Interfacing Multicellular Organisms with Polyelectrolyte Shells and

ARTICLE pubs.acs.org/Langmuir

Interfacing Multicellular Organisms with Polyelectrolyte Shells and Nanoparticles: A Caenorhabtidis elegans Study Renata T. Minullina, Yuri N. Osin, Dilara G. Ishmuchametova, and Rawil F. Fakhrullin* Biomaterials and Nanomaterials Group, Department of Biochemistry, Kazan (Idel buye\Volga region) Federal University, Kreml uramı 18, Kazan, Republic of Tatarstan, RF 420008

bS Supporting Information ABSTRACT: We report the surface modification of microscopic live multicellular nematodes Caenorhabtidis elegans with polyelectrolyte multilayers (pure and doped with 20 nm gold nanoparticles) and the direct magnetic functionalization of nematodes with biocompatible magnetic nanoparticles. Magnetically functionalized “ironoxideclad” nematodes can be effectively separated and moved using an external magnetic field. The surface-functionalized nematodes preserve their viability and reproduction.

’ INTRODUCTION Currently, the fabrication of hybrid biomimetic structures consisting of biological components (enzymes, DNA, live cells, etc.) integrated with a range of colloid particles has attracted increased attention from researchers. During the past decade, polymer microcapsules have been reported to enclose and controllably release DNA,1 enzymes,2 and drugs3 and are believed to be versatile and nontoxic drug delivery vehicles.4 The layer-by-layer (LbL) assembly of polyelectrolytes utilizes the sequential deposition of polycations and polyanions onto a sacrificial core, which can be decomposed once the polymer deposition is over, yielding in hollow multilayer microcapsules.5 Biological cells have been utilized as templates for polymer microcapsule fabrication,6 which allows for the fabrication of microcapsules of unusual shapes, resembling those of original templates. Furthermore, living microbial and mammal cells have been encapsulated with polymer multilayers alone7 or using a combination of polyelectrolytes and noble metal,8 silica9 or magnetic nanoparticles,10 and carbon nanotubes.11 Just recently, a novel technique utilizing the hydrogen-bonded LbL assembly of neutral polymers on yeast has been introduced.12 Practically, the modification of cells with semipermeable polymer multilayers and nanoparticles can be used for patterning the functional surfaces with living cells,7 magnetically facilitated manipulations with cells,10 cellular metabolism studies,13 identification of cells using surface-enhanced Raman scattering spectroscopy,8 and fabrication of novel whole-cell biosensors and toxicity screening tools.14 The carefully selected combination of polymers and nanoparticles deposited onto living cells may result in other, still undiscovered applications, since both polymers and nanoparticles are functionally tunable, whereas the diversity of living cells offers the almost unlimited range of potential applications. Although the modification and engineering of the surfaces of cells with polymers/nanoparticles became a versatile tool to r 2011 American Chemical Society

control the physiological parameters and the spatial distribution of the modified cells, so far this technique has been limited exclusively to unicellular organisms. To the best of our knowledge, there were no attempts to engineer the surface of small multicellular organisms (i.e., worms), which are evolutionarily closer to humans and thus can be used as models in toxicity screening, drug testing and behavioral studies. Since the multicellular organisms are regarded as considerably more complex than unicellular species, both in terms of physiology and behavior, the encapsulation of multicellular organisms with polyelectrolytes and nanoparticles may provide the scientific community with a powerful instrument to study ecology, behavior, and motility of microscopic invertebrates via tailoring functional moieties using LbL assembly. Here we focused our attention on a free-living soil nematode Caenorhabtidis elegans which, having its genome fully sequenced and being very easy to cultivate, became a popular object in biology.15 Particularly, C. elegans worms are extensively studied in engineered nanoparticle toxicity assays16 and remotely controlled behavior.17 In this article, we report for the first time the LbL encapsulation of living C. elegans nematodes with polyelectrolyte shells alone and doped with gold nanoparticles (NPs). In addition, we demonstrate the magnetic functionalization of the living nematodes. We have chosen C. elegans as a well-studied experimental object in neurobiology, toxicology, and physiology studies.15
17 Here we show that C. elegans worms can be successfully coated with consecutive layers of polycations and polyanions alone, or doped with gold nanoparticles. We observed no alterations in viability and reproduction in surface-modified worms. Received: February 22, 2011 Revised: April 3, 2011 Published: May 17, 2011 7708

dx.doi.org/10.1021/la2006869 | Langmuir 2011, 27, 7708–7713

Langmuir

ARTICLE

Furthermore, we applied biocompatible polyelectrolyte-stabilized iron oxide magnetic nanoparticles (MNPs) to magnetically functionalize C. elegans worms and found that the MNP-coated nematodes were viable and could be spatially manipulated by an external magnetic field. We show here that such “ironoxideclad” worms can be selectively separated from river sand and yeast suspensions and then cultivated, which makes this technique potentially important in environmental toxicity assays based on nematodes.

’ MATERIALS AND METHODS Materials. Poly(allylamine hydrochloride) (PAH, MW ca. 15 000 Da), poly(fluorescein isothiocyanate allylamine hydrochloride) (FITCPAH, MW ca. 15 000 Da), and poly(styrene sulfonate sodium salt) (PSS, MW ca. 70 000 Da) were purchased from Sigma-Aldrich (U.K.). Gold nanoparticles (AuNPs, mean diameter 20 nm) were purchased from BBInternational (U.K.). The original gold colloids were 4 times concentrated by centrifugation/sonication, and the resulting concentration of AuNPs was 7 mg mL
1. Bandelin Sonoplus sonifier and Eppendorf MiniSpin Plus centrifuge were used throughout. Magnetic nanoparticles (MNPs, mean diameter 15 nm) stabilized with PAH were synthesized as described elsewhere18 and redispersed in water at 0.5 mg mL
1. Other reagents were of analytical grade and used without further purification. Milli-Q water purified by reverse osmosis was used throughout the study. All experiments were performed at room temperature. C. elegans Culture. C. elegans wild type strain (N2 Bristol) was obtained from the collection of Habitat Institute, Academy of Sciences of the Republic of Tatarstan. The strain was maintained at room temperature on nematode growth medium (NGM) plates with Escherichia coli bacteria as a food source. Before the experiments, the worms were collected from the dishes and washed several times with water prior to coating with polyelectrolytes. In order to synchronize the culture, we washed the worms from the Petri dishes with M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85.5 mM NaCl, 1 mM MgSO4), centrifuged, and then redispersed the pellet with a mixture containing 2% NaOCl and 0.45 M NaOH and incubated for 10 min while shaking. This resulted in the complete destruction of all adult and larvae worms while leaving the eggs intact. The eggs were washed with M9 buffer several times and then transferred into NGM plates inoculated with E.coli. For coating procedures, the worms were collected after 48 h of cultivation. This provided us with worms of the same age. Encapsulation of Nematodes with Polyelectrolytes Multilayers Doped with AuNPs. The worms were introduced into 1 mL

of PAH aqueous solution (1 mg mL
1), incubated for 15 min while shaking, and then separated from the excess of polyelectrolyte solution and washed two to three times with water. Then the procedure was repeated using 1 mL of PSS aqueous solution (1 mg mL
1). The polyelectrolyte coating was performed until a desired number of PAH/ PSS bilayers was obtained. In several experiments, we used FITC-PAH (0.5 mg mL
1). For the deposition of gold nanoparticles, the nematodes already coated with PAH/PSS/PAH shell were introduced into 1 mL of 4 times concentrated AuNPs and incubated for 30 min, and then the worms were isolated by centrifugation and washed with water.

Surface Modification of Nematodes with PAH-Stabilized Magnetic Nanoparticles. For the surface modification, the nematodes were directly introduced into the suspension of MNPs and incubated for 30 min, and then the worms were separated magnetically using a permanent neodymium
iron
boron (NdFeB) magnet. Then the magnetically functionalized nematodes were washed with water.

Magnetically Facilitated Separation of Magnetically Functionalized Nematodes from Yeast and Sand Suspensions. Magnetically functionalized nematodes were mixed with aqueous yeast

Figure 1. (A) Optical microscopy image of C. elegans nematodes; (B) bright field and (C) fluorescence microscopy images of a C. elegans nematode coated with (FITC-PAH/PSS)5 multilayers. (50 mg mL
1) and sand (100 mg mL
1) suspensions and incubated for 30 min. Then the worms were separated from the yeast and sand with a permanent NdFeB magnet during 10 min. Characterization Techniques. Optical and Fluorescence Microscopy. Optical and fluorescence microscopy images were obtained using a Carl Zeiss AxioScope microscope equipped with an AxioCam MRc5 CCD camera. Real-time footage was recorded using a Carl Zeiss Jenamed microscope equipped with a Webbers M320 CCD camera. The real-time footage was recorded using dimpled glass slides. Living worms are motile; therefore, in several experiments, we used fixed worms in order to demonstrate the magnetic manipulations with MNP-coated nematodes. The worms were fixed with 2.5% aqueous glutaraldehyde for 7709

dx.doi.org/10.1021/la2006869 |Langmuir 2011, 27, 7708–7713

Langmuir

ARTICLE

Transmission Electron Microscopy (TEM). The TEM images of the thin-sectioned nematodes were obtained using a Jeol 1200 EX microscope (Japan) operating at 80 kV. The nematodes were fixed with 2.5% aqueous glutaraldehyde for 1 h, dehydrated with ethanol, and embedded into resin, and then thin sections were cut using a LKB ultramicrotome (Sweden) and mounted on copper grids, Finally, the samples were stained with 2% aqueous uranyl acetate and lead citrate. Viability Test. After coating with polyelectrolytes and nanoparticles, the viability of the worms was investigated microscopically. Typically, the motility of the worms was observed under the microscope and compared with the intact worms. Furthermore, to study the long-term effect of polyelectrolyte shells and nanoparticles on the reproductive functions of the coated nematodes, they were cultivated during 3 days after coating under normal conditions. Polyelectrolyte/nanoparticle coated worms were inoculated (100 μL) into NGM plates containing E. coli bacteria and cultivated at room temperature along with intact controls. Then the cultures were examined microscopically, and special attention was given to the density of the population, the motility of the worms, and the presence of eggs and pregnant nematodes.

Figure 2. (A) Optical microscopy image of C. elegans nematode coated with PAH/PSS multilayers doped with AuNPs; (B) TEM image of a C. elegans cuticle thin section coated with PAH/PSS multilayers and AuNPs; SEM images of intact (C) and PAH/PSS/AuNPs coated (D) C. elegans nematodes; (E) EDX spectrum of the sample shown in (D), demonstrating Au peaks (highlighted). 1 h, washed, and then placed onto glass slides. In several experiments the NGM plates inoculated with worms were directly observed using lowmagnification objectives (10, 20). Low-Vacuum Scanning Electron Microscopy (SEM) and EnergyDispersive X-ray (EDX) Spectroscopy. The low-vacuum SEM images were taken using a Carl Zeiss EVO 50XVP instrument. EDX spectra were obtained using an INCA 350 EDX spectrometer (Oxford Instruments, U.K.) fitted to the scanning electron microscope. The samples were fixed with 2.5% aqueous glutaraldehyde for 1 h, then dehydrated with ethanol and acetone, and finally placed on clean glass stubs and dried at 50 °C overnight. The samples were imaged “as is”, without sputter coating with gold layer. SEM images were obtained at 47
49 Pa working pressure and 20 keV accelerating voltage.

’ RESULTS AND DISCUSSION Figure 1A shows the optical microscopy image of intact nematodes cultivated for 3 days. These worms were collected from the nematode growth medium (NGM) plates, washed several times and redispersed in Milli-Q water, and then coated with polyelectrolytes PAH and PSS in the same way as has been reported previously for yeast and bacteria cells.7,8 The relatively small size of the worms (up to 1 mm) allows collecting them using a low speed centrifugation. We started the formation of the polyelectrolyte shells with deposition of cationic PAH since the surface cuticle of C. elegans is built mostly from glycoproteins providing the worms with a negative surface charge. The worms are apparently not affected by the procedure, and the deposition of up to (PAH/PSS)5 does not jeopardize the motility of the nematodes. We used FITC-PAH to confirm the polyelectrolyte shell formation on the worms using fluorescence microscopy. As shown in Figure 1B and C, the polyelectrolyte deposition resulted in a uniform shell formation on the coated worms. During microscopic observations, we found that the flexibility of the PAH/PSS multilayers allows the worms to move exactly as those worms which were not coated with polyelectrolytes. Next, we demonstrate that the nematodes’ surface can be further functionalized with gold NPs incorporated into polyelectrolyte multilayers. We followed our previously reported technique8 to introduce and immobilize the 20 nm spherical gold nanoparticles on the C. elegans worms. As the citratestabilized gold NPs are negatively charged, we coated the nematodes with a PAH/PSS/PAH shell and then introduced the polyelectrolyte-modified worms into the preconcentrated gold colloid (7 mg mL
1). After the incubation, the worms were separated from the excess of NPs, washed with Milli-Q water, and further coated with another PAH/PSS/PAH/PSS layer to seal the gold NPs on the surface of the worms. As shown in Figure 2A, the large aggregates of gold NPs are clearly seen in the optical microscopy image. TEM confirmed the gold NP layer on the outer part of the worms’ cuticle (Figure 2B). In addition, low vacuum SEM combined with EDX spectroscopy was applied to demonstrate the deposition of gold NPs on the large areas of the surface-functionalized worms (Figure 2D) in comparison with bare intact nematodes (Figure 2C). The samples were not sputter-coated with gold prior to imaging, and thus, the gold 7710

dx.doi.org/10.1021/la2006869 |Langmuir 2011, 27, 7708–7713

Langmuir

Figure 3. (A) Optical microscopy image of a C. elegans nematode coated with PAH-stabilized magnetic nanoparticles; (B, C) TEM images of a typical thin cross-section of C. elegans nematode coated with PAHstabilized magnetic nanoparticles (at lower and higher magnifications); (D) typical EDX spectrum of a C. elegans nematode coated with PAHstabilized magnetic nanoparticles (inset shows the SEM image of a spot where the spectrum was obtained).

peaks shown in Figure 2E originate from the gold NPs attached to the worms as a component of the composite polyelectrolyteNP shell. By analogy with microorganisms, a number of applications can be found for the thus modified worms. For example, polyelectrolyte shells deposited onto the worms can be used as surface barriers in physiology studies; in addition, the incorporation of gold NPs will make possible the selective heat treatment of the worms with microwave or infrared irradiation. However, the most important advantage of the surface-modified multicellular organisms would be the ability to spatially arrange and move the worms using magnetically facilitated manipulation. Accordingly, next we demonstrate the surface functionalization of C. elegans nematodes with biocompatible 15 nm iron oxide MNPs attached to the cuticle. We followed the previously reported direct technique for the magnetic functionalization of microbial cells with PAH-stabilized positively charged MNPs which readily adhere to yeast, algae, and bacteria cells in a simple single-step procedure.10,18 We introduced the intact worms into a MNP solution (0.4 mg mL
1) and incubated for 30 min.

ARTICLE

After the separation of the MNP-functionalized worms, they became magnetically responsive and could be manipulated externally with a permanent magnet, as one can see in Movie 1 (Supporting Information). As the worms are motile, we fixed the samples with 2.5% glutaraldehyde to demonstrate the magnetically facilitated deflection of “ironoxideclad” worms toward the permanent magnet positioned near the microscopy slide. Optical microscopy image of the MNP-coated nematode shows the characteristic brown color of MNPs (Figure 3A). TEM (Figure 3B and C) and SEM (Figure 3d inset) images along with the EDX spectra (Figure 3D) further confirm the uniform distribution of iron oxide MNPs on the cuticle of the nematodes, which is in good agreement with the previous studies.10,18 C. elegans nematodes are extensively used in toxicity screening16 and animal behavior17 studies, where the spatial manipulation, the remote control of ion channels and neurons, and the selective extraction of the nematodes (i.e., after incubation in a complex media containing toxicants and/or other unicellular or multicellular species) are deemed important. We propose here an easy and effective approach for the fabrication of living MNP-coated “ironoxideclad” worms which can be extracted from complex environments using a permanent magnet and then cultivated on NGM plates. As a proof-of-principle, we demonstrate the magnetic separation of the MNP-functionalized worms from yeast and river sand suspensions. The MNP-coated worms were mixed with the suspensions, and after a short incubation separated by a permanent magnet. As one can see in Figure 4A, the MNP-functionalized nematodes mixed with river sand after a short period concentrate near the permanent magnet positioned near the vial. Typical optical microscopy images of the MNPfunctionalized nematodes mixed with yeast cells and the same worms after the separation are shown in Figure 4B and C. This approach allows for the selective extraction of “ironoxideclad” worms from the media containing nonmagnetic colloid particles or cells, which might be important if one desires to separate the magnetic worms from the multicomponent mixture, that is, after the toxicity assay of soil or wastewater samples. Obviously, the preserved viability is the most important feature of the surface-functionalized nematodes, because the main applications in toxicity screening and behavioral experiments require viable worms with fully functional reproduction cycles. We found that the worms coated with polyelectrolytes (PAH/ PSS)n alone, (PAH/PSS)n doped with gold NPs and with PAHstabilized MNPs remain viable and their reproduction is not affected by coating. We inoculated the NGM plates with 100 μL of polyelectrolytes and NP-coated worms and incubated them at room temperature for 3 days. Figure 5A
C shows the agar plates where the viable worms of different ages can be seen along with numerous eggs. These worms originate from those coated with (PAH/PSS)5 (Figure 5A), (PAH/PSS/PAH/AuNPs/PAH/ PSS/PAH/PSS), and MNPs. The number of the worms and the eggs in all samples was not different from the same in the intact controls. The pregnant animals, as shown in Figure 5D, also indicate the preserved viability of the MNP-coated nematodes; the same results were obtained for (PAH/PSS)5 and gold NP-doped polyelectrolyte shell coated worms. Real-time footage of the viable surface-functionalized worms is given in Movies 2 (PAH/PSS and gold NPs) and 3 (MNPs) (Supporting Information). We suppose that the polyelectrolyte and NP composite shells are quite soft and therefore the coated animals free themselves 7711

dx.doi.org/10.1021/la2006869 |Langmuir 2011, 27, 7708–7713

Langmuir

ARTICLE

Figure 4. (A) Photograph demonstrating the concentration of the magnetic worms mixed with river sand near the permanent magnet; (B, C) optical microscopy images of magnetically functionalized nematodes before and after magnetic separation from yeast cells.

Figure 5. Optical microscopy images of a C. elegans nematodes and eggs (3 days incubation) originated from the samples coated with (a) polyelectrolyte shells (PAH/PSS)5; (b) composite polyelectrolytenanoparticle shell (PAH/PSS/PAH/AuNPs/PAH/PSS/PAH/PSS) and (c) PAH-stabilized magnetic nanoparticles; (d) optical microscopy image demonstrating pregnant worms originating from those coated with MNPs (3 days incubation); (e) fluorescence microscopy image demonstrating the fluorescence of agar plates after the inoculation of nematodes coated with polyelectrolyte shell containing FITC-PAH; (f) optical microscopy image demonstrating the aggregated gold NPs on agar plates after the inoculation of nematodes coated with polyelectrolyte shells doped with gold NPs.

from the coatings by gliding on the agar plates, while the shells are mechanically removed and remain on agar. Figure 5E shows the fluorescing agar where the (FITC-PAH/PSS)5 coated worms have been incubated for 3 days. It is worth mentioning, that we did not find any fluorescent worm on the same plate, which indicates that the polyelectrolyte shell is removed from the worms. The control samples did not exhibit any agar fluorescence (data not shown). Similar results were obtained with gold NP-doped polyelectrolyte shells; after 3 days incubation, the numerous NPs aggregates having the characteristic violet color

Figure 6. Typical optical microscopy images of pregnant adult C. elegans worms, obtained from control (A) and MNPs-coated (B) larvae after 48 h incubation on NGM plates (note the oval eggs inside the nematodes); (C) distribution of the eggs in adult C. elegans worms originating from control and MNPs-coated larvae.

were found on agar plates populated with the coated worms (Figure 5F). We were able to find the fluorescent-coating-free worms after 24 h of incubation after the coatings were deposited, but apparently the rate of coating removal depends on the individual activity of the worms. However, the removal of the coatings, as we suppose, depends on the media (i.e., worms suspended in aqueous media will maintain the shells for longer). The detailed investigation of coatings durability is currently 7712

dx.doi.org/10.1021/la2006869 |Langmuir 2011, 27, 7708–7713

Langmuir underway and will be reported later. We suppose that the worms are able to remove the coatings from the pharynx (head) part of the body and keep feeding on bacteria, meanwhile the gradual removal of the remaining shell occurs during the gliding on agar. These data is in good agreement with the previous reports suggesting that the polyelectrolyte multilayers can be pierced by dividing cells.7,8 Still, before the coating is removed, the required manipulations can be performed with the surfacefunctionalized worms (i.e., magnetic separation, etc.), thus making it possible to use these worms in soil toxicity assessments. In addition, the magnetically functionalized worms may find potential applications associated with the treatment of several severe disorders such as inflammatory Crohn’s disease, where the nematodes act as immune system regulators.19 To investigate the long-term effect of the MNPs coating onto the adult hermaphrodite nematodes hatched from the magnetized larvae, we magnetized the worms from the synchronized larvae cultures with MNPs as described before and after 48 h incubation on NGM plates counted the number of eggs in pregnant worms. In control samples, we used Milli-Q water instead of MNPs. The results along with the typical images of pregnant nematodes from control and magnetized batches are given in Figure 6. As one can see, there is a small decrease in the number of the eggs per worm in magnetically functionalized worms (9.7 ( 2.9) in comparison with intact nematodes (13.8 ( 5.6), which might be caused by MNPs, although the effect is not profound, since the total number of pregnant worms was the same in both control and magnetized samples. Nevertheless, the apparent effect of MNPs on reproduction of worms shown here must be taken into account and studied further in more detail. Taken into account the application of magnetic nanoparticles as magnetic resonance imaging (MRI) contrast agents,20 we anticipate another promising application of MNP-functionalized worms. The preserved viability of the magnetically functionalized C. elegans suggests that a similar approach for the magnetic functionalization might be utilized in studies aimed to investigate the distribution of parasite nematodes in the human body using MRI.

’ CONCLUSIONS To conclude, we have demonstrated that microscopic living multicellular organisms (C. elegans) can be encapsulated with polyelectrolyte multilayers (pure and doped with gold NPs) and magnetically functionalized with MNPs. The surface-functionalized nematodes remain viable and reproduce normally. We believe that the polymer-mediated surface engineering is not limited to C. elegans and can be applied to other microscopic invertebrates. ’ ASSOCIATED CONTENT

bS

Supporting Information. Real-time footage and additional figure, as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: þ 78432315246. E-mail: [email protected].

ARTICLE

technical help with TEM, and anonymous reviewers for their useful comments. The financial support from the Academy of Sciences of the Republic of Tatarstan is gratefully acknowledged.

’ REFERENCES (1) Shchukin, D.; Patel, A.; Sukhorukov, G.; Lvov, Y. J. Am. Chem. Soc. 2004, 126, 3374. Borodina, T.; Markvicheva, E.; Kunizhev, S.; M€ohwald, H.; Sukhorukov, G. B.; Kreft, O. Macromol. Rapid Commun. 2007, 28, 1894. (2) Tiourina, O.; Antipov, A.; Sukhorukov, G.; Lvov, Y.; M€ohwald, H. Macromol. Biosci. 2001, 1, 209. Ghan, R.; Shutava, T.; Patel, A.; John, V.; Lvov, Y. Macromolecules 2004, 37, 4519. (3) Teng, X. R.; Shchukin, D. G.; M€ohwald, H. Langmuir 2008, 24, 383. (4) Angelatos, A. S.; Katagiri, K.; Caruso, F. Soft Matter 2006, 2, 18. (5) Lvov, Y.; Decher, G.; M€ohwald, H. Langmuir 1993, 9, 481. Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; M€ohwald, H. Angew. Chem., Int. Ed. 1998, 37 (16), 2202. (6) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Baumler, H.; Kiesewetter, H.; Mohwald, H. Chem.—Eur. J. 2002, 8, 5481. Ai, H.; Fang, M.; Jones, S.; Lvov, Y. Biomacromolecules 2002, 3, 560. (7) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir 2002, 18, 5047. Krol, S.; Nolte, M.; Diaspro, A.; Mazza, D.; Magrassi, R.; Gliozzi, A.; Fery, A. Langmuir 2005, 21, 705. (8) Fakhrullin, R. F.; Zamaleeva, A. I.; Morozov, M. V.; Tazetdinova, D. I.; Alimova, F. K.; Hilmutdinov, A. K.; Zhdanov, R. I.; Kahraman, M.; Culha, M. Langmuir 2009, 25, 4628. Kahraman, M.; Zamaleeva, A. I.; Falhrullin, R. F.; Culha, M. Anal. Bioanal. Chem. 2009, 395, 2559. (9) Ai, H.; Fang, M.; Jones, S. A.; Lvov, Y. M. Biomacromolecules 2002, 3, 560. Balkundi, S. S.; Veerabadran, N. G.; Eby, D. M.; Johnson, G. R.; Lvov, Y. M. Langmuir 2009, 25, 14011. (10) Fakhrullin, R. F.; García-Alonso, J.; Paunov, V. N. Soft Matter 2010, 6, 391. (11) Zamaleeva, A. I.; Sharipova, I. R.; Porfireva, A. V.; Evtugyn, G. A.; Fakhrullin, R. F. Langmuir 2010, 26, 2671. (12) Kozlovskaya, V.; Harbaugh, S.; Drachuk, I.; Shchepelina, O.; Kelley-Loughnane, N.; Stone, M.; Tsukruk, V. V. Soft Matter 2011, 7, 2364. (13) Franz, B.; Balkundi., S. S.; Dahl, C.; Lvov, Y. M.; Prange, A. Macromol. Biosci. 2010, 10, 164. (14) García-Alonso, J.; Fakhrullin, R. F.; Paunov, V. N.; Shen, Z.; Hardege, J. D.; Pamme, N.; Haswell, S. J.; Greenway, G. M. Anal. Bioanal. Chem. 2011, 400, 1009. (15) Kaletta, T.; Hengartner, M. O. Nat. Rev. Drug Discovery 2006, 5, 387. Kim, H.; Rogers, M. J.; Richmond, J. E.; McIntire, S. L. Nature 2004, 430, 891. (16) Wang, H.; Wick, R. L.; Xing, B. Environ. Pollut. 2009, 157, 1171. Kim, J.; Shirasawa, T.; Miyamoto, Y. Biomaterials 2010, 31, 5849. (17) Al-Atar, U.; Fernandes, R.; Johnsen, B.; Baillie, D.; Branda, N. R. J. Am. Chem. Soc. 2009, 131, 15966. Huang, H.; Delikanli, S.; Zeng, H.; Ferkey, D. M.; Pralle, A. Nat. Nanotechnol. 2010, 5, 602. (18) Fakhrullin, R. F.; Shlykova, L. V.; Zamaleeva, A. I.; Nurgaliev, D. K.; Osin, Y. N.; García-Alonso, J.; Paunov, V. N. Macromol. Biosci. € 2010, 10, 1257. Zhang, D.; Fakhrullin, R. F.; Ozmen, M.; Wang, H.; Wang, J; Paunov, V. N.; Li, G.; Huang, W. E. Microb. Biotechnol. 2011, 4, 89. (19) Summers, R. W.; Elliott, D. E.; Urban, J. F., Jr.; Thompson, R.; Weinstock, J. V. Gut 2005, 54, 87. (20) Jaganathan, H.; Hugar, D. L.; Ivanisevic, A. ACS Appl. Mater. Interfaces 2011, 3, 1282.

’ ACKNOWLEDGMENT We thank Dr. T. B. Kalinnikova and Mr. N.N. Khaertdinov for C. elegans culture; Dr. V. V. Salnikov and Dr. A. I. Zamaleeva for 7713

dx.doi.org/10.1021/la2006869 |Langmuir 2011, 27, 7708–7713