International Journal of
Molecular Sciences
Article
Enhanced and Extended Anti-Hypertensive Effect of VP5 Nanoparticles
Ting Yu 1,2,3,†, Shengnan Zhao 2,3,†, Ziqiang Li 3,†, Yi Wang 4, Bei Xu 3, Dailong Fang 3, Fazhan Wang 3, Zhi Zhang 3, Lili He 2, Xiangrong Song 3,* and Jian Yang 1,*
1 School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen 518055, China; tingyu419@126.com
2 College of Pharmacy, Southwest University for Nationalities, Chengdu 610041, China; zhaoshn11@163.com (S.Z.); lilihes@163.com (L.H.)
3 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and Collaborative Innovation Center for Biotherapy, Sichuan University, Chengdu 610041, China; ziqiangli21@gmail.com (Z.L.); xb1990625@126.com (B.X.); fangdailongtwozero@126.com (D.F.); FazhanWang_16@163.com (F.W.); zhangzhi02@gmail.com (Z.Z.)
4 Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA; Yi_Wang@dfci.harvard.edu
* Correspondence: songxr@scu.edu.cn (X.S.); jiany@szpt.edu.cn (J.Y.); Tel.: +86-28-8550-3817 (X.S. & J.Y.) † These authors contributed equally to this work.
Academic Editor: Qinghua Qin Received: 29 September 2016; Accepted: 18 November 2016; Published: 25 November 2016
Abstract: Hypertension has become a significant global public health concern and is also one of the most common risk factors of cardiovascular disease. Recent studies have shown the promising result of peptides inhibiting angiotensin converting enzyme (ACE) in lowering the blood pressure in both animal models and humans. However, the oral bioavailability and continuous antihypertensive effectiveness require further optimization. Novel nanoparticle-based drug delivery systems are helpful to overcome these barriers. Therefore, a poly-(lactic-co-glycolic) acid nanoparticle (PLGANPs) oral delivery system, of the antihypertensive small peptides Val-Leu-Pro-Val-Pro (VLPVP, VP5) model, was developed in this study and its antihypertensive effect was investigated in spontaneously hypertensive rats (SHRs) for the first time. The obtained VP5 nanoparticles (VP5-NPs) showed a small particle size of 223.7 ± 2.3 nm and high entrapment efficiency (EE%) of 87.37% ± 0.92%. Transmission electronic microscopy (TEM) analysis showed that the nanoparticles were spherical and homogeneous. The optimal preparation of VP5-NPs exhibited sustained release of VP5 in vitro and a 96 h long-term antihypertensive effect with enhanced efficacy in vivo. This study illustrated that PLGANPs might be an optimal formulation for oral delivery of antihypertensive small peptides and VP5-NPs might be worthy of further development and use as a potential therapeutic strategy for hypertension in the future.
Keywords: PLGA nanoparticles; antihypertensive peptide; oral administration; sustained release; continuously antihypertensive effect

1. Introduction
Hypertension (high blood pressure), as a global public health risk, is highly associated with heart disease, stroke, kidney failure, premature death and disability [1]. Currently, many anti-hypertensive therapeutic drugs have been developed and are now available for clinical treatment, including thiazide diuretics, beta-blockers, renin-angiotensin-aldosterone system (RAAS) inhibitors and calcium channel blockers. However, side effects have been reported with these treatments. Thiazide diuretics could potentially result in insulin resistance, dyslipidemia, and hyperuricemia, which would accelerate

Int. J. Mol. Sci. 2016, 17, 1977; doi:10.3390/ijms17121977

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diabetes progression in patients with obesity and/or metabolic syndromes [2]. Traditional β-blockers appear to have a significantly higher risk of diabetes. RAAS inhibition, by an angiotensin converting enzyme (ACE) or an angiotensin receptor blocker (ARB) along with calcium channel blockers, appears to have improved safety properties, but some of them still have various side effects such as coughing, taste disturbances and skin rashes [3]. Clearly, future investigations for novel and safe antihypertensive drugs are still required.
Recently, a number of peptides inhibiting ACE have been demonstrated to reduce systolic (SBP) and diastolic blood pressure (DBP) in both animal models and humans [4]. Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP), the best characterized antihypertensive peptides, have been used as active ingredients in blood pressure control [5]. However, the effectiveness and the active duration of treatment are greatly compromised by their poor oral bioavailability. The major challenge is the peptide degradation in the gastrointestinal (GI) tract and poor permeation [6]. To overcome these barriers, peptide structural modifications, enzyme inhibitors, absorption enhancers, multifunctional polymers and different carrier systems have also been exploited to prevent proteolysis and to enhance systemic uptake. Among these different strategies, nanoparticles encapsulation is attractive due to its protective effect against degradation, enhanced uptake and sustained release [7].
Poly-(lactic-co-glycolic) acid (PLGA) is a well-known bio-degradable and biocompatible polymer approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA) [8]. It has been used in a variety of biomedical devices and tissue engineering scaffolds, which are proven to be safe for clinical applications [9]. PLGA nanoparticles (PLGANPs) have gained great attention in the field of drug delivery [10,11]. Controllable release can be achieved by using PLGANPs to effectively protect encapsulated material from degradation in the GI tract [12–14]. However, due to the hydrophobic nature of the PLGA polymer it is challenging to achieve entrapment of hydrophilic peptides with high efficiency, especially with peptides of low molecular weight.
In this study, PLGANPs were investigated as a potential oral delivery system for the antihypertensive small peptides Val-Leu-Pro-Val-Pro (VLPVP, VP5) model. VP5 was previously prepared using deoxyribonucleic acid recombinant technology and its bioactivity had been proven by in vitro inhibitory activity of ACE (IC50 1.8 µmol/L) and in vivo antihypertensive effect [15]. The formulation parameters of VP5 loaded PLGANPs (VP5-NPs) were systematically studied. The optimized VP5-NPs were then characterized in vitro and in vivo antihypertensive efficacy was analyzed with spontaneously hypertensive rats (SHRs).
2. Results
2.1. Optimization of Preparation Variables of VP5-NPs
VP5-NPs were successfully produced by systematically optimizing the factors that could affect the features of NPs, including the amount of PLGA, the volume of acetone, pH and the volume of inner aqueous phase, the concentration and volume of poly (vinyl alcohol) (PVA), and the sonication time. Firstly, the effects of the PLGA amount on entrapment efficiency (EE%), particle size, and particle distribution were investigated. As shown in Figure 1a, both the EE% and the particle size were positively correlated with the PLGA amount within the range of 10 to 50 mg. Figure 1b showed that the particle size decreased as the acetone increase. However, the EE% was significantly compromised by the presence of acetone. In Figure 1c, the particle size was not significantly affected by the pH of internal phase, and the EE% was observed to fluctuate as pH changed. As seen in Figure 1d, EE% of VP5 was significantly affected by the volume of inner aqueous phase, but negligibly by particle size.
The influence of the PVA concentration is illustrated in Figure 1e. As PVA concentration increased, the mean diameter first decreased dramatically and then stabilized over 0.5%. In contrast, the EE% of VP5-NPs exhibited an elevating trend with the increasing of PVA concentration. The effect of PVA volume on EE% and particle size were also studied and were similar to that of the PVA concentration. In Figure 1f, as the volume of PVA increased, the particle size was observed to decrease at low volume

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and then increase beyond 4 mL, while the EE% showed an opposite trend. In Figure 1g, as internal phaseI/nto. Jr.gMaonl.iSccip. 2h01a6s,e17(,W19717/O) sonication time was prolonged, the EE% increased rapidly first3aonf 1d3 then
droppFeigdu,rwe 1hgi,laespinatretrinclael pshizaesew/oargsarneilcaptihvaesley(Wsta1b/Ole).soFnuicrathtieornmtiomree,wtahseperfofleocntgoedf ,tthhee sEoEn%iciantciroenasteidme of internraalppidhlyasfeir/stoargndanthicenphdarosep/peexdt,ewrnhailleppharatsiecle(Wsiz1e/wOa/sWre2la)toivnelEyEs%tabalne.dFpuratrhteicrlmeosriez,ethweaesffaelcstoofexthpelored. It wassosnhiocawtinonintimFiegoufrien1tehrnthalapt,htahsee /EoEr%ganwicapshimaspe r/eoxvteedrnualnpdhearsesh(Wor1t/sOo/nWic2a)toionnE,Eb%utanwdapsacrotimclepsriozme ised once wthaes saolsnoiceaxptiloonredti.mItewwasasshboewynonindF2i0gusr,ew1hhitlheat,htehepaErEt%iclwe assiziemdpreocvredasuenddaers sthhoertsosonniiccaattiion, time
was pbruotlownagsecdo.mpromised once the sonication time was beyond 20 s, while the particle size decreased as Bthaesesodnoicnattihoen atibmoevwe aosbpsreorlvoantgioedn.s, the optimized preparation of VP5-NPs can be summarized as
follows. ABatosetadl oonf t2h0emabgoovfe PoLbsGerAvawtiaosnsd, itshseoolvpetidmiinzeddicphrleopraormatieotnhaonf eV.PT5h-Ne Posbctaainnbeedsourmgamnaircizpehdaasse was emulsfoifilloewd sw. Aithto3t3alµoLf 2o0f mVPg5ofsoPlLuGtiAonwbays dpirsosoblevesdoninicdaticiohnloraotm80ethWanfeo.rT3h0e soibntaiicneedbaotrhg.anTihceprheassuelting pforrim20awrsreosyasnausienclemtadimtneugtudlhlspsefiroionfirimner2doaw0rtwyasarsaieytnmahddeu3vdtl3hsaeiepµdononLtrrowoaottf4aaisoVrmyanPdLe5udvonseaofddpl1ouet%orrtai4ovt(inamwocn/bLuyvuuo)nfpmPd1rVo%ewrbA(evawasss/coovuuln)usuiPectmVidaotAwintoosandosrrleauoutmspti8eowod0nvitWsedoerd.rofeioTpcmrhwho3eliov0smeers.odiTmxiinhcteuheitlrcmhoeeraiwoxbnmtaeauteshrate.sthwoTa3nhn7aieesc◦aCtetdo obt aiant 3th7 e°Cnatonoobptaaritnictlheesnuasnpoepnasrtiiocnle. sTuhsproenusgiohnu. Tlthrarocuegnhtruiflutrgacaetniotrnifautgaatisopneaetdaosfp5ee0d,0o0f05r0p,0m00froprm1 h to removfoer P1VhAtoarnedmothvee PuVnAenacnadpsthuelautneednVcaPp5s,utlhateedVVPP5-5N, tPhes VwPe5r-eNfiPnsawlleyrecofinnsatlrlyuccotendst.ructed.

FigurFeig1u. rEef1fe. cEtffoefctvoafrvioaurisoupsropcroecsessinsigngpparaarammeetteerrss oonn tthheeppaarrtitcilcelesisziez(en(mn)man) danedntreanptmraepnmt eefnfitcieefnficcieiesncies (EE%()EoEf%n)aonfonpaanrotipcalertsi,cilensc,liundcliundginthgethaemaomuonutnotfopfoployl-y(-l(alcatcitcic-c-coo--ggllyyccoolliicc)) aacciidd((PPLLGGAA) )(a()a,)t,htehveovluomlueme of acetonofea(cbe)t,otnhee(pb)H, th(ce)paHnd(cv)oalnudmveol(udm) oef(din)noefrinanqeureaoquuseopuhsapseh,atshee, tchoenccoenncternattriaotnion(e()ea)nadndvvoolulummee(f) of poly ((vf)inofypl oallyco(vhionly)l(aPlVcoAh)o,l)an(PdVtAh)e, asnodnitchaetisoonnitciamtioenotfimWe1o/fOW(1g/O) a(ngd) aWnd1W/O1//OW/W22(h(h).).((nn == 33))..

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2.2. C2h.2a.rCachtaerraicztaetriiozantioofnVofPV5P-N5-PNsPs
OverOavlle, rtahlel, otphteimoapl tVimPa5l-NVPPs5w-NePres dwemeroensdtermatoendsttroasteigdnitfiocasnigtlnyifiimcapnrtloyveimthperodvrue gtihnecodrpruogration with ianncoErpEo%ratoiof n87w.3it7h%an±EE0%.9o2f%87a.3n7d%d±r0u.9g2l%oaadndindgrucgaploaacditinyg(cDaLpa%ci)tyof(D2L.1%0)%of±2.100.%17±%0..1T7%he. Tahveerage particaTlvheeersaicgzoeellpowaidrataislcl2es2os3iluz.7etiwo±nas2w2.32a3sn.7mo±b2s(e.F3rivngemudr(eFais2gaus)rliegw2haitt)lhywaibthlnuaaernroaoprwraolewssicszeieznecdediisswttrriiitbbhuutstitiorononn(Pg(DPTDI y=In0=d.1a20ll.±1e02f.f0e±1c)t.0.01). The c(oFlilgouidrea2l cs)o. lAustisohnowwnasinotbhseetrrvaendsmaissssiloignhetlleyctbroluneicompiaclreosscceonpcye(TwEiMth)simtroagnegiTnyFnigduarlel e2dff,eVctP(5F-NigPusre 2c). As shwowerne ginenthereatllryanspsmheirsisciaolnanedlehctormonoigcemneiocuros,swcohpicyh(TwEaMs in) igmoaogdeaignreFeimguernet 2wdit,hVtPh5e-nNaPrrsowwepraergtiecnleerally sphersiczaeldainstdribhuotmiono.geneous, which was in good agreement with the narrow particle size distribution.

FigurFeig2u. rPeh2a.rmPhaacermutaicceaul tpicraolpeprrtoipeserotifeVs Po5f-NVPP5s.-N(aP)sS. iz(ae)dSisiztreibduistitorinb;u(tbio)nζ; p(bo)teζntipaol;te(nc)tiTalh; e(ca)ppTehaerance and Taypnpdeaarlal necffeeacnt dofTVynPd5a-lNl ePfsfe; c(tdo)fTVEPM5-NimPsa;g(ed)oTfEVMP5im-NaPges.of VP5-NPs.

A differential scanning calorimetry (DSC) analysis is shown in Figure 3a, which shows that free AVPd5ipffreerseennttieadl asncaennndiontghecramloicripmeaektrayt (1D67S.0C°)CanfoalllyowsiesdisbsyhaonwenxoitnheFrimguicrpee3aak, pwrohbicahblsyhdouwe stothitast free VP5 pderegsraednatetidona. nTheensdamoteheenrdmoitchepremaikc paeta1k6w7a.0s a◦bCsefnotllinowDSeCd tbhyeramnogerxaomthoefrVmPi5c-NpPesa,kinpwrohbicahbtlhyerdeue to

its dewgerraedaotniloyn.twToheensadmotheeernmdicotpheearkms iactp1e1a8k.2waansda1b9s3e.n6 t°iCn DcoSrCrestphoenrdminoggrtaomthoef gVlPas5s-NtrPanss,iitnionwhich

theretewmepreeroatnulrye t(wTgo) eonf dthoethPeLrGmAicapndeaPkVsAa,t r1e1s8p.e2ctaivnedly1. 9H3o.6w◦eCvecr,otrhreespphoynsdicianl gmtioxttuhree ogflaVsPs5traanndsition

tempbelraantukrnea(nTogp)aortfictlhees PshLoGwAedatnhdrePeVeAnd, orethsperemctiicvpeelya.kHs wowhiecvhecro, rtrheesppohnydseicdatlomthixetufrreee oVfPV5Pa5ndantdheblank

nanoTpgarotfictlheesPsLhGowAeadndthPrVeeA.endothermic peaks which corresponded to the free VP5 and the Tg of the

PLGAVPa5n-dINnPPvsVitwAroa. srerleelaesaesepdropfirloegsroefssVivPe5lyfrwomithVoPu5t -oNbPvsiowuserbeuprrset sreenleteadseininFaiglluorfe

3b. the

The VP5 loaded in release media. The

Irnelevaistreos orfeflreeaesVe Pp5roanfidleVsPo5f-NVPPs5bofrtohmexhVibPi5te-dNtPyspiwcael rpeHp-dreepseenndteedntirnelFeaigsiunrgeb3ehba. vTiohr.eWViPth5inloaaded

in VP152-hNpPesriwoda,scormelpeaarseeddwpitrhogthrees7s8i.v0e%lyrewleaitsheoouf tfroeebvViPo5uast bpuHrs7t.4r,eolnelays5e2i.7n%aollf oVfPt5hwearserleelaesaesemd edia.

The rferolemasVePs5-oNf Pfsr.ee VP5 and VP5-NPs both exhibited typical pH-dependent releasing behavior.

Within a 1V2Ph5-NpePrsioddis,plcaoymedpaargeododwsittahbitlihtye w78it.h0%nordeeleteacsteabolef cfhreaengVesPi5n aptarptiHcle7s.i4z,eso,nζlypo5t2en.7ti%al orf VP5

was rEeEle%asfeodr aftrolemastV1Pw5-eNekP(sF.igure 3c), which was benefit from the negatively charges on nanoparticles
V(FPig5u-rNe P2bs)d. WisphelanyVePd5a-NgPosowdesrteasbtoilrietyd bweiytohnndo2dweeteekcst,aabnleenclhaargnegmesenint opf apratritciclelessiizzeesa,nζdpaodteronptial or EE%offoEr Ea%t leoaf sVtP15wweeereko(bFsigeruvreed3. Oc)v, ewrahlli,cVhPw5-aNsPbsewneefiret sfurogmgestthede ntoebgeatsitvaebllye icnh4ar°gCefsoron1 wnaeenkopanadrticles (Figubree f2ubr)t.hWerhinevneVstiPg5a-tNedPtsowdeevreelsotporaefdrebeezye-odnrdyin2gwfoeremksu,laatnioennfloarrgoeraml eandtmoinf ipstarrattiicolne.size and a drop of EE% of VP5 were observed. Overall, VP5-NPs were suggested to be stable in 4 ◦C for 1 week and be

further investigated to develop a freeze-drying formulation for oral administration.

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FigurFeig3u.reC3h. Carhaacrtaecrteizriaztaitoionnooff VVPP55--NNPPssbybydifdfeifrfeenrteianltsiacalnsncianngncianlogricmaelotrryim(DeStCry), (inDvSiCtr)o, rienlevaisteroanrdelease and ssttaabbiilliittyy.. ((aa))DDSSCCccuurvrvesesoof ffrfereeeVVP5P,5b,lbanlaknnkannaonpoarptiacrlteisc,lethse, tphheypsihcaylsimcaixltmuriextoufrVePo5f VanPd5balnandkblank nanonbpuaanfrfoetpircsalr(eptsiHc,lea1sn.,0da, n6V.d8PaV5nP-dN5-7PN.4sP);;s((;bc()b)C)RhReaelnelegaaesseienppsrrizooeffiialleenssdooEffEf%rfereeoefVVVPP5P55a-NnadPnsdViPnV52P-Nw5P-eNsekPisns. diniffedriefnfetrpehnotspphhaotsephate buffers (pH 1.0, 6.8 and 7.4); (c) Change in size and EE% of VP5-NPs in 2 weeks.
2.3. In Vivo Antihypertensive Efficacy 2.3. In Vivo Antihypertensive Efficacy
As seen in Figure 4 and Table 1, VP5 exhibited a significant blood pressure-lowering (BPL) effect iAnsasdeeonsei-ndeFpiegnudreen4t amnadnnTearb. lAed1m, VinPis5treaxtihoinbiotef d0.a4 smiggn/kigficoafnVt Pb5losoigdnpifriceasnsutlyred-leocwreearsiendgs(yBsPtoLli)ceffect in a dbloosoed-dperepsesnudree(nStBmP)abnyn1e0r..0 AmdmmHingiasttr2ahtipoonsto-afd0m.4inmisgtr/aktigono(fpV<P0.501s)i.gVnPifi5caatnatdlyosdeeocfr0e.a8smedg/ksygstolic blooddepcrreesassuedreS(BSPBbPy) 1b3y.41a0n.0dm20m.2 mHmg Hatg2aht 2paonsdt-a4dhmpionsits-atrdamtiionnist(rpat<io0n.,0r1e)s.pVecPt5ivaetlya. Tdhoesedeocfr0ea.8semdg/kg decreSaBsPedbeStwBPeebny21t3o.412anhdp2o0st.2amdmminHisgtraattio2nanwdas4ohbspeorvste-dadafmteirntirsetaratetdiobny, r1e.6spmecgt/ikvgelVyP. 5T,haenddetchreeased SBP mbeatxwimeeunm2BtPoL1e2ffhectp(o2s0t.9admmmiHngis)trwaatsioant w8 haspoobstseadrvmeidnisatfrtaetriotnr.eaSBtePdrbetyur1n.e6dmtog/thkeguVntPre5a, taendd the maxilwmeveuefmlusrwBthPietLrhiiennfvf2ee4cstthig(2pa0toe.s9dt-mathdmemHBinPgiLs)tewraffateisoctnaotif8nVhaPlpl5o-tNhsrtPease.dg0m.r8oinumipsgst/rkwagtiitoohfnvV. aPSrB5ia-PNtiorPenstsustirnrniketiidnmgteolydtdhueeracurtienoatnsr.eedTathSeeBdnP,levels withibny2246.h7 mpomsHt-agdamt 2inhisptorsattiaodnmiinniasltlratthioreneagnrdosuhposwweditha mvaarxiiamtiiozends BinPLtimeffeecdtuarta4tihonby. T31h.e7nm, wmHe gfu. rther investigated the BPL effect of VP5-NPs. 0.8 mg/kg of VP5-NPs strikingly decreased SBP by 26.7 mmHg
at 2 h post administration and showed a maximized BPL effect at 4 h by 31.7 mmHg. 4 h post treatment,
SBP ascended slowly and smoothly, even after 72 h, significant BPL effect was still observed as a
reduction of 18.7 mmHg (p < 0.01). SBP returned to the initial level at 96 h post administration.

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4 h post treatment, SBP ascended slowly and smoothly, even after 72 h, significant BPL effect was Isntti.lJl. oMbosl.eSrcvi.e2d01a6,s1a7, 1r9e7d7uction of 18.7 mmHg (p < 0.01). SBP returned to the initial level at 96 h6 pofo1s3t administration.

FFiigguurree 44.. AAnnttiihhyyppeerrtteennssiivveeeeffffeeccttssooffddififffeerreennttddoosseessooffVVPP55aannddVVPP55-N-NPPs sininSHSHRRs swwithitihnin9696h hbybya asisnignlgeleoroarlaal damdminiinsitsrtartaitoino.n.

TTaabbllee11. .TThhe ededcerceraesainsginagmapmliptulidtue doef soyfstsoylisctoblliocobdloporedsspurreesosuf srpeoonftasnpeoonutsalnyehoyupsleyrtehnyspiveretreantssiv(SeHrRasts) a(St HdiRffse)reant tdtiifmfeerepnotitnimtseafptoerinatssianfgteler aorsainl dgloeseor(aXl ±doSsDe (,Xn ±=S6D) (,mn m= 6H)g()m. mHg).

Time Saline Group 0.4 mg/kg VP5 2Thime S4a.l5in±e3.G2roup 100..04 ±m1g.3/k*g**VP5

Group Control 0.8 Gmrgo/ukpg CVoPn5trol 1.6 mg/kg VP5 0.8 mg/kg VP5-NPs 01.83.m4 ±g/2k.2g *V**P5 1.6 1m7g.7/k±g3V.8P*5** 0.8 mg2/6k.7g ±V3P.57-*N**Ps

4 8

42hh

h h

128hh

2412h h

11..2014..±±25 04±±..6703..62 8.21.±0 1±.24.7 1.28.±2 1±.51.2

581.2.052..±02±1±±2.9.611*..39

*** *

9.48.±21±.32.6

1.69.±41±.61.3

2120301...2242.4±±±±332.26..62.3********* 1152..84 ±±32.7.3 135.5.8±±0.38.7

1187..215780±±..59 ±±53..5488..82************ 20.199±.5 ±4.32.0****** 19.5 6±.03±.03*.*4*

3216..237781±±..97 23±±..3712..**93********** 28.293±.6 1±.93.*8***** 23.261±.5 3±.80.*7*****

4824h h

2.21.±2 1±.61.5

−0.15.6± ±1.81.6

23..45 ±±10.2.8

6.01±.5 ±3.24.5

21.250±.4 0±.71.*0****

7248h h

0.32.±2 1±.51.6

−2−.70±.51±.1 1.8

32..24 ±±11.6.2

1.5−0±.12±.51.0

20.148±.7 1±.03.*9****

9672h h

0.60.±3 0±.51.5

−2−.62±.72±.7 1.1

43..42 ±±01.7.6

−0−.11.±6 ±1.10.4

18.7−±0.63.±9 3**.5*

9O6 hne-way a0n.6al±ys0is.5of varian−ce2.6w±as2u.7sed for c4a.l4cu±la0ti.n7g statistic−a1l.6si±gn1if.4icance, whic−h0.w6 a±s 3s.e5t at

* pOn<e0-.w05ay, *a*n*aply<si0s.0o1f.vVarPia5n-NcePws aasnudstehdrfeoerccraulcduelaVtiPng5 gstraotiuspticsavl esrigsnuisficcoanncter,owl ghriochupw.as set at * p < 0.05,
*** p < 0.01. VP5-NPs and three crude VP5 groups versus control group.

3. Discussion 3. Discussion
VP5-NPs were prepared by a simple and controllable double emulsification method [16]. As prevVioPu5s-lyNrPespowrteerde [p1r7e]p, eanretrdapbmyeantseifmfipcileencaynd(EEco%n)traonldlasbilzee daroeuibmlepoermtaunltsiifincdaetxioens fomreetvhaoldua[t1in6g]. Athsepqreuvailoituysloyfrnepanoortpeadr[t1ic7l]e,se.nHtriagphmEeEn%t ecffiacnieinmcpyr(oEvEe%u)tailnizdastiizoenaorfe tihmepdorrtuagntainnddesxmesalfloerr esvizaelucaotuinlgd tehnehqanucaelittyhoefanbasnoorpptairotnicloefs. iHntiegshtinEaEl%epciatnheimliapl rcoevlelsu. tTilhizearetifoonreo, fhtihgehdEruEg%anadndsmbaeltlteerr ssiizzeecowuelrde eonphtiamncizeetdhebaybsvoarrpytiinogn soufcinhtepsatrinamaleetpeirtsh,eilniacllucedlilns.gTthheereafmoroeu, hnitgohfEPEL%GaAn,dthbeetvteorlusimzeewoefraecoeptotinme,izpeHd bayndvathryeinvgolsuumche poafrianmneertearqsu, ienoculusdpihnagsteh,ethaemcoounnctenoftrPaLtiGonAa, tnhdetvhoeluvmoleumofeaoceftPoVneA, ,paHndanthdethsoenvioclautmione otifminen. eFrirasqtluye, othues ipnhfaluseen, tcheeocfotnhceenamtraotuionnt oafndPLthGeAvoolnumEEe%ofaPnVdAs,izaendpottheenstioanlliycadtiuoenttoimthee. Fhiirgshtleyr, tvhiescionsflituyenincethoef tohregaamniocupnhtaosfePaLsGthAeoPnLEGEA%caonndcesniztreaptiootnenitnicarlleyasdeude. tMo othree hviisgchoeursvoisrcgoasnitiyc ipnhtahsee omrgigahnticrepshualsteinaslatrhgeerPdLrGoAplceotsnacenndtrleastisonneitnschreeaasresdtr.eMssoirnetvhiesceomuus losrigoann, aicspwhealsleasmdigehcrteraesseudltdiniffluarsgioenr dorfoVpPle5tfsraonmdinlensesrnaeqtusehoeuasr tsotroersgsainnicthpeheamseu. lTshiouns,, athsewinecllreaassdinegcrPeLasGeAd damiffouusniotnwoafsVshPo5wfrnotmo rinesnuelrt ainquleaorguesrtopaorrtgicalneicsipzheaasen.dThhuigsh, ethr eEiEn%cre[a1s8in–2g0P].LTGuAnianmgotuhnet pwraospsohrtoiwonnstoofredsiuchltloinrolamrgetehrapnaertaicnlde saiczeetoanned hinightheer EoErg%an[1ic8–p20h]a.sTeunminayg tphreopdruocpeorntaionnospoafrtdicilcehslowroimthetmhaonree adnedsiaracebtloenperionptehretioesrga[2n1i]c. pAhcacsoerdminayg ptorooduurceprnealinmopinaarrtyicleexspweritihmmenotrse[d22e]s,irtahbelevoplruompeertoiefsa[c2e1t]o.nAecwcoarsdionpgtitmo iozuedr ptroelgimenienraartye eVxPp5e-rNimPsenwtsit[h22h]i,gthheervEoElu%maenodfbaectetteornpeawrtiacsleopsitzime, iwzehdiletodgicehnleorraotme VetPh5a-nNePwsaws iftihxehdig. hTehreEpEr%eseanncde boeftatecretpoanreticinle tshizeeo, rwghaniliecdpichhalsoerocmoueltdharnedeuwceasthfiexeidn.teTrhfaeciparleesennercgeyofaat ctehteonoeil/inwtahteeroirngtaenrfiaccpehaansde ceonuhladnrceedtuhcee sthtaebiinlitteyrfoafciadlroenpelertgsy, awthtihcehocilo/nwtraibteurteindtetrofatcheeansmd aelnlehransiczeetsheofstnaabniloitpyaortficdlreosp[l2e3ts]., wHhoiwchevceorn,trthibeupterdesteontcheeosfmaacleletornseizemsiogfhnt aennohpaanrcteicltehse[2V3P].5Hdoiswpeevrseiro, nthferopmrestehneceinonfearcetototnheemoiugthetr eanqhuaenocuestphheaVsePa5nddiscpaeursseioansfhraormp dtheecrienanseeritnotthheeEoEu%terofaqVuPe5o[u2s4]p.hDausee taondthceaeufsfeecatssohnarspolduebcirleitayse[2i5n] tahnedEiEo%nizoifnVgPs5ta[t2u4s].oDf uVePt5o, tthhee epfHfecotsf othnesionlnuebriliatqyu[e2o5u] asnpdhiaosneizwinags fsutarttuhserofoVptPim5,itzheedp. HThoef othbeseinrvneedr aqueous phase was further optimized. The observed tendency probably resulted from the different
status of VP5 in the internal phase. When the pH was greater than the isoelectric point (pI) of the

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VP5 peptide (pI = 6.0), the charge distribution in the peptide could be changed and had an impact on the encapsulation of peptide [26]. The effect of inner aqueous phase volume was also investigated. The increase in W1/O volume ratio was able to enhance the entrapment of the internal phase by the organic phase, which led to the minimal particle size and maximal EE% [27,28]. When W1/O volume ratio exceeded a threshold, the organic phase failed to entrap the internal phase sufficiently and resulted in an increase in the size of droplets and a decrease in the EE%.
In this study, PVA, as the emulsifying agent, was used as a stabilizer during the emulsion formation and to produce stabilized organic droplets [19]. The impacts of the concentration and volume of PVA were then evaluated. The decrease of the particle size was probably caused by the excess amount of PVA residing at organic solvent/aqueous interface, and reduced the surface tension [29–31]. However, the increase in PVA concentration enhanced the outer aqueous viscosity [19], which impeded the diffusion of VP5 in the inner aqueous phase into outside and thereby improved the incorporation. The increase of the volume of PVA could enhance the net sheer stress and resulted in smaller size of particles [32]. On the other hand, the viscosity in the external phase could increase with higher PVA volume, which led to a bigger particle size. The EE% could be maximized when the interaction of primary emulsion with external phase was optimal. Peptides are generally subjected to denaturation under various stress conditions, such as sonication and homogenization [33]. Hence, the sonication duration was expected to be critical for the VP5-NP preparation. The short W1/O sonication time was suggested to cause VP5 incompletely being entrapped in the primary emulsion and lower down the EE%. Longer sonication time was speculated to break the coarse emulsion drops into nanodroplets, which could cause the observed particle size of VP5-NPs and the improved EE% [34]. However, when the formation of primary emulsion was good, longer sonication time would lead to the leakage of VP5 from the internal phase to the external phase, causing a decrease in EE%. Similar to the effect observed with W1/O, prolonged W1/O/W2 sonication time could enhance the EE%, and it could lead to an increasing net sheer force and result in a decreasing particle size at the same time.
To investigate the existing state of VP5 and verify the successful entrapment of VP5 into nanoparticles, DSC analysis was performed [35]. The Tg of PVA was in agreement with the previous research [18], but that of PLGA was higher than that reported in the literature most probably due to the different ratio of lactic acid / glycolic acid (LA:GA) and molecular weight of PLGA [36].
The release of VP5 out of nanoparticles was dramatically lower than the unincorporated VP5 when the same dialysis bag was used, although there might be some interaction of VP5 itself with the applied dialysis. Theoretically, the dialysis bag for the in vitro release study should let the free agent with small molecular weight get through freely. However, many dialysis bags would influence the passage of the agents investigated, probably because of some interaction including adsorption or hydrophobic interaction [37]. Only about 90% of the free VP5 can diffuse into the dispersed medium after 12 h, thus other methods for the in vitro release test of VP5 need to be developed in the future. The in vitro sustained release in all of the release media indicated a prolonged release of VP5-NPs and might lead to a potent and prolonged therapeutic efficacy of VP5-NPs. The pH-dependent releasing behavior was most probably caused by the degradation of PLGA [38]. The release percentages in media with different pH may provide some evidence that VP5-NPs could provide sustainable release of VP5 in both stomach and intestine and protect VP5 from premature degradation [39].
It is recognized that oral delivery of peptide has been a great continuous challenge due to its poor stability in the GI tract and low permeability through the intestinal epithelium membrane [40–42]. Numerous promising strategies have been utilized to circumvent these obstacles [43], including enzyme inhibitors [44] and solid lipid NPs [45] to protect these drugs from enzymatic degradation. Chemical absorption enhancers [46] were also used to improve intestinal epithelium membrane permeability. However, these approaches had only achieved limited success so far.
PLGANPs exhibit a wide range of erosion times, tunable biodegradation and mechanical properties [47] and have been widely used in academic and industrial research [7,10,11]. Here, we presented the encapsulation of VP5 into PLGANPs, which could expect to enhance the stability of

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VP5 and prolong the active ingredient release for a better therapeutic efficacy. In our study, VP5-NPs exhibited a four-time prolonged BPL effect than high-dose of free drug (Figure 4) that was partially supported by the in vitro sustained release experiment (Figure 3c). Moreover, VP5-NPs of small size could enhance the intestinal absorption and subsequently reach the systemic circulation and gradually release VP5, which might contribute a long-term antihypertensive effect [48]. It was previously reported PLGA is not a gastro-resistant polymer [49], which presented some conflicts with our results. The inconsistent observation could be explained by the incomplete degradation of VP5-NPs, where the remaining intact nanoparticles could still proceed with the fluid to the small intestine and be uptaken by the intestinal tissue. Another possible explanation could be the short VP5 oligopeptide being resistant to the digestion of gastrointestinal juices [50]. Even if being released from nanoparticles and losing shields, intact VP5 could be absorbed by intestinal epithelial cells to produce a response.
Currently, the mechanism of enhancement in therapeutic efficacy is still under investigation. The efficiency of VP5 being absorbed into systemic circulation requires further quantification. The association between elevated therapeutic efficacy and the area under concentration-time curve (AUC) or bioavailability needs further elucidation.
Overall, we systematically optimized the factors that could affect the features of NPs, and successfully constructed VP5-NPs with a high entrapment efficiency and small particle size. Then, we evaluated its antihypertensive efficiencies in vivo, including the crude VP5 and VP5-NPs, found out that VP5 decreased blood pressure in a dose dependent manner. Much to our surprise, a medium dose of VP5-NPs showed stronger BPL effect than a high dose of crude VP5, and maintained a prolonged BPL effect even up to 72 h. This may be partly explained by the controlled release of VP5 and the protection effect of the nanoparticle shield avoiding enzymatic degradatio, and subsequently elevating the bioavailability of VP5. This may provide some methodological clues and insights for the therapy of hypertension.
4. Materials and Methods
4.1. Materials
VP5 (purity > 99%) was purchased from Phtdpeptides Co., Ltd. (Zhengzhou, China). PLGA (MW = 15 kDa; LA/GA = 75:25) was purchased from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). Poly (vinyl alcohol) (PVA, MW = 30–70 kDa; HD, 80%) was procured from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and were used as supplied.
40-week-old male spontaneously hypertensive rats (SHRs) weighing between 350 to 400 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The trial was approved by the Animal experimental ethics committee of State Key Laboratory of Biotherapy of Sichuan University (20150356, 28 December 2015).
4.2. Preparation of VP5-NPs
VP5-NPs were prepared by a double-emulsion (W1/O/W2) solvent evaporation method. Briefly, VP5 was dissolved in deionized water to form the inner aqueous phase. The organic phase containing PLGA was emulsified with the inner aqueous phase by probe sonication in ice bath to form the primary W1/O emulsion. The obtained primary emulsion was then added to PVA solution and further sonicated to gain the final W1/O/W2 double emulsion. The organic phase was rapidly removed by evaporation under vacuum at 37 ◦C. The VP5-NPs were obtained through ultracentrifugation at a speed of 50,000 rpm for 1 h to remove PVA and the unencapsulated VP5. The blank nanoparticles were also prepared using the similar process.

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4.3. Characterization of VP5-NPs

4.3.1. Particle Size and ζ Potential
The mean particle size, size distribution and ζ potential were measured using a Zetasizer (Zetasizer Nano-ZS 90; Malvern Instruments Ltd., Malvern, UK) at 25 ◦C. The prepared VP5-NPs were dispersed in deionized water with 10 times and experiments were conducted in triplicate. All the data were presented as mean ± SD.

4.3.2. Entrapment Efficiency and Drug Loading
The supernatant after centrifuging the colloidal solution during the preparation of VP5-NPs was used to determine EE% and DL% of VP5-NPs as previously described [51] with minor modifications. Briefly, the amount of untrapped VP5 in supernatant was calculated by high performance liquid chromatography (HPLC, Waters Alliance 2695). A reverse-phase C18 column (150 mm × 4.6 mm, pore size 5 µm, Cosmosil, Nacalai, Japan) was used for the chromatographic separation with a mobile phase consisting of a mixture of acetonitrile/0.1% TFA water (75/25, v/v) at a flow rate of 0.8 mL/min. VP5 was detected at a wavelength of 220 nm. EE% and DL% of VP5 were calculated by the following formula:

EE% =

Total VP5 amount − the amount of VP5 in supernatant Total VP5 amount

× 100

DL% =

Total VP5 amount − the amount of VP5 in supernatant Total weight of nanoparticles

× 100

4.3.3. Appearance
The morphology of the VP5-NPs was examined by transmission electronic microscopy (TEM, H-600, Hitachi, Japan). Before analysis, the prepared samples were diluted with deionized (DI) water and negatively stained with 2 wt % phosphotungstic acid solution for 30 s, and then they were placed on a copper electron microscopy grids with a thin film for observation.

4.3.4. Differential Scanning Calorimetry (DSC)
The physical state of VP5 loaded in VP5-NPs was investigated by differential scanning calorimetry (DSC, 200PC, Netzsch, Karlsruhe, Germany) under nitrogen atmosphere at a flow rate of 20 mL/min. Freeze-dried VP5-NPs, blank nanoparticles, VP5 and the physical mixture of the latter two samples with the same mass ratio as those in VP5-NPs were heated from 50 to 250 ◦C at speed of 10 ◦C/min.

4.3.5. In Vitro Release
The in vitro release profiles of VP5 from VP5-NPs were carried out in PBS buffer at physiological pH condition (pH 7.4), in simulated intestinal fluid (pH 6.8), and in simulated gastric fluid (pH 1.0) by the dialysis method. Briefly, both VP5-NPs and free VP5 solution were first dispersed in release media for dialysis (MWCO 3000), which were shaken at 37 ◦C with a speed of 100 rpm. At a given time point, 1 mL buffer was removed and replaced by another 1 mL fresh release medium. The content of VP5 was measured by HPLC as described in the Section 4.3.2.

4.3.6. Stability
VP5-NPs were stored at 4 ◦C for two weeks to investigate the preliminary stability. The particle size, size distribution, and EE% of VP5 were measured as described above and the changes over time was analyzed.

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4.4. In Vivo Antihypertensive Efficacy
The in vivo pharmacodynamics studies were performed using SHRs, which were allowed for one-week acclimatization in independent cages. They were given free access to commercial laboratory feed (MF; Beijing HFK Bioscience Co., Beijing, China) and tap water in a controlled temperature room (1–22 ◦C) with a 12 h light-dark cycle. Four groups of SHRs, 6 rats each, were administered a single oral dose of crude drug VP5 or VP5-NPs. The crude drug VP5 was dissolved in saline, and the control group received only saline. Then, the dosing volume was calculated from the bodyweight of SHRs. The change in blood pressure caused by different dosage was indirectly recorded using the tail cuff method (Softron BP-2010A; Softron Beijing Biotechnology, Beijing, China) at 0, 2, 4, 8, 12, 24 h and every one day after administration.
4.5. Statistical Analysis
The statistical analysis was performed using the Statistical Product and Service Solutions software (SPSS V19.0, IBM Corp., New York, NY, USA). Data were analyzed by one-way analysis of variance. p < 0.05 was considered a statistically difference, and p < 0.01 was considered a statistically significant difference.
5. Conclusions
PLGANPs loaded with the antihypertensive small peptide model VP5 were successfully prepared using a simply double-emulsion solvent evaporation method. The optimal VP5-NPs showed desirable pharmaceutical properties through systematical process-parameter investigation, including small size, high EE% and sustained release. Moreover, VP5-NPs exhibited an enhanced antihypertensive function with a longer duration, up to 96 h in SHRs. Altogether, PLGANPs can serve as a compelling strategy for oral delivery of antihypertensive small peptides, and VP5-NPs might be a potential strategy for hypertension treatment in the future.
Acknowledgments: This research has been received financial support from the Innovation Plan of the Science and Technology Plan of Shenzhen (JCYJ20130331151204151).
Author Contributions: Xiangrong Song, Jian Yang and Lili He conceived and designed the experiments; Ting Yu prepared the VP5-NPs; Shengnan Zhao and Ziqiang Li performed the in vivo antihypertensive efficacy of VP5-NPs; Yi Wang and Bei Xu carried out the characterization of VP5-NPs; Dailong Fang, Fazhan Wang, and Zhi Zhang analyzed the data.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ACE VP5 RAAS ARB SBP DBP IPP VPP GI BPL

Angiotensin converting enzyme Val-Leu-Pro-Val-Pro Renin-angiotensin-aldosterone system Angiotensin receptor blocker Systolic blood pressure Diastolic blood pressure Ile-Pro-Pro Val-Pro-Pro Gastrointestinal Blood pressure-lowering

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