Research Article Organic Nanovesicular Cargoes for...

Research ArticleOrganic Nanovesicular Cargoes for Sustained Drug DeliverySynthesis Vesicle Formation Controlling lsquolsquoPearlingrsquorsquo Statesand Terfenadine LoadingRelease Studies

Ajay Kumar Botcha1 Balakrishna Dulla2 E Ramanjaneya Reddy2

Keerthana S Chennubhotla3 Pushkar Kulkarni34

Rajadurai Chandrasekar1 and Marina S Rajadurai2

1 Functional Molecular NanoMicro Solids Laboratory School of Chemistry University of Hyderabad Hyderabad 500046 India2Organic amp Medicinal Chemistry Dr Reddyrsquos Institute of Life Sciences University of Hyderabad Campus Hyderabad 500046 India3 Biology Department Dr Reddyrsquos Institute of Life Sciences University of Hyderabad Campus Hyderabad 500046 India4 Zephase Therapeutics Dr Reddyrsquos Institute of Life Sciences University of Hyderabad Campus Hyderabad 500046 India

Correspondence should be addressed to Pushkar Kulkarni kkpushkargmailcomRajadurai Chandrasekar chandrasekar100yahoocom and Marina S Rajadurai marinardrilsorg

Received 25 February 2014 Accepted 7 June 2014 Published 12 August 2014

Academic Editor Paresh Chandra Ray

Copyright copy 2014 Ajay Kumar Botcha et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

ldquoSustained drug delivery systemsrdquo which are designed to accomplish long-lasting therapeutic effect are one of the challenging topicsin the area of nanomedicine We developed an innovative strategy to prepare nontoxic and polymer stabilized organic nanovesicles(diameter 200 nm) from a novel bolaamphiphile where two hydrogen bonding acetyl cytosine molecules connected to 4410158401015840-positions of the 26-bispyrazolylpyridine through two flexible octyne chains The nanovesicles behave like biological membraneby spontaneously self-assembling into ldquopearl-likerdquo chains and subsequently forming long nanotubes (diameter 150 nm) whichfurther develop into various types of network-junctions through self-organization For drug loading and delivery applications thenanovesicleswere externally protectedwith biocompatible poly(ethyleneglycol)-2000 to prevent them from fusion and ensuing tubeformation Nontoxic nature of the nanovesicles was demonstrated by zebrafish teratogenicity assay Biocompatible nanovesicleswere loaded with ldquoterfenadinerdquo drug and successfully utilized to transport and release drug in sustained manner (up to 72 h) inzebrafish larvae which is recognized as an emerging in vivo model system

1 Introduction

Synthetic nano-microscale vesicles are of great interestbecause they emulate complex properties of biological cellmembranes [1ndash4] Most of the biological membranes areflexible therefore they show multidimensional shape trans-formation to form zero-dimensional (0D) microspheres onedimensional (1D) cylinders and long flexible tubular struc-tures Hence mimicking the properties of naturally occurringvesicles by polymer peptide or dendrimer based syntheticvesicles is of great interest since they are potential tool fornanomedicine biosensors and biophysical and bioelectronic

applications [5ndash8] For instance in the budding area ofnanomedicine nontoxic vesicles act as tiny spherical con-tainers for storage and delivery of chemicalsreagents in bio-chemical reactions Nanodrug delivery systems protect drugfrom degradation and deliver drug at specific sites therebyincreasing efficacy of treatment [5 9 10] Sometimes multi-ple vesicles fuse due to the interaction of surface moleculesbetween many vesicle membranes a phenomenon knownas ldquopearlingrdquo which leads to the formation of nanotubularvesicles (NTVs) [11] Synthetic NTVs are also proposed forthe transportation of chemicals between vesicles Hence lab-oratory synthesis of nontoxic bioinspired molecules capable

Hindawi Publishing CorporationJournal of NanotechnologyVolume 2014 Article ID 369139 13 pageshttpdxdoiorg1011552014369139

2 Journal of Nanotechnology

PEG 2000TFN

Vesicles

DMSOwater

Pearling vesicles Nanotubes

DMFwater

PEG-coatedTFN-loadedvesiclesvesicles

Sustained drugdelivery

No pearling of vesicles

Bolaamphiphile 1

NN NN N

NN

O

NH

O

NN

O

NH

O

Figure 1 Illustration of the nanovesicles formation from bolaamphiphile 1 and their subsequent self-assembly into pearl chains and finallynanotubes The nontoxic vesicles fusion (pearling) is prevented by biocompatible polymer coating and used in terfenadine (TFN) drugdelivery in a sustained manner (72 h) in zebrafish model system

of mimicking the biological membrane is a challenging tasksince they can act as life-saving drug delivery and sustaineddrug release cargoes Although a plethora of block copolymerbased vesicles are explored for drug delivery applications [5]vesicles from small biologically inspired designer moleculesare rare Earlier we have found that aromatic rigid core 26-bispyrazolylpyridine (BPP) connected with long alkyl groupsforms vesicles [12] Hence for drug loading application theuse of rigid molecules decorated with one of the base pairsfound in DNA and RNA is a promising starting point toachieve nontoxic drug delivery systemsOne of the ingredientproblems with soft vesicles is their tendency to form tubesthrough fusion mechanism

In this paper we present our strategy to prepare non-toxic and polymer stabilized vesicles from a novel bolaam-phiphile 1 that is two hydrogen bonding N4-acetyl cytosinemolecules connected to 4410158401015840-positions of the BPP using twoflexible octyne chains The vesicles are loaded with terfen-adine drug and successfully utilized to transport and releasedrugs in zebrafish larvae which is utilized as an emergingin vivo model system (Figure 1) In this paper we presentthe synthesis of 1 and prevention of fusion driven nanotubeformation by protecting the nanovesicles (NV) with biocom-patible PEG 2000 terfenadine (TFN) drug loading and itssustained drug releasing tendency The nanovesicles werethoroughly characterized using scanning electron micro-scope (SEM) transmission electron microscope (TEM) andatomic force microscopy (AFM)

2 Materials and Methods

21 Synthesis of Compound 1 Molecule 1 was synthesized insix steps from BPP (Scheme 1) compound 2 was synthesizedas per our reported procedure [13 14]

10101015840-(111015840-(Pyridine-26-diyl)bis(1H-pyrazole-41-diyl))bis(dec-9-yn-1-ol) (3) Compound 2 (05 g 107mmol) Pd(PPh

3)2Cl2

(0075 g 0106mmol) PPh3

(0050 g 0214mmol) CuI(0027 g 0144mmol) were taken in a 100mL two-neck roundbottom flask To this solid mixture high vacuum was appliedthrough condenser for deoxygenating Subsequently solventsdry triethylamine (15mL) 14 dioxane (3mL) was addedto the deoxygenated solid mixture under N

2atmosphere

After five minutes 9-dec-yn-1-ol (0217 g 141mmol) wasadded and stirred for 8 h under N

2atmosphere at 80∘C

The solvents were removed under reduced pressure andsolid product was purified by column chromatography using5 methanolDCM After washing the compound withdiethyl ether pure 3 was obtained as a white solid (80yield) 1H-NMR (400MHz CDCl

3) 120575 = 86 (s 2H) 79 (m

1H) 78 (d 2H) 77 (s 2H) 36 (m 2H ndashOCH2) 24 (m

4H) 16 (m 12H) 14 (m 12H) ppm 13C-NMR (100MHzCDCl

3) 120575 = 1494 1446 1415 1290 1095 1065 926 706

629 327 319 297 292 288 257 226 194 ppm FT-IR(KBr) 361686 339888 314618 305552 292821 285105172645 160106 148146 14971 140238 135415 126542121912 118633 105516 102815 97221 95292 8969886419 80053 73880 70601 65586 LC-MS analysis mz

Journal of Nanotechnology 3

NNNN

N NN NN N

I I

NN NN

NHO OH

NN NN N

O OS SO

OO

O

NN NN

N

NBr Br

NN

NH

O

O

NNNH

O

O

N NN N

BPP

b

c

d

e

2

3

4

5

1

6 6

6

6 6

66

6

a

Scheme 1 Synthesis of cytosine decorated to 26-bispyrazolyl-pyridine derivative (1) General reaction conditions (a) as per reportedprocedure [13 14] (b) 2 (107mmol) Pd(PPh

3)2Cl2(0106mmol) PPh

3(021mmol) CuI (014mmol) Et

3N14 dioxane N

2 9-dec-yn-1-

ol (141mmol) 80∘C 8 h 80 (c) 3 (058mmol) tosyl chloride (232mmol) DCMNEt3 RT 2 h 75 (d) 4 (024mmol) LiBr (097mmol)

dry acetone RT 2 h 89 (e) N4-acetyl cytosine (140mmol) K2CO3(181mmol) dry DMF 5 (047mmol) 80∘C 16 h 55

experimental value = 5150 calculated value = 5142 AnalCalcd for C

31H41N5O2 C 7220 H 801 N 1358 found C

7236 H 812 N 1349

10101015840-(111015840-(Pyridine-26-diyl)bis(1H-pyrazole-41-diyl))bis(dec-9-yne-101-diyl)bis(4-methylbenzene-sulfonate) (4) To thesuspension solution of 3 (03 g 058mmol) in DCM (15mL)and NEt

3(5mL) tosyl chloride (0443 g 232mmol) was

slowly added The reaction mixture was stirred for 2 hat room temperature All the solvents were evaporatedunder reduced pressure and crude was purified by columnchromatography with EtOAchexane (2 8) Colorless solidcompound 4was obtained in 75 yield 1H-NMR (400MHzCDCl

3) 120575 = 85 (s 2H) 79 (m 1H) 78 (s 4H) 77 (s 2H)

77 (s 2H) 73 (m 4H) 40 (m 2H ndashOCH2) 24 (s 6H)

235 (m 4H) 16 (m 8H) 15 (m 4H) 14 (m 4H) 12 (m12H) ppm 13C-NMR (100MHz CDCl

3) 120575 = 1494 1446

1415 1332 1298 1289 1278 1096 1065 925 706 289286 253 216 194 ppm FT-IR (KBr) 3483 3148 31093055 2932 2856 1718 1604 1469 1398 1358 1288 12191176 1099 1024 951 806 777 731 661 LC-MS analysis mzexperimental value = 6846 calculated value = 6838 AnalCalcd for C

45H53N5O6S2 C 6559 H 648 N 850 found

C 6548 H 642 N 861

26-Bis(4-(10-bromodec-1-ynyl)-1H-pyrazol-1-yl)pyridine (5)To the suspended solution of compound 4 (020 g0242mmol) in dry acetone lithium bromide (00842 g


0970mmol) was added After 2 h of stirring at RT acetonewas evaporated under reduced pressure and redispersedin water and crude compound was extracted with DCMAfter evaporation of DCM compound 5 was obtained as awhite solid (89 yield) 1H-NMR (400MHz CDCl

3) 120575 =

85 (s 2H) 79 (m 1H) 78 (d 2H) 77 (s 2H) 34 (m 2HndashBrCH

2) 24 (m 4H) 19 (m 4H) 15 (m 4H) 14 (m 8H)

13 (m 8H) ppm 13C-NMR (100MHz CDCl3) 120575 = 1494

1446 1415 1290 1095 1065 925 707 340 327 289 288264 194 ppm FT-IR (KBr) 3437 2924 2854 1720 16511599 1481 1390 1101 1014 954 800 LC-MS analysis mzexperimental value = 502 calculated value = 5012 AnalCalcd for C

31H39Br2N5 C 5804 H 613 N 1092 found C

5812 H 617 N 2487

NN1015840-(111015840-(10101015840-(111015840-(Pyridine-26-diyl)bis(1H-pyrazole-41-diyl))bis(dec-9-yne-101-diyl))bis(2-oxo-12-dihydropyrimi-dine-41-diyl))diacetamide (1) N4-acetyl cytosine (0333 g1404mmol) in dry DMF (15mL) was taken in 100mL roundbottom flask K

2CO3(0258 g 1814mmol) was added to

this solution to convert N-acetyl cytosine into salt Afterstirring at room temperature for about 15min compound 5(03 g 0468mmol) was added and heated to reflux for 16 hat 80∘C DMF was removed under reduced pressure and thecrude product was redispersed in water and extracted withchloroformThe organic fraction was washed with 01MHCl(20mL) and saturated NaCl solution (10mL) Compound1 was obtained after evaporation of chloroform in a liquidform and purified by silica gel column chromatography with5 methanolDCM Pure compound 1 was obtained as awhite solid with 55 yield 1H-NMR (400MHz CDCl

3) 120575

= 104 (s ndashNH) 85 (s 2H) 79 (m 1H) 78 (d 2H) 77 (s2H) 76 (d 1H) 74 (d 1H) 38 (m 2H ndashNCH

2) 24 (m

4H) 23 (s 6H) 17 (m 4H) 15 (m 4H) 14 (m 16H) ppm13C-NMR (100MHz CDCl

3) 120575 = 1713 1628 1557 1494

1488 1446 1415 1290 1095 1069 967 925 706 511365 314 297 289 264 248 212 194 ppm FT-IR (KBr)3325 3136 2926 2852 1697 1662 1556 1479 1429 1381 13071219 1178 1028 952 866 800 655 617 LC-MS analysis mzexperimental value = 6460 calculated value = 6452 AnalCalcd for C

43H51N11O4 C 6571 H 654 N 1960 found C

6586 H 651 N 1951

22 Preparation of Nanovesicles

Synthesis of Nanovesicles Stabilized by PEG-400 Compound1 (05mg 000063mmol) was dissolved in 05mL of DMSOand rapidly injected at vigorous stirring in 50mL ofdeionized water containing predissolved PEG-400 (06 g00003mmol) After 5 minutes of stirring at RT solutionwas drop casted on silica and analyzed by TEM AFM andmicro-Raman spectroscopy Remaining solution was left atmoderate stirring at RT for 120 h and sample was collectedand analyzed every 24 h

Synthesis of Nanovesicles Stabilized by PEG-2000The experi-ment was performed as described above PEG-2000 was usedinstead of PEG-400

Synthesis of TFN-Loaded Stabilized Nanovesicles Stock solu-tion of terfenadine (TFN) was prepared by dissolving 2mgof TFN in 1mL of deionized water 1 (05mg 000063mmol)was dissolved in 045mL of DMSO mixed with 005mLof TFN stock solution and rapidly injected at vigorousstirring in 50mL of deionized water containing predissolvedPEG-2000 (06 g 00003mmol) After 12 h of stirring at RTsolutionwas filtered through syringewith hydrophilic syringefilter (Millipore pores size 045120583m hydrophilic PVDF) toremove an excess of TFN and PEG-2000 and then vesicleswere washed with deionized water and final volume wasadjusted to 10mL (solution V1-TFN) This solution was usedto identify concentration of TFN in vesicles stability of drug-loaded vesicles and for zebrafish studies See also schematicrepresentation of the experiments (Figures 2 and 3)

23 Characterization of Nanovesicles Size and morphologyof the nanostructures were examined by scanning electronmicroscope (SEM) atomic force microscopy (AFM) andtransmission electron microscope (TEM) SEM studies wereperformed using a Philips XL30 ESEM scanning electronmicroscope with a beam voltage of 20 kV FESEM measure-ments were performed on a Hitachi S-4500 SEN instrumentoperating at 20 kV TEM studies were done using TecnaiG2 FEI F12 transmission electron microscope (TEM) at anaccelerating voltage of 200 kV Carbon coated TEM grids(200mesh type-B) were purchased from TED PELLA INCUSA Atomic force microscopy (AFM) imaging was carriedout on NT-MDT model solver Pro M microscope usinga class 2R laser of 650 nm wavelength having maximumoutput of 1mW All calculations and image processing werecarried out by a software NOVA 10261443 provided by themanufacturer The images were recorded in a semicontactmode using a noncontact super sharp silicon cantilever (NSG10 DLC) diamond like carbon (DLC) tip purchased fromNT-MDT Moscow The dimension of the tip is as followscantilever length = 100 (plusmn5)120583m cantilever width 35 (plusmn5) 120583mand cantilever thickness = 17ndash23 120583m resonate frequency =190ndash325 kHz force constant = 55ndash225Nm chip size = 36 times16times 04mm reflective side =Au tip height = 10ndash20120583mDLCtip curvature radius = 1ndash3 nm

AFM LCFM SEM FESEM and TEM-Samples PreparationTwo drops of the sample solution were drop casted on a cleanglass slide by a capillary for AFM studies Two drops of thesample solution were drop casted on a carbon coated coppergrid (200Mesh) with different time intervals for TEM studiesof nanostructures

Stability of Drug-Loaded Vesicles Study Solution V1-TFN wasleft at moderate stirring at RT for 120 h and sample wascollected and analyzed every 24 h to identify time point whennanovesicles disintegration will begin

Determination of Concentrations of TFN in SurroundingMedia (Figure 3) TFN concentration in solution resultingfrom leakage from the nanovesicles was investigated at differ-ent time points following procedure described below 1mL ofsolution V1-TFN was collected filtered through hydrophilic


TFN stock solution Compound 1 solution

Rapid injection

PEG-2000 solution

Filtration and wash

Adjust volume

Solution V1-TFN

Determination of TFN concentration

Stability study

Zebrafish study

005mL 045mL

in water (06 g in 50mL)

Stirring at RT 12h

to 10mL

(2mgmL 4mM) (2mgmL 4mM)

Figure 2 Schematic representation of TFN-loaded nanovesicles preparation

Residue Filtrate

Redisperse in

Filtration through hydrophilic syringe

filter and wash

Adjust volume

Record UV absorbance


Correlate with calibration curve

2mL of water

Extract with Et2O

Extract with Et2O

2mL of DDQ solution in ACN

Mix with water 2mL of DDQ solution in ACN

to 2mL

30min

1mL

Solution V1-TFN

Mix with

Ultrasonication

2mL of ACNRedissolve in


Figure 3 Determination of TFN concentration inside vesicles and in surrounding media (repeated at different time points)

syringe filter and washed 1 time with 1mL of deionizedwater TFNwas extracted from filtrate with diisopropyl ethersolvent was evaporated and remaining TFN was redissolvedin 2mL of acetonitrile Concentration of TFN in this solutionwas measured according to modified published procedure

[15 16] TFN solution in acetonitrile (2mL) was mixed with2mL of DDQ solution (5mgmL) The colored species weregenerated and absorbance intensity was measured immedi-ately at 457 nm and compared with calibration graphs whichwere constructed by plotting the absorbance of the formed


CT complexes versus the final concentration of the drug(120583gmL) This study was repeated for the samples collectedevery 24 h up to 120 h

Determination of Concentrations of TFN inside Nanovesicles(Figure 3) Concentrations of TFN inside vesicles at dif-ferent time points were investigated following proceduredescribed below 1mL of solution V1-TFN was collectedfiltered through hydrophilic syringe filter and washed 1 timewith 1mL of deionized water to remove excess of the drugand PEG-2000 and residue was redispersed in 2mL ofwater and subjected to sonication for 30min at RT to breakdown all vesicles and release TFN The ability of ultrasoundto induce localized and controlled drug release from lipo-somes synthetic diblock copolymers and polymersomes iswell known phenomena [17 18] Next TFN was extractedfrom this solution with large volume of diisopropyl ethersolvent was evaporated and remaining TFN was redissolvedin 2mL of acetonitrile Replacement of solvent with ACNwas necessary because it showed super priority over othersolvents for the formation of CT complex at the sametime facilitating charge-transfer from donor to acceptorAbsorbance intensity of TFN solution in ACN was thenmeasured at selected absorption maximum (457 nm) andobtained value was comparedwith values of calibration curveobtained using solutions of TFNDDQ complex of knownconcentrations (see also Figures 9 and 10)The concentrationof TFN calculated applying Beer-Lambert law was 375 120583Mwhich allowed us to find out drug loading efficiency using thefollowing equation

Loading Efficiency = 11986211198620

times 100 (1)

where 1198620is an initial concentration of TFN in solution

whereas 1198621is concentration of TFN in vesicles after loading

This study was repeated for the samples collected every 24 hup to 120 h

24 Zebrafish Studies

Zebrafish Teratogenicity Assessment Zebrafish maintenancebreeding and embryo collection was carried out by meth-ods described previously [19 20] Briefly wild-type adultzebrafish (5-6 months old) were maintained at 28∘C undera 14 10 h light and dark cycle Fish were allowed to breed(in a ratio of 2 females 3 males) in the morning under thestimulation of light Embryos were collected into petridishcontaining embryo medium and incubated at 28∘C tempera-tureThe teratogenicity studywas performedusing 6 embryosthatwere incubatedwith vehicle positive control and variousconcentrations of test compound 1 until 5 dpf The embryoswere then anesthetized using 0008 tricaine solution andvarious phenotypic parameters were evaluated namely bodyshape somites notochord tail intestine fins brain upperjaw heart lower jaw liver and swim bladder were observedfor defects in morphology [21] Each embryo was scoredbased on their level of toxicity from 5 being nontoxicand 05 being highly toxic The scoring was conducted by

Table 1 Results of zebrafish embryo toxicity study with toxicolog-ical indices and major organssystems affected in positive controland at MTC in test compounds (mdash no effect x slightly toxic xxmoderately toxic and xxx severely toxic)

Testedcompound (1) Phenobarbital

Test concentrations (120583M) 003 01 03 13 10 30 3000

Statistically significant toxicconcentration (120583M) mdash

PositiveControlNo observed adverse

effect level (NOAEL) (120583M) 30

Minimal toxicconcentration (MTC) (120583M) mdash

Parameters of toxicity atMTC

Body shape x xxxSomites mdash xxxNotochord mdash xxxTail mdash xxxFins mdash xxxBrain mdash xxxUpper jaw mdash xxxHeart mdash xxIntestine x xxxLower jaw mdash xxLiver mdash xxxSwim bladder x xxx

blinded observers and was done according to the proceduresdescribed earlier [22]

Protocol for Zebrafish Treatment with TFN-Loaded Nanovesi-cles For TFN-NV assessment 3 dpf (days post fertilization)larvae were distributed in 24-well plate along with 250120583L of01 DMSO solution Each well contained 6 embryos withvarious treatments and the larvae were observed at timepoints of 24 h 48 h and 72 h Embryos were then treatedwith different concentrations of TFN loaded microvesicles(5 120583M 10 120583M 20120583M and 40 120583M) TFN alone at 20120583Mconcentration was used as a positive control and 01 DMSOwas used as a negative control The embryos were incubatedin 24-well plate and atrial and ventricular rates were observedat different time points after treatment All embryos wereobserved for mortality arising out of effect of TFN Resultsof zebrafish embryo toxicity study are summarized in Table 1

3 Results and Discussion

31 Synthesis and Characterization of Compound 1 Molecule1 was synthesized in six steps from BPP (Scheme 1) Iodi-nation of the 4410158401015840-positions of the BPP to synthesize 2 wascarried out as per our reported procedure [13 14] Attachmentof ldquolinkermoleculerdquo decyn-1-ol to 2was achieved by adoptingSonogashira cross-coupling conditions to get 3 in good


Figure 4 SEM micrographs of nanofibrous network assembliesformed from compound 1 in DMFwater

yields (80) Compound 3 was tosylated in DCM solventin presence of triethyl amine as base to give 4 in 75 yieldCompound 5 was prepared in excellent yield (89) upontreatment of 4with LiBr in acetone at room temperatureThetarget bolaamphiphile 1 containing water soluble end groupsconnected to a hydrophobic alkyl chains was obtained in 55yield by treating 5 with N4-acetyl cytosine in presence ofpotassium carbonate at 80∘C Variable temperature 1H-NMRstudies of 1 in CDCl

3showed a clear down field chemical shift

of the cytosine ndashNHndash group upon decreasing the temperature(Supplemental Information Figure S1 available online athttpdxdoiorg1011552014369139)This observation indi-cates the operating intermolecular H bonding interaction ofthe cytosine Furthermore slow addition of water to theDMFsolution (10minus3M) of 1 showed a clear blue shift during UVtitration indicating its J-aggregation behaviour

32 Investigation of Nanostructures Formation To studythe aggregation driven nanostructure formation a solutioncontaining 2mg of compound 1 in DMF (12mL)water(6mL) was prepared and stabilized for an hour Drop castingthis solution on silica followed by SEM analysis showedthe formation of clear fibrous networks of several micronslong (Figure 4) To unravel the mechanism of formationof nanofibrous network assemblies the same solution of1 was drop casted immediately without stabilization on acarbon coated TEMgrid Interestingly the TEMmicrographsshowed the presence of tiny vesicles of size in the rangeof sim200 nm (Figure 5(a)) and some aggregated pearl-chainlinked vesicles of size in the range of sim150 nm (Figure 5(b))Additionally the generation of 1D pearl chain-like assemblyby the fusion of vesicles indicates the formation of nanotubu-lar network (Figures 5(c) and 5(d)) To further verify thefusion of spherical vesicles into nanotubes on silica surfaceAFM studies were undertaken The AFM topography clearlyrevealed the presence of nanovesicles (NVs) of sizes sim200 nm(Figures 6(a) and 6(b)) Interestingly some of the samplesshowed spontaneous formation of pearl-chain like vesicularassemblies or nanotube integrated vesicles pointing out tovesicles fusion For every 1120583mchain length ca 5 fused vesicleswere found which is matching with the average diameter

of the vesicles (Figures 6(c) and 6(d)) Continuous AFMmeasurement revealed the evolution of smooth nanotubularstructures of diameter sim160 nm from the pearl-chain likevesicular assemblies due to the molecular reorganization(Figures 6(e) and 6(f)) The finally formed tubes diameter(sim160 nm) is less than the initial vesicle diameter of a singleisolated vesicle (sim200 nm) due to the stretching of the fusedvesicles during the 1D tube formation The diameter of thetube is determined by the balance between surface tensionand tube bending rigidity preventing membrane collapseTheouter surface of the tubes is very smoothwith a roughnesssmaller than lt1 nm (Figures 6(g) and 6(h)) This resultdemonstrated the flexible nature of the presented syntheticmembrane composed of 1 Another interesting finding of thenanotubes is their tendency to form various types of network-junctions through self-organization (Figure 6(e) insert)

33 Control over Nanostructure Using Stabilizing Agents Todemonstrate application of NVs for drug delivery in the firststep different conditions were screened to stabilize vesiclesin solution state and prevent formation of tubular structuresWe intended to use polyethylene glycol (PEG) as stabilizingagent since it is biologically neutral and furthermore FDA-approved biocompatible polymer well suitable for pharma-ceutical applications [23] PEG-400 and PEG-2000 wereemployed for stabilization of nanovesicles In a typical proce-dure a DMSO solution of compound 1 (05mg in 05mL) wasrapidly injected into a deionized water containing PEG (06 gin 50mL) under vigorous stirring for 5 minutes at RT NVsPEGylated with polymer of lower molecular weight (PEG-400) did not demonstrate high stability however the use ofPEG-2000 stabilized the vesicles up to 108 h Even at that timepoint a significant amount of vesicles retained their structurealthough their surface was not as smooth as it was at 24 and48 hoursThe size distribution of the NVs was in the range oflt60 nm to 130 nm an insignificant amount of bigger vesicles(sim250 nm) was also observed (Figures 7(a) and 7(b))

34 Toxicity Investigation of Stabilized Nanovesicles UsingZebrafish Teratogenicity Assay For NVs based drug deliverystudies zebrafish was selected as a model organism sinceit is easy to maintain is relatively inexpensive gives quickresults and is a good predictor of efficacy and safety ofchemicals [24 25] In order to explore toxicity of compound1 zebrafish teratogenicity assay was performed Treatment of24 hpf (hours after fertilization) embryos with solution of1 at different concentrations and study of their growth anddevelopment for 5 days show no abnormalities (Figure 8 andTable 1) Figure 5 represents mean lesion score of all param-eters different treatment groups This experiment confirmedthe nontoxic nature of 1 at all tested concentrations

35 Terfenadine Loading into Nanovesicles For drug deliverystudy a well-known antihistamine drug terfenadine (TFN)was selected as a model drug TFN is also known to affectheartbeat regularity in zebrafish leading to bradycardiaatrioventricular blockage and ultimately death in less than12 h [26] Selection of this drug facilitates easy monitoring of


(a) (b) (c)

Random vesicles Vesicle dimer Fused dimer with pores

I II III

A smooth tube Cylindrical vesicle Fusion of cylindrical vesiclewith a vesicle

VI V IV

200nm

150

nm

(d)

Figure 5 TEMmicrographs of nanostructures obtained from compound 1 in DMFwater mixture (a) distribution of nanovesicles (b) pearl-chain like fused nanovesicles (c) nanotubes (d) proposed mechanism of nanotube formation by vesicle fusion

50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100

Vesicle diametersim200nm

35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)

Tube diametersim160nm

50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16

Surface roughnesssim1nm

270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)

Nanovesicles ldquoPearl staterdquo vesicles Nanotube vesicles

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions

Figure 6 AFMmechanistic analysis of the formation of nanotubes from nanovesicles (a) randomly distributed vesicles (c) fusion of vesiclesto form pearl-chain like structures (e) nanotubes and (g) roughness of a tube The corresponding profile analyses are shown in (b) (d) (f)and (h) respectively


(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)

Figure 7 (a) TEM micrograph image of nanovesicles stabilized by PEG-2000 (b) size distribution histogram obtained for stabilizednanovesicles

6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment

Teratogenicity assay

lowastlowastlowast

Figure 8 Mean (plusmnSD) lesion score of all parameters differenttreatment groups (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)Statistical significance was analyzed as control group versus allgroups

its effect on zebrafish by observing the endpoint of mortalityat different time points after treatment As the nontoxicnature of 1 in the zebrafish teratogenicity assay is alreadytested any mortality of zebrafish is only attributable to theeffect of TFN Additionally this procedure is simple rapidand noninvasive method of detection of drug action To pre-pare drug-encapsulated nanovesicles TFN and compound1 were dissolved in DMSO and rapidly injected in solutionof PEG-2000 in water with vigorous stirring The drug-encapsulated nanovesicles solutionwas left for 12 h for ageing

resultant solution was subsecuently subjected to study theTFN-loading efficiency and nanovesicles stability

Determination of TFN concentration in biological sam-ples is a challenging analytical task Straightforward appli-cation of spectroscopic methods (for instance measuringintensity of UV absorption and fluorescent emission) isnot applicable as there is no correlation between obtainedvalues and drug content The majority of current reportedmethods (eg HPLC or NMR) lack sensitivity to measurethe drug level at concentrations below 10 120583gmL [15 16]Hence spectrophotometric determination method based onthe charge-transfer (CT) reactions of TFN with 23-dichloro-56-dicyano-14-benzoquinone (DDQ) was opted [15] Thistechnique is one of the most simple rapid and accuratemethodologies developed till now In this method TFNacts as n-electron donor which forms a highly coloredcharge transfer (CT) complex upon spontaneous reactionwith strong 120587-acceptor DDQ In order to implement thisprocedure and find out initial concentration of the drugloaded in vesicles we have developed an in-house protocol(Figures 3 9 and 10) Using this method the calculated drugloading efficiency into the vesicles was sim35 plusmn 1 Further inorder to prove that there is no drug leakage from vesiclesinto surrounding media over time we performed stabilitycontrol experiment drug-loaded vesicles were gently stirredat RT up to 120 h collecting sample every 24 h and detectingconcentration of TFN inside vesicles and in mother liquid(solution collected prior to sonication) at every time-pointas described in Figure 3 The experiment revealed that thereis no major leakage observed up to 72 h (Figure 11) andconcentration of TFN in media remains below 10 120583M whichis zebrafish sensitivity threshold (fish development and heart-beat are not affected below this concentration of TFN) Sub-sequently drug burst release was observed after 72 h whenTFN concentration in mother liquid suddenly increased andcrossed 10 120583M Simultaneously TFN concentration inside


2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04

Concentration of TNF (mmolL)

Calibration457nm

(b)

Figure 9 (a) Absorbance of TFN at different concentrations in acetonitrile as complex with DDQ (DDQ concentration was maintainedconstant 22mM for all samples) and (b) calibration graphs derived from (a) according to Beer-Lambert law

300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h

Figure 10 Absorbance of TFNDDQ complex in acetonitrile atdifferent time points (to determine concentration the value ofabsorbance intensity at 457 nm was recorded and compared withvalue in calibration curve)

NVs decreased dramatically after 72 h We assumed thatslow dissolution of PEG coating resulted in deformation ofNVs and sudden drug release (Figure 6 cartoon) These datacorrelate well with TEMdata recorded for vesicles at differenttime points Figures 12(a) and 12(b) represent TEM imagesof the vesicles at 24 and 72 h respectively At 24 h NVs have

0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles

In vesiclesZebrafish sensitivity

threshold

TFN

In mother liquid

Figure 11 TFN concentration change inside vesicles and in motherliquid at different time points Cartoon represents slow deformationof vesicles and TFN ldquoburstrdquo drug release after 72 h

well-structured defined shape (Figure 12(a)) while at 72 h weobserve degradation of NVs and decrease of NVs amount(Figure 12(b))This explains well drug burst release after 72 han effect commonly seen in case of nano-microsphere ornano-microvesicles systems [27 28]

36 Sustained Drug Delivery of TFN-Loaded Nanovesicles inZebrafish Larvae Zebrafish larvae were treated with TFN-loaded nanovesicles (TFN-NV) at four different concen-trations (5120583M 10 120583M 20120583M and 40 120583M of TFN) and


24h

6876nm

6816nm

(a)

72h

(b)

Figure 12 TEMmicrograph images of TFN-loaded nanovesicles (a) at 24 h scale bar is 100 nm (b) at 72 h scale bar is 100 nm cartoon showsvesicles deformation over time

100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active

Figure 13 Percentage mortality in zebrafish larvae exposed tovarious concentrations of TFN-loaded nanovesicles (TFN-NV) atdifferent time points

were observed at the time points of 24 h 48 h and 72 h toinvestigate mortality caused by TFN after drug treatment(Figure 13) TFN alone at a concentration of 20120583Mwas usedas positive control The observations showed that all larvaein positive control (TFN alone 20 120583M) died within 12 hFor larvae exposed to TFN-NV at low concentrations (5ndash10 120583M)first cases ofmortality (and therefore drug effect) wereobserved at 24 h and all fish died within 48 hours Still furtherprolongated effect was observed at 20120583M mortality wasslowly increasing starting from 24 h and complete mortalitydetected only by 72 h

No cases of death were observed for larvae treatedwith the highest drug concentration up to 48 h but all ofthem were dead at 72 h time point The explanation of thisaberration at highest concentration can be based on the factthat the TFN-NV at 40 120583M form aggregates (Figure 14) andthereforemight not enter the systemic circulation in the larvalzebrafish however by 72 hours TFN-NV would release allTFN causing late mortality Vesicles stability study and TEMdata (Figures 11 12 and 14) support this explanation Apartfrom that we observed precipitation of nanovesicles from40 120583Msolution if left undisturbedThedelayedmortality seenin other concentrations is suggestive of a sustaineddelayedrelease of TFN from TFN-NV in vivo which can be con-trolled by changing concentration of nanovesicles As we did

0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40

TFN concentration (120583M)

Highaggregation

aggregationLow

Figure 14 Graphical representation of increased aggregation andtherefore slow drug release depending on time and TFN concentra-tion Inserts show TEM micrographs of TFN-loaded nanovesiclesscale bar is 02 120583M

not observe major leakage of the drug from nanovesicles upto 72 h (the concentration of the drug in mother liquid wasbelow zebrafish sensitivity threshold) we conclude that effectof TFN below 72 h is observed only after the drug is releasedinside zebrafish organism

4 Conclusion

Nontoxic and polymer stabilized organic nanovesicles asdrug delivery cargoes are prepared from a novel bioinspired26-bispyrazolylpyridine derivative decorated with cytosinemoieties Spontaneous self-assembly of nanovesicles intoldquopearl-likerdquo chains and subsequent fusion into nanotubes areinvestigated and protocol is developed to prevent pearlingby protecting nanovesicles with biocompatible PEG-2000Further the vesicles are loadedwith terfenadine drug and suc-cessfully utilized to transport into zebrafish larvae and releasethe drug in sustainedmanner up to 72 h Although polymericor block copolymer based vesicles are extensively exploredfor drug delivery applications nanovesicles prepared fromsmall bioinspired organic molecule were utilized for drug


delivery for the first time We demonstrated that TFN releasein vivo at desired time point can be controlled by alteringnanovesicles concentration The mechanism of the drug-release in vivo is a subject of separate study Neverthelesscurrent experiment undoubtfully demonstrated possibility ofnanovesicles application as sustained-release drug deliverysystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ajay Kumar Botcha and Balakrishna Dulla contributedequally to this work

Acknowledgments

This work was supported by DST (Fast Track Scheme noSRFTCS-0832009) NewDelhi AKB and ERR thankCSIR-New Delhi for SRFs

References

[1] E Soussan S Cassel M Blanzat and I Rico-Lattes ldquoDrugdelivery by soft matter matrix and vesicular carriersrdquo Ange-wandte ChemiemdashInternational Edition vol 48 no 2 pp 274ndash288 2009

[2] O C Farokhzad and R Langer ldquoImpact of nanotechnology ondrug deliveryrdquo ACS Nano vol 3 no 1 pp 16ndash20 2009

[3] R Langer ldquoNewmethods of drug deliveryrdquo Science vol 249 no4976 pp 1527ndash1533 1990

[4] F ZhaoM LMa and B Xu ldquoMolecular hydrogels of therapeu-tic agentsrdquo Chemical Society Reviews vol 38 no 4 pp 883ndash8912009

[5] J Du and R K OrsquoReilly ldquoAdvances and challenges in smart andfunctional polymer vesiclesrdquo SoftMatter vol 5 no 19 pp 3544ndash3561 2009

[6] S K Sahoo and V Labhasetwar ldquoNanotech approaches to drugdelivery and imagingrdquo Drug Discovery Today vol 8 no 24 pp1112ndash1120 2003

[7] Q Duan Y Cao Y Li et al ldquoPH-responsive supramolecularvesicles based on water-soluble pillar[6]arene and ferrocenederivative for drug deliveryrdquo Journal of the American ChemicalSociety vol 135 no 28 pp 10542ndash10549 2013

[8] D Shi ldquoIntegrated multifunctional nanosystems for medicaldiagnosis and treatmentrdquo Advanced Functional Materials vol19 no 21 pp 3356ndash3373 2009

[9] G P Kumar and P Rajeshwarrao ldquoNonionic surfactant vesic-ular systems for effective drug delivery an overviewrdquo ActaPharmaceutica Sinica B vol 1 no 4 pp 208ndash219 2011

[10] S C Jang O Y Kim C M Yoon D S Choi T Y Roh and JPark ldquoBioinspired exosome-mimetic nanovesicles for targeteddelivery of chemotherapeutics tomalignant tumorsrdquoACSNanovol 7 no 9 pp 7698ndash7710 2013

[11] L Gao L Shi W Zhang et al ldquoPolymerization of sphericalpoly(styrene-b-4-vinylpyridine) vesicles to giant tubesrdquoMacro-molecules vol 38 no 11 pp 4548ndash4550 2005

[12] Y S L V Narayana and R Chandrasekar ldquoTriple emission fromorganicinorganic hybrid nanovesicles in a single excitationrdquoChemPhysChem vol 12 no 13 pp 2391ndash2396 2011

[13] G Zoppellaro andM Baumgarten ldquoOne-step synthesis of sym-metrically substituted 26-bis(pyrazol-1-yl) pyridine systemsrdquoEuropean Journal of Organic Chemistry vol 2005 no 14 pp2888ndash2892 2005

[14] Y S L V Narayana S BasakM Baumgarten KMullen and RChandrasekar ldquoWhite emitting conjugated polymerinorganichybrid spheres phenylethynyl and 26-bis (pyrazolyl)pyridinecopolymer coordinated to Eu (tta)

3rdquo Advanced Functional

Materials vol 23 no 47 pp 5875ndash5880 2013

[15] E Khaled ldquoSpectrophotometric determination of terfenadinein pharmaceutical preparations by charge-transfer reactionsrdquoTalanta vol 75 no 5 pp 1167ndash1174 2008

[16] A A Al-Majed J Al-Zehouri and F Belal ldquoUse of mixedanhydrides for the determination of terfenadine in dosageforms and spiked human plasmardquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 281ndash289 2000

[17] A Schroeder J Kost andY Barenholz ldquoUltrasound liposomesand drug delivery principles for using ultrasound to controlthe release of drugs from liposomesrdquo Chemistry and Physics ofLipids vol 162 no 1-2 pp 1ndash16 2009

[18] G D Pangu K P Davis F S Bates and D A Hammer ldquoUltra-sonically induced release from nanosized polymer vesiclesrdquoMacromolecular Bioscience vol 10 no 5 pp 546ndash554 2010

[19] R K Banote S Koutarapu K S Chennubhotla K Chatti andP Kulkarni ldquoOral gabapentin suppresses pentylenetetrazole-induced seizure-like behavior and cephalic field potential inadult zebrafishrdquo Epilepsy and Behavior vol 27 no 1 pp 212ndash219 2013

[20] M WesterfieldThe Zebrafish Book A Guide for the LaboratoryUse of Zebrafish (Danio rerio) University of Oregon PressEugene Ore USA 4th edition 2000

[21] A Nakhi M S Rahman G P K Seerapu et al ldquoTransitionmetal free hydrolysiscyclization strategy in a single pot synthe-sis of fused furo N- heterocycles of pharmacological interestrdquoOrganic and Biomolecular Chemistry vol 11 no 30 pp 4930ndash4934 2013

[22] J M Panzica-Kelly C X Zhang T L Danberry et alldquoMorphological score assignment guidelines for the dechorion-ated zebrafish teratogenicity assayrdquo Birth Defects Research BmdashDevelopmental and Reproductive Toxicology vol 89 no 5 pp382ndash395 2010

[23] J Milton Harris and R B Chess ldquoEffect of pegylation onpharmaceuticalsrdquo Nature Reviews Drug Discovery vol 2 no 3pp 214ndash221 2003

[24] S Basu and C Sachidanandan ldquoZebrafish a multifaceted toolfor chemical biologistsrdquo Chemical Reviews vol 113 no 10 pp7952ndash7980 2013

[25] E R Reddy R K Banote K Chatti P Kulkarni and M SRajadurai ldquoSelective Multicolour Imaging of Zebrafish MuscleFibres by Using Fluorescent Organic Nanoparticlesrdquo Chem-BioChem vol 13 no 13 pp 1889ndash1894 2012


[26] P K Chan C C Lin and S H Cheng ldquoNoninvasive techniquefor measurement of heartbeat regularity in zebrafish (Daniorerio) embryosrdquo BMC Biotechnology vol 9 article 11 2009

[27] K Fu R Harrell K Zinski et al ldquoA potential approach fordecreasing the burst effect of protein fromPLGAmicrospheresrdquoJournal of Pharmaceutical Sciences vol 92 no 8 pp 1582ndash15912003

[28] P B OrsquoDonnell and J W McGinity ldquoPreparation of micro-spheres by the solvent evaporation techniquerdquo Advanced DrugDelivery Reviews vol 28 no 1 pp 25ndash42 1997

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


PEG 2000TFN

Vesicles

DMSOwater

Pearling vesicles Nanotubes

DMFwater

PEG-coatedTFN-loadedvesiclesvesicles

Sustained drugdelivery

No pearling of vesicles

Bolaamphiphile 1

NN NN N

NN

O

NH

O

NN

O

NH

O

Figure 1 Illustration of the nanovesicles formation from bolaamphiphile 1 and their subsequent self-assembly into pearl chains and finallynanotubes The nontoxic vesicles fusion (pearling) is prevented by biocompatible polymer coating and used in terfenadine (TFN) drugdelivery in a sustained manner (72 h) in zebrafish model system

of mimicking the biological membrane is a challenging tasksince they can act as life-saving drug delivery and sustaineddrug release cargoes Although a plethora of block copolymerbased vesicles are explored for drug delivery applications [5]vesicles from small biologically inspired designer moleculesare rare Earlier we have found that aromatic rigid core 26-bispyrazolylpyridine (BPP) connected with long alkyl groupsforms vesicles [12] Hence for drug loading application theuse of rigid molecules decorated with one of the base pairsfound in DNA and RNA is a promising starting point toachieve nontoxic drug delivery systemsOne of the ingredientproblems with soft vesicles is their tendency to form tubesthrough fusion mechanism

In this paper we present our strategy to prepare non-toxic and polymer stabilized vesicles from a novel bolaam-phiphile 1 that is two hydrogen bonding N4-acetyl cytosinemolecules connected to 4410158401015840-positions of the BPP using twoflexible octyne chains The vesicles are loaded with terfen-adine drug and successfully utilized to transport and releasedrugs in zebrafish larvae which is utilized as an emergingin vivo model system (Figure 1) In this paper we presentthe synthesis of 1 and prevention of fusion driven nanotubeformation by protecting the nanovesicles (NV) with biocom-patible PEG 2000 terfenadine (TFN) drug loading and itssustained drug releasing tendency The nanovesicles werethoroughly characterized using scanning electron micro-scope (SEM) transmission electron microscope (TEM) andatomic force microscopy (AFM)

2 Materials and Methods

21 Synthesis of Compound 1 Molecule 1 was synthesized insix steps from BPP (Scheme 1) compound 2 was synthesizedas per our reported procedure [13 14]

10101015840-(111015840-(Pyridine-26-diyl)bis(1H-pyrazole-41-diyl))bis(dec-9-yn-1-ol) (3) Compound 2 (05 g 107mmol) Pd(PPh

3)2Cl2

(0075 g 0106mmol) PPh3

(0050 g 0214mmol) CuI(0027 g 0144mmol) were taken in a 100mL two-neck roundbottom flask To this solid mixture high vacuum was appliedthrough condenser for deoxygenating Subsequently solventsdry triethylamine (15mL) 14 dioxane (3mL) was addedto the deoxygenated solid mixture under N

2atmosphere

After five minutes 9-dec-yn-1-ol (0217 g 141mmol) wasadded and stirred for 8 h under N

2atmosphere at 80∘C

The solvents were removed under reduced pressure andsolid product was purified by column chromatography using5 methanolDCM After washing the compound withdiethyl ether pure 3 was obtained as a white solid (80yield) 1H-NMR (400MHz CDCl

3) 120575 = 86 (s 2H) 79 (m

1H) 78 (d 2H) 77 (s 2H) 36 (m 2H ndashOCH2) 24 (m

4H) 16 (m 12H) 14 (m 12H) ppm 13C-NMR (100MHzCDCl

3) 120575 = 1494 1446 1415 1290 1095 1065 926 706

629 327 319 297 292 288 257 226 194 ppm FT-IR(KBr) 361686 339888 314618 305552 292821 285105172645 160106 148146 14971 140238 135415 126542121912 118633 105516 102815 97221 95292 8969886419 80053 73880 70601 65586 LC-MS analysis mz


NNNN

N NN NN N

I I

NN NN

NHO OH

NN NN N

O OS SO

OO

O

NN NN

N

NBr Br

NN

NH

O

O

NNNH

O

O

N NN N

BPP

b

c

d

e

2

3

4

5

1

6 6

6

6 6

66

6

a




3N14 dioxane N

2 9-dec-yn-1-




31H41N5O2 C 7220 H 801 N 1358 found C

7236 H 812 N 1349




3) 120575 = 85 (s 2H) 79 (m 1H) 78 (s 4H) 77 (s 2H)



3) 120575 = 1494 1446


45H53N5O6S2 C 6559 H 648 N 850 found

C 6548 H 642 N 861




3) 120575 =


2) 24 (m 4H) 19 (m 4H) 15 (m 4H) 14 (m 8H)



31H39Br2N5 C 5804 H 613 N 1092 found C

5812 H 617 N 2487




3) 120575


2) 24 (m


3) 120575 = 1713 1628 1557 1494


43H51N11O4 C 6571 H 654 N 1960 found C

6586 H 651 N 1951











Rapid injection

PEG-2000 solution

Filtration and wash

Adjust volume

Solution V1-TFN


Stability study

Zebrafish study

005mL 045mL


Stirring at RT 12h

to 10mL



Residue Filtrate

Redisperse in


filter and wash

Adjust volume




2mL of water

Extract with Et2O

Extract with Et2O



to 2mL

30min

1mL

Solution V1-TFN

Mix with

Ultrasonication










times 100 (1)






























(a) (b) (c)


I II III


VI V IV

200nm

150

nm

(d)


50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100


35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)


50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16


270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions



(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




NNNN

N NN NN N

I I

NN NN

NHO OH

NN NN N

O OS SO

OO

O

NN NN

N

NBr Br

NN

NH

O

O

NNNH

O

O

N NN N

BPP

b

c

d

e

2

3

4

5

1

6 6

6

6 6

66

6

a




3N14 dioxane N

2 9-dec-yn-1-




31H41N5O2 C 7220 H 801 N 1358 found C

7236 H 812 N 1349




3) 120575 = 85 (s 2H) 79 (m 1H) 78 (s 4H) 77 (s 2H)



3) 120575 = 1494 1446


45H53N5O6S2 C 6559 H 648 N 850 found

C 6548 H 642 N 861




3) 120575 =


2) 24 (m 4H) 19 (m 4H) 15 (m 4H) 14 (m 8H)



31H39Br2N5 C 5804 H 613 N 1092 found C

5812 H 617 N 2487




3) 120575


2) 24 (m


3) 120575 = 1713 1628 1557 1494


43H51N11O4 C 6571 H 654 N 1960 found C

6586 H 651 N 1951











Rapid injection

PEG-2000 solution

Filtration and wash

Adjust volume

Solution V1-TFN


Stability study

Zebrafish study

005mL 045mL


Stirring at RT 12h

to 10mL



Residue Filtrate

Redisperse in


filter and wash

Adjust volume




2mL of water

Extract with Et2O

Extract with Et2O



to 2mL

30min

1mL

Solution V1-TFN

Mix with

Ultrasonication










times 100 (1)






























(a) (b) (c)


I II III


VI V IV

200nm

150

nm

(d)


50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100


35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)


50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16


270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions



(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





3) 120575 =


2) 24 (m 4H) 19 (m 4H) 15 (m 4H) 14 (m 8H)



31H39Br2N5 C 5804 H 613 N 1092 found C

5812 H 617 N 2487




3) 120575


2) 24 (m


3) 120575 = 1713 1628 1557 1494


43H51N11O4 C 6571 H 654 N 1960 found C

6586 H 651 N 1951











Rapid injection

PEG-2000 solution

Filtration and wash

Adjust volume

Solution V1-TFN


Stability study

Zebrafish study

005mL 045mL


Stirring at RT 12h

to 10mL



Residue Filtrate

Redisperse in


filter and wash

Adjust volume




2mL of water

Extract with Et2O

Extract with Et2O



to 2mL

30min

1mL

Solution V1-TFN

Mix with

Ultrasonication










times 100 (1)






























(a) (b) (c)


I II III


VI V IV

200nm

150

nm

(d)


50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100


35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)


50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16


270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions



(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





Rapid injection

PEG-2000 solution

Filtration and wash

Adjust volume

Solution V1-TFN


Stability study

Zebrafish study

005mL 045mL


Stirring at RT 12h

to 10mL



Residue Filtrate

Redisperse in


filter and wash

Adjust volume




2mL of water

Extract with Et2O

Extract with Et2O



to 2mL

30min

1mL

Solution V1-TFN

Mix with

Ultrasonication










times 100 (1)






























(a) (b) (c)


I II III


VI V IV

200nm

150

nm

(d)


50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100


35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)


50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16


270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions



(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







times 100 (1)






























(a) (b) (c)


I II III


VI V IV

200nm

150

nm

(d)


50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100


35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)


50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16


270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions



(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














(a) (b) (c)


I II III


VI V IV

200nm

150

nm

(d)


50

50

40

40

30

30

20

20

(120583m

)

(120583m

)

(120583m) (120583m)

10

10 50403020

(120583m)100


35

35

30

25

25

20

15

15

10

05

05

(A)

(B)(C)

(D)


50

40

30

20(120583m

)

10

0

140

120

100

80Hei

ght (

nm)

02 06 10 14

Diameter (120583m)020 04 06 08 10

727068666462

2826242220181614

Hei

ght (

nm)

Hei

ght (

nm)

Length (120583m)02 06 10 14

Diameter (120583m)

313029282726

24

07 09 11

2322212019

200 400 600

(nm)

(120583m

)(120583

m)

(120583m

)(120583

m)

(120583m)

17161514131211

12 14 16

232221201918

39 41 43

(120583m)

(120583m)

38

36

34

32

30

28

26

04 08 12 16


270

265

260

255

0 02 04 06 08 10

(120583m)

Hei

ght (

nm)

(120583m

)

Length (120583m)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(A)

(B)

(C)

(D)

Vesic

les j

unct

ions



(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

60

50

40

30

20

10

0

0 50 100 150 200 250

Num

ber

Diatmeter (nm)

(b)


6

4

2

0

Scor

e

Con

trol

Phe3

mM

Test003120583

M

Test01120583

M

Test03120583

M

Test1120583

M

Test3120583

M

Test10120583

M

Test30120583

M

Treatment


lowastlowastlowast






2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




2

1

0

400 500 600 700

Abso

rban

ce (a

u)

Wavelength (nm)

457nm

005mmol01mmol02mmol

03mmol04mmol

(a)Ab

sorb

ance

(au

)

16

14

12

10

08

06

04

02

00 01 02 03 04


Calibration457nm

(b)


300 350 400 450 500 550 6000

1

2

3

4

Wavelength (nm)

Abso

rban

ce (a

u)

457nm

0h24h48h

72h96h120h



0

20

40

60

80

100

Aver

age c

onc

(uM

)

0 20 40 60 80 100 120

Time (h)

TFN-loadedvesicles


threshold

TFN

In mother liquid





24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




24h

6876nm

6816nm

(a)

72h

(b)


100

80

60

40

20

0

Mor

talit

y (

)

0 12 24 36 48 60 72

Time (h)

TFN20120583M

TFN-NV5 10 120583M

TFN-NV20120583M

TFN-NV40120583M

Heartbeat

Heartbeat

stopped

active




0

12

24

36

48

60

72

Tim

e (h)

0 10 20 30 40


Highaggregation

aggregationLow



4 Conclusion








Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









Acknowledgments


References







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article Organic Nanovesicular Cargoes for...

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