Urotensin-II-TargetedLiposomesasaNewDrugDelivery

Research ArticleUrotensin-II-Targeted Liposomes as a New Drug DeliverySystem towards Prostate and Colon Cancer Cells

Silvia Zappavigna 1 Marianna Abate1 Alessia Maria Cossu12 Sara Lusa3

Virginia Campani 3 Lorena Scotti3 Amalia Luce1 Ali Munaim Yousif3

Francesco Merlino 3 Paolo Grieco 3 Giuseppe De Rosa 3 and Michele Caraglia12

1Department of Precision Medicine University of Campania ldquoL Vanvitellirdquo Via L de Crecchio 7 80138 Naples Italy2Biogem Scarl Institute of Genetic Research Laboratory of Molecular and Precision Oncology 83031 Ariano Irpino Italy3Department of Pharmacy University of Naples ldquoFederico IIrdquo Via D Montesano 49 80131 Naples Italy

Correspondence should be addressed to Giuseppe De Rosa gderosauninait

Received 27 June 2019 Accepted 26 October 2019 Published 17 December 2019

Guest Editor Ana Espinosa

Copyright copy 2019 Silvia Zappavigna et al (is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Urotensin-II (UT-II) and its receptor (UTR) are involved in the occurrence of different epithelial cancers In particular UTR wasfound overexpressed on colon bladder and prostate cancer cells (e conjugation of ligands able to specifically bind receptorsthat are overexpressed on cancer cells to liposome surface represents an efficient active targeting strategy to enhance selectivityand efficiency of drug delivery systems (e aim of this study was to develop liposomes conjugated with UT-II (LipoUT) forefficient targeting of cancer cells that overexpress UTR (e liposomes had a mean diameter between 150 nm and 160 nm with anarrow size distribution (PIle 01) and a doxo encapsulation efficiency of 96 Moreover the conjugation of UT-II to liposomesweakly reduced the zeta potential We evaluated UTR expression on prostate (DU145 PC3 and LNCaP) and colon (WIDR andLoVo) cancer cells by FACS and western blotting analysis UTR protein was expressed in all the tested cell lines the level ofexpression was higher in WIDR PC3 and LNCaP cells compared with LoVo and DU145 MTT cell viability assay showed thatLipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR (PC3 LNCaP andWIDR) while in LoVo and DU145 cell lines the activity was similar to or lower than that one of Lipo-doxo respectivelyMoreover we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT than the not-armedcounterparts but at higher extent in UTR overexpressing PC3 cells (about 2-fold higher) as evaluated by both confocal and FACSIn conclusion the encapsulation of doxo in UT-II-targeted liposomes potentiated its delivery in UTR-overexpressing cells andcould represent a new tool for the targeting of prostate and colon cancer

1 Introduction

Urotensin-II (UT-II) has been described as the most potentvasoconstrictor superior to other vasoactive molecules suchas endothelin-1 noradrenalin and serotonin [1 2] In ad-dition to vascular effects UT-II and its receptor UTR havebeen found to be involved in the development of severaldiseases such as cardiorenal diseases heart failure carotidatherosclerosis and portal hypertension cirrhosis Severalstudies have investigated the involvement of UT-II and UTRin human cancers [3] In particular UTR was overexpressedin colon [4 5] bladder [6] and prostate [7] cancer cells

Recently UTR has been proposed as a prognostic marker inprostate carcinoma since UTR expression was correlatedwith well-known pathological indicators of aggressivecancers such as Gleason score Interestingly UTR was al-ways expressed at low intensity in hyperplastic tissues and athigh intensity in well-differentiated carcinomas (Gleason 2-3) Similarly UT-IIUTR axis plays a key role in coloncarcinogenesis [8] UTR expression was low in normal colontissues and increased in adenomas and colon cancersmoreover our previous data suggest that UTR regulatedmotility and invasion of colon and bladder cancer cells [4 6]All these works clearly demonstrated that UTR represents a

HindawiJournal of OncologyVolume 2019 Article ID 9293560 14 pageshttpsdoiorg10115520199293560

potentially useful target for innovative therapy against colonand prostate cancer together with the revival of its pre-viously reported modulators [9ndash11] Liposomes are one ofthe most common vehicles proposed for drug targeting Inparticular liposomes with stealth properties allow the en-capsulated drug to be addressed such as doxorubicin (doxo)towards tissues characterized by vessels with an enhancedpermeability of the endothelium such as tumors HoweverPEGylated liposomes containing doxo (marketed as Doxilregor Caelyxreg) have demonstrated reduced toxicity although ahigher delivery into the tumor has not been reported[12 13] To improve their target ability liposomes have beenfunctionalized with specific ligands or molecules such asantibodies peptides and oligonucleotides able to enhancethe selectivity of liposomes towards cancer cells [14ndash16]Several strategies can be approached for coupling ligand tothe surface of stealth liposomes in order to achieve a highinteraction of ligand with the receptor expressed on tumorcells Among all commonly used approaches PEG derivatesinclude maleimide groups that react with cysteine residuesor thiol groups on ligands to form stable thioether bondsbetween liposomes and ligands [17] (us receptor ligandssuch as transferrin [18 19] folic acid [13 20 21] or differentmonoclonal antibodies raised against tumor-associatedantigens (TAA) including receptors [22 23] have beenlargely investigated to target different cancer cells Herestealth liposomes were proposed as systems to target cancercells overexpressing UTR and to promote drug delivery of ananticancer agent ie doxorubicin into colon and prostatecancer cells

2 Materials and Methods

21Materials 12-Dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) and [N-(carbonyl-methoxypolyethylenglycol-2000)-12-distearoyl-snglycero-3-phosphoethanolamine sodium salt](DSPE-PEG2000) were purchased from Lipoid GmbH (CamSwitzerland) 23-(Dipyrrometheneboron difluoride)-24-norcholesterol (Bodipy-cholesterol) and 12-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide-(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide)were obtained from Avanti Polar Lipids (Alabaster AlabamaUSA) Doxorubicin hydrochloride (doxo) cholesterol (chol)sodium chloride [4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid N-(2-hydroxyethyl)piperazine-Nprime-(2-ethane-sulfonic acid)] (Hepes) ethylenediaminetetraacetic acid(EDTA) iron thiocyanate (FeSCN3) sodium citrate potas-sium chloride citric acid and Sepharose CL-4B were pur-chased from Sigma Chemical Co (St Louis Mo USA) AllFmoc-amino acids were purchased from GL Biochem(Shanghai China) with their corresponding side chain pro-tections for the solid-phase peptide synthesis (SPPS) Ace-tonitrile methanol (HPLC degree) and chloroform (ACSgrade) were obtained from Carlo Erba (Milan Italy) RPMIDMEM and FBS were purchased from FlowLaboratories(Milan Italy) Tissue culture plasticware was acquired fromBecton Dickinson (Lincoln Park NJ USA) Rabbit antiseraraised against α-tubulin and UTR were purchased from SantaCruz Biotechnology (Santa Cruz CA USA) UT-II peptide

was synthetized and purified by the research group of ProfPaolo Grieco (Department of Pharmacy University of NaplesFederico II)

22 Peptide Synthesis Peptide Cys-UT-II (H-Cys-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) was synthesized byadopting the solid-phase peptide synthesis (SPPS) using theFmoctBu orthogonal strategy as reported elsewhere [24]Upon cyclization procedure performed in solid phase [25]an additional Cys residue was added in the N-terminal byfollowing standard procedures [26] (e product was pu-rified and characterized by reverse-phase HPLC (RP-HPLC)

23 LiposomePreparation Liposomes composed of DPPCcholDSPE-PEG2000 or DPPCcholDSPE-PEG2000-mal-eimide at a 65 30 5 molar ratio were prepared by hy-dration of a thin lipid film followed by extrusion Brieflylipids were dissolved in 1mL of a mixture chloroformmethanol (2 1 vv) the resulting solution was added to a50mL round-bottom flask and the solvent was removedunder reduced pressure by using a rotary evaporator(Laborota 4010 digital Heidolph Schwabach Germany)(en the resulting lipid film was hydrated with 1mL ofcitrate buffer at pH 4 (150mM sodium citrate 150mMcitric acid) and the resulting suspension was gently mixedin the presence of glass beads until the lipid layer wasremoved from the glass wall after that the flask was left at50degC for 2 h (e liposome suspension was then extrudedat 50degC using a thermobarrel extruder system (NorthernLipids Inc Vancouver BC Canada) passing repeatedlythe suspension under nitrogen through polycarbonatemembranes (5 times for each membrane) with decreasingpore sizes from 400 to 100 nm (Nucleopore TrackMembrane 25mm Whatman Brentford UK) (e ex-ternal buffer was removed by ultracentrifugation (OptimaMax E Beckman Coulter USA rotor TLA 1202) at80000 rpm at 4degC for 40min and the liposomes wereresuspended with 1mL of phosphate buffer at pH 74(140mM sodium chloride 25mM of HEPES 01 mM ofEDTA) After preparation liposomes were stored at 4degCEach formulation was prepared in triplicate For in-tracellular penetration studies fluorescent-labeled lipo-somes containing 01 (ww) of Bodipy-cholesterolwere prepared similarly by adding the fluorescent cho-lesterol in the lipid mixture of DPPCcholDSPE-PEG2000or DPPCcholDSPE-PEG2000-maleimide during the li-posome preparation

24 Preparation of Liposomes Conjugated with UT-IICys-UT-II (H-Cys-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) was conjugated with liposomes prepared with mal-eimide-modified polyethylene glycol Briefly the UT-IIpeptide (0250mM) was conjugated to liposomes containingDSPE-PEG2000-maleimide with gentle magnetic agitation atroom temperature overnight (e resulting liposomes so-called LipoUT were then chromatographed on a Sepharose

2 Journal of Oncology

CL-4B column in phosphate buffer at pH 74 All liposomepreparations were stored at 4degC (e formulations wereprepared in triplicate

25 Preparation of Liposomes Encapsulating DoxorubicinDoxo was encapsulated into liposomes by remote loading[27] Briefly the liposome suspension was combined withthe doxo in a druglipid ratio of 02 (ww) and then in-cubated at 50degC for 1 h (e un-encapsulated doxo wasremoved by ultracentrifugation at 80000 rpm at 4degC for40min After purification the formulation was conjugatedwith UT-II as described above to obtain liposomes en-capsulating doxo and conjugated with UT-II (LipoUT-doxo)

26 Determination of Lipid Concentration (e concentra-tion of lipids present in the suspensions after preparationwas determined using the Stewart assay [28] Briefly analiquot of the suspension was added to a two-phase systemconsisting of an aqueous ammonium iron thiocyanate(FeSCN3) solution or iron thiocyanate solution (01N) andCHCl3 (e concentration of DPPC was obtained by mea-suring the absorbance at 485 nm into the organic layer withan ultraviolet-visible spectrophotometer (UV VIS 1204Shimadzu Corporation Kyoto Japan) Quantification ofDPPC was carried out by means of a calibration curve withstandard DPPC samples

27 Liposome Size and Zeta Potential Dimensional analysiswas performed at 20degC by photon correlation spectroscopy(PCS) (N5 Beckman Coulter Miami USA) Each samplewas diluted in deionizerfiltered water (022 μm pore sizepolycarbonate filters MF-Millipore Microglass Heim Italy)and analyzed with a detector at 90deg angle For measuring theparticle size distribution polydispersity index (PI) was usedFor each formulation the mean diameter and PI werecalculated as the mean of three different batches(e averagediameter and the size distribution of each formulation weredetermined (e results were expressed as liposome meandiameter (nm) and polydispersity index (PI) (e zeta po-tential (ζ) of liposomes was performed by the Zetasizer NanoZ (Malvern UK) Briefly an aliquot of each sample (10 μL)was diluted in filtered water and analyzed (e results werecalculated by the average of 20 measurements

28 Determination of Doxo Concentration (e amount ofun-encapsulated doxo was determined as follows 1mL ofliposome dispersion was ultracentrifugated at 80000 rpm at4degC for 40min Supernatants were carefully removed anddoxo concentration was determined by using a UVvisiblespectrophotometer at 480 nm (e results were expressed asencapsulation efficiency and were calculated as follows

1 minusTSdoxo minus ASdoxo( 1113857

TSdoxotimes 1001113890 1113891 (1)

where TSdoxo is the theoretical doxo in the supernatant andASdoxo is the actual doxo concentration in the supernatant

29 UT-II Binding to the Liposome (e amount of UT-IIlinked to the liposomes was carried out by a high-perfor-mance liquid chromatography (HPLC) method (e HPLCsystem consisted of an isocratic pump (LC-10A VP Shi-madzu Kyoto Japan) equipped with a 7725i injection valve(Rheodyne Cotati USA) and SPV-10A UV-Vis detector(Shimadzu) set at the wavelength of 254 nm(e system wascontrolled by a SCL-10A VP System Controller (Shimadzu)connected to a computer Chromatograms were acquiredand analyzed by a Class VP ClientServer 721 program(Shimadzu Scientific Instruments Columbia MD USA)(e quantitative analysis of UT-II was performed by reverse-phase chromatography (RP-HPLC) on a Luna 5 μm C18column (250times 460mm 110 A Phenomenex Klwid USA)equipped with a security guard (e mobile phase was amixture 40 60 (vv) of acetonitrile and filtered water (eanalyses were performed in isocratic condition at a flow rateof the mobile phase of 1mLmin and at room temperature(e amount of UT-II present on the surface of nanocarrierswas determined as follows Briefly 1mL of conjugated li-posomes dispersion was ultracentrifugated (Optima Max EBeckman Coulter USA) at 80000 rpm at 4degC for 40minSupernatants were carefully removed and the amount ofUT-II unlinked to liposomes was determined by RP-HPLC(e amount of UT-II was calculated by means of a cali-bration curve with UT-II samples (e results have beenexpressed as follows

1 minusUT-IIactual minus UT-IItheor( 1113857

UT-IItheortimes 1001113890 1113891 (2)

where UT-IIactual is the UT-II found in the supernatant andUT-IItheor is the theoretical UT-II added to the liposomes

210 Cell Culture Prostate (DU145 PC3 and LNCaP) andcolon (WIDR and LoVo) cancer cell lines were obtainedfrom American Type Culture Collection (ATCC RockvilleMD httpwwwatccorg) PC3 DU145 and WIDR celllines were grown in DMEM while LNCaP and LoVo weregrown in RPMI 1640 Both cell media were supplementedwith 10 heat-inactivated fetal bovine serum 20mMHEPES 100UmL penicillin 100mgmL streptomycin 1L-glutamine and 1 sodium pyruvate All the cells werecultured at a constant temperature of 37degC in a humidifiedatmosphere of 5 carbon dioxide (CO2)

211Cell ProliferationAssay After trypsinization all the celllines were plated in 100 μL of medium in 96-well plates at adensity of 2times103 cellswell One day later the cells weretreated with free doxo plain Lipo LipoUT Lipo-doxo andLipoUT-doxo at concentrations ranging from 20 μM to0156 μM

Cell proliferation was evaluated by MTT assay Brieflycells were seeded in serum-containing media in 96-wellplates at the density of 2times103 cellswell After 24 h

Journal of Oncology 3

incubation at 37degC the medium was removed and replacedwith fresh medium containing all developed formulations atdifferent concentrations Cells were incubated under theseconditions for 72 h (en cell viability was assessed by MTTassay (e MTT solution (5mgmL in phosphate-bufferedsaline) was added (20 μLwell) and the plates were incubated

for further 4 h at 37degC (e MTT-formazan crystals weredissolved in 1N isopropanolhydrochloric acid 10 solution(e absorbance values of the solution in each well weremeasured at 570 nm using a BioRad 550 microplate reader(BioRad Laboratories Milan Italy) Percentage of cell via-bility was calculated as

100lowast (3)(absorbance of the treated wells minus absorbance of the blank control wells)

(absorbance of the negative control wells minus absorbance of the blank control wells) (3)

(en the concentrations inhibiting 50 of cell growth(IC50) were obtained and these values were used for sub-sequent experiments MTTassay was carried out by triplicatedetermination on at least three separate experiments Alldata are expressed as meanplusmn SD

212 Evaluation of UTR Expression by FACS Analysis andWestern Blot Untreated cells were fixed for 20min with a3 PFA solution and permeabilized for 10min with 01 (vv) Triton X-100 in phosphate buffered saline at roomtemperature To prevent nonspecific interactions of anti-bodies cells were treated for 2 h in 5 (wv) BSA inphosphate-buffered saline and then were incubated with aspecific rabbit Ab raised against UTR (1 50 in blockingsolution 3 BSA in TBS-Tween 01) for 2 h at 37degC Afterseveral washes cells were incubated with a secondary goatanti-rabbit IgG-FITC (Life Technologies Carlsbad CA)diluted at 1 50 in blocking solution for 1 h at room tem-perature (e FITC fluorescence was measured with aFACSCalibur flow cytometer (Becton Dickinson) on10000 cells for each sample using the CellQuest software(Becton Dickinson) Mean fluorescence intensity (MFI) ofanti-rabbit-FITC sample was considered as 100

Equal amounts of cell proteins from untreated cells wereseparated by SDS-PAGE electrophoresis (BioRad Cal-ifornia USA) Proteins were electrotransferred to nitrocel-lulose membrane by using Trans Blot Turbo (BioRadCalifornia USA) Membrane were washed in TBST (10mMTris pH 80 150mM NaCl 005 Tween 20) and wereincubated with specific MAbs and later with horseradishperoxidase-conjugated secondary antibodies (e rabbitantibodies raised against UTR and the mouse antibodyraised against α-tubulin were purchased from Santa CruzBiotechnology Blots were then developed using enhancedchemiluminescence detection reagents ECL ((ermo FisherScientific Rockford IL) and exposed to X-ray film All filmswere scanned by using Quantity One software (BioRadChemi Doc)

213Analysis of Liposome IntracellularDistributionbyFACSandConfocalMicroscopy BD FACSCalibur fluorescent-activatedflow cytometer and the BD CellQuest software (BD Bio-sciences) were used to perform flow cytometry analysis ofliposome intracellular distribution For these experimentsBodipy-labeled liposomes were used on DU145 and PC3

cells Bodipy is a bright green-fluorescent dye with similarexcitation and emission to fluorescein (FITC) or Alexa Fluor488 dye Green fluorescent-labeled liposomes were added tothe medium of DU145 or PC3 cells at a fixed concentrationAfter 6 24 48 and 72 h of incubation at 37degC removal ofunattached liposomes was accomplished by washing the cellswith phosphate-buffered saline at pH 74 for 1min Cellswere then detached by trypsinization (e samples weresubsequently washed twice and for each sample 10000 cellswere measured by using FACSCalibur flow cytometer(Becton Dickinson) using the CellQuest software (BectonDickinson) After 6 h of incubation with a fixed concen-tration (10 μM) of Bodipy-labeled liposomes DU145 andPC3 cells were fixed for 20min with a 3 PFA solution andpermeabilized for 10min with 01 (vv) Triton X-100inphosphate-buffered saline at room temperature To preventnonspecific interactions of antibodies the cells were treatedfor 2 h in 5 (wv) BSA in phosphate-buffered saline andthen were incubated with a specific mouse Ab raised againstvimentin (1 50 in blocking solution 3 BSA in TBS-Tween01) for 2 h at 37degC After several washes the cells wereincubated with a secondary IgG goat anti-mouse-AlexaFluor 633 (Life Technologies Carlsbad CA) diluted at 1 50in blocking solution for 1 h at room temperature (e slideswere mounted onmicroscope slides byMowiol(e analyseswere performed with a Zeiss LSM 510 microscope equippedwith a plan-apochromat objective X 63 (NA 14) in oilimmersion (e fluorescence of the Bodipy-labeled lipo-somes and vimentin was collected in the multitrack modeusing BP550-625 and LP650 as emission filters respectively

3 Statistical Analysis

All data were expressed as mean valuesplusmn SD of at least threeindependent experiments Statistical analysis was obtainedusing ANOVA and significant differences were determinedat ple 001 Graphs were obtained using SigmaPlot v110(Systat Software San Jose CA USA)

4 Results

41 Liposome Characterization Here we used a shortpeptide of eight amino acids (peptide 4-11) of the UT-II withonly one binding site In particular a cysteine was added tothe NH2-terminal of the peptide to provide a unique site forthe conjugation with the nanocarrier (e conjugation hasbeen carried out at the extremity of PEG chain present on the


liposome surface by using maleimide groups that react withcysteine residues on ligands to form stable thioether bondsTo this aim liposomes with DSPE-PEG2000-maleimide wereused for the conjugation For comparison purpose lipo-somes with DSPE-PEG2000 were also prepared (e char-acteristics of LipoUT ie mean diameter PI and zetapotential are summarized in Table 1 (e mean diameter ofnonconjugated liposomes was 14784 nm (e conjugationwith the peptide significantly influenced liposome charac-teristics with a slight increase of the mean diameter(1684 nm) and a higher PI due to the presence of the peptideon the surface (Table 1) Moreover after conjugation of UT-II LipoUT compared with the unconjugated liposomesshowed a significant zeta potential decrease due to thepresence of peptide linked on the PEG extremity In fact thepercentage of UT-II present on the surface of liposomeswas about 61 (en the nanocarriers were loaded with acytotoxic agent doxo widely used in the clinical settingLiposomes with an internal aqueous phase containing acitrate buffer at pH 4 were prepared (e same liposomeswere then purified and resuspended in a phosphate buffer atpH 74 to create a pH transmembrane gradient PEGylatedliposomes encapsulating doxo (Lipo-doxo) and UT-II-conjugated PEGylated liposomes encapsulating doxo (Lip-oUT-doxo) were prepared (e characteristics of theseformulations are reported in Table 2 (e encapsulation ofdoxo into the liposomes did not affect their characteristicsIndeed Lipo-doxo had a mean diameter of about 150 nmwith a narrow size distribution (PIle 01) As expected doxoencapsulation efficiency was very high in both formulationsIndeed Lipo-doxo and LipoUT-doxo had a doxo encap-sulation efficiency of 90 and 96 respectively (Table 2) (eencapsulation of doxo did not affect the ζ of liposomessuggesting that negligible amount of the drug was associatedwith the liposome surface following purification (e dif-ferent surfaces of Lipo and LipoUT due to the presence ofDSPE-PEG2000 and DSPE-PEG2000-maleimide respectivelyhad only a slight influence on the doxo encapsulationprocess

42ExpressionofUTRonCancerCellLines In order to assessthe ability of UT-II-conjugated liposomes to efficientlydeliver doxo in cancer cells that express UTR we evaluated

the expression of the receptor in different human prostate(DU145 PC3 and LNCaP) and colon (WIDR and LoVo)cancer cell lines

(eUTR expression was investigated by western blottingin colon cancer cells In detail both cell lines expressed thereceptor but the level of expression was higher in WIDR(about 4-fold) compared to LoVo (Figure 1(a)) FACSanalysis was used to detect UTR expression on cell mem-brane of prostate cancer cells Also in this case the receptorwas expressed in all prostate cancer cells but at higher levelsin PC3 (25-fold) and LNCaP (15-fold) cells compared toDU145 (Figure 1(b))

In conclusion UTR protein was expressed in all theassessed cell lines the level of expression was higher inWIDR PC3 and LNCaP cells compared with LoVo andDU145

43 Liposome Uptake and Intracellular Distribution To in-vestigate if UT-II peptide conjugation to liposomes canenhance uptake and improve intracellular distribution of theformulations FACS and confocal laser scanning microscopy(CLSM) were performed on DU145 and PC3 cells treatedwith fluorescent-labeled liposomes (e fluorescence asso-ciated to the cells was evaluated by FACS after 6 24 48 and72 h of incubation with Bodipy-labeled liposomes used at afixed concentration

In particular in both cell lines (DU145 and PC3) after6 h of incubation with LipoUT or Lipo FACS analysis de-tected the of mean fluorescence intensity (MFI) ascompared with the untreated controls (Figure 2) MFI ofcontrol was considered as 100 In both cell lines we ob-served a higher internalization of LipoUTcompared to Lipobut at a major extent in PC3 In detail in DU145 that haslower UTR expression LipoUT induced about 30 of MFIincrease (2000) compared with Lipo (1700) while in PC3that has higher UTR expression LipoUT induced about 70of MFI increase (1700) compared with Lipo (1000)(Figure 2) In both cases MFIs gradually decreased in a time-dependent manner after 24 h

(e intracellular distribution of Bodipy-labeled lipo-somes after 6 h of incubation was studied by confocal mi-croscopy analysis CLSM showed a higher widespread andintense green fluorescence intensity both in DU145 and PC3

Table 1 Mean diameter PI and zeta potential (ζ) of plain liposomes (Lipo) and liposomes conjugated with UT-II (LipoUT)

Formulation Diameter (nmplusmn SD) PIplusmn SD ζ (mVplusmn SD) UT-II on the surface of liposomes ()Lipo 1478plusmn 690 0098plusmn 003 minus 1958plusmn 590 mdashLipoUT 1684 + 955 0203 + 007 minus 2610 + 434 61

Table 2 Mean diameter PI and zeta potential (ζ) of liposomes encapsulating doxo conjugated (LipoUT-doxo) and unconjugated (Lipo-doxo)

Formulation Encapsulation efficiency doxo()

Diameter(nmplusmn SD) PIplusmn SD ζ (mVplusmn SD) UT-II on the surface of liposomes ()

Lipo-doxo 90 1489plusmn 827 0102plusmn 006 minus 1876plusmn 374 mdashLipoUT-doxo 96 1909plusmn 1742 0247plusmn 011 minus 2889plusmn 215 60


cells incubated with UT-II-conjugated liposomes (LipoUT)compared to Lipo (Figures 3(a) and 3(b)) Moreover whencomparing the fluorescence into DU145 and PC3 cellshigher fluorescence was observed in PC3 cells (about 2-foldhigher) (is was likely due to the higher expression of UTRin PC3 cell line

44 Effects of Liposome Encapsulating Doxorubicin Conju-gated or Not with UT-II on Cancer Cell Lines (e effects offree doxo Lipo and liposome encapsulating doxo andconjugated (LipoUT-doxo) or not (Lipo-doxo) with UT-IIwere evaluated on the proliferation of prostate (DU145 PC3and LNCaP) and colon (WIDR and LoVo) cancer cell lines

lowastlowast

WIDR LoVo

WIDR LoVo

UTR

α-Tubulin

0

50

100

150

200

o

f arb

itrar

y un

its

(a)

CTR

Anti-rabbit-FITCAnti-UTR

CTR


DU145

FL1-H

Cou

nts

101100 103102 104

101100 103102 104

101100 103102 104

200

160

120

80

40

0

PC3

Cou

nts

FL1-H

200

160

120

80

40

0

MFI

DU

145

PC3

LNCa

P

lowastlowast

700

600

500

400

300

200

100

0

CTR


LNCaP

FL1-H

Cou

nts

200

160

120

80

40

0

(b)

Figure 1 (a) Evaluation of UTR expression on colon (WIDR and LoVo) cancer cells by western blotting analysis (e expression ofα-tubulin was assessed as loading control (left panel)(e intensities of the bands were expressed as arbitrary units and detected by QuantityOne software (BioRad Chemi Doc) (right panel) Error bars showed standard deviation from the mean in at least three independentexperiments Bars SDs lowastlowastple 001 (b) Evaluation of UTR expression on DU145 PC3 and LNCaP cell lines by FACS analysis (left panel andtop right panel) (e MFIs of control were calculated as described in Materials and Methods and represented as columns (bottom rightpanel) (e experiments were performed at least three times and the results were always similar Bars SDs lowastlowastple 001


by MTT assay as reported in ldquoMaterials and MethodsrdquoDoxo Lipo-doxo and LipoUT-doxo induced a dose-de-pendent growth inhibition in all the cell lines after 72 hwhile treatment with Lipo did not produce signicant cy-totoxic eects (Figures 4 and 5)

In Table 3 results are reported as concentrationsinhibiting 50 of cell growth (IC50) after 72 h of treatmentAfter 72 h IC50 of free doxo were equal to 06 μM 4 μM1 μM 06 μM and 125 μM for DU145 PC3 LNCaP WIDRand LoVo respectively (see Table 3) e doxo encapsulatedin liposomes (Lipo-doxo) induced a 50 growth inhibitionat a concentration of 2 μM 5 μM and gt20 μM in DU145PC3 and LNCaP respectively (Table 3) and 10 μM and7 μM in WIDR and LoVo respectively (Table 3) On theother hand LipoUT-doxo inhibited the 50 of cell growth ata concentration of 15 18 and 10 μM on DU145 PC3 andLNCaP respectively and 25 μM and 5 μM on WIDR and

LoVo respectively Although PC3 expresses high levels ofUTR LipoUT-doxo resulted less cytotoxic than Lipo-doxobased on the evaluation of IC50 However it is worthy of notethat the activation of UT-II can induce cell proliferation infact we found that liposomes conjugated with UT-II (Lip-oUT) increased cell proliferation in particular in PC3LNCaP andWIDR that overexpress UTR On these bases wetested all the formulations by using a very high concentrationof doxo to antagonize the eect of UT-II on cell proliferation(Figure 6) erefore comparing the eects of LipoUTcontaining doxo with empty LipoUT (155-fold decrease ofcell growth) these eects were stronger than those induced byLipo containing doxo compared to empty Lipo (216-folddecrease of cell growth) also in PC3 cells Similarly also on theother cell lines overexpressing UTR the dierence in cellgrowth inhibition was markedly increased considering theproliferative eects caused by empty LipoUT

DU145

CtrLipo 6hLipoUT 6h

Lipo 24hLipoUT 24hLipo 48h

LipoUT 48hLipo 72hLipoUT 72h

100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT

Figure 2 FACS analysis ofDU145 andPC3 cells after 6 24 48 and 72h of incubation at 37degCwith Bodipy-labeled liposomes conjugated or notwithUT-II (left panel) e mean centuorescence intensity (MFI) of control was calculated as described in ldquoMaterials and Methodsrdquo and represented ascolumns (right panel) e experiments were performed at least three times and the results were always similar Bars SDs lowastlowastple 001


5 Discussion

Liposomal nanoparticles represent the most investigatednanotechnology platform with a plethora of novel appli-cations still to discover (e success of liposomes as drug

delivery systems is due to different factors such as bio-compatibility and biodegradability (when using phos-pholipids from natural source) versatility of theformulation possibility to encapsulate drugs with differentlipophilicity design relatively easy and possible scale-up

PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy

RED = VimentinGREEN = Lipo-Bodipy

(a)

Control0

20

40

60

80

100

DU 145 PC3

Lipo-Bodipy LipoUT-Bodipy

lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)

Figure 3 (a) Confocal microscopy images of DU145 and PC3 after 6 h of incubation at 37degC with fluorescent-labeled liposomes Representativeimages of control unconjugated liposomes (Lipo-Bodipy) and liposomes conjugated with UT-II (LipoUT-Bodipy) For each cell line images onthe left panel report that the red fluorescence associated with the Ab against vimentin images on the right panel report the green fluorescenceassociatedwith Bodipy(e cells were visualizedwith a confocal microscope atmagnification 100x (b) Quantification of fluorescence intensity wasreported as arbitrary units (e experiments were performed at least three times and the results were always similar Bars SDs lowastlowastple 001


for large production in GMP quality Liposomes have beenextensively used in cancer therapy to change drug bio-distribution In particular liposomes allow addressing theencapsulated drug towards tissues characterized by vesselswith an enhanced permeability of the endothelium such astumors [29 30] Stealth properties are obtained by mod-ication of the liposome surface eg by including poly-ethylene glycol (PEG) PEG has a centexible chain thatoccupies the space immediately adjacent to the liposomesurface (ldquoperiliposomal layerrdquo) thus excluding othermacromolecules eg blood plasma opsonins from thisspace erefore the presence of PEG prevents liposomeinteraction with the reticulo-endothelial system (RES) andincreases the accumulation in solid tumors due to anincreased permeability of tumor vasculature [31] for the

presence of fenestrated endothelium in tumor blood vessels(passive targeting) [32] A longer circulation time results ina higher probability of liposome extravasation in tissueswhere capillaries are characterized by fenestrated endo-thelium ie liver and spleen or in tumors in which thehigh permeability of the vessels is associated to a reducedlymphatic drainage (enhanced permeability retention orEPR eect) [33]

Here stealth liposomes were proposed as systems totarget cancer cells overexpressing UTR and to promote drugdelivery of anticancer agents ie doxorubicin into colonand prostate cancer cells UT-II is a vasoactive peptide ableto bind the UTR a receptor involved in a number ofphysiological processes in dierent organs is receptor hasbeen found to be involved in proliferation motility and

001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo

Free doxoBlank liposomeLipo-doxo

lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)

Figure 4 Eect of all developed formulations on prostate cancer cell lines (DU145 PC3 and LNCaP) proliferation Prostate cancer cellswere seeded in serum-containing media in 96-well plates at the density of 2times103 cellswell After 24 h incubation at 37degC cells were treatedwith increasing concentrations of free doxo Lipo Lipo-doxo LipoUT and LipoUT-doxo (20-016 μM) for 72 h Cell viability was assessedby MTT assay as described in Materials and Methods lowastlowastple 001


Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo

Figure 5 Eect of all developed formulations on colon cancer cell lines (WIDR Lovo) proliferation Colon cancer cells were seeded inserum-containing media in 96-well plates at the density of 2times103 cellswell After 24 h incubation at 37degC cells were treated with increasingconcentrations of free doxo Lipo Lipo-doxo LipoUT and LipoUT-doxo (20-0 16 μM) for 72 h Cell viability was assessed byMTTassay asdescribed in Materials and Methods lowastlowastple 001

Table 3 IC50 values after 72 h treatment with free doxo doxo encapsulated in liposomes (Lipo-doxo) and doxo encapsulated in liposomesconjugated with UT-II (LipoUT-doxo)

Cell lines Doxo (IC50μm) Lipo-doxo (IC50μm) LipoUT-doxo (IC50μm) UTR expressionDU145 06plusmn 0016 2plusmn 0013 15plusmn 0076 LowPC3 4plusmn 0031 5plusmn 0018 18plusmn 0089 HighLNCaP 1plusmn 0011 gt20plusmn 0091 10plusmn 0045 HighWIDR 06plusmn 0018 10plusmn 0087 25plusmn 0018 HighLoVo 125plusmn 0013 7plusmn 0064 5plusmn 0013 Low

0

50

100

150

200

250

DU145 PC3 WIDR LoVo LNCaP

o

f cel

l gro

wth

CTRdoxoLipo

LipoUTLipo-doxoLipoUT-doxo

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

Figure 6 Cell proliferation of all developed formulations in prostate (DU145 PC3 and LNCaP) and colon (WIDR and LoVo) cancer celllines Cancer cells were seeded in serum-containing media in 96- well plates at the density of 2times103 cellswell After 24 h incubation at 37degCcells were treated with 10 μM of free doxo Lipo plain liposomes LipoUT PEGylated liposomes conjugated with UT-II peptide Lipo-doxoPEGylated liposomes-encapsulated doxo LipoUT-doxo PEGylated liposomes conjugated with UT-II peptide and encapsulated with doxoLipo Lipo-doxo LipoUT and LipoUT-doxo for 72 h Cell viability was assessed by MTT assay as described in Materials and Methodslowastlowastple 001


invasion of several cancer cells among them are the colon[34] and human prostate [7] cancer cells Moreover UTRwas overexpressed in prostate and colon cancers and itsexpression was correlated to the clinical outcome of neo-plastic patients representing a negative prognostic factor

(e conjugation of ligands able to specifically bind re-ceptors that are overexpressed on cancer cells to liposomesurface represents an efficient active targeting strategy toenhance selectivity and efficiency of drug delivery systems(is is especially important to reduce the toxic effect of theantitumor therapies (us specific ligands (ie transferrinfolic acid monoclonal antibodies and peptides) able to bindreceptors overexpressed on the surface of cancer cells havebeen coupled on the liposome surface

It is worthy of note that when considering proteins suchas transferrin or monoclonal antibodies different bindingsites are present on each molecules (erefore their con-jugation with nanovectors can lead to a heterogeneousconjugate with a quality of the final product difficult tocontrol Based on these considerations we used a shortpeptide of eight amino acids (peptide 4-11) of the UT-II withonly one binding site In particular a cysteine was added tothe NH2-terminal of the peptide to provide a unique site forthe conjugation with the nanocarrier (e introduction ofthe cysteine residue was followed by purification andcharacterization (by reverse-phase HPLC as reported in theMaterials andMethods section) thus excluding the presenceof by-products ie products derived by the reduction of thedisulfide bridge Moreover the formulation proposed in thisstudy has been developed following an optimization study tofind the experimental conditions for the highest liposomeUT-II conjugation degree In our case a conjugation degreeof about 61 was achieved (checked by HPLC) It is worthyof note that a significant percentage of the maleimide res-idues are not exposed on the external surface of the lipo-somes thus suggesting that the percentage of conjugationobtained here is close to the highest possible (en theobtained nanocarriers were loaded with a cytotoxic agentdoxorubicin (doxo) widely used in the clinical settingDoxorubicin (doxo) is an anthracycline with significantanticancer properties Unfortunately its use is limited by theinduction of severe cardiotoxicity that occurs throughpoorly investigated mechanisms Recently it was found [35]that doxorubicin induces senescence in human vascularsmooth muscle cells (VSMC) also at low calcium concen-trations On the other hand UTR activated by UT-II pro-duces second messengers ie inositol triphosphate anddiacylglycerol which induce calcium release (Ca2+) andconsequently activate Ca2+-dependent kinases (CaMKs)leading to cell proliferation [36] In this light UT-IIUTRcomplex formation in our experimental model (UT-IIarmed liposomes) could counteract the doxo-mediateddetrimental effects on VSMC(erefore we selected doxo inorder to demonstrate the high efficiency of liposomes intargeting UTR-expressing cancer cells but on the basis of thehistotype of the selected tumors other drugs with lowercardiovascular side effects could be encapsulated (e firstliposomal formulation encapsulating doxo Doxil was ap-proved by FDA in 1995(e encapsulation of doxorubicin in

stealth liposomes showed a significant decrease of the car-diotoxicity and enhanced the efficacy of doxorubicin due toa higher accumulation in tumors in drug-resistant tumorscompared with free doxorubicin [12]

Doxo was also chosen because it can be easily encap-sulated into liposomes by the well-known remote-loadingmethod [27] (is technique allows the achievement of highdrug encapsulation efficiency by using a transmembrane pHgradient Moreover doxo is a fluorescent molecule easilyassessable in cells We preferred the use of indirect methodfor a faster determination of doxorubicin and conjugatedpeptide due to the presence of the new lipid-peptide con-jugate in the formulation Indeed the newly synthesizedPEGylated lipid conjugated with UT-II was considered asource of bias due to the not well-known solubility in organicsolvents (cosolubilization of lipid mix with doxorubicin) andto a difficult-to-predict interaction with surfactants such asTriton X-100 (eg method described by A Fritze et al) [37](us a rapid determination of the doxorubicin encapsu-lation was achieved by an indirect method as previouslyreported by others [38] Similarly the dithiotreitol cleavageshould be validated for the newly-synthesized PEGylatedlipid conjugated with UT-II while we used an indirectdetermination by the HPLC method largely validated inprevious studies on the same peptide [10](e encapsulationof doxo into the liposomes did not affect their character-istics As expected doxo encapsulation efficiency was veryhigh in both formulations (e encapsulation of doxo didnot affect the ζ of liposomes suggesting that negligibleamount of the drug was associated to the liposome surfacefollowing purification On the other hand the conjugation ofUT-II to liposomes did not significantly affect the meandiameter of the liposomes when compared to Lipo-doxo butproduced a PI increase and a significant zeta potential de-crease Despite this liposomes with a mean diameter ofabout 169 nm can still be considered suitable for intravenousadministration especially when considering the PI of about02 Moreover after conjugation of UT-II with liposomesLipoUTcompared with the unconjugated liposomes showeddifferent ζ due to the presence of peptide linked on the PEGextremity In fact the percentage of UT-II present on thesurface of liposomes was about 61

In order to assess the ability of UT-II-conjugated lipo-somes to efficiently deliver doxo in cancer cells that expressUTR we evaluated the expression of the receptor in differenthuman prostate (DU145 PC3 and LNCaP) and colon(WIDR and LoVo) cancer cell lines

UTR protein was expressed in all the assessed cell linesthe level of expression was higher in WIDR PC3 andLNCaP cells compared with LoVo and DU145 To in-vestigate if the UT-II peptide conjugation to liposomes canenhance uptake and improve intracellular distribution of theformulations FACS and confocal laser scanning microscopy(CLSM) were performed on DU145 and PC3 cells treatedwith fluorescent labeled liposomes In particular we foundthat cell uptake of Bodipy-labeled liposomes in PC3 andDU145 was higher for LipoUT than the not armed coun-terparts but at major extent in PC3 cells that express higherUTR levels


Many efforts have been made to increase selectivity andthe efficacy of the liposome delivery (e strategy of activetargeting has been successfully used to specifically deliverdrugs to cancer cells Moreover it is possible to target re-ceptors able to be internalized upon binding with the li-gands thus promoting the intracellular delivery of the drugassociated with the ligand with consequent increase of thecytotoxicity [14 15] UTR is expressed in several humantissues such as heart brain aorta and kidney It has beenreported [39] that UT-II dissociation from UTR is an ir-reversible mechanism In fact when an excess of unlabeledUT-II was added in human rhabdomyosarcoma cells only15 of [125I] UT-II was dissociated after 90min In-ternalization experiments performed with radioactive-la-beled UT-II (125I-UT-II) showed that about 70 ofactivated UTR receptors on the cell membrane of humanembryonic kidney 293 cells were internalized through en-docytosis mechanisms within 30 minutes (half life [th]56plusmn 02 minutes) (e internalization of UT-IIUTRcomplex was dynamin-dependent and arrestin-in-dependent After UT-II dissociated from UTR the receptorquantitatively recycled back to the plasma membrane within60 minutes (th 319plusmn 26 minutes) [40] (e low rate ofdissociation and the continuous externalization of UTRdetermine the long-lasting UT-II-mediated effects UTR forits specific internalization characteristics is a suitablemolecule for targeting purposes because it is easily in-ternalized rapidly dissociates from the ligand and is alsorecycled on the cell membrane avoiding downregulationeffects due to the binding with its own ligand

On these bases the effects of free doxo Lipo LipoUT-doxo and Lipo-doxo were evaluated on the proliferation ofprostate (DU145 PC3 and LNCaP) and colon (WIDR andLoVo) cancer cell lines LipoUT-doxo was more active thanLipo-doxo on the growth inhibition of cells that overex-pressed UTR (LNCaP andWIDR) while in LoVo and DU145cell lines the activity was similar to or lower than that one ofLipo-doxo respectively Although PC3 express high levels ofUTR LipoUT-doxo resulted to be less cytotoxic than Lipo-doxo based on the evaluation of IC50 (ese findings seem incontrast with observation of confocal microscopy in whichhigher liposome uptake was observed in UTR overexpressingcells However it is worthy of note that the activation of UTRby the agonist UT-II can induce cell proliferation as describedin our previous work [4] in fact we found that empty li-posomes conjugated with UT-II (LipoUT) increased cellproliferation in particular in PC3 LNCaP and WIDR thatoverexpress UTR On these bases we tested all the formu-lations by using a very high concentration of doxo to an-tagonize the effect of UT-II on cell proliferation Based uponthese considerations the growth inhibitory effects obtainedwith LipoUT-doxo was underestimated due to the counter-acting proliferative effect caused by UT-II expressed on thesurface of the liposomes (erefore comparing the growth ofLipoUT containing doxo with empty LipoUT the effects ofthe former were much stronger than those induced by Lipocontaining doxo as compared to empty Lipo in PC3 cellsSimilarly also on the other cell lines overexpressing UTR thedifference in cell growth inhibition was markedly increased

considering the proliferative effects caused by empty LipoUTIn these conditions the effects of LipoUT containing doxowere more potent also than those induced by free doxo in thecells overexpressing UTR

Different anticancer drugs have been incorporated innanocarriers to address several issues such as the poor solu-bility of drugs the drug instability in biological fluids andserious side effects due to the drug accumulation into thehealthy tissues together with low concentration at the actionsite [41] More recently nanomedicine has been proposed alsoto overcome the multidrug resistance (MDR) of cancer cells tochemotherapeutic agents that promote an overexpression orsuppression of certain molecular pathways with consequenttreatment failure [42ndash44] Similarly liposomes and in generallipid nanovectors modified with specific ligands were used toenhance drug delivery into tumors In particular the access ofdrugs to the central nervous system (CNS) is hampered by theblood-brain barrier (BBB) formed by brain endothelial cellsconnected by tight junctions However in presence of braintumors the increase of the intracranial pressure leads to partialalternation of the BBB permeability (us colloidal particlescan access in these conditions the CNS On the other handmodification of nanocarrier surface with specific endogenousor exogenous ligands eg transferrin promoted enhancedBBB crossing also in case of unaltered endothelium [45 46]On these bases and according to the obtained data targetingtumor-related receptors could represent a successful drugdelivery strategy against tumors Additional in vivo studies areneeded to better investigate the efficiency and specificity ofthese new delivery systems and we would have benefit oftransparent in vivo models such as zebrafish embryos toevaluate fluorescent-labeled liposome biodistribution [47ndash51]

6 Conclusion

In conclusion we have developed liposomes conjugated withUT-II (LipoUT) for efficient targeting of cancer cells thatoverexpress UTR and promoting drug delivery of an anti-cancer agent ie doxorubicin into colon and prostatecancer cells We demonstrated that LipoUT-doxo was moreactive than Lipo-doxo on the growth inhibition of cells thatoverexpressed UTR (PC3 LNCaP and WIDR) Moreoverwe found that cell uptake of Bodipy-labeled liposomes inPC3 and DU145 was higher for LipoUT Taken together thedata obtained in this work suggest that the encapsulation ofdoxo in UT-II-targeted liposomes potentiated its delivery inUTR overexpressing cells and could represent a new tool forthe targeting of prostate and colon cancer

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Disclosure

(e abstract was submitted for the poster presentation in the15th Naples Workshop on bioactive peptides ldquoPeptidesrecent developments and future directionsrdquo held in Naples


on June 23ndash25 2016 Moreover it was submitted for theposter presentation in the congress ldquo(erapeutic Nano-products from Biology to innovative Technologyrdquo held inRome on June 19-20 2019

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors thank Dr Marco Bocchetti (PhD student) forhis assistance with statistical analysis (is work was sup-ported by AIRC (IG 2017 code 20711)

References

[1] S A Douglas and E H Ohlstein ldquoHuman urotensin-II themost potent mammalian vasoconstrictor identified to date asa therapeutic target for the management of cardiovasculardiseaserdquo Trends in Cardiovascular Medicine vol 10 no 6pp 229ndash237 2000

[2] P Grieco P Rovero and E Novellino ldquoRecent structure-activity studies of the peptide hormone urotensin-II a potentvasoconstrictorrdquo Current Medicinal Chemistry vol 11 no 8pp 969ndash979 2004

[3] K Takahashi K Totsune O Murakami and S ShibaharaldquoExpression of urotensin II and urotensin II receptor mRNAsin various human tumor cell lines and secretion of urotensinII-like immunoreactivity by SW-13 adrenocortical carcinomacellsrdquo Peptides vol 22 no 7 pp 1175ndash1179 2001

[4] A Federico S Zappavigna M Romano et al ldquoUrotensin-IIreceptor is over-expressed in colon cancer cell lines and incolon carcinoma in humansrdquo European Journal of ClinicalInvestigation vol 44 no 3 pp 285ndash294 2014

[5] A Federico S Zappavigna M Dallio et al ldquoUrotensin-IIreceptor a double identity receptor involved in vasocon-striction and in the development of digestive tract cancers andother tumorsrdquo Current Cancer Drug Targets vol 17 no 2pp 109ndash121 2017

[6] R Franco S Zappavigna V Gigantino et al ldquoUrotensin IIreceptor determines prognosis of bladder cancer regulatingcell motilityinvasionrdquo Journal of Experimental amp ClinicalCancer Research vol 33 no 1 p 48 2014

[7] P Grieco R Franco G Bozzuto et al ldquoUrotensin II receptorpredicts the clinical outcome of prostate cancer patients and isinvolved in the regulation of motility of prostate adenocar-cinoma cellsrdquo Journal of Cellular Biochemistry vol 112 no 1pp 341ndash353 2011

[8] N K Janz P A Wren D Schottenfeld and K E GuireldquoColorectal cancer screening attitudes and behavior a pop-ulation-based studyrdquo Preventive Medicine vol 37 no 6pp 627ndash634 2003

[9] F Merlino E Billard A M Yousif et al ldquoFunctional se-lectivity revealed by N-methylation scanning of humanurotensin II and related peptidesrdquo Journal of MedicinalChemistry vol 62 no 3 pp 1455ndash1467 2019

[10] F Merlino A M Yousif E Billard et al ldquoUrotensin II(4-11)azasulfuryl peptides synthesis and biological activityrdquo Journalof Medicinal Chemistry vol 59 no 10 pp 4740ndash4752 2016

[11] F Merlino D Brancaccio A M Yousif et al ldquoStructure-activity study of the peptides P5U and urantide by the de-velopment of analogues containing uncoded amino acids at

position 9rdquo ChemMedChem vol 11 no 16 pp 1856ndash18642016

[12] A Gabizon and F Martin ldquoPolyethylenglykol-umhulltes(pegyliertes) liposomales Doxorubicinrdquo Drugs vol 54 no 4pp 15ndash21 1997

[13] A Gabizon H Shmeeda and Y Barenholz ldquoPharmacoki-netics of pegylated liposomal doxorubicinrdquo Clinical Phar-macokinetics vol 42 no 5 pp 419ndash436 2003

[14] T M Allen P Sapra and E Moase ldquoUse of the post-insertionmethod for the formation of ligand-coupled liposomesrdquoCellular ampMolecular Biology Letters vol 7 no 2 pp 217ndash2192002

[15] A Wu W Cheng Z Li J Jiang and E Wang ldquoElectrostatic-assembly metallized nanoparticles network by DNA tem-platerdquo Talanta vol 68 no 3 pp 693ndash699 2006

[16] S Arpicco C Lerda E Dalla Pozza et al ldquoHyaluronic acid-coated liposomes for active targeting of gemcitabinerdquo Eu-ropean Journal of Pharmaceutics and Biopharmaceuticsvol 85 no 3 pp 373ndash380 2013

[17] F J Martin and D Papahadjopoulos ldquoIrreversible coupling ofimmunoglobulin fragments to preformed vesicles An im-proved method for liposome targetingrdquo Journal of BiologicalChemistry vol 257 no 1 pp 286ndash288 1982

[18] T Kobayashi T Ishida Y Okada S Ise H Harashima andH Kiwada ldquoEffect of transferrin receptor-targeted liposomaldoxorubicin in P-glycoprotein-mediated drug resistant tumorcellsrdquo International Journal of Pharmaceutics vol 329 no 1-2 pp 94ndash102 2007

[19] M Yuan Y Qiu L Zhang H Gao and Q He ldquoTargeteddelivery of transferrin and TAT co-modified liposomes en-capsulating both paclitaxel and doxorubicin for melanomardquoDrug Delivery vol 23 no 4 pp 1171ndash1183 2016

[20] A A Lohade R R Jain K Iyer et al ldquoA novel folate-targetednanoliposomal system of doxorubicin for cancer targetingrdquoAAPS PharmSciTech vol 17 no 6 pp 1298ndash1311 2016

[21] A Gabizon H Shmeeda A T Horowitz and S ZalipskyldquoTumor cell targeting of liposome-entrapped drugs withphospholipid-anchored folic acid-PEG conjugatesrdquoAdvancedDrug Delivery Reviews vol 56 no 8 pp 1177ndash1192 2004

[22] A S Manjappa K R Chaudhari M P Venkataraju et alldquoAntibody derivatization and conjugation strategies appli-cation in preparation of stealth immunoliposome to targetchemotherapeutics to tumorrdquo Journal of Controlled Releasevol 150 no 1 pp 2ndash22 2011

[23] J W Park D B Kirpotin K Hong et al ldquoTumor targetingusing anti-her2 immunoliposomesrdquo Journal of ControlledRelease vol 74 no 1-3 pp 95ndash113 2001

[24] A Carotenuto L Auriemma F Merlino et al ldquoLead opti-mization of P5U and urantide discovery of novel potent li-gands at the Urotensin-II receptorrdquo Journal of MedicinalChemistry vol 57 no 14 pp 5965ndash5974 2014

[25] S Di Maro R-C Pong J-T Hsieh and J-M Ahn ldquoEfficientsolid-phase synthesis of FK228 analogues as potent antitu-moral agentsrdquo Journal of Medicinal Chemistry vol 51 no 21pp 6639ndash6641 2008

[26] D Brancaccio F Merlino A Limatola et al ldquoAn in-vestigation into the origin of the biased agonism associatedwith the Urotensin II receptor activationrdquo Journal of PeptideScience vol 21 no 5 pp 392ndash399 2015

[27] R J Lee S Wang M J Turk and P S Low ldquo(e effects of pHand intraliposomal buffer strength on the rate of liposomecontent release and intracellular drug deliveryrdquo BioscienceReports vol 18 no 2 pp 69ndash78 1998


[28] R R C Stewart and J D Bewley ldquoLipid peroxidation as-sociated with accelerated aging of soybean axesrdquo PlantPhysiology vol 65 no 2 pp 245ndash248 1980

[29] D B Fenske and P R Cullis ldquoLiposomal nanomedicinesrdquoExpert Opinion on Drug Delivery vol 5 no 1 pp 25ndash44 2008

[30] L Cattel M Ceruti and F Dosio ldquoFrom conventional tostealth liposomes a new Frontier in cancer chemotherapyrdquoJournal of Chemotherapy vol 16 no 4 pp 94ndash97 2004

[31] D Papahadjopoulos T M Allen A Gabizon et al ldquoStericallystabilized liposomes improvements in pharmacokinetics andantitumor therapeutic efficacyrdquo Proceedings of the NationalAcademy of Sciences vol 88 no 24 pp 11460ndash11464 1991

[32] O C Farokhzad and R Langer ldquoImpact of nanotechnologyon drug deliveryrdquo ACS Nano vol 3 no 1 pp 16ndash20 2009

[33] H Maeda ldquo(e enhanced permeability and retention (EPR)effect in tumor vasculature the key role of tumor-selectivemacromolecular drug targetingrdquo Advances in Enzyme Reg-ulation vol 41 no 1 pp 189ndash207 2001

[34] F Merlino S Di Maro A Munaim Yousif M Caraglia andP Grieco ldquoUrotensin-II ligands an overview from peptide tononpeptide structuresrdquo Journal of Amino Acids vol 2013Article ID 979016 2013

[35] M Hodjat H Haller I Dumler and Y Kiyan ldquoUrokinasereceptor mediates doxorubicin-induced vascular smoothmuscle cell senescence via proteasomal degradation of TRF2rdquoJournal of Vascular Research vol 50 no 2 pp 109ndash123 2013

[36] M Iglewski and S R Grant ldquoUrotensin II-induced signalinginvolved in proliferation of vascular smooth muscle cellsrdquoVascular Health and RiskManagement vol 6 pp 723ndash734 2010

[37] A Fritze F Hens A Kimpfler R Schubert and R Peschka-Suss ldquoRemote loading of doxorubicin into liposomes drivenby a transmembrane phosphate gradientrdquo Biochimica etBiophysica Acta (BBA)-Biomembranes vol 1758 no 10pp 1633ndash1640 2006

[38] B Sabeti M I Noordin S Mohd R Hashim A Dahlan andH A Javar ldquoDevelopment and characterization of liposomaldoxorubicin hydrochloride with palm oilrdquo BioMed ResearchInternational vol 2014 p 765426 2014

[39] V Camarda M Spagnol W Song et al ldquoIn vitroandinvivopharmacological characterization of the novel UT re-ceptor ligand [Pen5DTrp7Dab8]urotensin II(4-11) (UFP-803)rdquo British Journal of Pharmacology vol 147 no 1pp 92ndash100 2006

[40] G Giebing M Tolle J Jurgensen et al ldquoArrestin-in-dependent internalization and recycling of the urotensinreceptor contribute to long-lasting urotensin II-mediatedvasoconstrictionrdquo Circulation Research vol 97 no 7pp 707ndash715 2005

[41] M Caraglia M Marra G Misso et al ldquoTumour-specificuptake of anti-cancer drugs the future is hererdquo Current DrugMetabolism vol 13 no 1 pp 4ndash21 2012

[42] N R Jabir S Tabrez G M Ashraf S ShakilG A Damanhouri andM A Kamal ldquoNanotechnology-basedapproaches in anticancer researchrdquo International Journal ofNanomedicine vol 7 pp 4391ndash4408 2012

[43] J Kopecka S Porto S Lusa et al ldquoSelf-assembling nano-particles encapsulating zoledronic acid revert multidrug re-sistance in cancer cellsrdquo Oncotarget vol 6 no 31pp 31461ndash31478 2015

[44] J Kopecka S Porto S Lusa et al ldquoZoledronic acid-encap-sulating self-assembling nanoparticles and doxorubicin acombinatorial approach to overcome simultaneously che-moresistance and immunoresistance in breast tumorsrdquoOncotarget vol 7 no 15 pp 20753ndash20772 2016

[45] G De Rosa G Salzano M Caraglia and A AbbruzzeseldquoNanotechnologies a strategy to overcome blood-brainbarrierrdquo Current Drug Metabolism vol 13 no 1 pp 61ndash692012

[46] M Caraglia G De Rosa G Salzano et al ldquoNanotech revo-lution for the anti-cancer drug delivery through blood- brain-barrierrdquo Current Cancer Drug Targets vol 12 no 3pp 186ndash196 2012

[47] G Gaudenzi and G Vitale ldquoTransplantable zebrafish modelsof neuroendocrine tumorsrdquoAnnales drsquoEndocrinologie vol 80no 3 pp 149ndash152 2019

[48] G Vitale G Gaudenzi L Circelli et al ldquoAnimal models ofmedullary thyroid cancer state of the art and view to thefuturerdquo Endocrine-Related Cancer vol 24 no 1 pp R1ndashR122017

[49] R Wurth F Barbieri A Pattarozzi et al ldquoPhenotypical andpharmacological characterization of stem-like cells in humanpituitary adenomasrdquo Molecular Neurobiology vol 54 no 7pp 4879ndash4895 2017

[50] G Gaudenzi M Albertelli A Dicitore et al ldquoPatient-derivedxenograft in zebrafish embryos a new platform for trans-lational research in neuroendocrine tumorsrdquo Endocrinevol 57 no 2 pp 214ndash219 2017

[51] G Vitale G Gaudenzi A Dicitore F Cotelli D Ferone andL Persani ldquoZebrafish as an innovative model for neuroen-docrine tumorsrdquo Endocrine-Related Cancer vol 21 no 1pp R67ndashR83 2014


Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

potentially useful target for innovative therapy against colonand prostate cancer together with the revival of its pre-viously reported modulators [9ndash11] Liposomes are one ofthe most common vehicles proposed for drug targeting Inparticular liposomes with stealth properties allow the en-capsulated drug to be addressed such as doxorubicin (doxo)towards tissues characterized by vessels with an enhancedpermeability of the endothelium such as tumors HoweverPEGylated liposomes containing doxo (marketed as Doxilregor Caelyxreg) have demonstrated reduced toxicity although ahigher delivery into the tumor has not been reported[12 13] To improve their target ability liposomes have beenfunctionalized with specific ligands or molecules such asantibodies peptides and oligonucleotides able to enhancethe selectivity of liposomes towards cancer cells [14ndash16]Several strategies can be approached for coupling ligand tothe surface of stealth liposomes in order to achieve a highinteraction of ligand with the receptor expressed on tumorcells Among all commonly used approaches PEG derivatesinclude maleimide groups that react with cysteine residuesor thiol groups on ligands to form stable thioether bondsbetween liposomes and ligands [17] (us receptor ligandssuch as transferrin [18 19] folic acid [13 20 21] or differentmonoclonal antibodies raised against tumor-associatedantigens (TAA) including receptors [22 23] have beenlargely investigated to target different cancer cells Herestealth liposomes were proposed as systems to target cancercells overexpressing UTR and to promote drug delivery of ananticancer agent ie doxorubicin into colon and prostatecancer cells

2 Materials and Methods

21Materials 12-Dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) and [N-(carbonyl-methoxypolyethylenglycol-2000)-12-distearoyl-snglycero-3-phosphoethanolamine sodium salt](DSPE-PEG2000) were purchased from Lipoid GmbH (CamSwitzerland) 23-(Dipyrrometheneboron difluoride)-24-norcholesterol (Bodipy-cholesterol) and 12-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide-(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide)were obtained from Avanti Polar Lipids (Alabaster AlabamaUSA) Doxorubicin hydrochloride (doxo) cholesterol (chol)sodium chloride [4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid N-(2-hydroxyethyl)piperazine-Nprime-(2-ethane-sulfonic acid)] (Hepes) ethylenediaminetetraacetic acid(EDTA) iron thiocyanate (FeSCN3) sodium citrate potas-sium chloride citric acid and Sepharose CL-4B were pur-chased from Sigma Chemical Co (St Louis Mo USA) AllFmoc-amino acids were purchased from GL Biochem(Shanghai China) with their corresponding side chain pro-tections for the solid-phase peptide synthesis (SPPS) Ace-tonitrile methanol (HPLC degree) and chloroform (ACSgrade) were obtained from Carlo Erba (Milan Italy) RPMIDMEM and FBS were purchased from FlowLaboratories(Milan Italy) Tissue culture plasticware was acquired fromBecton Dickinson (Lincoln Park NJ USA) Rabbit antiseraraised against α-tubulin and UTR were purchased from SantaCruz Biotechnology (Santa Cruz CA USA) UT-II peptide

was synthetized and purified by the research group of ProfPaolo Grieco (Department of Pharmacy University of NaplesFederico II)

22 Peptide Synthesis Peptide Cys-UT-II (H-Cys-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) was synthesized byadopting the solid-phase peptide synthesis (SPPS) using theFmoctBu orthogonal strategy as reported elsewhere [24]Upon cyclization procedure performed in solid phase [25]an additional Cys residue was added in the N-terminal byfollowing standard procedures [26] (e product was pu-rified and characterized by reverse-phase HPLC (RP-HPLC)

23 LiposomePreparation Liposomes composed of DPPCcholDSPE-PEG2000 or DPPCcholDSPE-PEG2000-mal-eimide at a 65 30 5 molar ratio were prepared by hy-dration of a thin lipid film followed by extrusion Brieflylipids were dissolved in 1mL of a mixture chloroformmethanol (2 1 vv) the resulting solution was added to a50mL round-bottom flask and the solvent was removedunder reduced pressure by using a rotary evaporator(Laborota 4010 digital Heidolph Schwabach Germany)(en the resulting lipid film was hydrated with 1mL ofcitrate buffer at pH 4 (150mM sodium citrate 150mMcitric acid) and the resulting suspension was gently mixedin the presence of glass beads until the lipid layer wasremoved from the glass wall after that the flask was left at50degC for 2 h (e liposome suspension was then extrudedat 50degC using a thermobarrel extruder system (NorthernLipids Inc Vancouver BC Canada) passing repeatedlythe suspension under nitrogen through polycarbonatemembranes (5 times for each membrane) with decreasingpore sizes from 400 to 100 nm (Nucleopore TrackMembrane 25mm Whatman Brentford UK) (e ex-ternal buffer was removed by ultracentrifugation (OptimaMax E Beckman Coulter USA rotor TLA 1202) at80000 rpm at 4degC for 40min and the liposomes wereresuspended with 1mL of phosphate buffer at pH 74(140mM sodium chloride 25mM of HEPES 01 mM ofEDTA) After preparation liposomes were stored at 4degCEach formulation was prepared in triplicate For in-tracellular penetration studies fluorescent-labeled lipo-somes containing 01 (ww) of Bodipy-cholesterolwere prepared similarly by adding the fluorescent cho-lesterol in the lipid mixture of DPPCcholDSPE-PEG2000or DPPCcholDSPE-PEG2000-maleimide during the li-posome preparation

24 Preparation of Liposomes Conjugated with UT-IICys-UT-II (H-Cys-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) was conjugated with liposomes prepared with mal-eimide-modified polyethylene glycol Briefly the UT-IIpeptide (0250mM) was conjugated to liposomes containingDSPE-PEG2000-maleimide with gentle magnetic agitation atroom temperature overnight (e resulting liposomes so-called LipoUT were then chromatographed on a Sepharose








TSdoxotimes 1001113890 1113891 (1)




UT-IItheortimes 1001113890 1113891 (2)

















4 Results




















lowastlowast

WIDR LoVo

WIDR LoVo

UTR

α-Tubulin

0

50

100

150

200

o

f arb

itrar

y un

its

(a)

CTR


CTR


DU145

FL1-H

Cou

nts

101100 103102 104

101100 103102 104

101100 103102 104

200

160

120

80

40

0

PC3

Cou

nts

FL1-H

200

160

120

80

40

0

MFI

DU

145

PC3

LNCa

P

lowastlowast

700

600

500

400

300

200

100

0

CTR


LNCaP

FL1-H

Cou

nts

200

160

120

80

40

0

(b)






DU145

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT



5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























TSdoxotimes 1001113890 1113891 (1)




UT-IItheortimes 1001113890 1113891 (2)

















4 Results




















lowastlowast

WIDR LoVo

WIDR LoVo

UTR

α-Tubulin

0

50

100

150

200

o

f arb

itrar

y un

its

(a)

CTR


CTR


DU145

FL1-H

Cou

nts

101100 103102 104

101100 103102 104

101100 103102 104

200

160

120

80

40

0

PC3

Cou

nts

FL1-H

200

160

120

80

40

0

MFI

DU

145

PC3

LNCa

P

lowastlowast

700

600

500

400

300

200

100

0

CTR


LNCaP

FL1-H

Cou

nts

200

160

120

80

40

0

(b)






DU145

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT



5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























4 Results




















lowastlowast

WIDR LoVo

WIDR LoVo

UTR

α-Tubulin

0

50

100

150

200

o

f arb

itrar

y un

its

(a)

CTR


CTR


DU145

FL1-H

Cou

nts

101100 103102 104

101100 103102 104

101100 103102 104

200

160

120

80

40

0

PC3

Cou

nts

FL1-H

200

160

120

80

40

0

MFI

DU

145

PC3

LNCa

P

lowastlowast

700

600

500

400

300

200

100

0

CTR


LNCaP

FL1-H

Cou

nts

200

160

120

80

40

0

(b)






DU145

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT



5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




































lowastlowast

WIDR LoVo

WIDR LoVo

UTR

α-Tubulin

0

50

100

150

200

o

f arb

itrar

y un

its

(a)

CTR


CTR


DU145

FL1-H

Cou

nts

101100 103102 104

101100 103102 104

101100 103102 104

200

160

120

80

40

0

PC3

Cou

nts

FL1-H

200

160

120

80

40

0

MFI

DU

145

PC3

LNCa

P

lowastlowast

700

600

500

400

300

200

100

0

CTR


LNCaP

FL1-H

Cou

nts

200

160

120

80

40

0

(b)






DU145

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT



5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















lowastlowast

WIDR LoVo

WIDR LoVo

UTR

α-Tubulin

0

50

100

150

200

o

f arb

itrar

y un

its

(a)

CTR


CTR


DU145

FL1-H

Cou

nts

101100 103102 104

101100 103102 104

101100 103102 104

200

160

120

80

40

0

PC3

Cou

nts

FL1-H

200

160

120

80

40

0

MFI

DU

145

PC3

LNCa

P

lowastlowast

700

600

500

400

300

200

100

0

CTR


LNCaP

FL1-H

Cou

nts

200

160

120

80

40

0

(b)






DU145

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT



5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















DU145

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200

0

500

1000

1500

2000

2500

M

FI

Lipo

lowastlowast

6h 24h

48h 72h

LipoUT

PC3

CtrLipo 6hLipoUT 6h



100 101 102 103 104

FL1-H

Cou

nts

0

40

80

120

160

200 lowastlowast

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6h 24h

48h 72h

M

FI

Lipo LipoUT



5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















5 Discussion



PC3

Con

trol

Lipo

-Bod

ipy

Lipo

UT-

Bodi

py

DU 145C

ontro

lLi

po-B

odip

yLi

poU

T-Bo

dipy


(a)

Control0

20

40

60

80

100

DU 145 PC3


lowastlowast

Fluo

resc

ence

inte

nsity

of

fluor

esce

nt-la

bele

d lip

osom

es(a

rbitr

ary

units

)

(b)






001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















001 01 1 10 1000

20

40

60

80

100

120

140DU145

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(a)

001 01 1 10 10020

40

60

80

100

120

140

160

180PC3

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(b)

001 01 1 10 1000

20

40

60

80

100

120

140

160LNCaP

o

f cel

l gro

wth

Log microM

LipoUTLipoUT-doxo


lowastlowast

(c)



Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Log microM

o

f cel

l gro

wth

0001 01 1 10 100

20

40

60

80

100

120

140 WIDR

lowastlowast

001 01 1 10 100Log microM

o

f cel

l gro

wth

20

40

60

80

100

120

140 LoVo

lowastlowast


LipoUTLipoUT-doxo


LipoUTLipoUT-doxo




0

50

100

150

200

250


o

f cel

l gro

wth

CTRdoxoLipo


lowastlowast

lowastlowast

lowastlowast
















6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























6 Conclusion


Data Availability


Disclosure






Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Acknowledgments


References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Urotensin-II-TargetedLiposomesasaNewDrugDelivery