Polyelectrolyte Nanocontainers

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Langmuir 2008, 24, 383-389

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Articles A Novel Drug Carrier: Lipophilic Drug-Loaded Polyglutamate/ Polyelectrolyte Nanocontainers Xin Rong Teng,*,†,‡ Dmitry G. Shchukin,*,† and Helmuth Mo¨hwald† Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany, and School of Materials Science and Engineering, Tongji UniVersity, 200092, Shanghai, China ReceiVed August 2, 2007. In Final Form: October 10, 2007 A novel lipophilic drug carrier, “oil-in-water” multifunctional composite nanocontainers, is developed by combining ultrasonic technique and layer-by-layer assembly. Polyglutamate/polyethyleneimine/poly(acrylic acid) nanocontainers loaded with the lipophilic drug, rifampicin, dissolved in soybean oil were fabricated. Raman confocal microscopy and scanning electron microscopy proved the successful incorporation of rifampicin into composite water-dispersible polyglutamate/polyelectrolyte nanocontainers. Transmission electron microscopy and confocal laser scanning microscopy indicated that the drug can be released by changing the pH value of the media due to the pH-responsive properties of the polyglutamate/polyelectrolyte shell.

Introduction Many studies of pharmaceutical delivery systems have been carried out to develop drug carriers that are suitable for delivering a drug to the injured site. Among them, more attention has been paid to polymeric micelles,1 lipid nanocapsules,2-3 liposomes, or lipid microspheres.4-6 Emulsion delivery systems have been widely used to encapsulate drugs. Unfortunately, the solubility of the shell material (i.e., polymers and lipids) in both organic solvent and water is frequently required, high quantities of surfactants and co-surfactants like butanol increasing a potential toxicity. Lipid microspheres (LM), usually including soybean oil and lecithin, are widely used in clinical medicine for parenteral nutrition. They are very stable and have no adverse effects. Drugs can be incorporated either into lipid microspheres, if they are soluble in vegetable oil, or retained within the single lipid membrane. Because of the avoidance of any harmful organic solvent or toxic products, the lipid microspheres formed by dispersion of the LM particles in aqueous solution appear to be safe and efficient drug carriers.5 However, preparation of lipid microspheres usually needs a special high-pressure setup to emulsify oil solutions, which increases processing costs. To avoid harmful byproducts and, simultaneously, to explore some new functionalities of the potential drug carrier, we developed a simple method to fabricate a novel drug carrier system by combining ultrasonic technique and layer-by-layer (LbL) assembly protocol. This new method is the continuation of our previous work, which describes the formation of water-dispersed polyglutamate/ * Corresponding authors. E-mail: [email protected]; [email protected]. † Max Planck Institute of Colloids and Interfaces. ‡ Tongji University. (1) Jones, M. C.; Leroux, J. C. Eur. J. Pharm. Biopham. 1999, 48, 101-111. (2) Malzert-Fre´on, A.; Vrignaud, S.; Saulnier, P.; Lisowski, V.; Benoıˆt, J P.; Rault. S. Int. J. Pharm. 2006, 320, 157-164. (3) Lamprecht, A.; Bouligand, Y.; Benoit, J. J. Controlled Release 2002, 84, 59-68. (4) Yamaguchi, T. AdV. Drug DeliVery ReV. 1996, 20, 117-130. (5) Igarashi, R.; Takenaga, M.; Matsuda, T. AdV. Drug DeliVery ReV. 1996, 20, 147-154. (6) Mizushima, Y. AdV. Drug DeliVery ReV. 1996, 20, 113-115.

polyelectrolyte nanocontainers filled with toluene/water-insoluble dye.7 Herein, we use a lipophilic drug (rifampicin) and vegetable oil to fabricate a new type of bio-friendly drug carrier. Rifampicin is a semisynthetic antibiotic, which is derived from a form of rifamycin that interferes with the synthesis of RNA and is used to treat bacterial and viral diseases. It is widely used together with isoniazid and streptomycin for the chemotherapy of tuberculosis.8 The core of the presented drug carrier contains the lipophilic drug and vegetable oil as well as lecithin as an emulsifier, which is similar to that for lipid microspheres. The shell of the containers is made of polyglutamate/polyethyleneimine (PEI)/poly(acrylic acid) (PAA) multilayers. The hydrophobic drug can be released by switching the polyglutamate/polyelectrolyte shell permeability through variation of environmental conditions, such as pH value9,10 and ionic strength.11 Figure 1 shows the scheme of the formation of composite containers and the release of drugs by changing the pH value. The polyelectrolyte shell can also be modified to have some additional functionality. For example, if the containers are coated with appropriate antigens, they can be targeted to specific regions in the body. Despite the high cost, lipid microspheres have already proved to be a safe carrier for drug targeting and have been put into the market, such as lipo-prostaglandin E1, Lipo-steroid.5 However, composite drug carriers based on layer-by-layer deposition technology have not been reported yet. Fluorescein particles were used as a model system for permeability studies of LbL assembled multilayers.12 Poly(styrenesulfonate) (PSS)/poly(allylamine) (PAH) multilayers formed a polyelectrolyte shell on the fluorescein core. It was found that increased layer numbers (7) Teng, X.; Shchukin, D. G.; Mo¨hwald, H. AdV. Funct. Mater. 2007, 17, 1273-1278. (8) Kamat, B. P.; Seetharamappa, J. J. Chem. Sci. 2005, 117, 649-655. (9) Antipov, A. A.; Sukhorukov, G. B.; Leporatti, S.; Radtchenko, I. L.; Donath, E.; Mo¨hwald, H. Colloids Surf. A 2002, 198, 535-541. (10) De´jugnat, C.; Sukhorukov, G. B. Langmuir 2004, 20, 7265-7269. (11) Ibarz, G.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. AdV. Mater. 2001, 13, 1324-1327. (12) Antipov, A. A.; Sukhorukov, G. B.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 2001, 105, 2281-2284.

10.1021/la702370k CCC: $40.75 © 2008 American Chemical Society Published on Web 12/12/2007

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Figure 1. Schematic illustration of the formation of the LbL-stabilized drug-loaded polyglutamate containers and drug release after pH changes (PG, polyglutamate; PEI, polyethyleneimine; PAA, poly(acrylic acid); LbL, layer-by-layer). (a) Formation of oil-loaded polyglutamate containers in ultrasonic field; (b) layer-by-layer assembly of PEI/PAA shell; (c,d) swelling and dissolution of the container shell by changing the pH value of the surrounding media.

decreased the shell permeability and resulted in prolonged dye dissolution. Besides, encapsulation of ibuprofen and furosemide microcrystals into the polyelectrolyte capsules with chitosan/ dextran and PSS/gelatin shell, respectively, also resulted in prolonged release of the drugs at different pH values.13-14 Many anticancer drugs and photosensitive drugs are lipophilic ones15 and can be encapsulated into layer-by-layer assembled containers. They can be protected by the nanocontainer shell from chemical-, enzymatic-, and photodegradation to facilitate their delivery efficiency. Lipophilic drugs are poorly absorbed after oral administration; however, their absorption may be enhanced in combination with lipids. Although such a kind of containers has largely been confined to lipophilic drugs, there are still plentiful opportunities for their application to modified hydrophilic drugs. For example, when drugs are not sufficiently lipophilic, derivation by alkyl esters could be achieved to obtain a hydrophobic analogue (e.g., hydrophobic mitomycin C derivatives).6,16 Experimental Section Materials. Poly-L-glutamic acid sodium salt (polyglutamate, Mw ∼ 50000-100000), rifampicin, soybean oil, lecithin (soybean phosphatidylcholine), polyethyleneimine (PEI, Mw ∼ 25000), poly(acrylic acid) (PAA, Mw ∼ 50000) were purchased from SigmaAldrich (Germany). All compounds were used without any further purification. PEI-Rhodamine B isothiocyanate was synthesized as described elsewhere.17 (13) Qiu, X.; Leporatti, S.; Donath, E.; Mo¨hwald, H. Langmuir 2001, 17, 5375-5380. (14) Ai, H.; Jones, S. A.; Villiers, M. M.; Lvov, Y. M. J. Controlled Release 2003, 86, 59-68. (15) Konan, Y. N.; Gurny, R.; Alle´mann, E. J. Photochem. Photobiol., B 2002, 66, 89-106. (16) Sasaki, H.; Takakura, Y.; Hashida, M.; Kimura, T.; Sezaki, H. J. Pharmacodynam. 1984, 7, 120-130. (17) Kaschak, D. M.; Mallouk, T. E. J. Am. Chem. Soc. 1996, 118, 42224223.

Preparation of Drug-Loaded Polyglutamate/Polyelectrolyte Nanocontainers and Release of Drug. The encapsulated drug, rifampicin, was first dissolved in soybean oil (5 wt % of rifampicin in soybean oil) and then the drug oil solution was placed over a 5% polyglutamate aqueous solution containing 1 mg/mL lecithin as emulsifier. A high-intensity ultrasonic horn was positioned at the aqueous-organic interface. The mixture was sonicated for 3 min in an ice bath employing an acoustic power of 150 W/cm2 at 20 kHz frequency (Figure 1a). The unreacted oil solution was removed from the resulting drug-loaded emulsion by centrifugation and filtered through a membrane filter with 3 µm diameter pore size (Millipore, Germany) several times. The polydispersity index of the resulting oil-loaded polyglutamate containers is 0.15. For fabrication of the layer-by-layer assembled polyelectrolyte shell, we used the technique of subsequent addition of the polyelectrolyte solution (Figure 1b).18 PEI (0.05 mL) labeled with Rhodamine B or PAA solution (2 mg/ mL in 0.5 M NaCl) was mixed with a 1 mL suspension of drugloaded polyglutamate containers at pH ) 7. The mixture was gently shaken for 8 min. The next layers were deposited in the same manner. After eight alternating deposition steps, the final (PEI/PAA)4 layered structure was formed on top of the polyglutamate shell. Resulting polyglutamate/(PEI/PAA)4 containers are stable for at least 4 months at 4 °C. Drug release was studied by incubating the container suspension at pH ) 1.4, pH ) 11.4, and pH ) 7.4, 37 °C, 0.2 M phosphate buffer solution (Figure 1c,d). Characterization. Scanning electron microscopy (SEM) measurements were conducted using a Gemini Leo 1550 instrument. Samples were prepared by applying a drop of the suspension onto glass wafer with sequential drying and gold sputtering. Transmission electron microscopy (TEM) (Zeiss EM 912 Omega) was used for the visualization of the containers. Coated copper grids were employed to support the samples. Raman spectra were recorded by a confocal Raman microscope (CRM200, WITec) with diode-pumped Green laser excitation (532 nm) and a 100× oil (NA ) 1.25, Nikon) microscope objective. Confocal images were taken with a confocal (18) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Polym. AdV. Technol. 1998, 9, 759-767.

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Figure 2. CLSM images of soybean oil-loaded (without drug) polyglutamate/(PEI/PAA)4 containers without using lecithin as emulsifier: (a) Original containers in transmission mode; (b) containers after 3 weeks storage at 4 °C in fluorescence mode. laser-scanning system TCLS attached to an inverse microscope from Leica (Wetzlar, Germany) and equipped with a 100× oil immersion objective having a numerical aperture of 1.4. The ζ-potential of the coated containers was measured by Zetasizer Nanoinstrument Nano Z equipment. Each value was averaged from three parallel measurements.

Results and Discussion Formation and Stability of Drug-Loaded Polyglutamate/ Polyelectrolyte Containers. A core-shell structure was formed with rifampicin-loaded soybean oil inside and polyglutamate shell outside after ultrasonic treatment of the aqueous polyglutamate/oil-rifampicin two-phase system. The mechanism of core-shell structure formation by ultrasonic treatment has been discussed in more detail previously.7,19-21 Addition of lecithin as an emulsifier can drastically increase the stability of containers. Figure 2 shows confocal laser scanning microscopy (CLSM) images of (PEI/PAA)4-coated oil-loaded (without drug) containers with and without using lecithin as emulsifier. As compared with containers with lecithin, containers without lecithin were inclined to aggregate after 3 weeks storage at 4 °C and have larger size. The reason is that the lecithin has a negative charge and may strongly interact with the polyglutamate shell, increasing the surface charge of the polyglutamate shell, which results in better electrostatic repulsion between containers with lecithin/polyglutamate shell.22 Another probable mechanism is that the miscibility between soybean oil and lecithin is critically important for the stability of the dispersed containers. Lecithin is known to spontaneously form a bilayer at the oil/water interface.23 Soybean oil has limited solubility in lecithin bilayer membranes. It is often stabilized by the closely packed lecithin monolayer. When the lecithin content is under the solubility threshold in soybean oil, the lecithin monolayer does not completely cover the hydrophobic surface of soybean oil droplets, which causes the aggregation of the containers. However, when the content of lecithin is high (in our work, 1 mg/mL lecithin content was used), the lecithin monolayer covers the soybean oil completely and stabilizes the dispersion. Thus, the obtained oil-lecithin (19) Dibbern, E. M.; Toublan, F. J.; Suslick, K. S. J. Am. Chem. Soc. 2006, 128, 6540-6541. (20) Suslick, K. S.; Grinstaff, K. J.; Kolbeck, K. J.; Wang, M. Ultrasonics Sonochem. 1994, 1, S56-S68. (21) Avivi, S.; Gedanken, A. Biochem. J. 2002, 366, 705-707. (22) Washington, C. AdV. Drug DeliVery ReV. 1996, 20, 131-145. (23) Asai, Y. J. Oleo Sci. 2003, 52, 359-365.

Figure 3. ζ-potential as a function of the layer number for containers during PEI/PAA layer-by-layer deposition.

Figure 4. Structure of rifampicin.

containers can be stored at least for 2 months at 4 °C, which is especially important for long-term drug delivery system. Although lecithin-stabilized containers have sufficient surface charge to produce better long-term stability, they cannot provide controlled release of the encapsulated drug. To obtain this specific functionality of the containers, eight alternating monolayers (PEI/ PAA) were deposited on the container surface via layer-by-layer

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Figure 5. Raman spectra of polyglutamate containers without polyelectrolyte multilayers: (a) Empty containers; (b) rifampicin-loaded containers.

assembly after formation of the oil-loaded polyglutamate shell. The emulsion became diluted after adding polyelectrolytes and a small quantity of flocculation was observed. When the first PEI layer (2 mg/mL in 0.5 M NaCl) deposited on the uncoated oil-lecithin surface, the ζ-potential of the containers changed from -32 to +33 mV (Figure 3). Following alternating PEI/ PAA layer depositions results in surface recharging between -3 and 21 mV, which indicates electrostatic assembly of polyelectrolyte multilayers. Encapsulation of Rifampicin into Polyglutamate/Polyelectrolyte Containers. The lipophilic drug rifampicin (the structure is in Figure 4) was added to soybean oil to test the encapsulation procedure. Lecithin (1 mg/mL solution) was used as an emulsifier dissolved in polyglutamate aqueous solution before mixing with

the oil phase. Raman confocal microscopy studies of the container interior (Figure 5) show a number of peaks corresponding to the pure soybean oil within the range 1100-1700, 2900 cm-1 for both the empty and rifampicin-loaded containers, which indicates that soybean oil was successfully incorporated into the containers. For rifampicin-loaded containers, peaks at 1300 and 1380 cm-1 can be assigned to the vibrations of υ (C-C) band and the vibration of δ (CH3) band of rifampicin, respectively,24 which is not observed for empty containers. Moreover, the small intensity of the broad band at 3200-3600 cm-1 corresponding to the vibration of the OH group of water is observed for all samples. This can be interpreted as a signal from water entrapped in the polyglutamate shell of containers. Hence, we can conclude that

Figure 6. SEM images of empty and rifampicin-loaded polyglutamate containers with and without polyelectrolyte multilayers: (a) Empty containers without polyelectrolyte layers; (b) rifampicin-loaded containers without polyelectrolyte layers; (c) empty containers with polyelectrolyte layers; (d) rifampicin-loaded containers with polyelectrolyte layers.

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Figure 7. TEM images of polyglutamate containers without polyelectrolyte multilayers incubated at different pH for 16 h: (a) Containers incubated at pH 1.4; (b) containers incubated at pH 11.4.

rifampicin was successfully incorporated in the hydrophobic interior of the containers, which have a hydrophilic polyglutamate shell. SEM images show that containers without rifampicin are inclined to become clamp-shaped, especially when there are no polyelectrolytes as additional protective layers (Figure 6a, such as arrows indicate) and, in contrast, drug-loaded containers with or without additional polyelectrolyte shell keep their integrity (Figure 6b,d, arrows). This can be caused by drying during preparation of the SEM probes. The polyglutamate container shell is invaded by water molecules evaporating from the inner container, which results in rupture. The broken surface of oilloaded containers tends to contract along the crack of the polyglutamate shell, resulting in formation of clamp-shaped containers. When using polyelectrolytes as additional layers, containers mostly collapse instead of rupture and the amount of clamp-shaped containers decreases consequently (Figure 6c, arrows). In a comparison of empty and drug-loaded containers, the latter ones keep their integrity, reducing the size to 200-600 nm due to the drug entrapped inside (Figure 6b,d, arrows). Release of Rifampicin from Polyglutamate/Polyelectrolyte Containers. Multilayers of weak polyelectrolytes deposited onto the container shell have semipermeable properties.9,10 The inner charge of the polyelectrolyte shell depends on the pH value, which can be utilized for the pH-controlled release of encapsulated

material. To investigate the possibility of controlled rifampicin release, rifampicin-loaded containers with and without (PEI/ PAA)4 multilayers were analyzed by TEM and CLSM after treatment at different pH values: pH ) 1.4 (close to stomach pH) and pH ) 11.4. One hundred microliters of container suspension was incubated in 200 µL of HCl (pH 1.4) or NaOH (pH 11.4) for 16 h to demonstrate pH-controlled release. For the containers without polyelectrolyte shells, drug release was observed only when incubating at pH 1.4 (Figure 7a). Figure 7a shows more drug microcrystals (arrow) near a spherical container. This is because the hydrogen bonds or ion pairs between two carboxylates ([RCO2-...M+...-O2CR] where M+ ) H+ or Na+) are destroyed in an acidic environment,19 which leads to instability of the polyglutamate/lecithin composite. However, it is interesting that intact polyglutamate/lecithin containers were observed after treatment at pH 11.4 without evidence of rifampicin release (Figure 7b). This is probably because the added alkaline increases the ionization ratio of polyglutamate, provoking stronger intermolecular hydrogen bonds between two carboxylates and electrostatic interactions between polyglutamate and lecithin, which keeps the stability of the containers and prevents the integrity of the shell and, as well, release of the encapsulated drug. To investigate the influence of additional polyelectrolyte multilayers, the containers with polyelectrolyte shell were monitored by CLSM after incubation for 16 h in both acid and

Figure 8. Confocal images of polyglutamate/(PEI/PAA)4 containers incubated at different pH conditions: (a) Original containers in transmission mode; (b) containers incubated at pH 1.4 for 16 h in fluorescence mode; (c) containers incubated at pH 11.4 for 16 h in fluorescence mode.

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Figure 9. Release of the rifampicin from polyglutamate (1, 4) and polyglutamate/(PEI/PAA)4 (2, 3) containers in acidic (pH ) 1.4) and alkaline (pH ) 11.4) media.

alkaline conditions (Figure 8). CLSM images show that most containers disappeared and only a few containers were observed after treatment at pH 1.4 (Figure 8b). On the other hand, an increase of the container size was found when incubating at pH 11.4 (Figure 8c). The kinetics of the rifampicin release is represented in Figure 9. Both polyglutamate and polyglutamate/polyelectrolyte containers release rifampicin quite rapidly at low pH (pH ) 1.4) due to the destruction of the polymer shell. However, the stabilizing effect of the electrostatically adsorbed PEI/PAA shell is observed and the release time for polyglutamate/(PEI/PAA)4 containers is 3 times longer than that for pure polyglutamate ones. In contrast, the release of the rifampicin is not observed in alkaline pH for pure polyglutamate containers while deposition of the polyelectrolyte shell leads to the expansion of the polymer shell followed by formation of the pores permeable for rifampicin, demonstrating the release of the 25% of loaded drug within 16 h. Such behavior of containers with polyelectrolyte multilayer decomposition at pH 1.4 and the size increase at pH 11.4 can be interpreted as follows: At pH 1.4 most carboxylate groups of the PAA are highly protonated and cannot compensate the charge of PEI uncompensated ammonium groups. The electrostatic repulsion between the positive charges results in a swelling of the whole structure. If the swelling pressure of the container increases, the remaining stabilizing electrostatic interactions are too weak to maintain the intact shell at such a low pH, resulting in container rupture due to Laplace’s law followed by dissolution of the container shell and drug release. In contrast, in the case of pH 11.4, PAA remains fully charged and PEI is deprotonated.25 Although the negative charges of PAA are repelled from each other, resulting in swelling of containers, there are still some ionic pairs of PEI and PAA due to the relatively weak basic environment; thus, the size of most containers increases, which could result in drug release from the pores of polyelectrolyte shell because the interior of polyglutamate/lecithin membrane could be defected by the swelling pressure. Similar results26 were reported for PAH/PMA polyelectrolyte capsules with aqueous interior, which were stable in the pH range from 2.5 to 11.5. (24) Schrader, B. Raman Infrared Atlas of Organic Compounds, 2nd ed.; VCH: Weinheim, 1989. (25) Lulevich, V. V.; Vinogradova. O. I. Langmuir 2004, 20, 2874-2878. (26) Mauser, T.; De´jugnat, C.; Sukhorukov, G. B. Macromol. Rapid Commun. 2004, 25, 1781-1785.

Figure 10. TEM images of polyglutamate containers with and without polyelectrolyte multilayers incubated at pH 7.4, PBS, 37 °C for 21 days: (a) Containers without (PEI/PAA)4 shell incubated in PBS; (b) containers with (PEI/PAA)4 shell incubated in PBS.

Finally, rifampicin release from containers with and without polyelectrolyte shell was investigated under physiological pH conditions in pH 7.4 (close to blood pH) phosphate buffer solutions (PBS) to simulate drug release in vivo. One milliliter of container suspension was incubated in 5 mL of 0.2 M PBS, 37 °C, with gentle stirring. After incubation, the containers were studied by TEM (Figure 10). Figure 10a shows that most containers without polyelectrolyte layers were destroyed even in such a neutral pH environment, resulting in strong drug release after 21 days of incubation. This effect could be caused by a synergetic effect of both H+ and phosphate on the stability of the polyglutamate shell, particularly on the possibility of hydrogen bond formation. However, with polyelectrolytes as protective layers, most containers kept their integrity after incubation for 21 days in phosphate buffer (Figure 10b). Even after 40 days of incubation, there were still many intact containers (results not shown). Thus, the nanocontainers can maintain better integrity while polyelectrolytes are being used as protective layers, which helps to perform slow release of lipophilic drugs from the hydrophobic container interior. Such protective properties of polyelectrolyte shell are important for controlled delivery and release, especially for long-term drug delivery.

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Conclusion A novel drug carrier, “oil-in-water” multifunctional composite nanocontainers, were prepared by combined ultrasonic technology and layer-by layer assembly protocol. A lipophilic drug, rifampicin, was successfully incorporated into composite waterdispersible polyglutamate/polyelectrolyte nanocontainers and it can be released in different pH conditions due to the pH-responsive properties of the polyglutamate/polyelectrolyte shell. With use of rifampicin as a model, any other lipophilic drug can be encapsulated into the functional containers following the described procedure, which is especially important for long-term drug delivery with controlled release. In addition, we can utilize such composite nanocontainers as artificial cell membranes by changing the lipid composition or use them to deliver genes

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into target cells by regulating the charge on the polyelectrolyte surface as well as to target specific organs or tumor types by incorporating biochemical functions, e.g., proteins, receptors, or DNA sequences as monolayer constituent. This opens the way for creating shells with specific adhesion properties that are of great potential for water-insoluble drug-delivery applications and for diagnostics. Acknowledgment. Dr. X. R. Teng thanks the China Scholarship Council for a research fellowship and we also thank Rona Pitschke for electron microscopy analysis and Gabi Wienskol for Raman microscopy measurement. This work was supported by the BMBF project “NanoFuture”. LA702370K