Architectonics of Phage-Liposome Nanowebs as Optimized Photosensitizer Vehicles for Photodynamic Cancer Therapy

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Themembity, abtory hotherphoto(PDT)toxicin dioand Pas PTto treation oto inistroyinitiattransfnearblightof phlogicathrou(form

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Mol C2524

Molecular
Cancer
apeutics

apeutic Discovery

hitectonics of Phage-Liposome Nanowebs as Optimized

Ther

tosensitizer Vehicles for Photodynamic Cancer Therapy

m Kalarical Janardhanan1, Shoba Narayan1, Gopal Abbineni1, Andrew Hayhurst2, and Chuanbin Mao1

ractFila

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s' Affiliatiosity of Okly and Immch, San Ant

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American A

ancer Ther

mentous M13 phage can be engineered to display cancer cell–targeting or tumor-homing peptidesh phage display. It would be highly desirable if the tumor-targeting phage can also carry anticancerto deliver them to the cancer cells. We studied the evolution of structures of the complexes betweenc filamentous M13 phage and cationic serum-stable liposomes that encapsulate the monomeric pho-itizer zinc naphthalocyanine. At specific phage-liposome ratios, multiple phage nanofibers and lipo-are interwoven into a “nanoweb.” The chemical and biological properties of the phage-liposomeeb were evaluated for possible application in drug delivery. This study highlights the ability of
nanow
phage-liposome nanowebs to serve as efficient carriers in the transport of photosensitizers to cancer cells.Mol Cancer Ther; 9(9); 2524–35. ©2010 AACR.

most2,11,2whichpenetwavenineor ramembulumtion otissuerelativhighin sinmostand tventsand city anencapmedia(type

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use of liposomes for drug delivery across cellranes is ubiquitous, owing to their biocompatibil-ility to protect encapsulant, and improve circula-alf-life and release profiles (1–3). Compared withcancer therapies, photomedicine, which comprisesthermal therapy (PTT) and photodynamic therapy, has many advantages, including the absence ofside effects or disfigurement (3). Developmentsde lasers and optical fibers have introduced PTTDT as emerging cancer treatment methods. Where-T relies on the ability of electromagnetic radiationt cancer cells, PDT takes advantage of the interac-f light with drug (in this case, the photosensitizer)tiate apoptosis or necrosis of cancer cells and de-the tumor (1, 4). In PDT, cytotoxic reactions areed by reactive oxygen species generated due toer of triplet state energy from photosensitizer toy oxygen molecules present when activated by(5). The effect of PDT is dependent on the abilityotosensitizer to bring about photooxidation of bio-l matter through a type I (radical formation

sensitized electron transfer) or type IIsinglet oxygen) mechanism. One of the

cur ebe dibranesitizerthe incomeChemylene(20), pleculeinto lleakagHer

cation

ns: 1Department of Chemistry and Biochemistry,ahoma, Norman, Oklahoma and 2Department ofunology, Southwest Foundation for Biomedicalonio, Texas

ary material for this article is available at Molecularcs Online (http://mct.aacrjournals.org/).

thor: Chuanbin Mao, Department of Chemistry andersity of Oklahoma, Norman, OK 73019. Phone: 405--325-6111. E-mail: [email protected]

7163.MCT-10-0253

ssociation for Cancer Research.

; 9(9) September 2010

promising photosensitizers used in PDT is zinc0,29-tetra-tert-butyl-2,3-naphthalocyanine (ZnNC),has improved properties such as increased tissueration arising from strong absorbance at longlengths (700–1,000 nm; refs. 6–9). 2,3-Naphthlocya-has been reported to bring about photonecrosisndom necrosis, with direct photodamage to therane, mitochondria, and rough endoplasmic retic-in the neoplastic cells and delayed photodestruc-f the endothelial cells surrounding the tumor(10–12). Its phototoxicity is enhanced due to theely long-lived excited singlet and triplet states inquantum yields, and there is nearly 100% increaseglet oxygen efficiency (13). However, ZnNC, likephotosensitizers used in PDT, is insoluble in waterends to aggregate in biologically compatible sol-, which further reduces photosensitizing efficiencyauses low tumor selectivity (14, 15). The insolubil-d aggregation of the drugs can be overcome bysulating them inside liposomes (16). In liposomal, energy transfer and singlet oxygen formationI and type II mechanism) have been found to oc-fficiently (17). However, liposomal structure maysrupted by serum proteins and biological mem-s, resulting in the nonspecific release of photosen-into the bloodstream (14, 16). This drawback andability to deliver drugs to target sites can be over-through chemical or physical modification (18).ical methods include surface coating with polyeth-glycol chains (19) and ligands such as antibodieseptides (2), sterol modification (21), or small mo-s (22). However, incorporating targeting motifsiposomes has been shown to result in increasede of encapsulated drugs (23, 24).
e, we show a new drug delivery system in whichic liposomes (which encapsulate ZnNC) and

filamepeptidtary Sweb-l

tractesearch

Figuregeneticpeptidecationicresult, aand lipo(not to

Phage-Based Photodynamic Cancer Therapy

www.a

ntous M13 phage (which can codisplay anionices and cell- or tissue-specific peptides; Supplemen-
cheme S1) are electrostatically assembled into aike nanostructure (Fig. 1). The M13 phag
delive

scale).

acrjournals.org

d worldwide attention in medical and materials re-(25). It has the potential for gene transfer, drug

ry, and vaccine delivery due to its plasticity, low
e has at- cost, stability, and safety (26–28). It is a rod-like virus
1. Filamentous phages, which wereally engineered to display an anionicon the side wall, were mixed withliposomes that encapsulate ZnNc. As acomplex was formed in which phagesomes were interwoven into a nanoweb

Mol Cancer Ther; 9(9) September 2010 2525

(∼880bactersembcalled(e.g., pmentaby thepeptidthe phdisplathe siand ethe lipphagesomesdelivecan b

Mate

ChemMT

hexadpyl-L-Bertacarboacid,ride,minorwitho3-phonoyl)DOTAlaminCF), aInc., afied mfromfrom

DisplpVIIIThe

was ddisplaresidutein oXL 1GLU-digescleasePCRM13KGAACAAA5′-GCrestric

BriePCR pthe dwas d1% agTris-bthenDNAout atransfCaCl2spreaphenithe p(plus

Kalarical Janardhanan et al.

Mol C2526

-nm long and ∼7-nm wide) that specifically infectsia and is nontoxic to humans. Its side wall is as-led from ∼2,700 copies of a major coat proteinpVIII, whereas a few copies of minor coat proteinsIII) form the two distal ends of the phage (Supple-ry Scheme S1). Because the coat protein is encodedDNA encapsulated inside the protein coat, foreignes can be displayed on the tip and/or side wall ofage by genetic means (25, 29–31). In this work, weyed an anionic peptide with a sequence of Glu8 onde wall of phage to make it anionic (Scheme S1)ncapsulated ZnNC in the hydrophobic region ofid bilayers of cationic liposomes. Thus, the anioniccan electrostatically interact with cationic lipo-to form a novel nanoweb (Fig. 1) for targeted drug
ry through PDT because target-specific peptides overn
ed uscordithe redigeszymephoreThe cDNAon thephagestrainThe tcontaamphwas itaininbatedshakiunits)incubtube wum caddedisoprofinalincub

EnginThe

for 10perfored anThen,chloristoredcollec4°C, d10 miamineand it

e codisplayed on the phage.

rials and Methods

icals/materialsT, chloramphenicol, dimethylsulfoxide (DMSO),ecyltrimethylammonium bromide (CTAB), isopro-thio-β-D-galactopyranoside, kanamycin, Luria-ni broth, N-(3-dimethylaminopropyl)-N′-ethyldiimide hydrochloride (EDC), phosphotungsticpolyethylene glycol, rhodamine B, sodium chlo-tetracycline, trehalose, Tris, ZnNC, and otherchemicals were from Sigma-Aldrich, and used

ut purification. 1,2-Di-(9Z-octadecenoyl)-sn-glycero-sphoethanolamine (DOPE), 1,2-di-(9Z-octadece--3-trimethylammonium-propane (chloride salt;P) and 1,2-dioleoyl-sn-glycero-3-phosphoethano-e-N-(carboxyfluorescein) (ammonium salt; DOPE-ll in chloroform, were from Avanti Polar Lipids,nd used as received. DMEM, McCoy's 5a modi-edium, and other cell culture requirements werethe American Type Culture Collection. KSFM wasInvitrogen.

ay of the E8 peptide on the major coat protein,(i.e., side wall), of the M13 phagemolecular cloning technique used in this workone as described in the literature (32). The phagey technique was used to fuse eight glutamic acides (E8) to the NH2 terminus of the major coat pro-f the M13 bacteriophage. By using Escherichia coliblue bacteria and pecan49 as a phagemid, thepecan49 was constructed. For this, pecan49 wasted with NcoI and HindIII restriction endonu-s and ligated with similarly digested GLU-g8pproduct, which was amplified by PCR of07. The primers used were 5′-ATCCATGGCGGAA-GAAGAAGAAGAAGAAGAAGATCCCG-AGCG-3′ (NcoI restriction site is underlined) and
AAGCTTTTATCAGCTTGCTTTCGAG-3′ (HindIIItion site is underlined).
callybance

ancer Ther; 9(9) September 2010

fly, the PCR products were purified using QIAgenroduct purification kit (QIAgen, Inc.) according toescribed procedures in the kit. The purified DNAigested, and the DNA fragments were loaded intoarose gel and isolated by electrophoresis in 0.5×orate EDTA buffer. The digested fragment wasextracted from the agarose gel using QIAgen gelextraction kit. The ligation reaction was carried

t 25°C for 2 hours using T4 ligase. DNA was thenected into competent E. coli TG1 cells by using themethod. The transfected bacteria (100 μL) was

d onto agar plate containing 30 μg/mL chloram-col and 2% glucose. A well-separated clone fromlate was inoculated into 3 mL Luria-Bertani broth2% glucose) and incubated at 250 rpm at 37°Cight. The recombinant phagemid DNA was isolat-ing QIAgen Miniprep plasmid extraction kit ac-ng to the manufacturer's protocols. To analyzecombinant phagemid DNA, phagemid DNA wasted with both NcoI and HindIII restriction en-s. The digested products were analyzed by electro-sis in agarose gel and observed under UV light.orrectly inserted fragments were verified bysequencing (carried out MCLAB). To display E8major coat of the M13 phage, 0.5 μL recombinantmid DNA was transfected into competent hostXL1 blue E. coli by using the CaCl2 method.

ransfected bacteria were spread on an agar plateining 20 μg/mL tetracycline and 35 μg/mL chlor-enicol. Then, a well-spread clone from the platenoculated into 3 mL Luria-Bertani medium con-g the appropriate antibiotics. The culture was incu-in the tube for 4 to 6 hours at 37°C with moderateng. M13KO7 virions (1.2 × 1010 plaque-formingwere added to the tube, and the tube was furtherated for 30 minutes at 37°C. The mixture in theas transferred to 150 mL of Luria- Bertani medi-

ontaining appropriate antibiotics. Kanamycin wasto reach a final concentration of 70 μg/mL, andpyl-L-thio-β-D-galactopyranoside was added to aconcentration of 0.1 mmol/L. The mixture wasated overnight at 37°C with vigorous shaking.

eered phage purificationovernight culture was first centrifuged at 2,400 × gminutes at 4°C by using a Beckman Coulter high-mance centrifuge, and the supernatant was collect-d recentrifuged at 6,700 × g for 10 minutes at 4°C.one-fifth volume of polyethylene glycol/sodiumde solution was added, and the suspension wasin a refrigerator overnight. Phage precipitates were

ted by centrifugation at 10,100 × g for 60 minutes atissolved in TBS, and centrifuged at 10,100 × g fornutes until clear. The engineered phage was then ex-d under a transmission electron microscope (TEM),s concentration was determined spectrophotometri-
(Shimadzu UV2101 PC) by measuring the absor-at 269 nm.
Molecular Cancer Therapeutics

PrepaThe

and astockthat ttwo l4 mL)as sumixedweighcrolitemixtuunderand dwas dlipid.[10 mchloriflaskfor aba cleaextenstons)excessed thboundbuffewerebonatvesiclbufferform

PrepaThe

spectrdureswereand mlipid t2 to 40mol/L0 to 3tion bto staassemquentdark a

DeterliposoThe

spectrin absapproand w(as devariou

mericUV-Vsome,

CharThe

compstrumequat

Size aphagSiz

scatteInstrusomeplexethe sastaintrehallutioncoppewickelowedliposo2 houexam(200,0

FluorTo

enginstudieconceand vto 30ZnNCplexescenceused700 tslit wof Zn(L/PscribeDMSO

wheresity oand Adard,constaand sA s


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ration of ZnNC-encapsulated liposomesliposome was made from a cationic lipid DOTAPhelper lipid DOPE in a molar ratio of 30:70. Asolution of ZnNC was prepared in DMSO suchhe effective concentration was 6 mmol/L. Theipids, DOPE (1 g/50 mL) and DOTAP (1 mg/, were procured as chloroform solutions and usedch without purification. The two lipids werein a mole ratio of 70:30 such that the total molet of the lipids was 4.10 × 10−3 g. Six hundred mi-rs of ZnNC in DMSO were added to the lipidre, shaken well, and the chloroform was removednitrogen atmosphere in a narrow glass beakeresiccated under vacuum for 6 hours. Hydrationone using three parts of CTAB for every part ofCTAB stock solution was prepared in PBSmol/L NaH2PO4 (pH 7.0), 100 mmol/L sodiumde]. After addition of CTAB, the contents of thewas vortexed for 3 minutes, allowed to hydrateout 30 minutes, and finally sonicated to obtainr solution. The solution was then subjected toive dialysis (dialysis bag MWCO 6,000–8,000 dal-against phosphate buffer to remove most of theCTAB. The dialyzed vesicle suspension was elut-

rough a Sephadex G-50 column to remove un-ZnNC, lipids, and surfactant, with phosphate

r as solvent. The formed multilamellar vesiclesthen extruded through a 100-nm-diameter polycar-e membrane for 21 times to form small unilamellares. The final suspension (25 mL) was in phosphateand stored in the dark at 4°C. Residual chloro-

was analyzed by using gas chromatography.

ration of phage-liposome complexesconcentration of phage used in this study wasophotometrically determined as per reported proce-(33). Liposomes having known lipid concentrationsmixed with varying quantities of engineered phageade up to 3 mL in phosphate buffer such that theo engineered phage (L/P) weight ratio varied fromfor a lipid concentration of 4.92 × 10−5 g (1.65 × 10−11

) and engineered phage concentration varying from0 × 10−11 mol/L. After an initial mixing of the solu-y vortexing for 30 seconds, the mixture was allowednd for 30 minutes in room temperature for the self-bly of liposomes onto the engineered phage. Subse-ly, the phage-liposome complexes were stored in thet 4°C for characterization.

mination of the concentration of ZnNC inmes and in the phage-liposome complexamount of ZnNC encapsulated was determinedophotometerically after disruption of the liposomesolute ethanol. The absorbance of the sample afterpriate dilution in DMSO was measured at 769 nmith an extinction coefficient of 1.08 × 105 mol/L/cm
termined from a standard plot constructed usings concentrations of ZnNC in DMSO). The mono-
mineneere

acrjournals.org

nature of ZnNC was confirmed by comparing theis spectral characteristics of ZnNC in DMSO, lipo-and the phage-liposome complex.

ge of liposomes and phage-liposome complexesζ potential of the liposome and phage-liposome

lex was measured using Zetapals (Brookhaven In-ents Corporation) and theHemholtz-Smoluchowskiion.

nd morphology of liposomes ande-liposome complexese distribution was measured using dynamic lightring using a particle sizer (Zetapals, Brookhavenments Corporation). The morphology of the lipo-s, engineered phages, and phage-liposome com-s was evaluated under a TEM. For this, 10 μL ofmple were mixed with equal quantity of a negative(comprising of 5% phosphotungstic acid and 2.5%ose at pH 7.3) on a parafilm, and the resultant so-was dropped onto a formvar-coated 400-meshr grid. After 5 minutes, the leftover solution wasd away and the negatively stained sample was al-to dry under natural conditions. For the phage-

me complexes, the samples were examined withinrs of the preparation of the complex. The grids wereined under JEOL 2000-FX Intermediate Voltage00 V) scanning TEM.

escence spectroscopic studiesestimate the binding constant for liposomes to theeered phage, UV-Vis and fluorescence spectroscopics were carried out by maintaining the liposomentration constant (1.65 × 10−11 mol/L of total lipid)arying the engineered phage concentration from 0× 10−11 mol/L. The fluorescence spectra of the-encapsulated liposome and phage-liposome com-were obtained on a Shimadu RF5301 PC fluores-spectrophotometer. The excitation wavelength

was 680 nm and the emission was monitored fromo 900 nm, keeping the excitation and emissionidths at 3.0 nm. The fluorescence quantum yieldsNC in liposome and phage-liposome complexes= 2.2) were measured using the ratio method de-d by the Eaton equation (34, 35) using ZnNC inas a standard (φFStandard = 0.07; ref. 36).

’F ðXÞ ¼ �FStandardAStandard

ASample

� �FSample

FStandard

� ��Sample

�Standard

� �2

(1)

FSample and FStandard are the fluorescence inten-f the sample and standard, respectively; ASample

Standard are the absorbance of sample and stan-respectively; ηSample and ηStandard are the dielectricnt of the solvents used for preparation of sampletandard, respectively.pectroscopic titration technique was used to deter-
the binding constant (Kb) of liposome to the engi-d phage as per reported procedures (37). Briefly,

the fluwas maddedthe lipwas fthe pstudy

wherecentratensitsaturaKb islog[phtainedbindinlog[(Fdissocthe bilues wspectrThe qincorpof baca quethe awas ewas rrescenStern-

wher(meth[MV2

quencVolme

EffecLip

(0.3 m0.3 mcubattimes518 nby thepercenculatewere

whereed samF0 is t

incubty forestim

Cell cBre

Amer2008.firmewere90% cand ctant wof Mctakenverifiesquamand cCultuDiseaBostothaweas pewereKSFMobtainof pro

MTTAn

shoulwhenried ocells5 μmophageoctagservedall exof anythe psuredinto eat 37°solvedThe aand sin a SSimilmeasuThe d

Cellu


Mol C2528

orescence intensity of the ZnNC in the liposomeonitored as a function of the concentration of thephage, exactly after 60 minutes of incubation ofosome with phage. The intensity of fluorescenceound to increase with increasing concentration ofhage in all the concentrations investigated in this. The increase fulfills the expression

F0�FF�F1

¼ ½Phage�Kdiss

� �n

(2)

F is the fluorescence intensity for a given phage con-tion, and F0 and F∞ are the relative fluorescence in-ies of the ZnNC-liposome alone and liposometed with phage, respectively. The binding constantobtained by plotting log[(Fo − F)/(F − F∞)] versusage]. The slope of the double-logarithm plot ob-from experimental data is the number equivalentg capacity (n), whereas the value of log[phage] ato − F)/(F − F∞)] = 0 equals the logarithm of theiation constant (Kdiss). The reciprocal of Kdiss isnding constant Kb. The fluorescence intensity va-ere obtained from the area under the fluorescencea, in the range of our investigation (700–900 nm).uenching of the fluorescence emission by ZnNCorated into liposomes in the presence and absenceteriophages was studied using methyl viologen asncher. ZnNC-liposome solution (5.0 μmol/L) inbsence/presence of bacteriophages (L/P = 2.2)xcited at 680 nm, and their fluorescence emissionecorded in the range of 700 to 900 nm. The fluo-ce quenching data obtained were analyzed byVolmer formalism (15).

F0=F ¼ 1þKSV½MV2þ� (3)

e [MV2+] is the concentration of the quencheryl viologen), F0 is the fluorescence intensity at+] = 0, F the fluorescence intensity at a givenher concentration [MV2+], and KSV is the Stern-r quenching constant.

t of blood plasma on liposome stabilityosome fractions containing 6-carboxyfluoresceinL containing 3 mg total lipid) were mixed withL blood plasma (Innovative Research, Inc.) and in-ed for 3 hours at 37°C. Aliquots were diluted 500, and the fluorescence (λex = 492 nm and λem =m) was measured before and after liposome lysisaddition of 100 μL 10% (w/v) Triton X-100. Thetage of encapsulated carboxyfluorescein was cal-d from the expression below (38). The experimentsrepeated in triplicates.

%Encapsulation ¼ 100� ðFi�F0Þ�100Ft�F0

� �(4)

Fi and Ft are the fluorescence intensity of the dilut-
ple before and after lysis using Triton X-100, and
he fluorescence intensity of the same sample beforeThe

15 wa


ation with blood plasma. The blood plasma stabili-the phage-liposome complex (L/P = 6.7) was alsoated through the same procedure.

ulturesast cancer cell lines (SKBR-3) were obtained from theican Type Culture Collection around DecemberThe viability of the cells has been tested and con-d using trypan blue dye exclusion tests. The cellsinitially cultured in 25-cm2 flasks. On reachingonfluence, cells were detached using trypsin/EDTAentrifuged at 2,000 rpm for 5 minutes. The superna-as removed and a pellet was resuspended in 5 mLCoy's medium. Later, 100 μL of the cell sample wereand treated with trypan blue, and the viability wasd using a hemocytometer. A human head and neckous cell carcinoma cell line (SCC-15), established

haracterized by Rheinwald, was obtained from Cellre Core facility at BWHDermatology/Harvard Skinse Research Center, Harvard Institutes of Medicine,n, MA, in November 2008. The cells obtained wered, expanded, and cryopreserved in GIBCO KSFMr the protocols provided by the supplier. The cellssubsequently grown to ∼1/3 confluence in GIBCOmedium and used for the experiments. The cellsed from the suppliers were used within 6 monthscurement for the experiments in this study.

assayimportant prerequisite that a photosensitizerd have is the absence of toxicity, or low toxicity,not irradiated. Toxicity measurements were car-ut on SKBR-3 (3.6 × 103) and SCC-15 (2.9 × 103)plated onto 24-well plates and treated withl/L ZnNC encapsulated in liposomes, engineereds, and phage-liposome complexes (termed lip-lu) carrying 5 μmol/L ZnNC. Untreated cellsas control. Special care was taken to carry out

periments in dark conditions to avoid the influencestray light. After 6 hours of incubation in the dark,

ercentage of viable cells in each sample was mea-. For this, 0.5 mL MTT (0.5 mg/mL) was addedach well and samples were incubated for 3 hoursC. The MTTcrystals formed on incubation were dis-in 1 mL of HCl (0.04 mol/L) in isopropyl alcohol.bsorbance of the samples were read at 595 nmubtracted from a reference wavelength of 620 nmhimadzu UV-Vis spectrophotometer (Abssample).arly, the absorbance of untreated cells were alsored (Abscontrol). Data were collected in triplicate.ark toxicity/toxicity was calculated as follows (39):

%Cell viability ¼ Abssample

Abscontrol� 100 (5)

lar uptake
cellular uptake of ZnNC by SKBR-3 cells and SCC-s determined based on a previous method (40).

ZnNCoctgluamouriod otrationcell sucellulaside thin 3%tive o

FluorFlu

wereduresphageagentphageprocethrouextenslow mmolecthe dwereCF inand liner assuch tculturwith 324 hoslips,

PBS (mediuliposomentand thydeeral tslidewith caminerhodaZeisspus oof celand s

PhotoAt

with lto neathe cedeliveThe ceThe passaywereblue. C

Resu

Optic

Figureof ZnNcompleof engineered phages (0–30 × 10−11 mol/L).


www.a

(5 μmol/L)–encapsulated liposomes and Lip-containing 5 μmol/L ZnNC was used to study thent of ZnNC taken up by cells over an incubation pe-f 12 hours. To calculate intracellular ZnNC concen-s (Cintra), we used the concentration of ZnNCwithinspensions of known cell densities and assumed ar volume of 2.3 × 10−9 mL. The concentration out-e cell was assumed to be the concentration of ZnNCPBS (Cextra). The ratio of these two values is indica-f the tendency of ZnNC to be taken up by the cells.

escence microscopy studiesorescence-labeled bacteriophages and liposomesprepared according to previously reported proce-(41). Briefly, rhodamine B labeling of engineereds were done by mixing 0.2 g of the EDC couplingand 0.02 g of rhodamine B in 25 mL of engineeredstock solution in phosphate buffer (pH 5.6). This

dure results in permanent attachment of the dyegh EDC-assisted caging. The labeled phages wereively dialyzed for 5 days with stirring to removeolecular weight organic compounds and free dyeules. The modified phages were stored at 4°C inark. Carboxyfluorescein (CF)-labeled liposomesprepared by replacing 1% of DOPE with DOPE-the preparation of liposomes. The labeled phageposomes were allowed to interact in a similar man-that of engineered phage and ZnNC-liposomes

hat the L/P ratio was 6.7. Two sets of six-well tissuee plates carrying 25-mm coverslips were seeded.5 × 103 SKBR-3 and SCC-15 cells. After allowing
urs for the cells to attach and spread on the cover-unattached cells were removed by washing with
Theintrod

acrjournals.org

pH 7.4). The cells were then treated with culturem containing 500 μL of carboxyfluorescein-labeledmes and fluorescent lip-octaglu for 3 hours. Treat-was terminated by washing with PBS three timeshen fixed by incubating with 4% paraformalde-in PBS for 15 minutes, then washed with PBS sev-imes. The cells were mounted on a microscopeusing gelvatol. The green fluorescence associatedarboxyfluorescein-associated cell cultures were ex-d using a blue filter, and the red fluorescence ofmine B was examined using a green filter on aUniversal epiflourescence microscope with Olym-il immersion DApo UV objectives. Digital imagesls were collected using Olympus DP-70 cameraoftware.

dynamic therapy30 minutes after treatment of SKBR-3 cells (5 × 103)iposome-ZnNCor lip-octaglu, the cellswere exposedr-IR radiation for 4 minutes. External irradiation oflls was done through a fiber-optic cable capable ofring laser power at a fluence rate of 206 mW/cm2.lls were incubated for 3 hours following irradiation.ercentage of cell death was calculated using theMTTas described above. Trypan blue exclusion studiescarried out by staining the cells with 0.4% trypanell viabilitywas observed under a light microscope.

lts and Discussion

al properties of phage-liposome complexes

2. Fluorescence spectra (λex = 680 nm)C-encapsulated liposomes onxation with increasing concentration

entrapment of ZnNC in liposomes did notuce any additional band in the spectra of ZnNC


(Suppenviroic strunm dtary FZnNCdetectcentaestimain absomesformathe abing ththe liZnNC(Fig.proteoxygeQu

viologsion qwereintensthe pring. Ito thethe po(44).MV2+

structwhich

Fursomequenc(L/Picallychang

Figureof the wfrom st5.46 × 10 viruses/mL engineered phage. The horizontal line indicatescharge


Mol C2530

lementary Fig. S1), indicating that the liposomenment did not have an influence on the electron-cture of ZnNC. In addition, the Soret band at 337id not undergo any red shift or split (Supplemen-ig. S1), implying the absence of aggregation ofin liposomes (42). Residual chloroform was not

able in the final liposomal composition. The per-ge of encapsulation of ZnNC in liposomes wasted to be 68.9% after disruption of the liposomessolute ethanol. The absorption spectra of lipo-and phage-liposome complexes show that the

tion of phage-liposome complex did not changesorption spectra (Supplementary Fig. S1), indicat-at ZnNC is not aggregated inside the bilayers ofposomes. However, the fluorescence intensity ofis increased on increasing phage concentration

2), indicating that the phage-liposome complexcts ZnNC from water molecules and molecularn that may quench the fluorescence (43).enching of ZnNC fluorescence by using methylen (MV2+) was studied through steady-state emis-uenching experiments. Stern-Volmer plots (Fig. 3)constructed from relative integrated fluorescenceity at 778 nm. ZnNC-encapsulated liposomes, inesence of MV2+ at 25°C, had no significant quench-n liposomal systems, MV2+ is oriented coplanarlyliposomal surface to maximize contact betweenlar and nonpolar parts of quencher and liposomeLower Stern-Volmer constant (KSV) values forindicate the incorporation of ZnNC into the bilayer
ure toward the inner part of the liposomal structure,lowers the accessibility of MV2+ to ZnNC.
diculaobser


ther electrostatic repulsion between cationic lipo-s and divalent MV2+ reduces the accessibility ofher to ZnNC. In the presence of engineered phage= 2.2), both liposome and quencher are electrostat-immobilized on the phage surface, which maye the orientation of MV2+ from parallel to perpen-

neutrality (ξ potential = 0) at L/P = 1.63.

4. The ζ potential of the phage-liposome complex as a functioneight ratio of lipid and phage (L/P). The complexes were madeock solutions of 1.6 × 10−4 g/mL DOPE + DOTAP (70:30) and

11

r to liposomal surface. The KSV value of 350/mol/Lved in this study for the phage-liposome system is

ureluorsomintsomcenasurthecagivabsnche of the plot, with an intercept of 1.

Figof flipoThelipoconmeandwasat athequeslop

M

3. Stern-Volmer plots for the quenchingescence of ZnNC-encapsulatedes and the phage-liposome complex.egrated fluorescence intensity ofe and complex with increasingtration of methyl viologen (quencher) wased (λex = 680 nm, λem = 700–900 nm),relative fluorescence intensity (F0/F)

lculated from the fluorescence intensityen concentration of MV2+ (F) and inence of a quencher (F0). Stern-Volmering constant was obtained from the

olecular Cancer Therapeutics

similawiththrouvationefficieresceZnNC(L/Pwas foin thephageless rengincompThe

somes

technnumbmentaof maocta-aweigh(L/Pliposothenfirmsthe ca

Microcomp

Figureliposomand phaa functA, ZnNA′, a hiindividuliposoma singlephagesview ofphage-L/P ratithe phastructu


www.a

r to the case of ZnPC interacting perpendicularly9,10-anthraquinone-2,6-disulfonic acid (AQS−)gh an electron transfer process (15). This obser-suggests that engineered phages can serve as

nt carriers for electron transfer processes. The fluo-nce quantum yield of the liposome-entrappedin the absence and presence of engineered phage

= 2.2) determined as per standard procedures (36)und to be 0.072 and 0.076, respectively. An increasequantum yield of ZnNC due to the formation of-liposome complex can be attributed to radiation-elaxation and resonance energy transfer betweeneered phage and ZnNC, making phage-liposomelex an ideal vehicle for phototherapeutics (45).
binding constant for ZnNC-encapsulated lipo-onto phage, estimated by a spe
The

res.

acrjournals.org

ique (46), was found to be 1.1 × 1010/mol/L, wither equivalent binding capacity being 1.48 (Supple-ry Fig. S2). The binding constant is three ordersgnitude higher than that reported for hexa- andrginines onto anionic liposomes (47). When thet ratio of the total lipid to the engineered phagevalue) was increased, the ζ potential of the phage-me mixture changes from negative, to zero, andto positive (Fig. 4). The ζ potential change con-that the anionic phage is being assembled withtionic liposomes.

scopic characterization of phage-liposomelexes
lipid bilayer thickness was estimated to be around
ctroscopic titration 23 nm (Fig. 5A′). When the phage (Fig. 5B) is mixed with

5. TEM images of thees, engineered phages,ge-liposome complexes asion of L/P values.C-encapsulated liposomes.gh-magnification view of anal ZnNC-encapsulatede highlighting the bilayer ofliposome. B, engineered

. B′, high-magnificationphages. C to F,liposomes at differentos. Insets, schematics ofge-liposome complex


liposofew litary Fsomesliposo(Fig.on-robeadsweb ((L/Pwherringsliposodensi(49), aintera(L/P =somes

PlasmtheirWe

nanowplasmof thecapsu(38). Wyfluorplexedenhanbly dsimila

liposoany dsquammined5 μmoof lipchainat L/show

IntercancePD

as posion between negatively charged cell surface and anionicdelivery vectors hinders cellular uptake, we chose to use

FigureSKBR-with lippresento absefluoresc

FigureI, untreengineeliposom(L/P = 6SEMs wobserved by Student's t test when groups II, III, and IV were comparedagainst


Mol C2532

mes at a very low concentration (L/P = 40), veryposomes are bound to a single phage (Supplemen-ig. S3) due to the repulsion between cationic lipo-. At a higher phage concentration (L/P = 10), moremes were found to bind to an individual phage5C), similar to the previously observed “beads-d” structure (48, 49). At L/P = 6.7, several linear-on-rod structures were entangled to form a nano-Fig. 5D). At an even higher phage concentration= 2.9), short rings or spirals (Fig. 5E) were founde phages wrapped liposomes. The formation ofor spirals has been suggested in microtubule-me complexes owing to the mismatch of chargeties between microtubule and cationic liposomesnd in the DNA-liposome system due to electrostaticctions (43). When phage concentration is very high2.0), phages self-assembled into a matrix with lipo-embedded (Fig. 5F).

a stability of phage-liposome nanowebs andtoxicity to mammalian cellsentrapped carboxyfluorescein into phage-liposomeebs and measured their stability in the presence ofa. Generally, plasma stability is measured in termspercentage of carboxyfluorescein that remains en-lated within the liposome in the presence of plasmae observed an 18.2% higher percentage of carbox-escein encapsulation when liposomes were com-to phage than when liposomes were alone. The

cement in plasma stability for nanowebs is proba-

group I.

ue to the presence of phage, which functions in ar manner to cholesterol in liposomes (50). Both

fluorescnanoweobserv


me and nanoweb carriers of ZnNC did not showark toxicity within 6 hours to breast (SKBR-3) andous cell carcinoma (SCC-15) cell lines, as deter-by the MTT assay for a ZnNC concentration of

l/L (Fig. 6). This could be attributed to the stabilityosomes and nanowebs as well as to shorter sides in ZnNC (51). Therefore, the nanowebs formedP = 6.7 were found to be stable in serum anded no dark cell toxicity.

nalization of phage-liposome nanowebs inr cellsT requires that photosensitizers be delivered as closessible to targeted cells. Because electrostatic repul-

7. Fluorescence microscopic images of treated and untreated3 cells. A and B, untreated cells with no fluorescence. Cells treatedosomes, with green fluorescence from carboxyfluorescein tagt in the liposomes (C) and with no fluorescence observed duence of phage (D). Cells treated with nanowebs, with greenence from carboxyfluorescein-tagged liposomes (E) and with redence from the rhodamine-B tagged phages (F) present in the

6. Percentage cell viability observed for the different groups.ated SKBR-3 and SCC-15 cell lines. II, treated with 250 μLred phage (1.6 × 1011 viruses/mL). III, treated with 250 μLes (1.8 × 10−10 mol/L lipids). IV, treated with 250 μL nanowebs.7). Cell viability was measured by using the MTT assay.ere calculated using SPSS 10.0. No significant change was

bs. Left column (A, C, D) and right column (B, D, E) are imagesed under blue and green filters, respectively.


phage(Fig. 4to theof cansquamnanowplasmcancethrou(Cintra

reporZnNCthe cedeterconcethe (CliposoresperespecenhansomesCel

shownment,a greeliposo

regioncentlybodiimby dicompwebswith(Fig.fromthe nand pilar ocells (

PhotophagThe

photostudieencapirrad206 min cellcells when compared with liposome-treated cells (Fig. 8),indicating that the nanowebs can deliver more drugs into

Figurewithoutbefore

2

FigureI, SKBR4 min a[1.6 × 1(e.g., lipMTT asStudent's t test, where “a” indicates comparison of group I with groups IIIand IV,


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-liposome nanowebs (L/P = 6.7), which are cationic) and capable of carrying drug more effectively dueir web-like structure, to deliver ZnNC to two typescerous cell lines [i.e., breast cancer (SKBR-3) andous cell carcinoma (SCC-15) cell lines]. The cationicebs can be attracted by the negatively chargeda membrane to increase their uptake potential byr cells. The ratio of ZnNC internalized into the cellsgh liposomes or nanowebs to that outside the cells/Cextra) was determined by following a previouslyted procedure (40). To calculate the intracellular, the concentration of ZnNC that remained withinll was determined. The extracellular ZnNC wasmined from the difference between total ZnNCntration and that within the cell. It was found thatintra/Cextra) was higher for nanowebs than formes alone (8.19 and 5.13 for SCC-15 cell lines,ctively, and 6.13 and 3.95 for SKBR-3 cell lines,tively). This suggests that formation of nanowebsces drug delivery into the cancer cells because lipo-are embedded in the web-like nanostructures.

l internalization of phage-liposome nanowebs wasthrough fluorescence microscopy. For this experi-ZnNC in the bilayer of liposomes was replaced by

and “b” indicates comparison of group III with group IV.

n dye, carboxyfluorescein, to fluorescently label themes, because ZnNC fluorescence is in the near-IR

cm forblue aftaken u

acrjournals.org

and is invisible. Engineered phage was fluores-labeled with a red dye, rhodamine-B, through car-ide coupling, and the unbound dye was removed

alysis (41). Fluorescently tagged liposomes werelexed with rhodamine-tagged phages to form nano-(L/P = 6.7). After the nanowebs were incubatedSKBR-3 cells for 3 hours, the fluorescence images7) show that the red and green fluorescence arisethe same regions within the cells, suggesting thatanowebs containing both dye-labeled liposomeshages are now associated with the same cells. Sim-bservations were also made in the case of SCC-15Supplementary Fig. S4).

dynamic destruction of cancer cells bye-liposome complexesability of the nanowebs to function as an efficientsensitizer carrier was further shown by PDTs on SKBR-3 cells. Cells were treated with ZnNC-sulated liposomes and nanowebs separately andiated with a near-IR light at a fluence rate ofW/cm2 for a period of 4 minutes. A 20% increasedeath was observed in the case of nanoweb-treated

9. Light microscopic images of SKBR-3 cells treated with andZnNC-liposome and nanowebs (i.e., lip-octaglu at L/P = 6.7)

and after irradiation with near-IR light at a fluence rate of 206 mW/a period of 4 min. The cells were treated with 0.4% trypan

8. Percentage of cell viability observed for the different groups.-3 cells without irradiation. II, SKBR-3 cells with irradiation fort 206 mW/cm2. III, treated with 250 μL ZnNC-liposomes0−4 g/mL (1.8 × 10−10 mol/L) lipids]. IV, 250 μL nanowebs-octaglu at L/P = 6.7). Cell viability was measured using thesay. SEMs were calculated using SPSS 10.0. a,b, P < 0.05,

ter 3 h of incubation. Trypan blue was excluded by live cells butp by dead cells.


the ceshowiwas oThe

nanowsomesIt is ether enanowtides,niqueunderIn s

tion ochemitionsto codsion twall (can bwebsanticacan sp(Supp

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lls than the liposomes alone. Further evidenceng increased cell death on treatment with nanowebsbtained from trypan blue exclusion studies (Fig. 9).study highlights the ability of phage-liposomeebs to serve as a more efficient vehicle than lipo-alone to transport photosensitizers to target cells.

xpected that the cell delivery efficiency will be fur-nhanced if the tips or side walls of the phage in theebs are engineered to codisplay cell-targeting pep-which we have selected by phage display tech-recently (not shown here). This work is nowway.ummary, we have studied the morphologic evolu-f phage-liposome complexes and evaluated theircal and biological properties for possible applica-in drug delivery. Because phages can be engineeredisplay cancer-targeting peptides at the tip (by fu-o pIII) and liposome-binding peptides on the sideby fusion to pVIII, Scheme S1) and anticancer drugse loaded into liposomes, the phage-liposome nano-assembled from such cancer-targeting phage andncer liposomes will serve as novel carriers that
ecifically target the anticancer drugs to the tumors Rece
thesis, comparative photochemical and photobiological studies.hotochem Photobiol B 1996;35:167–74.opova M, Wohrle D, Stoichkova N, et al. Hydrophobic Zn(II)-

naca

13. RuDyinphso

14. Chag

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osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank the financial support from the National Institutes of Health,ment of Defense Congressionally Directed Medical Researchm, National Science Foundation, SFBR internal funds, NIH12087, and the Oklahoma Center for the Advancement of Sciencechnology (HR06-161S). We also thank Greg Strout (TEM),neth Dormer (ζ potential), Prof. B. Safiegko-Mroczka (fluorescencecopy), and Prof. Paul F. Cook (fluorescence spectroscopy) foring the facilities, and Dr. Pascaline Ngweniform for help duringly stages of this study. S. Kalarical Janardhanan thanks thement of Science and Technology, Government of India, for theAST fellowship.

Support

C06RR12087 (A. Hayhurst), NIH 1R21EB009909-01A1 (C.NSF CBET-0854465 (C. Mao), and DoD Breast Cancer Research

W81XWH07-1-0572 (C. Mao).costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 03/22/2010; revised 06/17/2010; accepted 07/09/2010;
lementary Fig. S5). published OnlineFirst 08/31/2010.
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Architectonics of Phage-Liposome Nanowebs as Optimized Photosensitizer Vehicles for Photodynamic Cancer Therapy