SELF-ASSEMBLY AND GROWTH OF SMART CELL ......uptake of a drug into the circulatory system. Thus, the mucin bound microtubes are very promising vehicles for drug delivery. The scheme

SELF-ASSEMBLY AND GROWTH OF SMART CELL-ADHESIVEMUCIN-BOUND MICROTUBES

Karen T. Johnson,1 Karl R. Fath,2 Marsiyana M. Henricus,1 and Ipsita A.Banerjee1

1Department of Chemistry, Fordham University, Bronx, New York, USA2Department of Biology, The City University of New York and The Graduate Center,Queens College, Flushing, New York, USA

% Microtubular structures were self-assembled in aqueous media from a newly synthesizedbolaamphiphile, bis(N-a-amido-threonine)-1,3-propane dicarboxylate. The self-assembly processwas examined at varying pH. The formed microtubes were then functionalized with the highlyglycosylated protein mucin. In nature, the O-linked saccharides of mucin are generallyassociated with Thr or Ser residues of protein scaffolds. In this work, peptide microtubes withthreonine functionality were prepared synthetically in order to enhance the affinity of themicrotubes toward mucin, thus mimicking natural proteins. After binding the mucin to themicrotubes, we investigated the biocompatibility of those materials by conducting in vitro cellattachment, cell proliferation, and cytotoxicity studies using normal rat kidney (NRK) cells. Thestudies revealed that the biomaterials were nontoxic, biocompatible, and showed significantadhesion to the cells. It is well known that natural mucins may degrade into their motifs;however, upon binding to the surface of microtubes, their stability may be increased. Becausemucin is one of the major components of mucoadhesion, and various types of mucins areubiquitous in human tissues, such mucin-bound microtubes may potentially be used asmucoadhesive materials for targeted drug delivery and improve the localization of drugs.

Keywords Biocompatible, Microtubes, Mucin, Peptide, Self-assembly

INTRODUCTION

Over the past few years, attention has been focused on the developmentof new biomaterials for various biomedical applications (1–8). The potentialuse of nanoparticles as drug carriers is of particular interest because drugscan be released in a suitable manner and the particle sizes can bemanipulated in order to target different tissues. Further, these particles canbe made in different forms to increase the bioavailability of the drugs and

Received 20 August 2008; Accepted 10 December 2008.Address correspondence to I. A. Banerjee, Department of Chemistry, Fordham University, 441

East Fordham Road, Bronx, NY 10458, USA. E-mail: [email protected]

Soft Materials, 7(1): 21–36, (2009)Copyright # Taylor & Francis Group, LLCISSN 1539-445X print/1539-4468 onlineDOI: 10.1080/15394450802693969

21

Downloaded At: 21:59 9 March 2009

reduce undesirable effects. Among the various types of nano andmicroparticles studied, liposomes are known to be important drug deliverysystems for the systemic administration of drugs, antisense oligonucleotides,and plasmids for gene therapy (9–11). Some of the advantages of liposomesinclude their ability to selectively accumulate at specific sites such as sites ofinflammation or tumors (12). Drug delivery systems, formed from varioustypes of nanoparticles, establish an alternative to liposomes. The adapt-ability of particulate technologies has allowed for the modification of thenanoparticle–based drug delivery systems with respect to the target, specificpharmacokinetic profile, and means of administration. Some examplesinclude polymeric nanoparticles (13–15), porous hollow silica nanoparticles(16–17), and cyclodextrin nanoparticles (18–19). It is understood that thesurface property of nanoparticles is the controlling factor for the fate ofnanoparticle drug complexes in vivo, including cellular interactions anddrug release properties. Therefore, the manipulation of surface propertiesof nanoparticles is an essential part of targeted drug delivery. Themodification of particle surfaces can be achieved by specifically functionaliz-ing the particles. Recently researchers functionalized gold nanoparticles foruse as carriers of anti-angiogenic and anticancer agents (20). Functionalizedcarbon nanotubes have also been found to be applicable for gene and drugdelivery (21–23).

Nanotubes and microtubes self-assembled from peptide bolaamphi-philes (amino acid head groups covalently bound via hydrocarbon chain),exhibit several different properties that make them promising biomaterialcandidates, including facile self-assembly in aqueous solutions andadaptability to functionalization for increased biocompatibility (24–34).The peptide head groups can be readily modified in order to manipulateand potentially alter the properties of the self-assembled micro andnanostructures. Although many biomedical applications related to peptide-based nanotubes (35–37) have been investigated, the immense potential ofpeptide nanotubes and microtubes has yet to be extensively investigated.

In this work, we have synthesized a new peptide bolaamphiphile, bis(N-alpha-amido-threonine)1,3-propane dicarboxylate. This bolaamphiphilecontains threonine head groups, which are connected by a propyl carbonchain. The self-assembly of the structures formed from this bolaamphiphilewas studied at different pH values. In general, we found that in a pH rangebetween 4 through 6 microtubulular structures were formed. Themicrotubes were then functionalized by covalently binding them to theglycosylated protein mucin. Recently, researchers have attached carbohy-drate-functionalized polymers that mimic the structures of mucin glyco-proteins to carbon nanotubes for specific receptor binding (38). It is wellknown that in nature, the carbohydrate chains of mucins are attached to theoxygen atom of threonine or serine (39) by N-acetyl galactosamine linkages.

22 K. T. Johnson et al.


Thus, in order to mimic nature, we have prepared microtubules withthreonine moieties, and have attached those microtubes to bovine mucin.Because mucin is one of the major components of mucoadhesion, it mayhelp to both improve the localization of drug delivery and deliver moleculesthat were previously problematic by other delivery methods (40–41).Several transmucosal drug carriers have been designed to adhere to themucosal surface of the nasal cavity and the buccal cavity (42). Natural mucinmay degrade into its motifs; however, upon binding to the tubule surface itsstability may be enhanced. Further, depending on the specific target, themicrotubes can be functionalized with different types of mucin.Mucoadhesive drug carriers have attracted considerable attention asmucous membranes are reasonably permeable and, hence allow rapiduptake of a drug into the circulatory system. Thus, the mucin boundmicrotubes are very promising vehicles for drug delivery. The scheme forfunctionalization of the microtubes with mucin is shown in Fig. 1. Theattachment of mucin to the microtubes was confirmed by Fouriertransformation infrared (FTIR) spectroscopy, transmission electron micro-scopy (TEM), and scanning electron microscopy (SEM) analysis. Finally thebiocompatibility and cell adhesive properties of the mucin-bound micro-tubes were examined in the presence of normal rat kidney (NRK) cells.Such mucoadhesive microtubes may be of potential importance in targetedcell adhesion, controlled drug delivery applications, enhanced bioavail-ability, and may provide a significant improvement in therapeutic efficacy.

EXPERIMENTAL

Materials

N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDAC), L-threo-nine-methyl ester hydrochloride, glutaric acid, N-hydroxysuccinimide

FIGURE 1 Scheme for functionalization of self-assembled microtubes with mucin and theirapplications in cell attachment and drug encapsulation and release (figure is provided in coloronline).

Smart Cell-Adhesive Mucin-Bound Microtubes 23


(NHS), triethylamine, DMF, methanol, acetone, DMSO-d6 with 0.1% v/vTMS, hydroxybenzotriazole, and bovine submaxillary mucin werepurchased from Sigma-Aldrich and used as received. Buffer solutionsof various pH values, hydrochloric acid, and sodium hydroxide pelletswere purchased from Fisher Scientific. Normal rat kidney cells werepurchased from the American Type Culture Collection (ATCC CRL-6509) and cultured in Dulbecco’s Modified Eagle’s Medium supplemen-ted with 100 units/ml penicillin, 100 mg/ml streptomycin and 10% fetalbovine serum at 37uC in a humidified atmosphere of 5% CO2.

Methods

Synthesis of bis(N-alpha-amido-threonine)-1,3 propane dicarboxylate

The bolaamphiphile bis(N-a-amido-threonine)-1,3 propane dicarbox-ylate was synthesized by modification of previously established peptidecoupling methods (43). The hydroxyl group of threonine-methyl ester wasprotected as Bzl (benzyl methyl ether). The product obtained was set asidebefore coupling with the activated glutaric acid. Glutaric acid (0.90 g,1.9 mmol) and 1-hydroxybenzotriazole (1.12 g, 4.1 mmol) were mixedtogether in DMF and were added to EDAC (1.72 g, 3.8 mmol) in 25 mLchlorofom with stirring at 22uC. After an hour, the protected threoninemethyl ester (2.72 g, 4.12 mmol) was added. Triethylamine (0.6 mL) wasadded immediately thereafter to the reaction mixture. The contents werestirred for 24 h at 2uC and then allowed to sit until it reached roomtemperature conditions. The intermediate obtained was washed with coldcitric acid and sodium bicarbonate and recrystallized from DMF. In orderto remove the protecting groups, the reaction mixture was subjected tobase hydrolysis at 90uC for 4 h. The reaction mixture was then broughtto room temperature, and treated with 0.1 M HCl. It was then rotavaped toobtain the final product. The product obtained was then washed with coldacetone, yielding a pale yellow product (62.5% yield), and re-crystallizedfrom methanol. The 1H NMR spectra were recorded with a Bruker300 MHz NMR spectrometer. The 1H NMR DMSO-d6 spectrum showedpeaks at d 1.2 (d, 6H) (–CH3 groups); 2.1 (m, 6H, three CH2 groups), 4.3(m, 2H) (–CH group); 4.5 (d) (2H) (–CH group); 4.9 (s) due to H fromalcohol –OH group; 8.3 (s) due to hydrogen from the amide peaks. Theelemental analysis of the C13H22N2O8 compound revealed the followingmass %: C 46.1; H 6.52, N 8.01.

Self-Assembly

Individual stock solutions (8 mM) of the bis(N-alpha-amido-threonine)-1,3 propane dicarboxylate monomers were prepared in buffer solutions



ranging from pH 4 to pH 10. In general, the materials were allowed to self-assemble for 7–10 days. Aggregates of various assemblies formed were thenwashed in nanopure water, followed by sonication for 30 min. The sizesand morphologies of the structures formed were examined by TEM, SEM,and atomic force microscopy (AFM). The formed microtubes were foundto be stable for months at room temperature.

Attachment of Mucin to Microtubes

The peptide microtubes (500 mL) were immersed in an aqueous solutionof 30 mM EDAC (200 mL) and 15 mM N-hydroxy succinimide (200 mL) andshaken vigorously for 1 h to activate the carboxyl groups of the microtubes.This solution was then mixed with 500 mL of mucin (0.1M) in nanopurewater and allowed to react for 24 h at 2uC in order to allow for reaction tooccur between the free carboxyl groups of the microtubes and the aminegroups of the glycoprotein mucin. It is to be noted that before addition themucin solutions were centrifuged at 4,000 rpm for 2 h and then filteredthrough 0.2 micron filters to remove any aggregates. The final concentra-tion of the mucin in solution was determined by uv-vis spectroscopy at almax of 280 nm (44).The microtubes were then washed exhaustively withnanopure water and centrifuged in order to remove any unbound mucin.This process was repeated thrice. The incorporation of mucin on to themicrotubes was confirmed by FTIR, TEM, and SEM analyses.

Cell Viability and Morphology Assays

NRK cells were collected using 0.25% Trypsin, 0.1% EDTA in Hank’sBalanced Salt Solution and plated in cell culture medium in 12-wellCostar plates at a density of 5 6 104 cells per well (1.3 6 104 cells/cm2).After allowing the cells to attach and spread for 5 h, mucin microtubes(25 mL) were added into wells containing 2 mL final volume of media andincubated with the cells for an additional 48 to 72 h. To determine cellviability, the adherent and any unattached cells were collected from eachwell by trypsinization and the number of live cells were determined bytrypan blue exclusion. The cells were counted using a hemocytometer.To document cell morphology, photomicrographs were taken using aninverted Olympus CK 30 phase contrast microscope directly through theeyepiece using a hand-held Olympus C-50 zoom digital camera.

Characterization

The particle sizes and morphologies of the materials were analyzed byTEM, SEM, and AFM. For TEM, the samples were vortexed for 5 min



following which 5 ml of the samples were pipetted onto a carbon coatedcopper grid and analyzed at various magnifications. TEM analyses werecarried out using a JEOL-120EX instrument, operated at 100 kV. AFMmeasurements were carried out using Quesant Instruments UniversalSPM. The samples were analyzed in tapping mode using silicon nitrideSPM cantilevers. SEM analyses was carried out using a Hitachi-2600NSEM operated at 25 kV.

Infrared Spectroscopy: Fourier transform IR (FTIR) spectra wererecorded using Mattson Infinity FTIR infrared spectrophotometer in therange of 4000–400 cm21. Samples were dried at room temperature andthen KBr pellets were prepared. The % transmittance of the samples wasmeasured.

NMR Spectroscopy was carried out using a Bruker 300 MHz highperformance digital NMR spectrometer.

Absorption Spectroscopy: The absorption spectra for the drugrelease studies were recorded in a range of 190–400 nm using a Cary-300Bio Varian Spectrophotometer.

RESULTS AND DISCUSSION

In order to mimic natural biological structures, many peptideassemblies have been synthesized thus far (45–48). It has been observedthat many such assemblies are promoted by protonation and/orhydrophobic interactions between the peptide moieties and they areformed either instantaneously or over a period of time. We havesynthesized a new bolaamphiphile, HOOC-Thr-NH-CO-(CH2)3-NH-CO-Thr-COOH, which has two carboxylic acid groups at the end of the headgroups. For this study, the alkyl spacer between the head groups waslimited to three methylene groups. The effect of varying the alkyl chainlength on the self-assembly process is being currently studied and will bereported separately. The bolaamphiphiles are predicted to be pHsensitive because of the presence of the carboxyl groups. Althoughhydroxyl groups are also present in the threonine moiety, they remainlargely uncharged (49) and are involved in hydrogen bonding interac-tions. After synthesis, the bolaamphiphile was mixed with solutions ofvarious pH to allow for the growth of various nano and microstructures.In general, assemblies at various pH appeared after 10 days at roomtemperature.

We observed high yields of tubular formations in a pH range of 4–6,in the size range of 500 nm to 2 microns in diameter. However, at higherpH (.8), globular structures were observed. The SEM and AFM imagesof the structures obtained are shown in Fig. 2. Fig. 2(a) shows the SEMimage of microtubes formed at pH 6. Similar structures were observed at



pH 4 and pH 5 (data not shown). Fig. 2b shows the amplitude AFM imageof globular microstructures formed at pH 9. The variation in types ofstructures formed is most likely due to the difference in the hydrogen-bonding pattern at different pH. The proposed three-dimensionalassembly mechanism is shown in Fig. 3. At pH ,7, hydrogen bondinginteractions exist between two COOH groups via acid-acid dimerinteractions, intermolecular amide-amide hydrogen bonds as well asintermolecular hydrogen bonding between the hydroxyl groups and theadjacent carboxyl groups and amide groups. However, at higher pH (pH.8), acid-anion interactions are involved due to deprotonation of thecarboxyl groups in addition to amide-amide hydrogen bonds andhydrogen bonding due to the hydroxyl groups. Thus, higher strengthof hydrogen bonding at lower pH leads to tubular structures, due totighter intra and intermolecular interactions, leading to sheetlikestructures (Fig. 3b—three- dimensional model), which can fold up toform tubes, while the acid-anion interactions of the carboxylic groups athigh pH reduces the hydrogen bonding between the monomers, anddoes not allow as much strong interactions leading to globular extendedstructures. The hydrogen bonding interactions were further explored byFTIR spectroscopy.

FTIR Spectroscopy

In order to elucidate the hydrogen bonding interactions involved,FTIR spectroscopy was used to analyze the various assemblies. The FTIRspectrum of tubular structures obtained at low pH (pH 4), is shown in

FIGURE 2 (a) SEM image of tubules formed at pH 6. Scale bar 5 10 mm; (b) AFM image of globularstructures formed at pH 9. (Scale bar 5 4mm) (figure is provided in color online).



Fig. 4(a). The peak at 1630 cm21 is assigned to the carbonyl group of theamide I stretching vibration and is indicative of acidic b-sheetlikestructure formation (50). The C5O group of carboxylic acid vibrationshave been observed in some bolaamphiphile assemblies as well as several

FIGURE 3 (a) Proposed assembly of microstructures at low pH and at high pH; (b) Three-dimensional assembly of structures at low and high pH(figure is provided in color online).



amphiphiles with gluconamide head groups around 1740 cm21 (51).However, in the case of the microtubes, this peak is red shifted to1720 cm21 because those C5O groups are involved in H-bondinginteractions with the N-H of the amide groups. In general, red shiftedcarboxylic acid frequencies indicate stronger hydrogen bonding due tothe –COOH groups (52). The peak at 2990 cm21 is assigned to thealiphatic C-H stretch due to the connecting methylene groups. A shortpeak is observed at 1537 cm21, which is assigned to the amide II bendingvibrations. The peak at 1260 cm21 is assigned to the stretching vibrationsdue to the C-O bonds in the carboxyl group, while peaks at 1410 cm21

and 1473 cm21 are assigned to the C-O-H bending vibrations due to thecarboxylic acid groups. The region between 2500–3700 cm21 is broaddue to the overlap of the carboxyl group as well as the –OH group fromthe alcohol group and the NH from amide group. Further, a very broadpeak in this range indicates strong hydrogen bonding interactions due to–OH groups.

At higher pH (Fig. 4b) (pH 9), peaks are observed for the amide Igroup at 1645 cm21, while a relatively less intense C5O peak due to thecarboxyl group is seen at 1735 cm21. Those peaks are blue shiftedcompared to low pH, due to diminished hydrogen bonding interactions.The peaks at 1407 cm21 and 1472 cm21 are assigned to C-O-H vibrations.

FIGURE 4 FTIR spectra of microstructures formed at (a) low pH and (b) high pH.



It is to be noted that the region between 3100 cm21 and 3500 cm21 is stillfairly broad due to the presence –OH groups and amide groups. Fromthe aforementioned analysis it appears that hydrogen bonding betweenacid-acid dimer and intermolecular amide-amide hydrogen bonds play acrucial role in the formation of microtubular structures.

Attachment of Mucin to Microtubes

The self-assembled microtubes formed were used for the incorpora-tion of mucin. Nano and microtubular structures have several advantagessuch as having a uniform surface with hollow ends, and may be used forencapsulation of enzymes, drugs, DNA, and various other materials. Theoutside walls of the tubes can also be coated with different types ofmaterials for several applications (53). The attachment of mucin to themicrotubes was confirmed by FTIR spectroscopy, as well as TEM andSEM. The TEM and SEM images of covalently bound mucin-coatedmicrotubes are shown in Fig. 5. Fig. 5(a) represents a low magnificationimage of mucin-coated microtubes while Fig. 5(b) represents a SEMimage of a single mucin-coated microtube. When compared to uncoatedtubes (Fig. 2a), it is clear that the surfaces of the tubes are uniformlycoated with mucin as the surfaces of the tubes are no longer smooth, butare replaced by furry mucin coating. Natural mucins are glycoproteinspresent on the surface of many epithelial cells. They have a proteinbackbone, which consists of both highly glycosylated and unglycosylated

FIGURE 5 (a) TEM image of mucin bound microtubes; (b) High magnification SEM image of asingle mucin bound microtube (Scale bar 5 2.5 mm).



regions (41, 54) having a high content of Ser and Thr along with otheramino acid groups. Mucin was coupled to the microtubes by coupling thefree carboxyl groups of the microtubes with the amino groups of mucin.Further, the presence of threonine moiety in the microtubes alsosupports stronger interactions between the microtubes and mucin. Theincorporation of mucin on the surface of the microtubes was alsoconfirmed by FTIR analysis as shown in Fig. 6. Prior to the immobiliza-tion of mucin, the IR frequencies observed for the microtube are shownin Fig. 4(a). However, after incorporation of mucin, significant changesare seen in the IR spectrum (Fig. 6b). The spectrum of the mucin-coatedmicrotube agrees well with the neat mucin spectrum (Fig. 6a). In additionit is to be noted that the peak at 1720 cm21, due to the free carboxylgroups present in the uncoated tubes, is absent in the mucin-coatedmicrotubes, indicating that those groups were involved in covalentbinding with mucin. These results indicate that mucin was incorporatedon the surface of the microtubes.

Cytotoxicity, Cell Adhesion, and Cell Morphology Analysis

To determine whether the mucin-bound microtubes could be used inbiomedical applications, we investigated their biocompatibility byconducting in vitro cytotoxicity and cell-proliferation studies with cultured

FIGURE 6 Comparison of IR spectra of (a) neat mucin and (b) mucin bound microtubes.



normal rat kidney (NRK) cells. Trypsinized cells were allowed to attachand spread in 12 well plates and 25 mL of microtube solutions were addedto the culture media. In the light microscope, we observed no cell lysis orchanges in cell morphology in the presence of mucin-coated microtubes.The cells remained relatively flat and well spread even when bound tolarge tubular aggregates (Fig. 7a). Note that in the light microscope,numerous, small microtubes and nanotubes are not visible. In some trials,the microtubes and suspended cells were added to the culture platesconcurrently. Under those conditions, many cells attached to and spreadalong large aggregates of microtubes. We also observed that the cells werecapable of dividing and growing in the presence of the mucin-coatedtubes. We found that the number of cells more than doubled after 24 h.We also examined the samples after a period of 72 h and the resultsindicated that the number of cells had tripled. Thus, the cells continued

FIGURE 7 (a) Low magnification photomicrograph indicating attachment of mucin boundmicrotubes on to NRK cells; (b) High magnification image of individual mucin bound microtubesattached to cells; (c) Cell proliferation and cytotoxicity studies. Arrows indicate microtubes.



to proliferate in the presence of the mucin-coated tubes. Although weobserved no cell lysis under the microscope, we determined thepercentage of viable cells using the trypan blue exclusion assay after24 h, 48 h, and 72 h of incubation. After duplicating the experiments, wecalculated the average percentage of viable cells after different periods oftime, as shown in Fig. 7b. We found that the percentage of viable cells wasalmost similar to control conditions lacking microtubes. These resultssuggest that not only are the mucin-coated microtubes non-toxic but alsoexcellent substrates for cell adhesion. Such materials can be potentiallyuseful for highly biocompatible mucoadhesive systems.

CONCLUSIONS

In this work, we have designed a new bolaamphiphile, bis(N-alpha-amido-threonine)-1,3-propane dicarboxylate and studied its self-assemblyprocess under aqueous conditions at varying pH. We observed that ingeneral, tubular structures were obtained in a pH range of 4–6. Theformed tubes were then functionalized with the glycosylated protein,mucin. We then examined the biocompatibility of those mucin-coatedtubes in the presence of NRK cells. Such mucin-bound microtubes mayplay a key role in developing novel therapeutic agents that could beimportant in targeting specific genes and tissues and address severalconcerns related to the basic challenges in the synthesis of novel,biocompatible and safe delivery and controlled release of drugs.

ACKNOWLEDGMENTS

The authors thank Dr. Areti Tsiola at the Queens College CoreFacility for Imaging, Cell and Molecular Biology for the use of thetransmission electron microscope and Dr. Patrick Brock at theDepartment of Earth and Environmental Sciences, Queens College,CUNY for the use of the scanning electron microscope. IB thanksFordham University Faculty Research Grant for financial support. KFthanks the Professional Staff Congress of The City University of NewYork for financial support.

REFERENCES

[1] Salata, O.V. (2004) Applications of nanoparticles in biology and medicine. J. Nanobiotechnol.,2(1):3–9.

[2] Nair, L.S., and Laurencin, C.T. (2008) Nanofibers and nanoparticles for orthopaedic surgeryapplications. J. Bone Joint Surg. (Am.), 90:128–131.

[3] Lu, Y., and Liu, J. (2007) Smart nanomaterials inspired by biology: Dynamic assembly of error-free nanomaterials in response to multiple chemical and biological stimuli. Acc. Chem. Res.,40(5):315–323.



[4] Wu, C., Fu, J., and Zhao, Y. (2000) Novel nanoparticles formed via self-assembly ofpoly(ethylene glycol-b-sebacic anhydride) and their degradation in water. Macromolecules,33(24):9040–9043.

[5] Lee, L.J. (2006) Polymer nanoengineering for biomedical applications. Ann. Biomed. Eng.,34(1):75–88.

[6] Penn, S.G., He, L., Natan, M.J. Nanoparticles for bioanalysis. (2003) Curr. Opin. Chem. Biol.,7(5):609–615.

[7] Hu, M., Chen, J., Li, Z., Au, L., Hartland, G.V., Li, X., Marquez, M, Xia, Y. (2006) Goldnanostructures: Engineering their plasmonic properties for biomedical applications. Chem. Soc.Rev., 35:1084–1094.

[8] Salata, O.V. (2007) Nanotechnology in therapeutics: Hydrogels and beyond. J. Nanobiotechnol.,5:5–6.

[9] Gregoriadis, G. (1995) Engineering liposomes for drug delivery: Progress and problems. TrendsBiotechnol., 13(12):527–537.

[10] Ostro, M.J., and Cullis, P.R. (1989) Use of liposomes as injectable-drug delivery systems. Am. J.Hosp. Pharm., 46(8):1576–1587.

[11] Lian, T., and Ho, R.J.Y. (2001) Trends and developments in liposome drug delivery systems.J. Pharm. Sci., 90(6):667–680.

[12] Maurer, N., Fenske, D., and Cullis, P.R. (2001) Developments in liposomal drug deliverysystems. Expert Opin. Biol. Ther., 1(6):923–947.

[13] Panyam, J., and Labhasetwar, V. (2003) Biodegradable nanoparticles for drug and genedelivery to cells and tissue. Adv. Drug Delivery Rev., 55(3):329–347.

[14] Duclairoir, C., Orecchioni, A.-M, Depraetere, P., Osterstock, F., and Nakache, E. (2003)Evaluation of gliadins nanoparticles as drug delivery systems: A study of three different drugs.Int. J. Pharm., 253(1–2):133–144; Qian, F., Cui, F., Ding, J., Tang, C., and Yin, C. (2006)Chitsoan graft copolymer nanoparticles for oral protein drug delivery: Preparation andcharacterization. Biomacromolecules., 7(10):2722–2727.

[15] LaVan, D.A., McGuire, T., and Langer, R. (2003) Small-scale systems for in vivo drug delivery.Nat. Biotechnol., 21(10):1184–1191; Emerich, D., and Thanos, C. (2006) The pinpoint promiseof nanoparticle based drug delivery and molecular diagnosis. Biomol. Eng., 23(4):171–184; Yih,T.C., and Al-Fandi, M. (2006) Engineered nanoparticles as precise drug delivery systems. J. CellBiochem., 97(6):1184–1190.

[16] Barbe, C., Bartlett, J., Kong, L., Finnie, K., Lin, H.Q., Larkin, M., Calleja, S., Bush, A.,and Calleja, G. (2004) Silica particles: A novel drug-delivery system. Adv. Mater., 16(21):1959–1966.

[17] Chen, J.-F., Ding, H-M., Wang, J-X., and Shao, L. (2004) Preparation and characterization ofporous hollow silica nanoparticles for drug delivery application. Biomaterials, 25(4):723–727.

[18] Memisoglu-Bilensoy, E., Vural, I., Bochot, A., Renoir, J.M., Duchene, D., and Hincal, A. (2005)Tamoxifen citrate loaded amphiphilic b-cyclodextrin nanoparticles: In vitro characterizationand cytotoxicity. J. Controlled Release, 104(3):489–496.

[19] Hirayama, F., and Uekama, K. (1999) Cyclodextrin-based controlled drug release system. Adv.Drug Delivery Rev., 36(1):125–141.

[20] Mukherjee, P., Bhattacharya, R., and Mukhopadhyay, D. (2005) Gold nanoparticles bearingfunctional anti-cancer drug and anti-angiogenic agent: A ‘‘2 in 1’’ system with potentialApplication in Cancer Therapeutics J. Biomed. Nanotechnol., 1(2):224–228.

[21] Bianco, A., Kostarelos, K., and Prato, M. (2005) Applications of carbon nanotubes in drugdelivery. Curr. Opin. Chem. Biol., 9(6):674–679.

[22] Liu, A., Sun, X., Nakayama-Ratchford, N., and Dai, H. (2007) Supramolecular chemistry onwater-soluble carbon nanotubes for drug loading and delivery. ACS Nano., 1(1):50–56.

[23] Prato, M., Kostarelos, K., and Bianco, A. (2007) Functionalized carbon nanotubes in drugdesign and discovery. Acc. Chem. Res., 41(1):60–68.

[24] Spear, R.L., Tamayev, R., Fath, K.R., and Banerjee, I.A. (2007) Templated growth of calciumphosphate on tyrosine derived microtubules and their biocompatibility. Colloids Surf. B.,60(2):158–166.

[25] Fairman, R., and Akerfeldt, K.S. (2005) Peptides as novel smart materials. Curr. Opin. Struct.Biol., 15(4):453–463.



[26] Matsui, H. (2004) Peptide nanotubes. In Encyclopedia of Nanoscience and Nanotechnology vol. 8;Nalwa, H.S., ed.; American Scientific Publishers, Stevenson Ranch, CA, 445.

[27] Claussen, R.C., Rabatic, B.M., and Stupp, S.I. (2003) Aqueous self-assembly of unsymmetricpeptide bolaamphiphiles into nanofibers with hydrophilic cores and surfaces. J. Am. Chem. Soc.,125(42):12680–12681.

[28] Reches, M., and Gazit, E. (2006) Molecular self-assembly of peptide nanostructures: Mechanismof association and potential uses. Curr. Nanosci., 2(2):105–111.

[29] Kogiso, M., Hanada, T., Yase, K., and Shimizu, T. (1998) Intralayer hydrogen-bond-directedself-assembly of nano-fibers from dicarboxylic valylvaline bolaamphiphiles. Chem. Commun.,17:1791–1792.

[30] Graveland-Bikker, J.F., Schaap, I.A., Schmidt, C.F., and de Kruif, C.G. (2006) Structural andmechanical study of a self-assembling protein nanotube. Nano Lett., 6(4): 616–621.

[31] Matsui, H., and Douberly, Jr., G.E (2001) Organization of peptide nanotubes into macroscopicbundles. Langmuir, 17(25):7918–7922.

[32] Porrata, P., Goun, E., and Matsui, H. (2002) Size-controlled self-assembly of peptide nanotubesusing polycarbonate membranes as templates. Chem. Mater., 14(10):4378–4381.

[33] Carloni, P., Andreoni, W., and Parrinelo, M. (1997) Self-assembled peptide nanotubes fromfirst principles. Phys. Rev. Lett., 79(4):761–764.

[34] Matsui, H., and Gologan, B. (2000) Crystalline glycylglycine bolaamphiphile tubules and theirpH sensitive structural transformation. J. Phys. Chem., 104(15):3383–3386.

[35] Gao, X., and Matsui, H. (2005) Peptide-based nanotubes and their applications inbionanotechnology. Adv. Mater., 17(17):2037–2050.

[36] Hartgerink, J.D., Clark, T.D., and Ghadiri, M.R. (1998) Peptide nanotubes and beyond. Chem.Eur. J., 4(8):1367–1372.

[37] Yemini, M., Reches, M., Gazit, E., and Rishpon, J. (2005) Peptide nanotube-modified electrodesfor enzyme-biosensor applications. Anal. Chem, 77(16):5155–5159; Yu, L., Banerjee, I.A., Gao,X., Nuraje, N., and Matsui, H. (2005) Fabrication and application of enzyme-incorporatedpeptide nanotubes. Bioconjugate Chem., 16(6):1484–1487.

[38] Chen, X., Lee, G.S., Zettl, A., and Bertozzi, C.R. (2004) Biomimetic engineering of carbonnanotubes by using cell surface mucin mimics. Angew. Chem. Int. Ed., 43:6111–6116; Chen, X.,Tam, U.C., Czlapinski, J. L., Lee, G.S., Rabuka, D., Zettl, A., and Bertozzi, C.R. (2006)Interfacing carbon nanotubes with living cells. J. Am. Chem. Soc., 128:6292–6293.

[39] Devine, P.L., and McKenzie, I.F.C. (1992) Mucins: Structure, function, and associations withmalignancy. BioEssays, 14(9):619–625.

[40] Smart, J.D. (2005) The basics and underlying mechanisms of mucoadhesion. Adv. Drug DeliveryRev., 57(11):1556–1568.

[41] Svensson, O., Thuresson, K., Arnebrant, T. (2008) Interactions between drug delivery particlesand mucin in solution and at interfaces. Langmuir, 24(6):2573–2579; Adikwu, M. (2006) Mucinsand their potentials. Trop. J. of Pharm. Res., 5(2):581–582.

[42] Junginger, H.E. (1991) Mucoadhesive hydrogels. Pharm. Ind., 53(11):1056–1060; Bodde, H.E.,De Vries, M.E., and Junginger, H.E. (1990) Mucoadhesive polymers for the buccal delivery ofpeptides, structure-adhesiveness relationships. J. Controlled Release, 13:225–231; Abd El-Hameed,M.D., and Kellaway, I.W. (1997) Preparation and in vitro characterisation of mucoadhesivepolymeric microspheres as intra-nasal delivery systems. Eur. J. of Pharm. Biopharm., 44(1):53–60.

[43] Kogiso, M., Ohnishi, S., Yase, K., Masuda, M., and Shimizu, T. (1998) Dicarboxylicoligopeptide bolaamphiphiles: Proton triggered self-assembly of microtubes with loose solidsurfaces. Langmuir, 14:4978–4986; Guzzo, P.R., Trova, M.P., Inghardt, T., and Linschoten, M.(2002) Synthesis of conformationally constrained threonine-valine dipeptide mimetic: Design ofpotential inhibitor of plasminogen activator inhibitor-1. Tetrahedron Lett., 43:41–43.

[44] Efremova, N.V., Huang, Y., Peppas, N.A., and Leckband, D.E. (2002) Direct measurement ofinteractions between tethered poly(ethylene glycol) chains and adsorbed mucin layers.Langmuir, 18:836–845.

[45] Reches, M., and Gazit, E. (2003) Casting metal nanowires within discrete self-assembled peptidenanotubes. Science, 300:625–627; Reches, M., and Gazit, E. (2006) Controlled patterning ofaligned self-assembled peptide nanotubes. Nature Nanotech., 1(3):195–200.

[46] Makabe, K., McElheny, D., Tereshko, V., Hilyard, A., Gawlak, G., Yan, S., Koide, A., andKoide, S. (2006) Atomic structures of peptide self-assembly mimics. Proc. Natl. Acad. Sci. USA,



103(47):17753–17758; Hartgerink, J.D., Granja, J.R., Milligan, R.A., and Ghadiri, M.R. (1996)Self-assembling peptide nanotubes. J. Am. Chem. Soc., 118:43–50.

[47] Haines-Butterick, L., Rajagopal, K., Branco, M., Salick, D., Rughani, R., Pilarz, M., Lamm,M.S., Pochan, D.J., and Schneider, J.P. (2007) Controlling hydrogelation kinetics by peptidedesign for three-dimensional encapsulation and injectable delivery of cells. Proc. Natl. Acad. Sci.USA, 104(19):7791–7796; Zhang, S. (2003) Fabrication of novel biomaterials through molecularself-assembly. Nature Biotech., 21:1171–1178.

[48] Vauthey, S., Santoso. S., Gong, H., Watson, N., and Zhang, S. (2002) Molecular self-assembly ofsurfactant-like peptides to form nanotubes and nanovesicles. Proc. Natl. Acad. Sci. USA,99(8):5355–5360; Hirata, T., Fujimura, F., Horikawa, Y., Sugiyama, J., Morita, T., and Kimura,S. (2007) Molecular assembly formation of cyclic hexa-b-peptide composed of acetylatedglycosamino acids. Peptide Sci., 88(2):150–156; Jagannadh, B., Reddy, M.S., Rao, C.L.,Prabhakar, A., Jagdeesh, B., and Chandrasekhar, S. (2006) Self-assembly of cyclic homo- andhetero-b-peptides with cis-furanoid sugar amino acid and b-hGly as building blocks Chem.Comm., 46:4847–4849.

[49] Mark, S., and Sanson, P. (1992) The roles of serine and threonine side chains in ion channels: Amodeling study. Eur. Biophys. J., 21(4):281–298.

[50] Segman-Magidovich, S., Grisaru, H., Gitli, T., Levi-Kalisman, Y., and Rapaport, H. (2008)Matrices of acidic b-Sheet peptides as templates for calcium phosphate mineralization. Adv.Mater., 20(11):2156–2161.

[51] Fuhrhop, J.-H., Demoulin, C., Rosenberg, J., and Boettcher, C. (1990) J. Am. Chem. Soc.,112:2827–2829.

[52] Lin-Vien, D., Colthup, N.B., Fateley, W.G., and Grasselli, J.G. (1991) The Handbook of Infraredand Raman Characteristic Frequencies of Organic Molecules; Academic Press: New York.

[53] Baughman, R.H., Zakhidov, A., and de Heer, W. (2002) Carbon nanotubes—The route towardapplications. Science, 297(5582):787–792; Heng, L.Y., Chou, A., Yu, J., Chen, Y., and GoodingJ. (2005) Demonstration of the advantages of using bamboo-like nanotubes for electrochemicalbiosensor applications compared with single walled carbon nanotubes. Electrochem. Comm.,7(12):1457–1462; Kohli, P., Martin, C.R. (2005) Smart nanotubes for biotechnology. Curr.Pharm. Biotechnol., 6(1):35–47; Huang, Y., Duan, X., and Lieber, C.M. (2005) Nanowires forintegrated multicolor nanophotonics. Small, 1(1): 142–147.

[54] Shogren, R., Gerken, T.A., and Jentoft, N. (1989) Role of glycosylation on the conformationand chain dimensions of o-linked glycoproteins: Light-scattering studies of ovine submaxillarymucin. Biochemistry, 28(13):5525–5536; Lang, T., Alexandersson, M., Hansson, G.C.,Samuelsson, T. (2004) Bioinformatic identification of polymerizing and transmembranemucins in the puffer fish Fugu rubripes. Glycobiology, 14(6):521–527.



PLEASE SCROLL DOWN FOR ARTICLE

This article was downloaded by:On: 9 March 2009Access details: Access Details: Free AccessPublisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

Soft MaterialsPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713597297

Self-Assembly and Growth of Smart Cell-Adhesive Mucin-Bound MicrotubesKaren T. Johnson a; Karl R. Fath b; Marsiyana M. Henricus a; Ipsita A. Banerjee a

a Department of Chemistry, Fordham University, Bronx, New York, USA b Department of Biology, The CityUniversity of New York and The Graduate Center, Queens College, Flushing, New York, USA

Online Publication Date: 01 January 2009

To cite this Article Johnson, Karen T., Fath, Karl R., Henricus, Marsiyana M. and Banerjee, Ipsita A.(2009)'Self-Assembly and Growthof Smart Cell-Adhesive Mucin-Bound Microtubes',Soft Materials,7:1,21 — 36

To link to this Article: DOI: 10.1080/15394450802693969

URL: http://dx.doi.org/10.1080/15394450802693969

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.

Documents

SELF-ASSEMBLY AND GROWTH OF SMART CELL ......uptake of a drug into the circulatory system. Thus, the mucin bound microtubes are very promising vehicles for drug delivery. The scheme