Article pubs.acs.org/Langmuir
Spontaneous Formation of a Vesicular Assembly by a Trimesic Acid Based Triple Tailed Amphiphile Soumik Dinda, Moumita Ghosh, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata 700 032, India
Langmuir 2016.32:6701-6712. Downloaded from pubs.acs.org by UNIV OF BRITISH COLUMBIA on 01/02/19. For personal use only.
S Supporting Information *
ABSTRACT: Trimesic acid based amino acid functionalized triple tailed amphiphiles (TMA-1 and TMA-2) were synthesized. The triskelion amphiphile TMA-1 with a neutral side chain self-assembled into a vesicle in 2:1 (v/v) DMSO−water, while the ammonium side chain decorated TMA-2 formed vesicles in pure water. Microscopic and spectroscopic characterizations were carried out to confirm the self-aggregated vesicular morphology and its size which is around 250−300 nm in the case of TMA-1 and around 100−150 nm for TMA-2 vesicles. The unique structure of these amphiphiles with an aromatic core and three hydrophilic side chains led to an interlamellar orientation of their hydrophobic (aromatic) domain, while hydrophilic terminals were directed toward the aqueous domain. These amphiphiles formed monolayered vesicles possibly through H-aggregation during the process of self-assembly, which is different from conventional bilayered vesicles formed by twin-chain lipid molecules. The time resolved decay curve of hydrophobic dye entrapped within these vesicles indicated that the hydrophobicity within the microenvironment of TMA-1 and TMA-2 vesicles is higher than that in pure water; however, at the same time, it is comparatively lower than that observed in bilayered phosphocholine vesicles. Furthermore, calcein dye was entrapped within these vesicles to ensure their encapsulation efficiency (65−85%). The ability to entrap dye molecules by these synthesized vesicles was utilized to encapsulate and deliver anticancer drug doxorubicin inside the mammalian cells. A simple synthetic procedure and facile aggregation to vesicular selfassembly with superior dye/drug encapsulation proficiency made these vesicles a potential cellular transporter. models at the air−water interface.16,17 The lipid vesicle was first reported in the pioneering work of Bangham et al. where it was termed spherulites.18 Thereafter, vesicle formation by single and twin chained amphiphiles, polymeric amphiphiles, phospholipids, and synthetic surfactants has been well reported.3,6,15,19,20 Stable vesicles also can be formed by mixing oppositely charged surfactants.21,22 Synthetic vesicles provide fundamental insight on the self-assembly phenomena, and they are utilized for a wide range of applications in tissue engineering, nanotechnology, gene therapy, drug delivery, and so on.5,6,13,23,24 Furthermore, Nature explores a triskelion based building block, clathrin, to regulate vesicle formation at the cell membrane during endocytosis and exocytosis.25 Thus, synthetic vesicles having a simple triskelion decorated microstructure could be beneficial for cellular internalization as well as for delivery of cargo inside cells. Triskelion based supramolecular aggregates like gel, microcapsules, discs, etc., have been previously reported for applications in adsorption, encapsulation, and release of dyes.26−30 To this end, trimesic acid
1. INTRODUCTION Over decades, supramolecular self-assembled systems have drawn attention due to their unique features. Liposomes or vesicles are such polymolecular aggregates which are in general formed upon association of amphiphilic molecules aided by simple binding forces like hydrophobic interactions, π-stacking, H-bonding, metal−ligand interactions, etc.1−5 Usually, small amphiphilic molecules having a polar hydrophilic headgroup and two long hydrocarbon tails (either fully saturated or partially unsaturated) are favorable for bilayered vesicle formation in aqueous medium above the critical aggregation concentration (CAC).6−8 Under the osmotic balanced condition, they can form a perfectly spherical aggregate with an inner aqueous core and an outer aqueous domain having a hydrophobic membrane in between these two polar mediums.7 Their unique microstructure having a hydrophobic membrane and an interior aqueous core is proficient for entrapping a large number of substances (hydrophobic as well as hydrophilic).6,9,10 This membrane mimetic system is finding enormous potentials as cellular transporters as well as in biocatalysis to biomedicine.11−13 Bilayered vesicles have gained notable importance due to their structural resemblance to that of natural phospholipid bilayers.14,15 Alternatively monolayered vesicles have also been utilized as free-standing membrane mimetic © 2016 American Chemical Society
Received: May 21, 2016 Revised: June 11, 2016 Published: June 14, 2016 6701
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Figure 1. Structure of synthesized triskelion amphiphiles.
(benzene-1,3,5-tricarboxylic acid) is a well-known symmetric molecule with an aromatic moiety at its center, which is known to facilitate the formation of a hexagonal network and 1D cylindrical aggregates by ordered stacking.31−36 Thus, its unique self-assembling behavior could be appropriately utilized for the design and development of a triskelion based vesicular structure suitable for encapsulation and cellular transportation of different cargo. Herein, the present work reports the synthesis of trimesic acid based triple tailed amphiphiles (TMA-1 and TMA-2, Figure 1) for the development of vesicular self-assembly. The vesicle formation by these triskelion amphiphiles took place possibly through an interlamellar orientation of their hydrophobic (aromatic) core, while the hydrophilic terminal was directed toward the aqueous medium. TMA-1 having neutral hydrophilic tails formed a vesicle in a 2:1 (v/v) DMSO−water mixture, while TMA-2 comprised of a cationic hydrophilic tail formed vesicular self-assemblies in pure water. All of the physicochemical characterizations of these vesicles were carried out using different microscopic and spectroscopic techniques.
The self-assembling mechanism of triskelion amphiphiles investigated by absorption and NMR spectroscopy showed monolayered vesicle formation through H-type aggregation. Dye encapsulation and its release efficiency by these vesicles were checked using calcein as well as a drug/fluorophore, doxorubicin. Moreover, TMA-2 vesicle was utilized to deliver entrapped doxorubicin inside mammalian cells.
2. EXPERIMENTAL SECTION 2.1. Materials. Silica gel of 60−120 mesh and 100−200 mesh, 6-amino caproic acid, triethylamine (Et3N), N,Ndicyclohexylcarbodiimide (DCC), 4-N,N-(dimethylamino)pyridine (DMAP), N-hydroxybenzotriazole (HOBT), trifluoroacetic acid (TFA), solvents, and all other reagents were procured from SRL, India. Water used throughout the study was Milli-Q water. Thin layer chromatography was performed on Merck precoated silica gel 60-F254 plates. Amberlite Ira 900 chloride ion exchange resins were obtained from Aldrich Chemical Co. Trimesoyl chloride, triethylene glycol monomethyl ether, 2,2′-(ethylenedioxy)bis(ethylamine), sephadex-G50, calcein, 1,6-diphenyl-1,3,5-hexatriene (DPH) dye, 8-anilino-1-naphthalenesulfonic acid (ANS), coumarin 153 (C153) dye, 1,2-dioleoyl-sn-glycero3-phosphocholine (PC), and MTT were purchased from Sigma-Aldrich. L-phenylalanine,
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2.3. Preparation of Vesicles. TMA-1 was found to be insoluble in pure water. Hence, TMA-1 (12 mg) was dissolved in DMSO to obtain a clear homogeneous solution and Milli-Q water was added to maintain a volume ratio of 2:1 (v/v) DMSO−water (1 mL) that produced a translucent vesicular solution of TMA-1. The vesicular solution of TMA-2 was prepared in pure water simply by dissolving 12 mg of TMA-2 in 1 mL of Milli-Q water. All of the microscopic and spectroscopic characterizations were performed with these vesicular solutions of TMA-1 and TMA-2. 2.4. Preparation of Phosphocholine (PC) Based Vesicle. PC (2 mg) was dissolved in a minimum amount of chloroform in a glass vial. The chloroform was evaporated by a thin flow of moisture free nitrogen gas, and a dried film of lipid was left behind. This film was further dried thoroughly under a high vacuum for 8 h. To this vacuumdried lipid film, 1 mL of Milli-Q water was added and the mixture was allowed to swell overnight. The vial was then vortexed for 2−3 min at room temperature and bath sonicated for 30 min followed by probe sonication for 30 s to get a clear translucent liposomal solution of PC in water.38 2.5. Determination of Critical Aggregation Concentration (CAC). The critical aggregation concentration (CAC) for both amphiphiles (TMA-1 and TMA-2) was determined by surface tension measurement using a tensiometer by applying the Du Noüy ring method at 25 ± 0.1 °C.6,39 Stock solutions of TMA-1 and TMA-2 were prepared (10 mg/mL) in 2:1 (v/v) DMSO−water and water, respectively, and the surface tension was measured of specific concentrations of each diluted solution prepared from the stock. The CAC values were determined by plotting surface tension versus concentration of amphiphiles with an accuracy of ±2% in triplicate experiments. 2.6. Characterization. 2.6.1. High-Resolution Transmission Electron Microscopy (HRTEM). For HRTEM studies, 3 μL of the TMA-1 solution in 2:1 (v/v) DMSO−water and TMA-2 solution in pure water was deposited on a 300-mesh carbon coated copper grid and allowed to adsorb for 1 min. The excess solution was then blotted with a filter paper. The copper grid was negatively stained with freshly prepared uranyl acetate solution (3 μL, 1% w/v), and the excess solution was immediately blotted with filter paper. The sample was then dried for 4 h in a vacuum before taking the image. The HRTEM images were obtained in a JEOL JEM 2010 microscope. 2.6.2. Field-Emission Scanning Electron Microscopy (FESEM). FESEM images were obtained on a JEOL-6700F microscope. Six μL of TMA-1 solution in 2:1 (v/v) DMSO−water as well as TMA-2 solution in aqueous medium was placed separately on a piece of coverslip and dried overnight. Then, it was kept for a few hours under a vacuum before imaging. 2.6.3. Atomic Force Microscopy (AFM). The AFM images of TMA-1 and TMA-2 were taken on a Veeco, model AP0100, instrument in noncontact mode. A 10 μL solution of TMA-1 in 2:1 (v/v) DMSO−water and an aqueous solution of TMA-2 were deposited separately on freshly cleaved (1 cm × 1 cm) mica and dried overnight before imaging. 2.6.4. Dynamic Light Scattering (DLS) and Zeta (ζ) Potential Measurements. Mean hydrodynamic diameters of the aggregates were determined by DLS using a fixed-angle apparatus (Zen 3690 Zetasizer Nano ZS instrument (Malvern Instruments Ltd.)). The scattering intensity was measured at an angle of 175°. TMA-1 and TMA-2 solutions of different concentrations were prepared in 2:1 (v/v) DMSO−water and water, respectively. The scattering intensity data collected in different solution concentrations were computed by a data processor with the necessary software. The zeta (ζ) potential measurements were performed with solutions of TMA-1 and TMA-2 having 12 mg/mL concentrations at room temperature. An average of three successive measurements was noted for each sample. 2.7. Spectroscopic Studies. 2.7.1. UV−vis Study. To investigate the aggregation pattern of TMA-1 and TMA-2, an UV−vis spectroscopic study was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS) as the probe. The solvent dependent UV−vis spectra of ANS (1 × 10−5 M) doped TMA-1 (ANS-TMA-1) and TMA-2 (ANS-TMA-2) were recorded on a PerkinElmer Lambda 25
Doxorubicin was extracted from doxorubicin hydrochloride. All materials used in the cell culture study such as Dulbecco’s Modified Eagles’ Medium (DMEM), heat inactivated fetal bovine serum (FBS), and trypsin from porcine pancreas were obtained from Himedia. Probe sonication was done using an Omni Sonic Ruptor 250 instrument. Bath sonication was performed with a Telsonic Ultrasonics bath sonicator. 1 H NMR spectra were recorded in an AVANCE 300 MHz (Bruker) spectrometer. The critical aggregation concentrations (CAC) of TMA-1 and TMA-2 were measured using a tensiometer (Jencon, India) by applying the Du Noüy ring method at 25 ± 0.1 °C in water. 2.2. Synthetic Procedure. 2.2.1. Synthesis of TMA-1. In a typical experiment, tert-butoxycarbonyl (Boc) protected 6-amino caproic acid was coupled with the N-terminus of the methyl ester of L-phenylalanine (Phe) using DCC (1.1 equiv) and DMAP (1.1 equiv) in the presence of 1.1 equiv of HOBT in dry DCM. The reaction mixture was then filtered, and the filtrate was concentrated in a rotary evaporator. The whole residue was extracted in DCM, and the product was washed with HCl (1 N), sodium carbonate (1 M), and brine to neutrality. The organic part was dried over anhydrous sodium sulfate and purified through column chromatography using 100−200 mesh silica gel where chloroform/methanol was used as the eluent. The obtained pure material was dissolved in methanol and subjected to alkaline hydrolysis by stirring for 14−16 h with NaOH (1 N). The reaction mixture was then acidified with HCl (1 N) and extracted with ethyl acetate followed by washing with brine to neutrality. It was dried over anhydrous sodium sulfate and concentrated to get the solid material. This was again purified through column chromatography using 60−120 mesh silica gel and chloroform/methanol used as the eluent to get the pure acid. The acid was coupled with triethylene glycol monomethyl ether using the same protocol of DCC coupling stated above. Purified product was subjected to deprotection of Boc by stirring with trifluoroacetic acid (4 equiv) in dry DCM for 3 h. Solvent was removed on a rotary evaporator, and the mixture was taken in ethyl acetate. The ethyl acetate part was thoroughly washed with 10% aqueous sodium carbonate (Na2CO3) solution followed by brine to neutrality. The organic part was dried over anhydrous sodium sulfate and concentrated to get the corresponding amine. The prepared amine (3.5 equiv) was then mixed with trimesoyl chloride (1 equiv) in freshly distilled anhydrous tetrahydrofuran (THF) in the presence of triethyl amine (3.5 equiv) under a nitrogen atmosphere for 6 h.37 The THF was then removed completely with a vacuum pump to get white gelatinous material. The final product, trimesic amide derivative (TMA-1), was then column purified with 100−200 mesh silica gel and chloroform/methanol used as the eluent. The schematic diagram of the synthesis of TMA-1 is shown in Scheme S1, Supporting Information. 2.2.2. Synthesis of TMA-2. The trimesoyl chloride (1 equiv) was added to methyl ester of L-phenylalanine (3.5 equiv) in freshly distilled anhydrous THF medium in the presence of triethyl amine (3.5 equiv).37 THF was removed completely by a vacuum pump and then purified through column chromatography using 60−120 mesh silica gel and chloroform/methanol as an eluent. This corresponding ester was hydrolyzed with NaOH, as described above to prepare the corresponding free acid. This acid was coupled with monoBoc-protected 2,2′-(ethylenedioxy)bis(ethylamine) by treatment with DCC (3.3 equiv), DMAP (3.3 equiv), and HOBt (3.0 equiv) in dry DCM. The product was purified by column chromatography on 100−200 mesh silica gel (chloroform/methanol as eluent). The pure Boc-protected compound was treated with trifluoroacetic acid (10 equiv) in dry DCM under magnetic stirring for 3 h to obtain the corresponding amine. The reaction mixture was concentrated in a rotary evaporator. The obtained yellow gummy material was thoroughly washed with hot ether to get purified triammonium trifluoroacetate salt. This salt was subjected to ion exchange on an Amberlite Ira-400 chloride ion-exchange resin column to get the pure chlorides (TMA-2) (Scheme S2, Supporting Information). Both TMA-1 and TMA-2 were characterized by 1H NMR and mass spectroscopy (data and corresponding spectra are provided in the Supporting Information, Figures S1 and S2 for TMA-1 and Figures S3 and S4 for TMA-2). 6703
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spectrophotometer. The solvents were varied from a non-self-assembled (CHCl3) state to a self-assembled one (2:1 DMSO−water (v/v)) for TMA-1 and form DMSO (non-self-assembled) to water (self-assembled one) for TMA-2. A similar procedure was done for PC liposomal solutions where ANS was doped to PC liposomes (ANS-PC), and the corresponding absorbance of these ANS-PC liposomes was checked in three different solvents like water, DMSO, and CHCl3. The amphiphile and PC concentrations were maintained at 0.5 mg/mL, whereas the ANS concentration was fixed at 10 μM in each case and the corresponding absorbance was measured. 2.7.2. X-ray Diffraction. X-ray diffraction (XRD) spectra of a dried film of vesicular solution were obtained on a Bruker D8 Advance diffractometer, and the source was Cu Kα radiation (α = 0.15406 nm) with a voltage of 40 kV and current of 30 mA. The vesicular solutions of TMA-1 and TMA-2 in their respective solvent systems were placed over a glass slide and dried to form a thin film. The samples were scanned from 0 to 50°. 2.7.3. Fluorescence Anisotropy. Fluorescence spectra were recorded with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5hexatriene (DPH) in a Varian Cary Eclipse fluorescence spectrophotometer. The steady-state anisotropy (r) of DPH was measured in individual solutions of TMA-1 in 2:1 (v/v) DMSO−water and aqueous solution of TMA-2 with varying concentrations. A stock solution of DPH (0.2 mM) was prepared in tetrahydrofuran (THF), and the final concentration of DPH was maintained at 1 μM in each solution (500 μL of each sample solution). The DPH included TMA-1 in 2:1 (v/v) DMSO−water and TMA-2 in water were excited at 370 nm. The emission intensity was measured at 450 nm using an emission cutoff filter at 430 nm to avoid any scattering due to turbidity of the solution. The excitation and emission slit widths were kept at 5 nm. The fluorescence anisotropy value (r) was calculated by the instrumental software following eq 122,40
r = (IVV − GIVH)/(IVV + 2GIVH)
where αi = ∑ iα which indicates the contribution of a decay compoi nent. 2.7.5. Solvent Dependent 1H NMR Measurements. Solvent dependent 1H NMR spectra of TMA-1 and TMA-2 were recorded in an AVANCE 300 MHz (Bruker) spectrometer where the compound concentrations were maintained at 0.5 mg/mL. The solvents were varied from CDCl3 (non-self-assembled) to 2:1 [D6]DMSO− D2O (v/v) (self-aggregated state) for TMA-1 and from [D6]DMSO (non-self-assembled) to D2O (self-assembled state) for TMA-2. Mixed solvents of [D6]DMSO−CDCl3 and [D6]DMSO−D2O were chosen for TMA-1 and TMA-2, respectively, in between the two extreme solvent systems. 2.8. Dye Entrapment and Release. TMA-1 (12 mg) was mixed with 20 μL of calcein (2 mM) solution in water, and the volume was made up to 1 mL with 2:1 (v/v) DMSO−water. The final concentrations of TMA-1 and calcein were 12 mg/mL and 40 μM, respectively, and it was kept overnight under stirring conditions. The solution was then loaded into a sephadex G-50 column (15 cm height and 1.2 cm diameter) pre-equilibrated with 2:1 (v/v) DMSO−water and eluted with the same solvent mixture. Vesicular solutions were eluted right after the void volume. The filtration was carried out until unentrapped calcein was gel-filtrated and removed completely. The eluent was collected in 2 mL fractions. The absorbance for all the fractions was taken at 490 nm to confirm the presence of calcein in each fraction. Eluent was collected until no detectable absorbance of calcein was obtained. Finally, 0.5% (v/v) Triton X-100 was used to rupture the vesicles and estimate the amount of the dye loaded.42 The percentage loading of the dye was determined using the standard calibration curve of free calcein and was found to be ∼85% for TMA-1 vesicle. On the other hand, for TMA-2, all experimental procedures for dye entrapment were carried out in a similar way in pure water (as TMA-2 is soluble in water). It was found that ∼65% of calcein loading took place in TMA-2 vesicles. The loading of calcein within vesicles was further ensured from the comparison of the fluorescence intensity of free calcein and entrapped calcein within TMA-1 and TMA-2 vesicles at λem = 520 nm (λex = 450 nm). For release experiments, 0.5% (v/v) Triton X-100 was mixed with the dye loaded vesicular solution of both TMA-1 and TMA-2 and the florescence spectra were taken before and after the addition of Triton X-100. Both solutions before and after Triton X-100 treatment were further examined under a fluorescence microscope. For DLS studies, 0.5% (v/v) Triton X-100 was added to the vesicular solution of TMA-2 (12 mg/mL) and scattering data was recorded accordingly before and after the treatment of Triton X-100. 2.9. Doxorubicin Loading and Release. The anticancer drug doxorubicin was loaded in TMA-2 vesicles prepared in water. In a typical experiment, TMA-2 (12 mg) and doxorubicin (2 mg) were taken in a glass vial. Milli-Q water (1 mL) was added to it, and the mixture was kept overnight under stirring conditions. This resulting solution was then loaded into a sephadex G-50 column (15 cm height and 1.2 cm diameter) pre-equilibrated with water. Elution was carried out with pure water, and vesicular solution was collected in 2 mL fractions. The absorbance for all of the fractions was taken at 570 nm to confirm the presence of doxorubicin. Eluent was collected until no detectable absorbance of doxorubicin was observed. Finally, 0.5% (v/v) Triton X-100 was used to rupture the vesicles and estimate the percent of drug loaded (∼49%) following the standard protocol using a calibration curve. The fluorescence spectra were taken before and after the addition of Triton X-100 to investigate the release studies of the doxorubicin. Drug loaded TMA-2 vesicular solution was also observed under a fluorescence microscope before and after the treatment of Triton X-100. 2.10. MTT Assay. The cell viability in the presence of TMA-2 was tested by microculture MTT reduction assay. This assay involves the reduction of a soluble tetrazolium salt by mitochondrial dehydrogenase of the viable cells to an insoluble colored formazan product. The amount of product formed was measured spectrophotometrically after dissolution of the reduced dye in DMSO. The enzyme activity and the amount of the formazan produced are proportional to the number of
(1)
where IVV and IVH are the intensities of the emission spectra obtained with vertical and horizontal polarization (for vertically polarized light), respectively, and G = IHV/IHH is the instrumental correction factor, where IHV and IHH are the emission intensities obtained with vertical and horizontal polarization (for horizontally polarized light), respectively. The fluorescence measurements were performed at least five times for each sample at 25 °C. 2.7.4. Time Resolved Study. Time correlation single photon count (TCSPC) measurement was performed in a picosecond diode laser IBH-405. A stock solution of coumarin 153 (C153) dye (1 mM) was prepared in dry THF and was added to vesicular solutions (TMA-1 and TMA-2) in such a way that the final concentration of each compound remains 0.5 mg/mL and the dye concentration was 2 μM. Another two solutions were prepared for the control experiment. Free C153 in pure water was prepared by adding 4 μL of stock solution (THF) to 2 mL of water. Also, C153 was encapsulated in bilayered PC liposomal solution (PC concentration 0.5 mg/mL) by following a similar procedure to that stated above. In both cases, the dye concentration was maintained at 2 μM. All samples were excited at 405 nm. The fluorescence decays were analyzed with IBH DAS6 software. Equation 2 was used to analyze the experimental time resolved fluorescence decay p(t)41 n
p(t ) = b +
⎛ t⎞ ⎟ ⎝ τi ⎠
∑ αi exp⎜− i
(2)
where n is the number of discrete emissive species, b is a baseline correction (“dc” offset), and αi and τi are pre-exponential factors and excited state fluorescence lifetimes associated with the ith component, respectively. For multiexponential decays, the average lifetime ⟨τ⟩ was calculated from eq 3 n
⟨τ ⟩ =
∑ αiτi i=1
(3) 6704
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Figure 2. Negatively stained HRTEM images of (a) TMA-1 prepared in DMSO−water (2:1, v/v) and (b) TMA-2 prepared in water and FESEM images of (c) TMA-1 prepared in DMSO−water (2:1, v/v) and (d) TMA-2 prepared in water. alive cells. The decrease in absorbance can be attributed to the killing of the cells or inhibition of the cell proliferation by TMA-2. B16F10 cells were seeded at a density of 20000 cells per well in a 96-well microtiter plate for 18−24 h before the assay. MTT assay with TMA-2 was performed over a concentration range of 100−500 μg/mL in the microtiter plate. The B16F10 cells were incubated with TMA-2 for 12 h at 37 °C under 5% CO2 atmosphere. MTT stock solution (10 μL from 5 mg/mL) in phosphate buffer saline was added to the above mixture, and the cells were further incubated for another 4 h. The precipitated formazan was dissolved thoroughly in DMSO, and absorbance at 570 nm was measured using a BioTek1 Elisa Reader. The number of surviving cells was expressed as percent viability = [A570(treated cells) − background/A570(untreated cells) − background] × 100. 2.11. Cellular Studies. Doxorubicin loaded TMA-2 vesicle was added into a 24-well chambered plate containing confluent B16F10 melanoma cells in 10% FBS−DMEM culture media for 3 h at 37 °C in 5% CO2 atmosphere. The concentration of doxorubicin in the well was 100 μg/mL. The final volume of the solution was 300 μL in the well. After 3 h of incubation, the cells were repeatedly washed with PBS buffer to ensure complete removal of the extracellular hybrids. The cells were then observed under the Olympus IX51 inverted microscope using an BP530-550 excitation filter and a band absorbance filter covering wavelengths below 570 nm. Bright red images were observed when doxorubicin was internalized inside the cells. The images were taken at 40× magnification.
TMA-2, Figure 1). Trimesic acid was chosen as one of the prime building blocks because of its trifunctionalized structure with an aromatic moiety at its center that could result in ordered stacking of the phenyl moieties during selfassembly.31−36 Initially, TMA-1 having neutral hydrophilic triethylene glycol monomethyl ether as a side chain of trimesic acid was synthesized. It was observed that TMA-1 was insoluble in pure water. However, TMA-1 formed a homogeneous translucent solution at 2 mg/mL in 2:1 (v/v) DMSO−water solvent mixture. This finding triggered our interest in synthesizing a more hydrophilic side chain based triskelion scaffold which will be soluble in pure water. To this end, TMA-2 was synthesized having a 2,2′-(ethylenedioxy)bis(ethylamine) unit terminated with a free −NH3+ group (Figure 1). As to our expectation, cationic TMA-2 was soluble in pure water and it formed a translucent solution in water at 2 mg/mL. Although TMA-1 and TMA-2 do not have a traditional surfactant-like structure with a hydrophilic headgroup and a hydrophobic tail, the central aromatic scaffold of these synthesized amphiphiles might play an important role in ordered stacking in the process of self-assembly. On the other hand, the hydrophilic side chains might have a specific orientation toward the bulk aqueous medium. In this context, Meijer and co-workers reported the formation of columnar aggregates with a C3-symmetrical benzene-1,3,5-tricarboxamide (BTA) unit which was restricted to discotic self-assembly by highly charged peripheral GdIII− DTPA (diethylene triamine pentaacetic acid) groups.31 It has also been reported that an optimum balance between the
3. RESULTS AND DISCUSSION With the objective of developing a triskelion based vesicular self-assembly, we have synthesized benzene-1,3,5-tricarboxylic acid or trimesic acid based tripodal amphiphiles (TMA-1 and 6705
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Figure 3. AFM images of (a) TMA-1 prepared in DMSO−water (2:1, v/v) and (b) TMA-2 prepared in water. Height profile (c) along p−q in part a and (d) along r−s in part b.
3.2. Microscopic Characterization of TMA-1 and TMA-2 Vesicles. The morphology of the self-aggregated systems formed by TMA-1 and TMA-2 was initially investigated by HRTEM. The negatively stained HRTEM images showed the spherical shaped vesicles with a thin wall and hollow core. The average diameter of the vesicles is 250−300 nm in the case of TMA-1 and around 100−150 nm for TMA-2 (Figure 2a and b and Figure S6a and b, Supporting Information). Hence, the formation of a vesicular self-assembly in both DMSO−water as well as water by trimesic acid based amphiphiles is evident from the HRTEM images. The vesicular aggregation of TMA-1 and TMA-2 was further confirmed from the FESEM investigation. The FESEM images of TMA-1 showed a perfectly spherical morphology with an average diameter of ∼300−400 nm (Figure 2c and Figure S6c, Supporting Information). Similar spherical aggregated structures were observed in the FESEM image for TMA-2 (Figure 2d and Figure S6d, Supporting Information). In concurrence with the HRTEM images, here also the sizes of TMA-2 vesicles were in the range of ∼100 nm. AFM study further confirmed the vesicular structures formed by TMA-1 and TMA-2 in their respective solvent systems (Figure 3a and b and Figure S6e and f, Supporting Information). The morphology from AFM images showed a spherical vesicle having a diameter of ∼350 nm for TMA-1, while it was ∼120 nm for TMA-2. Cross-section analysis of the AFM images (Figure 3c (p−q) and 3d (r−s)) provides an idea about the height of the vesicles which was ∼55 nm for TMA-1 and ∼40 nm for TMA-2 vesicles, indicating that these vesicles were hollow and flattened in shape.3,44
attractive noncovalent forces within the hydrophobic core of the polymerizing building block with the electrostatic repulsive interaction of the hydrophilic ring led to a switch from elongated, rod-like assemblies to small and discrete supramolecular aggregates by the C3-symmetrical self-assembling molecules.33 In a separate study, BTA derivatives having an amphiphilic chain also were reported to form fibrillar aggregates through selfassembly.43 Hence, we were curious to understand the molecular organization of these newly synthesized amphiphiles in their respective solvent systems, i.e., TMA-1 in 2:1 (v/v) DMSO− water and TMA-2 in pure aqueous medium. 3.1. Critical Aggregation Concentration (CAC). The critical aggregation concentration (CAC) of both amphiphiles was determined by the surface tension method in the respective solvent systems. The surface tension of a liquid gradually decreases upon increasing the concentration of a surface active agent. Upon reaching the CAC value, any further addition of surfactant molecules will just increase the number of aggregation. Herein, the surface tension of TMA-1 and TMA-2 was measured with a varying concentration range of 0−4 mg/mL (Figure S5, Supporting Information). The break point in both cases indicates that TMA-1 and TMA-2 have self-aggregation property. The CAC value of TMA-1 and TMA-2 was found to be ∼250 and 330 μg/mL, respectively, from where the amphiphile starts to form the self-aggregated structure. The self-aggregated systems were found to be stable for more than 3 months. These self-assemblies were consequently investigated by microscopic and spectroscopic studies to determine their morphology as well as aggregation mechanism. 6706
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Figure 4. Concentration dependent size distribution profiles of vesicles from DLS of (a) TMA-1 prepared in DMSO−water (2:1, v/v) and (b) TMA-2 prepared in water.
3.3. DLS Study and Zeta Potential. DLS experiments were carried out to obtain the mean hydrodynamic diameter and the size distribution of TMA-1 and TMA-2 vesicles. The size distribution (expressed in number percentage) profiles for both amphiphiles of varying concentrations (2−16 mg/mL) are shown in Figure 4. It is clearly observed that the size distribution for TMA-1 vesicles was in the range 200−500 nm, whereas TMA-2 aggregates showed a smaller hydrodynamic diameter in the range 70−150 nm (Figure 4). Moreover, it is also evident that the mean hydrodynamic diameter remains almost constant with varying concentrations of amphiphiles, indicating the stability of the vesicles with respect to concentration dependent phase transformation.6 Also, the DLS measurement data for both TMA-1 and TMA-2 vesicles are in good concurrence with the diameter of vesicles obtained from microscopic investigations (Figures 2 and 3). Zeta (ζ) potential is used to determine the stability of a colloidal suspension. Conventionally, a high ζ potential value indicates better stability of the colloids due to repulsion between the surface charges on components present in the solution. The ζ potential of TMA-1 and TMA-2 vesicles was found to be −8.8 and +24.9 mV, respectively. Despite having the neutral side chain, TMA-1 showed a considerable negative zeta potential while TMA-2 expectedly showed a high positive zeta potential value. These values indicate substantial stability of the corresponding vesicular aggregates. 3.4. Spectroscopic Studies for Determination of Aggregation Pattern of TMA-1 and TMA-2 Vesicles. 3.4.1. Steady-State Fluorescence Anisotropy. Steady state fluorescence anisotropy measurement is often used to investigate the microenvironment of an aggregated structure. DPH is a widely used fluorescence probe to determine the fluorescence anisotropy (r) for different aggregated structures like micelles, vesicles, bilayers, etc.22,40 Herein, the r-value was determined for both TMA-1 and TMA-2 self-aggregates using DPH (Table 1). The r value increased from 0.08 to 0.18 and 0.09 to 0.16 for TMA-1 and TMA-2 self-assemblies, respectively, over a concentration range from 4 to 25 mg/mL. DPH is a well-known membrane fluidity probe which can easily accommodate itself in the hydrophobic region of the vesicle because of its rigid, rod-like structure.40 The higher the restriction in the movement of DPH in the hydrophobic domain, the larger the anisotropy value. In general, DPH always exhibit greater fluorescence anisotropy in vesicles compared to that of micellar aggregates.
Table 1. Steady-State Fluorescence Anisotropy (r) of DPH with Varying Concentrations of TMA-1 in DMSO−Water (2:1, v/v) and TMA-2 in Water r-values concentration (mg/mL)
TMA-1
TMA-2
4 8 12 16 20 25
0.08 0.08 0.09 0.18 0.18 0.18
0.09 0.09 0.15 0.15 0.16 0.16
Here also, the observed r values of DPH (0.18 for TMA-1 and 0.16 for TMA-2) are considerably higher compared to those of micellar aggregates of sodium dodecyl sulfate (r = 0.054), which further confirms the formation of vesicular aggregates by TMA-1 and TMA-2.45 3.4.2. UV−vis Study and 1H NMR Study. At this instant, we were keen to understand the molecular level self-aggregation nature of TMA-1 and TMA-2 that led to the formation of vesicles. The aggregation pattern of TMA-1 and TMA-2 having a concentration of 0.5 mg/mL was investigated from solvent dependent UV−vis spectra of ANS (1 × 10−5 M) doped TMA-1 (ANS-TMA-1) and TMA-2 (ANS-TMA-2). In the case of TMA-1, solvent composition was varied from CHCl3 (non-selfaggregated state) to 2:1 (v/v) DMSO−water (self-aggregated state). Similarly for TMA-2, the solvent system was varied from a non-self-aggregated system (DMSO) to a self-aggregated system (water). The hydrophobic probe ANS is prone to localize itself at the hydrophobic domain of the vesicular aggregate. Herein, ANS-TMA-1 and ANS-TMA-2 showed blue-shifted UV absorption maxima upon moving from non-self-assembled to selfassembled state (Figure 5). Absorption maxima of ANS-TMA-1 blue-shifted from 378 to 372 nm upon changing the solvent from CHCl3 to 2:1 (v/v) DMSO−water (Figure 5a). Similarly, ANS-TMA-2 showed blue-shifted absorption maxima from 346 to 342 nm when the solvent system was varied from DMSO to water (Figure 5b). As reported earlier, blue-shifted absorption maxima correspond to self-organization of molecules through parallel plane-to-plane stacking forming a sandwichtype arrangement termed H-type aggregation.46−48 Thus, the observed blue-shifted absorption maxima clearly designate the H-type aggregation pattern for the self-assemblies of both TMA-1 6707
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Figure 5. UV−vis spectra of (a) ANS tagged TMA-1 and (b) ANS tagged TMA-2 in different solvents.
Scheme 1. Pictorial Representation of the Vesicular Self-Assembly through H-Aggregation from a Triple Tailed Amphiphile (with TMA-2 as an Example)a
a
The blue part represents the hydrophilic chain, and the red part represents the central aromatic core.
state), the NMR signals of aromatic protons almost diminished accompanied by further lowering in the δ value to 7.86 and 6.29−6.41 ppm, respectively. A similar upfield shift in the 1 H NMR signal was observed in the case of TMA-2 (Figure S8b, Supporting Information). The aromatic protons of the trimesic moiety showed a sharp peak at δ = 8.43 ppm in [D6]DMSO (non-self-assembled state) which got shifted to δ = 7.51 ppm with increasing content of D2O (self-assembled state). Similarly, in the case of aromatic protons of L-phenylalanine, the characteristic aromatic peak in the region of δ = 7.23−7.45 ppm in [D6]DMSO got shifted to δ = 6.75−6.84 ppm in D2O. Upon gradual increase in D2O content, the self-aggregation gets initiated and facilitates the intermolecular π−π interaction between the aromatic core of the adjacent amphiphile. As a consequence, upfield shift of aromatic protons was noticed in the 1H NMR signals. Both UV−vis and NMR spectroscopic experiments confirmed the participation of hydrophobic as well as π−π stacking interaction during self-aggregation of TMA-1 and TMA-2. 3.4.3. XRD Study. The X-ray diffraction data of TMA-1 and TMA-2 in the aggregated state showed the peak at 2θ ∼ 23° (Figure S9, Supporting Information), which demonstrates the well-defined and compact π−π stacking interaction between the aromatic rings of amphiphiles.50,51 The ordered π−π stacking might play a vital role in the formation of monolayered vesicular aggregates which is in good concurrence with the abovediscussed UV−vis and 1H NMR studies.
and TMA-2. On the contrary, the aggregation pattern of bilayered PC liposomes showed no such change in the absorption maxima of ANS from non-self-assembled (DMSO, CHCl3) to self-assembled state in water (Figure S7, Supporting Information). Thus, it is evident that the vesicular aggregates formed by TMA-1 and TMA-2 are different in nature from those of conventional bilayered liposomes of PC. The unique hydrophilic−hydrophobic− hydrophilic molecular backbone of TMA-1 and TMA-2 possibly resulted in the formation of a monolayered ordered arrangement of the amphiphiles (inset of Figure 5).16,17 The formation of vesicular aggregates through monolayer stacking of triple tailed amphiphiles in a parallel layer-by-layer fashion is depicted in Scheme 1. The 1H NMR study provides information about the participation of various interacting forces between the self-associating molecules.49 Accordingly, we performed solvent dependent 1 H NMR experiments (Figure S8, Supporting Information) for TMA-1 and TMA-2 to monitor the interactions between aromatic rings during the course of self-aggregation. In CDCl3, the aromatic protons of core trimesic acid and the L-phenylalanine at the side chain of TMA-1 showed a sharp peak in the region δ = 8.49 and 7.09−7.23 ppm, respectively (Figure S8a, Supporting Information). In CDCl3, TMA-1 is in a molecularly dissolved state without any self-aggregation. On addition of [D6]DMSO in the system ([D6]DMSO:CDCl3 (1:1, v/v)), the NMR signals of the aromatic protons got upfield shifted to δ = 8.11 and 6.42−6.57 ppm, respectively. Notably when TMA-1 was taken in [D6]DMSO− D2O (2:1, v/v) mixture (TMA-1 in self-assembled vesicular 6708
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Figure 6. Fluorescence microscopic images (λex = 450 nm) of encapsulated calcein in (a) TMA-1 vesicle prepared in DMSO−water (2:1, v/v), (b) fluorescence emission spectra of encapsulated calcein in TMA-1 vesicle and free calcein in 2:1 (v/v) DMSO−water, (c) fluorescence microscopic images (λex = 450 nm) of encapsulated calcein in TMA-2 vesicle prepared in water, and (d) fluorescence emission spectra of encapsulated calcein in TMA-2 vesicle and free calcein in water.
3.4.4. Time Resolved Study. To further understand the aggregation pattern of vesicular assemblies, we have performed a time-correlated single-photon counting (TCSPC) experiment where the decay time of C153 dye (excitation 405 nm) was analyzed in pure water and in vesicular solution (Figure S10, Supporting Information). The decay time of free C153 in water was found to be 1.62 ns, while it increased to 3.01 ns after encapsulation in TMA-1 vesicles prepared in DMSO−water (2:1, v/v). C153 encapsulated in TMA-2 vesicles in water showed an intermediate decay time of 1.73 ns. This increase in the decay time of C153 within TMA-1 and TMA-2 vesicles could be due to the encapsulation of the dye molecule in a more hydrophobic environment.52 It was further observed that the decay time of C153 in TMA-1 and TMA-2 vesicles is less than that of wellknown bilayered PC liposomes which is 4.65 ns (Figure S10, Supporting Information). Hence, the hydrophobicity within the microenvironment of TMA-1 and TMA-2 vesicles is comparatively lower than that observed in bilayered PC vesicles.52 This intermediate hydrophobicity of the synthesized vesicles distinguishes it from bilayered PC liposomes and further indicates the formation of monolayered vesicles by TMA-1 and TMA-2 (Scheme 1). 3.5. Dye Entrapment and Release Studies. To further shed light on the vesicular structure of the newly synthesized
amphiphiles, a dye encapsulation study was carried out for TMA-1 and TMA-2 vesicles. This study can confirm the presence of a confined water pool inside the self-assembly as well as the ability of the aggregate to entrap hydrophilic guest molecules in its aqueous core. To this end, water-soluble fluorescent calcein was chosen as a model drug. Calcein is known to accommodate itself in the inner aqueous compartment of the vesicles.6 The detailed procedures of entrapment experiments are described in the Experimental Section where calcein was encapsulated with the TMA-1 and TMA-2 vesicles prepared in 2:1 (v/v) DMSO−water and pure water, respectively. Dye encapsulated vesicles were separated from unentrapped calcein by size exclusion chromatography using sephadex G-50.6 The eluted solution of entrapped calcein showed a characteristic UV−vis absorbance peak at λmax = 490 nm, indicating the presence of the dye within the vesicles (Figure S11, Supporting Information). Dye entrapped self-aggregated solutions of both TMA-1 and TMA-2 were further examined under a fluorescence microscope which showed a green emitting sphere (Figure 6a and c), confirming the efficient calcein encapsulation inside the aqueous core of vesicles. Encapsulation of the dye was further ensured from the comparative fluorescence intensity of the free calcein and entrapped calcein in vesicles at 520 nm (λex = 450 nm). It was observed that the fluorescence 6709
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Langmuir intensity of vesicle entrapped calcein notably reduced compared to that of free calcein (Figure 6b and d) possibly due to its selfquenching property under confined conditions.3,53 The loading percentage of the entrapped dye was also determined by rupturing the vesicles with 0.5% (v/v) Triton X-100 solution. The calcein loading was about 85% for TMA-1 and 65% for TMA-2 vesicles, as calculated from the standard calibration curve (absorbance vs concentration) of free calcein. The release of entrapped calcein from the synthesized vesicles was also investigated by fluorescence spectroscopy. The emission intensity of entrapped calcein in TMA-1 as well as TMA-2 vesicles was found to be low (Figure 6 and Figure S12, Supporting Information). However, upon treating with Triton X-100, the emission intensity notably increased and became comparable to that of free calcein (Figure S12, Supporting Information). This observation supports the release of dye molecules from the inner aqueous compartment of the vesicle to the bulk solvent due to the rupturing of vesicular aggregates in the presence of Triton X-100. The disintegrated vesicles were also observed under fluorescence microscope which showed no well-defined spherical shape (Figure S13, Supporting Information). In the case of the TMA-2 vesicle, DLS measurement showed that, upon treating with Triton X-100, the hydrodynamic diameter of the vesicular assembly of around 100 nm had completely disappeared, which indicates the disintegration of vesicles (Figure 7). New scattering data
appeared at around 3−5 nm probably due to molecular aggregates of disintegrated amphiphiles or Triton X-100. 3.6. Doxorubicin Loading and Release Studies. Ensuring superior cargo encapsulation ability and stability of the vesicle, the anticancer drug doxorubicin was loaded within the TMA-2 vesicle in water following the same procedure as described for calcein (see the Experimental Section). TMA-1 could not be considered for cellular transportation, as it forms a vesicle in the DMSO−water (2:1, v/v) mixture. Entrapment of doxorubicin within the TMA-2 vesicle was confirmed from the fluorescence microscopic image (Figure 8a), and its loading efficiency was found to be 49% (calculated after treating the doxorubicin entrapped vesicle with Triton X-100 and followed by comparing the absorbance with the standard calibration curve).54 The release of encapsulated drug from the synthesized TMA-2 vesicles was also investigated by fluorescence spectroscopy. The emission intensity of entrapped doxorubicin in TMA-2 vesicles was found to be low enough with respect to the emission intensity of free doxorubicin having the same concentration (Figure S14, Supporting Information). The emission intensity of doxorubicin was revived upon addition of Triton X-100. This observation supports the release of drug molecules from the inner aqueous core of the vesicle to the bulk solvent due to the rupturing of vesicular aggregates in the presence of Triton X-100. The fluorescence microscopic images also show disintegrated vesicles where no spherical vesicular aggregates are seen (Figure S14, Supporting Information). 3.7. Cell Viability Determined by MTT Assay. To exploit the superior drug encapsulation ability of TMA-2 vesicles, one of the prerequisites is to ensure the cytocompatibility of the synthesized amphiphiles. The biocompatibility of TMA-2 was studied against mammalian cells (B16F10) by MTT assay.55 The critical aggregation concentration (CAC) of TMA-2 was found to be 330 μg/mL; hence, MTT assay with TMA-2 was performed over a concentration range from 100 to 500 μg/mL. After 12 h of incubation, around 80−85% cells were found to be alive even at 500 μg/mL concentration of TMA-2 (Figure S15, Supporting Information). Thus, TMA-2 vesicles showed substantial cell viability and were suitable to be used as cellular transporters. 3.8. Cellular Internalization. Doxorubicin loaded TMA-2 vesicle was then incubated with B16F10 melanoma cells for 3 h. These mammalian cells were found to be in spindle shape after the incubation period which indicates the healthy morphology of the melanoma cells (Figure 8b). Importantly, after 3 h of incubation, the red fluorescence images of the cells confirmed the successful internalization of doxorubicin within B16F10
Figure 7. Size distribution of aggregates formed by TMA-2 vesicles in water before (red) and after (black) treatment with Triton X-100 (0.5% v/v) in 1 mL of vesicle solution.
Figure 8. (a) Fluorescence microscopic images of doxorubicin encapsulated TMA-2 vesicle. (b) Bright field and (c) fluorescence microscopic images of B16F10 cells incubated with doxorubicin loaded TMA-2 vesicle for 3 h. 6710
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(2) Xu, Y. X.; Wang, G. T.; Zhao, X.; Jiang, X. K.; Li, Z. T. SelfAssembly of Vesicles from Amphiphilic Aromatic Amide-Based Oligomers. Langmuir 2009, 25, 2684−2688. (3) Mondal, T.; Dan, K.; Deb, J.; Jana, S. S.; Ghosh, S. HydrogenBonding-Induced Chain Folding and Vesicular Assembly of an Amphiphilic Polyurethane. Langmuir 2013, 29, 6746−6753. (4) Wang, J.; Song, A.; Jia, X.; Hao, J.; Liu, W.; Hoffmann, H. Two Routes to Vesicle Formation: Metal-Ligand Complexation and Ionic Interactions. J. Phys. Chem. B 2005, 109, 11126−11134. (5) Liu, S.; Gonzá lez, Y. I.; Kaler, E. W. Structural Fixation of Spontaneous Vesicles in Aqueous Mixtures of Polymerizable Anionic and Cationic Surfactants. Langmuir 2003, 19, 10732−10738. (6) Ghosh, R.; Dey, J. Vesicle Formation by L-Cysteine-Derived Unconventional Single-Tailed Amphiphiles in Water: A Fluorescence, Microscopy, and Calorimetric Investigation. Langmuir 2014, 30, 13516−13524. (7) Evans, E. A.; Waugh, R. Mechano-Chemistry of Closed, Vesicular Membrane Systems. J. Colloid Interface Sci. 1977, 60, 286−298. (8) Savsunenko, O.; Matondo, H.; Messant, S. F.; Perez, E.; Popov, A. F.; Rico-Lattes, I.; Lattes, A.; Karpichev, Y. Functionalized Vesicles Based on Amphiphilic Boronic Acids: A System for Recognizing Biologically Important Polyols. Langmuir 2013, 29, 3207−3213. (9) Xiao, M.; Xia, G.; Wang, R.; Xie, D. Controlling the SelfAssembly Pathways of Amphiphilic Block Copolymers into Vesicles. Soft Matter 2012, 8, 7865−7874. (10) Mondal, J. H.; Ahmed, S.; Ghosh, T.; Das, D. Reversible Deformation−Formation of a Multistimuli Responsive Vesicle by a Supramolecular Peptide Amphiphile. Soft Matter 2015, 11, 4912− 4920. (11) Summers, D. P.; Rodoni, D. Vesicle Encapsulation of a Nonbiological Photochemical System Capable of Reducing NAD+ to NADH. Langmuir 2015, 31, 10633−10637. (12) Boyer, C.; Zasadzinski, J. A. Multiple Lipid Compartments Slow Vesicle Contents Release in Lipases and Serum. ACS Nano 2007, 1, 176−182. (13) Soussan, E.; Cassel, S.; Blanzat, M.; Rico-Lattes, I. Drug Delivery by Soft Matter: Matrix and Vesicular Carriers. Angew. Chem., Int. Ed. 2009, 48, 274−288. (14) Evans, C. C.; Zasadzinski, J. Encapsulating Vesicles and Colloids from Cochleate Cylinders. Langmuir 2003, 19, 3109−3113. (15) Walde, P. Buidling Artificial Calls and Protocell Models: Experiments Approaches with Lipid Vesicles. BioEssays 2010, 32, 296− 303. (16) Palivan, C. G.; Goers, R.; Najer, A.; Zhang, X.; Cara, A.; Meier, W. Bioinspired Polymer Vesicles and Membranes for Biological and Medical Applications. Chem. Soc. Rev. 2016, 45, 377. (17) Zhou, Y. F.; Yan, D. Y. Supramolecular Self-Assembly of Amphiphilic Hyperbranched Polymers at all Scales and Dimensions: Progress, Characteristics and Perspectives. Chem. Commun. 2009, 1172−1188. (18) Bangham, A. D.; Horne, R. W. Negative Staining of Phospholipids and Their Structural Modification by Surface-active Agents as Observed in the Electron Microscope. J. Mol. Biol. 1964, 8, 660−668. (19) Antunes, F. E.; Marques, E. F.; Gomes, R.; Thuresson, K.; Lindman, B.; Miguel, M. G. Network Formation of Catanionic Vesicles and Oppositely Charged Polyelectrolytes. Effect of Polymer Charge Density and Hydrophobic Modification. Langmuir 2004, 20, 4647−4656. (20) Wall, S. L. D.; Barbour, L. J.; Gokel, G. W. Solid-state Bilayer Formation from a Dialkyl-Substituted Lariat Ether that Forms Stable Vesicles in Aqueous Suspension. J. Phys. Org. Chem. 2001, 14, 383− 391. (21) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Iampietro, D. J.; Kaler, E. W. The Origins of Stability of Spontaneous Vesicles. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 1353−1357. (22) Shome, A.; Kar, T.; Das, P. K. Spontaneous Formation of Biocompatible Vesicles in Aqueous Mixtures of Amino Acid-Based
cells (Figure 8c). Thus, TMA-2 vesicle holds immense potential to be utilized as a cellular transporter.
4. CONCLUSION In summary, trimesic acid based amino acid functionalized triple tailed amphiphiles have been synthesized. These triskelion amphiphiles can self-assemble into a vesicular morphology in 2:1 (v/v) DMSO−water for TMA-1 and also in pure water for TMA-2. Formation of vesicles by these amphiphiles was thoroughly investigated using microscopic as well as spectroscopic techniques. It was found that these synthesized amphiphiles form monolayered vesicles possibly through H-aggregation in the process of its self-assembly, which is different from the bilayered vesicles of twin-chain lipid molecules. Moreover, the ability to entrap dye/drug molecules by these synthesized vesicles was used to deliver doxorubicin inside the mammalian cells. Considering the ease of synthesis, possibilities of structural modification by introducing new functionality and facile aggregation to vesicular self-assembly, these triskelion based vesicles hold immense potential to be utilized as a delivery vehicle into cells.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01942. Synthetic schemes of TMA-1 and TMA-2, 1H NMR mass spectra and data of TMA-1 and TMA-2, plot of surface tension versus concentration of TMA-1 and TMA-2 in their respective solvent systems, HRTEM, FESEM, AFM of TMA-1 and TMA-2 vesicles, UV−vis spectra of ANS tagged PC in different solvents, solvent dependent 1H NMR spectra of TMA-1 and TMA-2, XRD of TMA-1 and TMA-2 vesicles, fluorescence decay curves of C153 dye encapsulated in PC liposomes, TMA-1 and TMA-2 vesicles and in pure water, UV−vis absorbance spectra of calcein entrapped TMA-1 and TMA-2 vesicles and free dye, emission spectra of calcein entrapped TMA-1 and TMA-2 vesicles and after treating with Triton X-100, fluorescence microscopic images of calcein encapsulated TMA-1 and TMA-2 vesicles and after treatment with Triton X-100, fluorescence emission spectra of doxorubicin entrapped TMA-2 vesicles and after treating with Triton X-100, fluorescence microscopic images of doxorubicin encapsulated TMA-2 vesicles after treatment with Triton X-100, and cell viability plot (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS P.K.D. is thankful to CSIR, India (ADD, CSC0302), for financial assistance. S.D. and M.G. acknowledge CSIR, India, for Research Fellowships. We acknowledge Dr. Subhra Kanti Mandal for helpful discussion.
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REFERENCES
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DOI: 10.1021/acs.langmuir.6b01942 Langmuir 2016, 32, 6701−6712
