Design and development of soft nanomaterials from biobased amphiphiles

Abstract

Design and development of different forms of soft matter from renewable (biomass) feedstocks is gaining attention in current research. This highlight summarizes our continuing efforts towards the effective utilization of renewable resources for new chemicals, fuels and soft materials, and selected successful stories in that direction. Cashew nut shell liquid, an industrial by-product, was used as a raw material to synthesize aryl glycolipids which upon self-assembly generated an array of soft materials such as lipid nanotubes, twisted/helical nanofibers, low-molecular-weight hydro/organogels and liquid crystals. These soft architectures were fully characterized by using different techniques. In another example, amygdalin, a by-product of the apricot industry, was used to develop novel amphiphiles, which showed unprecedented gelation properties in a wide range of solvents. To take these soft nanomaterials to a second level, we successfully demonstrated the utility of these hydrogels as drug delivery vehicles. Intriguingly, enzyme catalysis was used as a tool to make and break the hydrogels, which apparently triggered controlled drug delivery. We believe these results and this highlight will motivate us and others in the field of biobased materials research, green chemistry and soft material development through self-assembly processes, to design and develop new functional materials from plant/crop-based renewable resources, otherwise underutilized.

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George John

Professor George John received his PhD in Chemistry from University of Kerala, India in 1993. After a year of post-doctoral stay at the University of Twente, The Netherlands, he spent four years as a research scientist at the Agency for Advanced Industrial Science and Technology (AIST), Japan. In the fall of 2002 he joined the Rensselaer Nanotechnology Center as a research faculty and pursued his research interests in the area of soft nanomaterials. He joined the City College of the City University of New York as an Associate Professor of Chemistry in September 2004. His current research interests include biobased organic synthesis, self-assembled soft materials (vesicles, liquid crystals, helices, tubes and gel systems) for functional applications, understanding growth mechanisms of nanostructures and designing new structures and multifunctional nanocomposites.

Praveen Kumar Vemula

Dr. Praveen Kumar Vemula received his MSc degree in Organic Chemistry from Osmania University, Hyderabad and joined the Department of Organic Chemistry, Indian Institute of Science, Bangalore, India to pursue research towards his PhD degree under the guidance of Prof. Santanu Bhattacharya. He carried out his research in the area of developing novel catalysts for decontamination reactions in various supramolecular nanoaggregates, through detailed experimental and computational studies. He received his PhD degree in 2005; currently he is a postdoctoral fellow in Prof. George John’s laboratory at the City College of the City University of New York, New York. His current research interests includes design and development of novel self-assembled soft nanomaterials from renewable resources, molecular gels, liquid crystals, metal nanoparticles and organic–inorganic hybrid materials.

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1. Introduction

Soft matter exists in different forms; for example, self-assembled organic nanotubes, nanofibers, gels, liquid crystals and polymers.^1,2 In future research, developing soft materials from renewable feedstock would be an intriguing yet challenging task, which will have a direct impact on industrial development and the local economy. Recent consensus in the scientific community is that in the long run, renewable resources are the only workable and viable solution, and their catalytic processing will make it possible to replace oil and coal as basic feedstocks.³ ‘Technology Vision 2020: The US Chemical Industry’ envisages 25% of the production of organic chemical products from renewable feedstocks by 2020.⁴ More importantly, natural products are stereo- and regiochemically pure, thereby reducing dependence on expensive chiral catalysts and complex synthesis that are currently required for selective functionalization in chemical industry. A recent review by Metzger et al. addressed various synthetic protocols for value added products using oils and fats as renewable feedstock for the chemical industry.⁵ Furthermore, the authors stressed the importance of optimizing separation operations from renewable resources in order to reduce energy consumption.⁶ Biomass is rich in unsaturated fatty acids and thus it was used extensively for developing value added chemicals,⁵ and identified as the only practical source of renewable liquid fuel.⁷ Efforts have been put to develop various chemicals such as phenols and hydrocarbons from sugar platforms.^5,8 Plant/crop-based feedstocks are already having an impact on practical applications, including plastics, solvents, fragrances and lubricants. “Bioplastics” such as polylactic acid are drawing attention due to their biocompatibility and trivial hydrolytic degradation.^9–11 The above examples illustrate the urgency of developing value added materials from biomass. This highlight summarizes our recent efforts towards the preparation of soft nanomaterials from renewable plant-derived resources as an alternate feedstock for organic synthesis, utilizing the specificity of enzyme catalysis, green and supramolecular chemistry principles.

2. Soft nanomaterials from cashew nut shell liquid (CNSL): helical/twisted fibers, lipid nanotubes, gels and liquid crystals

The raw material was obtained from the cashew nut, a common household item. The tough husk that envelops the cashew nut is rich in natural long chain phenols; cardanol and cardol, which together are technically known as cashew nut shell liquid (CNSL). CNSL, a by-product of the cashew industry obtained from Anacardium occidentale L, is familiar for its industrial use,¹² and its derivatives are well known for various applications.^13,14 Cardanol, the distilled product of CNSL, consists of four meta-alkyl phenols, with alkyl chains differing in the degree of unsaturation. In order to develop glycolipids, we simply attached a sugar group on the phenol as a hydrophilic headgroup (Chart 1). We studied the self-assembly properties of glycolipids GL1 and GL2 in water (Chart 1).

Chart 1 Chemical structure of cardanol and diaminopyridine based amphiphiles and cartoon for the general structure of an amphiphile.

5 mg of powdered glycolipid samples were dispersed in Milli-Q water (100 mL) and gradually heated to boiling and kept for 30 min before thorough mixing and dispersing. The transparent aqueous dispersion was gradually cooled to room temperature and kept without shaking. While cooling to room temperature, these amphiphiles formed a self-assembled white fluffy cotton like material (Fig. 1a) within 12 hours. The fine fibrous structures of the self-assembly were confirmed by various tools including optical light microscopy, and scanning and transmission electron microscopy (SEM and TEM). Unstained samples were examined under energy-filtering TEM (EF-TEM) and found that the thinnest width of the fibers derived from GL1 was about 30–35 nm. Intriguingly, all fibers adopted helical morphologies with a width of ∼50 nm. Upon ageing coiled nanofibers gradually turned into structures that are hundreds of micrometers long and have internal diameters of 10–15 nm. The tubular structures are open-ended with a uniform shape, with an aspect ratio of ∼1000 (Fig. 1b,c).¹⁵ At this stage there are two important issues to understand and explore; the first is the role of the unsaturated tail in the self-assembly process and the second is to delineate the formation of nanotubes which contain inner hollowness from typical twisted ribbons. We believe that understanding one aspect of the above will give us the information about the second. In order to achieve that, detailed experiments were carried out using individual components, GL1-b and GL2. Self-assembly was performed by using mixtures of glycolipds with different ratios of GL1-b and GL2. Our results suggest that saturated glycolipid (GL2) produced stable twisted fibers which remained the same without turning into tubular structures.¹⁶

Fig. 1 Self-assembled organic nanotubes formed from cardanyl glucoside GL1, a) real picture and TEM images of b) lipid nanotube, c) twisted fiber, helical nanofiber and nanotube. d) Schematic representation of tube formation from helical fibers. e) Possible 3-point hydrogen bonding between DAP-amphiphile (GL3) and thymidine (hydrogen bond showed in dotted line).

In contrast, upon self-assembly in water, the pure monoene GL1-b resulted in nanotube formation including partly coiled nanofibres. The coiled fibres might be the precursor for nanotube formation.¹⁶ Self-assembly of mixed glycolipids generated a library of compositions and was analysed by EF-TEM and field emission SEM (FE-SEM). Typically the 90 : 10 (saturated–monoene) mixture self-assembled to form twisted ribbons in water; this might be expected for the 10% doping of the monoene component. The 80 : 20 compositions also showed twisted-ribbon morphology on FE-SEM analysis. No significant effect on the twisted morphology was observed by doping of the monoene component by up to 30–40%. On the other hand, an equimolar (50 : 50) composition gave loosely coiled ribbons morphology, in between the twisted and tight helical coil.

While increasing the monoene content in these libraries, the helical pitch decreased to give tubular morphologies with helical markings in comparison with the nanotubes obtained from pure monoene and from cardanyl glucoside (GL1), which had smooth surfaces.¹⁶ TEM images of twisted fibers, helical fibers and nanotubes are shown in Fig. 1c. A schematic illustration of the various helical morphologies’ formation is depicted in Fig. 1d for comparison and clarity. This result seems to signify that one could regulate the morphology of high-axial-ratio nanostructures (HARNs) by appropriate mixing and self-assembly of existing simple amphiphiles. Schematic representations for self-assembly of cardanol based saturated and unsaturated amphiphiles are shown in Fig. 2.

Fig. 2 Schematic representation of the proposed model for self-assembly of cardanol–DAP based amphiphiles with a) saturated and b) unsaturated alkyl chains (the alkyl chains does not represent the exact number of carbons, it is only representative).

Indeed, these results prompted us to design the analogous synthetic amphiphile GL3, using bench reagents equipped with complementary groups for further interaction and binding to generate functional soft materials by hierarchical assembly. On self-assembly of GL3 in water, as expected, helical ribbon morphologies were produced; upon aging for an additional 12 h these helical morphologies converted to lipid nanotubes with an outer diameter of 60–80 nm and an inner diameter of ca. 20 nm. Diaminopyridine (DAP) is known to form 3-point hydrogen bonding, which makes this functional group handy in making artificial receptors. In the present case, organic nanotubes which formed by self-assembly of GL3 are fluorescent in nature, hence one can imagine that suitable guest molecules with complementary hydrogen bonding capability to the DAP group would directly alter the intrinsic fluorescence properties of nanotubes.¹⁷ Indeed, addition of up to 10 mM of thymidine caused the nearly immediate quenching of fluorescence, which might be due to the formation of multiple hydrogen bonding as shown in Fig. 1e. In addition, thymidine analogues uracil and other anticancer compounds 5-fluorouracil and its prodrug derivative Tegafur also quenched fluorescence; importantly fluorescence quenching was selective for nucleosides over urea and β-D-glucose which can also form extensive hydrogen bonding. Hence, by using this design strategy it is possible to develop novel soft nanomaterials, which could be used in molecular recognition and other functional applications.¹⁷

We also developed other forms of soft materials such as low-molecular-weight gels and liquid crystals from glycolipids GL1 and GL2.^18,19 The effect of unsaturation on the gelation ability was thoroughly studied by conducting experiments with glycolipids substituted with different unsaturated chains in pure form as well as mixed form. Gels derived from GL2 in a water–ethanol system showed a characteristic fibrous network under SEM (Fig. 3a). Amphiphiles fully derived from renewable resources have shown excellent gelation behavior in water–alcohol mixtures as well as in pure organic solvents strongly dependent on the unsaturation of the substituted pentadecyl hydrocarbon chain. The liquid crystalline properties of glycolipids GL1 and GL2 were also studied by optical polarizing microscopy, differential scanning calorimetry and X-ray diffraction.¹⁹ All the phases were identified as lamellar in structure (Fig. 3b). Introduction of double bonds in the liphophilic part significantly decreased the phase transition temperatures, although these glycolipids exhibited common patterns in their phase transitions. In a nutshell, we could generate various forms of soft matter such as organic nanotubes, molecular gels and liquid crystals from renewable plant/crop-based resources, as an alternate feedstock for future materials development.

Fig. 3 a) SEM image of a water–ethanol gel from GL2. b) Optical micrograph of lamellar phases formed from GL2.

3. Soft materials from amygdalin: biocatalysis as a tool to make and break hydrogels

Continuing our quest towards developing value added soft materials from renewable resources, we focused our attention on plant/crop-based resources. However, challenges still exist equally in the development of environmentally friendly and sustainable production processes, coercing nature to work on the industrial scale. With a few exceptions, most applications have been for low volume, high value products, such as drugs and fine chemicals. Nevertheless, enzyme mediated feedstock conversion would be a suitable solution for the production of bulk value added materials with less production cost. In this direction, biocatalysis²⁰ would become a handy tool for generating soft materials from renewable raw materials in an environmentally benign approach.

Amygdalin is a by-product of the fruit industry, and a naturally occurring glycoside found in many food plants such as the kernels of apricots, almonds and apples.²¹ Amygdalin has been used as a main constituent in commercial preparations of laetrile,^22,23 a purported therapeutic agent. In particular, our aim is to synthesize amygdalin derivatives which can form nanoaggregates through self-assembly, encapsulation of hydrophobic drugs, followed by the release of the encapsulated drug upon enzyme mediated degradation (an enzyme triggered drug delivery model). Amygdalin has a phenyl ring and multiple hydroxyl groups which might enhance the intermolecular associations through π–π stacking and hydrogen bonding, respectively. In addition to this, the presence of hydrocarbon chains might enhance self-assembly through van der Waals interactions. Based on that assumption we designed amphiphiles AMG1–AMG3 (Chart 2).

Chart 2 Chemical structure of amygdalin based amphiphiles.

Herein, we demonstrate the utility of enzyme catalysis as a tool to generate amphiphiles from renewable resources. Biocatalysis offers supreme control on regioselective synthesis, by using this we synthesized various amygdalin derivatives where selectively an acyl moiety was introduced on the primary hydroxyl group in excellent yields. Amygdalin is a disaccharide containing one primary hydroxyl group, which formed ester bonds with fatty acids (vinyl esters were used as acyl donors). In general, multi-step synthesis, laborious separation procedures and lower yields often keep low-molecular-weight gelators away from commercial use due to high production costs.²⁴ Intriguingly, the hydrogelators we developed were synthesized from renewable resources in a single-step process in quantitative yields, and unpurified crude products showed unprecedented gelation abilities similar to the purified products. In particular, this property may give the opportunity to develop these gelators in industrial scales for various applications, which may have deep impact when it comes to commercialization.

AMG1–AMG3 showed unprecedented gelation properties in a broad range of solvents such as polar water to non-polar cyclohexane at extremely low concentrations (0.05–0.2 wt% (MGC)) while displaying excellent thermal and temporal stabilities.²⁵ All these gels were thermoreversible in nature. SEM images confirmed that hydrogels and organogels formed by these amphiphiles containined helical fibers and grass-like morphologies at the nanoscale level (Fig. 4a,b). The mode of self-assembly of these amphiphiles was delineated with the help of single crystal analysis and X-ray diffraction data. Single crystal structures provided preliminary information about the existing hydrogen bonding network in these amphiphiles (Fig. 4f).

Fig. 4 SEM images of a) morphology of a hydrogel formed from AMG3 and b) morphology of an organogel formed from AMG1. Real images of c) drug (curcumin) encapsulated hydrogel, d) gel with enzyme solution immediately after addition and e) after 12 h. Crystal structure of AMG1 in water f), the extended hydrogen bonding network can be viewed.

4. Enzyme triggered drug delivery

Dissolving hydrophobic drugs in aqueous solutions and developing suitable drug delivery systems is a challenging task in drug discovery.²⁶ Recently we demonstrated a conceptually novel approach where we encapsulated a hydrophobic drug molecule in a hydrogel, and subsequently released the drug by breaking the gel by using a hydrolase enzyme (Lipolase 100 L, Type EX).

Importantly, no chemical modification has been done on the drug; solubilization occurred exclusively due to the availability of hydrophobic pockets within the hydrogel nanoaggregates. Preformed hydrogel was degraded completely by the lipolase while releasing the encapsulated chemopreventive hydrophobic drug curcumin.^27,28 Curcumin has an extremely low water solubility,²⁹ by using amygdalin based hydrogels we could solubilise ∼33 000 times the amounts of curcumin, hence the resulting gel was yellow in colour (Fig. 4c) and due to the hydrophobic nature, curcumin might be located at hydrophobic pockets of the gel. Then the hydrolase enzyme, lipase, was added to the preformed gel and it was kept at 37 °C, which was lower than the gel melting temperature. Initially the added solution was colourless (Fig. 4d) and after 12 h visual changes had occurred (Fig. 4e), i.e., the complete gel had been degraded and the top solution had became yellow in color which indicated that upon enzyme mediated gel degradation, the encapsulated curcumin had been released into the solution. By performing similar experiments in absence of enzyme it was proved that the enzyme indeed plays a vital role in drug degradation. Drug release was monitored by absorbance spectra of the drug. Control on the drug release rate was achieved by manipulating the enzyme concentration and/or the temperature. The by-products formed after gel degradation were characterized and thus the cleavage site of the gelator by the enzyme was determined. Gel degradation occurred due to cleavage of the ester bond in the gelator by the hydrolase enzyme.

To obtain control on the rate of release, we investigated the role of enzyme concentration and temperature on gel degradation or controlled drug release. In the first set of experiments we changed the temperature while keeping the enzyme concentration constant. We observed rate enhancements in drug release while increasing the enzyme concentration at 37 °C. Similarly, enzyme concentration was kept constant while changing the temperature from 37 °C to 45 °C, and drug release rates were enhanced while increasing the temperature. Hence we could control the drug release by altering the temperature while keeping enzyme concentration constant and/or by changing the enzyme concentration at constant temperature.

5. Conclusion and outlook

Successfully we showed several examples of soft nanomaterials derived from plant/crop-based renewable feedstocks. Initially, lipid amphiphiles were generated from cardanol, a renewable resource and by-product of the cashew industry. Upon utilizing basic self-assembly properties of glycolipids, a wide range of soft materials such as lipid nanotubes, helical/twisted nanofibers, low-molecular-weight hydro/organogels and liquid crystals were generated; such materials would find applications in various fields such as biomaterials, templated synthesis and biosensors. We also developed a different set of amphiphiles from amygdalin, a by-product of the fruit industry. Biocatalysis was used to generate such amphiphiles in quantitative yields. Those amygdalin based amphiphiles showed unprecedented gelation behaviour in a wide range of solvents. Importantly, an enzyme triggered drug delivery model was demonstrated by using these supramolecularly organized hydrogels. In this study a well-known chemopreventive drug, curcumin, was used, and control on drug delivery was achieved by altering the enzyme concentration and/or temperature. Hence, enzyme catalysis was used as a tool to make and break the hydrogels; this may simplify the design of advanced drug delivery vehicles. Overall, various forms of soft nanomaterials were generated from different renewable resources, which would find applications in biosensors research, liquid crystal displays and in medicine. We believe that these prompting results and this highlight will encourage us and others in the field to explore the emerging science of soft materials from alternate sources.

Acknowledgements

GJ acknowledges the encouragement and support from Dr C. K. S. Pillai, Prof. T. Shimizu and Prof. J. S. Dordick.

References

1. P.-G. de Gennes, Soft Matter, 2005, 1, 16.
2. E. Carretti, L. Dei and R. G. Weiss, Soft Matter, 2005, 1, 17.
3. (a) W. A. Herrmann, in Chemie: Eine reife Industrie oder weiterhin Innovationsmotor? ed. U.-H. Felcht, Verlag der Universitatsbuchhandlung Blazek und Bergmann seit 1891, Frankfurt, 2000, pp. 97–122; (b) R. J. Koopmans, Soft Matter, 2007, 2, 537.
4. Biobased Industrial Products; Priorities for Research and Commercialisation, National Research Council, Washington, DC, 2000; see also: A. Thayer, Chem. Eng. News, 2000, 78, 40.
5. U. Biermann, W. Friedt, S. Lang, W. Luhs, G. Machmuller, J. O. Metzger, M. Rusch gen. Klaas, H. J. Schafer and M. P. Schneider, Angew. Chem., Int. Ed., 2000, 39, 2206 , and references therein.
6. M. Eissen, J. O. Metzger, E. Schmidt and U. Schneidewind, Angew. Chem., Int. Ed., 2002, 41, 414.
7. T. E. Bull, Science, 1995, 285, 1209.
8. J. O. Metzger, Angew. Chem., Int. Ed., 2006, 45, 696.
9. A. J. Ragauskas, C. K. Williams, B. H. Davison, G. Britovsek, J. Cairney, C. A. Eckert, W. J. Frederick, Jr., J. P. Hallett, D. J. Leak, C. L. Liotta, J. R. Mielenz, R. Murphy, R. Templer and T. Tschaplinski, Science, 2006, 311, 484 , and references therein.
10. R. Auras, B. Harte and S. Selke, Macromol. Biosci., 2004, 4, 835.
11. G. Partick and M. O'Brien, Biopolymers, 2002, 4, 235.
12. J. H. P. Tyman, Chem. Soc. Rev., 1979, 8, 499.
13. G. John and C. K. S. Pillai, Macromol. Rapid Commun., 1992, 13, 255.
14. G. John and C. K. S. Pillai, J. Polym. Sci., Part A: Polym. Chem., 1993, 31, 1069.
15. G. John, M. Masuda, Y. Okada, K. Yase and T. Shimizu, Adv. Mater., 2001, 13, 715.
16. G. John, J. H. Jung, H. Minamikawa, K. Yoshida and T. Shimizu, Chem.–Eur. J., 2002, 8, 5494.
17. G. John, M. Mason, P. M. Ajayan and J. S. Dordick, J. Am. Chem. Soc., 2004, 126, 15012.
18. G. John, J. H. Jung, M. Masuda and T. Shimizu, Langmuir, 2004, 20, 2060.
19. G. John, H. Minamikawa, M. Masuda and T. Shimizu, Liq. Cryst., 2003, 30, 747.
20. G. John, G. Zhu, J. Li and J. S. Dordick, Angew. Chem., Int. Ed., 2006, 45, 4772.
21. D. A. Jones, Phytochemistry, 1998, 47, 155.
22. J. W. Turczan and T. Medwick, Anal. Lett., 1977, 10, 581.
23. K. N. Syrigos, G. Rowlinson-Busza and A. A. Epenetos, Int. J. Cancer, 1998, 78, 712.
24. S. Zhang, Nat. Mater., 2004, 3, 7.
25. P. K. Vemula, J. Li and G. John, J. Am. Chem. Soc., 2006, 128, 8932.
26. T. Miyata, T. Uragami and K. Nakamae, Adv. Drug Delivery Rev., 2002, 54, 79.
27. A. Duvoix, B. Romain, D. Sylvie, S. Michael, M. Franck, H. Estelle, D. Mario and D. Marc, Cancer Lett., 2005, 223, 181.
28. M. Hergenhahn, S. Ubaldo, W. Annette, P. Axel, H. Chih-Hung, C. Ann-Lii and R. Frank, Mol. Carcinog., 2002, 33, 137.
29. H. H. Tønnesen, M. Másson and T. Loftsson, Int. J. Pharm., 2002, 244, 127.

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