Colloids and Surfaces B: Biointerfaces 111 (2013) 571– 578
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Colloids and Surfaces B: Biointerfaces
jou rn al hom epage: www.elsev ier .com/ locate /co lsur fb
hotosensitive nanocapsules for use in imaging fromoly(styrene-co-divinylbenzene) cross-linked withoumarin derivatives
algorzata Sierant, Piotr Paluch, Marcin Florczak, Artur Rozanski, Beata Miksa ∗
entre of Molecular and Macromolecular Studies, Polish Academy of Science, Sienkiewicza 112, 90-363 Lodz, Poland
r t i c l e i n f o
rticle history:eceived 12 December 2012eceived in revised form 29 June 2013ccepted 1 July 2013vailable online 17 July 2013
eywords:olymer vesicle
a b s t r a c t
The study objective was to generate biocompatible probes and develop a stable macromolecule imag-ing system that are based on nanolipopolymersomes and can be used in living cells. We synthesizednanolipopolymersomes with a fluorescent polymer wall surrounded by an outer phospholipid shell thatexhibits potential for the controlled delivery of diagnostic agents to cells. We describe a new type of probesuitable for dual detection methods (spectrophotometric and fluorescence). This aspect makes it uniqueamong currently available probes because allows it to be detected with greater accuracy. We developeda highly fluorescent coumarinated polymer to overcome the limited brightness of conventional dyes
anolipopolymersomesoumarin
n vitro imaging
with insufficient for long-term photostablility. Hydrophilic dyes (Lucifer yellow, Procion red, Procionblue) are entrapped in the aqueous core of stable polymeric nanocapsules with coumarin 6 embeddedin a nanometre-thick poly(styrene-co-divinylbenzene) wall. Target compounds can be incorporated intonanocapsules in a single step. The hydrophilic phospholipids outer shell ensures biocompatibility andfacilitates cell penetration. In this way, the novel fluorescent hybrid materials can help of nanotechnology.
. Introduction
The design of diverse nanometer-scale objects for use in therapy,rug delivery, and imaging [1,2] is a tremendous challenge [3–7].olymer capsules [8–10] are particularly important as nanovesselsapable of controlled transport of active compounds, enhancingermeability and efficacy [11–16]. A number of strategies aremployed to achieve this goal. Among the most interesting areanolipopolymersomes which are polymer vesicles based on lipo-omal templates [17]. Self-assembly is one of the most importantays to create a temporary template from phospholipids which
re soft spherical structures. An aqueous core gives them a supe-ior ability to encapsulate guest compounds: as a result they findany applications in analytical fields [18–21].However, liposomes are fragile. A number of approaches
ave been used to enhance the stability of phospholipids
esicles including polymeric phospholipids [22–25], surface graft-ng of hydrophilic polymers (particularly polyethylene glycolPEG)) [26–31], and the incorporation of polymeric hydrophobic
monomers (butyl methacrylate, 2-ethylhexyl methacrylate) intothe bilayer [32]. Inorganic silica is also considered biocompatibleand is an important element for a wide range of potential commer-cial applications [33–36]. Recently, Pinkhassic [37] developed analternative to the incorporation of hydrophobic monomers (tert-butyl styrene and divinylbenzene) into the hydrophobic, lamellarregion of the lipid bilayer. This method involved UV-initiated rad-ical polymerization to obtain cross-linked polymer capsules withrigid walls and a well-controlled shape [38]. This method reducesthe outer shell to a nanometer thickness which is vital for in vivo use,enabling polymer vesicles to retain their cargo capacity after lipidremoval. Due to the possibility of including well-defined nanoporesacross the polymer membrane [39] of capsules, they are well suitedto communication with diverse external environments (varying insuch factors as pH, temperature, or ionic strength). Thus, in thecreation of a neutral polymer system for use in functional nan-odevices this method may assist in the design of a multifunctionalcarrier for delivery of therapeutic and imaging agents via controlledencapsulation and the simultaneously release of compounds [36].
Here, we propose a new form of nanopolymersomes with theouter shell acting as an imaging agent and stabilizer without
the use of surfactants. We present an efficient synthesis of fluo-rescent polymer nanovesicles with a photo-trigger chromophore(coumarin 6) [40,41] embedded into a cross-linked organic poly-mer wall [42]. Surface functional groups [43] can be modified
ig. 1. Plausible structure highlighting the role of dye-sensitized coumarin 6 in freeadical UV polymerization (A). Coumarin 6 is incorporated in polymer host in neutralorm (B) [47–51].
epending on the intended use via nano/bioconjugated bonds.rowing interest in coumarin chemistry has been sparked by theseompounds versatility in many applications and their biologicalroperties (anticoagulant, anti-HIV, antibacterial, antihyperpro-
iferative) [44,45]. Coumarin derivatives have also been useds phototriggers in a photocontrolled biological release system46,47]. In the present work an outer hydrophilic shell of phos-holipids enables cell penetration. This biocompatibility is a crucialspect of in vivo medical diagnostics.
The goal of this work is the preparation of biocompatible probesased on nanolipopolymersomes suitable for application to liv-
ng cells. We investigated biomedical applications of these probes
n vitro using HeLa cells. Moreover, we tested the cellular uptakend cytotoxicity of the probes both of which are vital determinantsf the probes biomedical applications.
ig. 2. Self-assembled liposomes forming a phospholipids bilayer were saturated witolymerization produces a nanometer-thick fluorescent film. Dye solutions were encapsu
Biointerfaces 111 (2013) 571– 578
2. Materials and methods
2.1. Materials
Phospholipids (1,2-dimyristoyl-sn-phosphocholine (DMPC))were purchased from Avanti Polar Lipids Inc. (Alabaster, AL,USA) as a dry powder. All other reagents [divinylbenzene(DVB) styrene (ST), 2,2-dimethoxy-2-phenylacetophenon (DMPA),Procion red, Procion blue, Lucifer yellow, 3-(2-benzothiazolyl)-7-(diethylamino)coumarin, Sephadex G-25, Aluminum oxide,4′,6-diamidino-2-phenylindole and Triton X-100 (TX)] were pur-chased from Sigma–Aldrich (St. Louis, USA).
2.2. Synthesis of polymer capsules
Monomers were purified on a column of neutral alu-mina to remove inhibitors shortly before polymerization. Themonomers were dissolved in 2 mL of chloroform in a culturetube, in the following ratio [(ST/DVB 1:1)/coumarin 6/DMPC wasrespectively: 2 × 25 �L (21.5 × 10−5 mol + 17.4 × 10−5 mol)/3 mg(8.6 × 10−6 mol)/60 mg (8.8 × 10−5 mol)], with 0.3 mg(1.17 × 10−6 mol) of initiator. The solvent was evaporated undera stream of nitrogen leaving a lipid-monomer film. The dried filmwas hydrated with deionized water containing dyes (1 mg/mL) toresult in a dispersion of multilamellar vesicles. The dispersion wasextruded (21 times) at 35 ◦C through a polycarbonate nucleoporetrack-etch membrane (Whatman, with 0.1 �m pore size) usinga Lipex stainless steel extruder (Northern Lipids). To inducepolymerization, the sample was irradiated for 5 h with UV light(� = 310 nm) in a quartz tube in a photochemical reactor (RayonetRPR-200, Southern New England, USA) equipped with a stirrerand 10 × 32 lamps forming polymer nanocapsules. Non-entrappeddyes were separated using size-exclusion chromatography. Thesuspension of nanolipopolymersomes was passed through acolumn of Sephadex G-25 to remove dyes. Next the outer lipid
scaffold was removed by washing repeatedly with methanol untilit was completely colorless. The colored polymer nanocapsulesprecipitated in this manner can be freeze-dried from tert-butanolfor storage without loss of their contents. The cross-linking of
h hydrophobic monomers and coumarin 6. UV (� = 310 nm) irradiation-inducedlated in aqueous cave.
M. Sierant et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 571– 578 573
Fig. 3. TEM and SEM images of nanopolymersomes respectively: (A) precipitatedfrom methanol after removal of the outer lipid shell and (B) nanolipopolymersomesin water. (C) Hydrodynamic diameter of polymer capsules with an outer lipidsspi
ci
2
fmf4(to
◦
hell determined by PCS. (D) Zeta-potential measurements of polymer capsules sus-ended in aqueous NaCl (1.0 × 10−3 M) before and after removing lipids by washing
n methanol.
oumarinated poly(styrene-co-divinylbenzene) by UV (� = 310 nm)s shown in Fig. 1.
.3. 13C NMR spectroscopy
Before filling NMR rotors, the sample was dried under vacuumor 24 h and crushed to a fine powder. Solid-state cross-polarization
agic-angle spinning (CP MAS) 13C NMR experiments were per-ormed at a frequency of 100.6 MHz on a Bruker Avance III
00 spectrometer at a MAS speed of 8 kHz in a 4 mm rotorCRAMPS BL4, CortecNet). A sample of glycine was used to sethe Hartmann–Han condition, and adamantane was used as a sec-ndary chemical shift reference at 38.48 ppm in addition to external
Fig. 4. UV–vis spectra of a nanovesicle suspension with encapsulated Procionred. (a) Insoluble poly(styrene-co-divinylbenzene) capsules with TX and liposomesbefore (b) and after dissolution by TX (c).
tetramethylsilane (TMS: 29.46 ppm). The conventional spectrum13C NMR was recorded with a 90◦ pulse length of 3.5 �s and a con-tact time of 1 ms. The repetition delay was 8 s and the spectral widthwas 25 kHz. Free induction delay (FID) spectra were accumulatedwith a time domain size of 2000 data points. A RAMP pulse shapewas used during cross-polarization (CP). Two-pulse phase modu-lation (TPPM) with �p = 6.8 �s and a phase angle of 20◦ was usedduring acquisition. A total of 7850 scans were recorded for eachspectrum.
Zeta-potential and photon correlation spectroscopy (PCS) mea-surements were prepared using a Zetasizer Nano-Z (MalvernInstrument, UK). Analyses of liposomes and polymer capsuleswere performed at 25 ◦C dispersed in 1.0 × 10−3 mol/L aqueousNaCl. Data were analyzed using a cumulative method from sixmeasurements for each sample. The hydrodynamic diameter ofnanostructures was evaluated from the diffusion coefficient ofspherical nanoparticles calculated according to the Einstein–Stokesequation. Zeta-potential measurements were measured in a cellequipped with electrodes to which a potential (up to 400 V) wasapplied. Scanning electron microscopy (SEM) was performed usinga Quanta 200 (W) from FEI Company (USA). Fluorescence spec-tra of polymer capsule suspensions in methanol were measuredin quartz cuvettes (standard fluorimeter cell, 10 mm path lengthused Fluorolog HORIBA, Jobin Yvon Japan).
Transmission electron microscopy (TEM) was performed usinga TESLA BS500 electron microscope operating at 90 kV. A dispersionof nanolipopolymersomes in water was collected using carbon-film-covered copper grids for analysis.
Fourier transform infrared spectroscopy (FTIR) Samples weredispersed in KBr disks prior to analysis. Spectra were recorded forpolymer capsules and coumarin 6 bulk powder using an FTIR-ATIspectrometer (Mattsonn, USA). Resolution was 4 cm−1; 32 scanswere averaged per spectrum.
2.4. Evaluation of nanocapsule cytotoxicity
The cytotoxicity of nanocapsules toward HeLa cells wasmeasured using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma). Cells were plated in 96-wellplates at a density of 7000Ycells per well. After 24 h nanocap-sules (0–100 �g/mL) were added to the culture medium [RPMImedium, (Sigma), with 10% fetal bovine serum, (Gibco)]. Untreatedcells were used as control (100% of viability). After 24 or 48 h ofincubation at 37 ◦C and 5% CO2, MTT in PBS (25 �L of 5 mg/mL
per well) was added and incubated for an additional 2 h at 37 C.Finally, 95 �L of lysis buffer (20% SDS, 50% aqueous dimethylform-amide, pH 4.5) was added to each well and incubated overnightat 37 ◦C. Absorbance was measured at 570 nm and 630 nm (the
574 M. Sierant et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 571– 578
Fig. 5. Colored poly(styrene-co-divinylbenzene) nanocapsules were separated on a size-exclusion column to remove release probes. Upper panel: an aqueous suspensionof polymersomes with encapsulated dye mixtures. Middle panel: colored polymer vesicles precipitated in methanol; (A) violet (Procion red with Procion blue), (B) orange(Procion red with Lucifer yellow), (C) green (Procion blue with coumarin 6), and (D) coumarinated polymer capsules. Lower panel: orange and green polymersomes in visiblea aquer this a
rTf(Ac
2
mbwF
nd UV light (330 nm), revealing fluorescence. The graph represents UV spectra ofeferences to color in this figure legend, the reader is referred to the web version of
eference wavelength) using a plate reader (Synergy HT, BIO-EK). The percentage of living cells (PLC) was calculatedrom the equation PLC = AbsSpl × 100%/Abscontrol cells, where AbsSplAbs570–Abs630) is the absorbance of cells treated with siRNA andbscontrol cells (Abs570–Abs630) is the absorbance of the referenceells.
Nanocapsules (0-100 �g/mL) were added to HeLa cells in
edium (six-well plates, 3 ×Y105 cells per well). After 24 h of incu-
ation at 37 ◦C under 5% CO2, the cells were washed three timesith ice-cold PBS trypsinized and collected for flow cytometry (BD
ACS Calibur Flow Cytometry System Becton Dickinson) using an
ous suspensions of polymersomes with entrapped dyes. (For interpretation of therticle.)
Ar-ion laser (488 nm). Fluorescence dot plots and histograms weregenerated using Cell Quest software.
3. Results and discussion
3.1. Synthesis and characterization of polymer capsules
Polymer nanocapsules formation is illustrated in Fig. 2.We used lipids to simultaneously entrap hydrophilic dyes (Pro-
cion red, Procion blue, and Lucifer yellow) in the aqueous core,
and hydrophobic monomers (styrene and divinylbenzene) withcoumarin 6 in the bilayer. Benzothiazol-linked coumarins arenearly insoluble in water (<10 �M) but solubilization in styreneallows for the sequestration of dyes in the hydrophobic domains
M. Sierant et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 571– 578 575
Fig. 6. Normalized spectra: relative fluorescence intensities (I) versus emission for coumarin 6-containing lipid bilayer before (dotted pink) and after (solid red) UV irradiation(310 nm). Excitation wavelengths: �Ex = 330 nm (A), �Ex = 370 nm (B), �Ex = 400 nm (C). The solid red lines correspond to dimerization of chromophores in polymer capsulesa cifer
i s to co
oimt
drbi6osdTsddbIct�mtT
fter the removal of the outer lipid shell with methanol. (D) Fluorescence spectra of Lun polymer capsules (�max = 516 nm, black line). (For interpretation of the reference
f polymers. Thus, the solubility of coumarin 6 dye increasesn polymeric media. It may be doped or bound to hydrophobic
icrodomains and remains as the neutral-free base when boundo hypercoiled polymers [47,51,52].
Under UV light (� = 310 nm) cross-linked poly(styrene-co-ivinylbenzene) bonded to the phototrigger chromophoreesulting in photodimerization. Consequently the embeddedenzothiazole-linked coumarin dye is a fluorescent probe that is
ncorporated into the polymer backbone. Our studies of coumarin complexation by polymers have been extended to the structuresf liposomes. Furthermore, the incorporation of coumarin groups,ensitive to the cross-linking density determined by the degree ofimerization increased the leak-resistance of the polymer capsules.his combination of factors yielded colored capsules susceptible topectrophotometric or fluorescent detection. We found that theseyes, encapsulated in the aqueous core, did not undergo bleachinguring free-radical polymerization in the monomer-saturatedilayer and that the colors of the capsules did not fade in storage.
n this manner we formed capsules with cross-linked walls fromoumarinated poly(styrene-co-divinylbenzene) that retainedhe characteristic fluorescence of coumarin 6 (�Abs = 443 nm,
Em = 505 nm in ethanol). The outer lipid shell was removed withethanol, causing the capsules to precipitate. The morphology of
he nanopolymersomes was characterized using TEM and SEM.he capsules were spherical and similar to the parent liposomes.
yellow at �Ex = 330 nm in methanol (�max = 518 nm, dash blue line) and encapsulatedlor in this figure legend, the reader is referred to the web version of this article.)
Freeze-dried capsules (from tert-butanol) retained this sphericalshape. Most capsules were in the 200-250 nm range; polymernanovesicles were 200 nm on average (±SD = 30 nm, n = 100). Thespherical shape, confirmed by TEM and SEM is shown in Fig. 3,respectively.
The hydrodynamic diameter of liposomes and polymer capsuleswith an outer lipid shell was determined by PCS. The diameterof the nanostructures was similar before and after radical poly-merization and no aggregates were observed (Fig. 3C). Exposure ofthe interfacial layer to water changed the effective charge of poly-mer nanocapsules with an outer phospholipids shell (containing14-carbon atom hydrophobic chains) relative to naked polymercapsules from −19.1 mV to −35.5 mV (Fig. 3D). The presence ofpenetrating ions (1.0 × 10−3 M NaCl) within the shell resulted inswelling in contrast to the rigid and water impermeable polymercapsules.
Colored polymersomes were characterized by spectrophotom-etry after encapsulation of a mixture of dyes [Procion red (MW 615,�max. Abs = 538 nm) and Procion blue (MW 637, �max. Abs = 595 nm)]or fluorescent marker Lucifer yellow (MW 521, �Ex = 400 nm,�Em = 518 nm in methanol). This result unambiguously confirmed
permanent entrapment in impermeable, stable capsules. Retentionof hydrophilic molecular probes (similar in size but with differentcolors) inside the aqueous vesicle core caused a change in tintfrom orange to green and allowed the capsules to be used as
5 ces B: Biointerfaces 111 (2013) 571– 578
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Fig. 7. (A) The 13C CP/MAS NMR spectrum of polymer capsules with a cross-linkedcoumarinated poly(styrene-co-divinylbenzene) wall. (B) The FTIR absorption spec-tra (KBr disk): nanocapsules with coumarin 6-poly(styrene-co-divinylbenzene) wall
76 M. Sierant et al. / Colloids and Surfa
olecular containers amenable to fluorescence detection method.he wall from poly(styrene-co-divinylbenzene) formed in bilayerf liposomes effectively cut-off the internal space of capsules fromuter environment and no dye was found in the eluent after theirrecipitation and washing in methanol. The addition of Triton X-00 dissolved liposomes immediately, releasing the encapsulatedyes from the evanescent vesicles. As a result, light scatteringiminished because most of the liposomes disappeared. Thisrocess was monitored by measuring optical density: OD = log(I0/I)here I0 and I denote the intensity of incident and transmitted
ight, respectively (Fig. 4).Unlike liposomes, polymer capsules precipitated from methanol
due to the dissolution of the outer phospholipid shell), with theetention of entrapped molecules, as illustrated in Fig. 5. No leakageas detected after the addition of Triton X-100. The polymer cap-
ules did not dissolve in organic solvents chloroform or methylenehloride. To valuate long-term dye retention precipitated capsulesere left in methanol for one month. During this time, no dyeas observed in the supernatant. Moreover, SEM revealed thatashing polymer capsules with methanol caused no alternation
f the morphology. These results indicate that nanocapsules withanometer-thick polymer walls were stable under typical handlingonditions, such as solvent exchange. Thus, this method allowsor the entrapment of several different species in such structuresimultaneously. The retention of the dye mixture in nanocapsuless shown in Fig. 5.
As mentioned earlier, non-covalent association of coumarin with the poly(styrene-co-divinylbenzene) layer conferred flu-rescence and installed additional functional groups on theolymer surface. Freeze-dried nanocapsules with a cross-linkedoumarinated poly(styrene-co-divinylbenzene) wall exhibitedharacteristic yellow color of coumarin 6 (�Abs = 443 nm) and wereompletely impermeable to the dyes in the aqueous core. Thus,he probes Procion red and Procion blue yielded orange and greenolors respectively, due to overlap with the yellow polymer. Weompared the fluorescence spectra of polymer vesicles encapsu-ating hydrophilic Lucifer yellow with that of capsules featuring aoumarinated poly(styrene-co-divinylbenzene) shall. The normal-zed fluorescence spectra are presented in Fig. 6.
Fluorescence depends strongly on the local environmenthich influences efficiency. When coumarin 6 is bound to aolymer, the mobility of the dye moiety is hindered due ton increase in local viscosity; thus, high polarization valuesanisotropy) are obtained [53] and all dyes species are complexedith the polymer microdomain. Lucifer yellow encapsulated inoly(styrene-co-divinylbenzene) vesicles revealed the character-
stic emission maximum �Em = 516 nm at �Ex = 330 nm, a blue shift2 nm) relative to methanol solution (�Em = 518 nm). Coumarin 6ncorporated into a lipid bilayer yielded �Em = 492 nm at excita-ion: �Ex = 330, 370, and 400 nm. After dimerization of coumarin
under UV irradiation (310 nm), the fluorescent nanocapsuleolymer wall exhibited a slight red shift (10 nm) (�Em = 502 nm).his observation implies that coumarin 6 remained neutral [49].hus, coumarin 6 underwent cross-linking in the poly(styrene-o-divinylbenzene) shell via a single photochemical reaction:yclobutane formation and dimerization of coumarin 6 monomers.he exposure of coumarinated poly(styrene-co-divinylbenzene)apsules to UV light (� < 260 nm) resulted in an incomplete rever-al of crosslinking leaving coumarin dimers intact. This irreversiblehotochemical process reduces mobility and flexibility in the mainhains of poly(styrene-co-divinylbenzene) matrix. Thus, coumarin
retained its fluorescence during radical polymerization. Intense
V irradiation (5 h) neither destroyed nor bleached fluorescentolecules.We used solid state 13C CP/MAS NMR spectroscopy to
etermine the structure of capsules with coumarinated
(red), pure coumarin 6 (blue), and empty poly(styrene-co-divinylbenzene) capsules(black). (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)
poly(styrene-co-divinylbenzene) walls. The spectrum of cross-linked polystyrene in Fig. 7A exhibits peaks at 146 ppm (quaternaryaromatic carbon), 127 ppm (protonated aromatic carbon), and40 ppm (aliphatic methine carbon). The aliphatic methylene car-bons yielded a broad signal at 40-50 ppm due to configurationaland conformational heterogeneity. The signal of the cross-linkmethylene carbons (expected at 46 ppm) was not resolved fromthe backbone methine and methylene carbon signals [54]. Thesignal at 27 ppm suggests that fatty acid chains (DMPC) remained
in the inner wall of the capsules [55].
We also used FTIR method to confirm the presence of coumarin6 in the polymer wall of the nanocapsules (Fig. 7B). The FTIR spec-trum of coumarin 6 featured bands at 1261 and 1191 cm−1 that
M. Sierant et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 571– 578 577
Fig. 8. (A) Microscopic observation of HeLa cells after application of coumarinated poly(styrene-co-divinylbenzene) nanocapsules. (B) Capsules with encapsulated Luciferyellow. (C) Cytotoxicity of nanopolymersomes at different concentrations toward HeLa cells by MTT assay after incubation for 24 or 48 h. Reference samples: cells withoutnanopolymersomes representing 100% viability. (D) FACS technique for cellular uptake of nanopolymersomes (0-100 mg L−1) applied to HeLa cells. The error bars shows easure
mtaFc1oscttdc(ainbp
tandard deviation (±SD) with probability value (p < 0.05) for three independent m
ay be assigned to C C or C O ring stretching, C H bending, orhe C N stretching modes. A C C stretching mode at 1589 cm−1
nd C O stretching mode at 1712 cm−1 are clearly apparent inig. 7B [56]. The most intense infrared absorptions observed inoumarinated poly(styrene-co-divinylbenzene) capsules were at510, 1589 and 1614 cm−1. Incorporation of the functional groupsf coumarin 6 into a bulk polymer significantly broadens theignals in the range from 1440 to 1604 cm−1. These data areonsistent with embedding of chromophores into a nanometer-hick polymer film. As the polymerization reaction proceeded,he peaks at 1614, 1191, and 1133 cm−1 decreased in intensityue to the increased in C C double bonds. Moreover, due toyclobutane ring formation, the intensity of the peak at 1712 cm−1
carbonyl C O stretching vibrations for neutral species) decreasedfter exposure to UV irradiation (� > 310 nm). This observation
ndicates that cyclobutane ring formation generated crosslinkedetworks. Concominantly, the intensity of the 1614 and 1589 cm−1
ands decreased as double bonds were broken by UV-inducedhotodimerization.
ments (n = 3).
3.2. Permeability of HeLa cells to polymer capsules
To be acceptable as imaging agents, polymer capsules must bebiocompatible, so it is of great importance to evaluate the poten-tial toxicity of materials. Here, HeLa cells were used to determinethe in vitro cytotoxicity of these capsules. We used flow cytometryto evaluate the cell permeability and bioactivity of nanolipopoly-mersomes for biomedical applications. The viability of cells after24 or 48 h of incubation with different capsule concentrations wasdetermined using the MTT method. FACS flow cytometry resultsfor uptake of nanopolymersomes (0-100 mg L−1) into HeLa cells areshown in Fig. 8 (all values represent average of three independentmeasurements). Polymer capsules had low cytotoxicity: approxi-mately 80% of cells were viable at capsules concentrations up to100 mg L−1. This result was mainly due to the excellent biocompat-
ibility of these nanolipopolymersomes, which appear to have greatpotential for biomedical applications.
DAPI (4′,6-diamidino-2-phenylindole) a fluorescent dye thatbinds strongly to A–T rich regions of DNA, was used for the imaging
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78 M. Sierant et al. / Colloids and Surfa
f cellular nuclei. The nanocapsules did not enter the nucleus:nstead they mainly localized in the space around the nucleus,ikely in the cellular membranes (see Fig. 8A; Nikon Eclipse Ti-Uuorescent imaging).
The ability to conjugate biologically active ligands to a polymerurface containing reactive groups will provide a further meansf targeting therapies and assist in the use of imaging to monitoriodistribution. Thus, our polymersomes hold enormous potentials nanostructured biocompatible materials for diagnostic imaging.
. Conclusion
We demonstrate a prospective useful synthetic strategy via theadical polymerization of a hollow structure capable of encapsu-ating compounds within a strong fluorescent wall. Furthermore,elective incorporation of coumarin 6 into nanometer-thick poly-er membranes creates fluorescent vesicles with numerous
otential applications. Such coumarinated polymer nanocapsulesrovide the framework for new molecular imaging devices thatnhance cross-linked biocompatible materials. To our knowledge,his is the first report of synthetic nanolipopolymersomes with ahotostable fluorescent wall that is not susceptible to quenchingnd does not to release encapsulated species. This is a promisingpproach to the development of devices for use in studies of theransport routes in living cells. In vitro cytotoxicity assays revealedhat the polymer capsules have excellent biocompatibility. In con-lusion, we anticipate that this methodology may be adapted to aroad range of monomers, particularly those with reactive func-ional groups which may be used for the attachment of antibodiesor controlled delivery of drugs and imaging agents. Coumarin-functionalized polymer capsules proved suitable for use as pho-ostable probes for fluorescence measurements in cells. Thus, theseapsules can be used for the fluorescent labeling of membranes.
cknowledgment
We acknowledge financial support from the National Centre forcience in Poland, Grant no. NN 209 762 440.
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