome bufferingfunctionality for gene delivery systemsrdquo Journal of ControlledRelease vol 152 no 1 pp 110ndash119 2011
[21] S Son K Singha and W J Kim ldquoBioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrierrdquoBiomaterials vol 31 no 24 pp 6344ndash6354 2010
[22] G Basu Ray I Chakraborty and S P Moulik ldquoPyrene absorp-tion can be a convenient method for probing critical micellarconcentration (cmc) and indexing micellar polarityrdquo Journal ofColloid and Interface Science vol 294 no 1 pp 248ndash254 2006
[23] G Lluri andDM Jaworski ldquoRegulation of TIMP-2MT1-MMPandMMP-2 expression duringC2C12 differentiationrdquoMuscleampNerve vol 32 no 4 pp 492ndash499 2005
[24] C Chetty S S Lakka P Bhoopathi and J S Rao ldquoMMP-2alters VEGF expression via 120572V1205733 integrin-mediated PI3KAKTsignaling in A549 lung cancer cellsrdquo International Journal ofCancer vol 127 no 5 pp 1081ndash1095 2010
[25] M T Malik and S S Kakar ldquoRegulation of angiogenesisand invasion by human pituitary tumor transforming gene(PTTG) through increased expression and secretion of matrixmetalloproteinase-2 (MMP-2)rdquoMolecular Cancer vol 5 article61 2006
[26] A Kichler C Leborgne E Coeytaux and O Danos ldquoPoly-ethylenimine-mediated gene delivery a mechanistic studyrdquoJournal of Gene Medicine vol 3 no 2 pp 135ndash144 2001
[27] K Kim K Ryu and T-I Kim ldquoCationic methylcellulose deri-vativewith serum-compatibility and endosome buffering abilityfor gene delivery systemsrdquo Carbohydrate Polymers vol 110 pp268ndash277 2014
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
CrystallographyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CoatingsJournal of
Advances in
Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Smart Materials Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MetallurgyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
MaterialsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Nano
materials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal ofNanomaterials
Submit your manuscripts athttpwwwhindawicom
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CorrosionInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Polymer ScienceInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CeramicsJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CompositesJournal of
NanoparticlesJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Biomaterials
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
NanoscienceJournal of
TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Journal of
NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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