**1. Introduction**

#### **1.1. Nano particles and drug delivery**

Nanostructured materials, due to the 'size effect' [1] and flexible surface modifications, demonstrate unique properties. The surface chemistry and quantum effects of nanomaterials give rise to novel electronic, optical and magnetic properties and thus have become attractive for their applications *in vivo* imaging and diagnostics, regenerative medicine, infection biology, neuroelectronics and biosensors [2, 3]. Functional organic molecules self-assemble into welldefined nanostructures have become a fast developing field due to its similarity with the natural biological processes but also to produce a new range of materials with many applica‐ tion possibilities. Studies have shown that the self-assembling molecular scaffolds like peptides, lipids and other organic scaffolds, on the basis of different non-covalent interactions (π-π interaction, van der Waals, hydrogen bonding, hydrophilic/hydrophobic and electro‐ static) can spontaneously associate to form nanomaterials with various morphologies, like nanotubes, nanospheres, nanofibrils, nanorods, nanotapes, and nanovesicles under different conditions [4, 5, 6]. These nanomaterials made from simple building blocks, can be tuned to present interesting physicochemical properties owing to their biocompatibility, capability of specific molecular recognition, easy availability, and functional flexibility. They offer several advantages as mass delivery agents due to their size, which allows them to cross biological barriers and their chemical versatility makes them suitable for loading a wide range of substances enabling multifunctionality [7].

Several biomaterials have been studied that show a greater promise in nanomedicine including quantum dots [8, 9], carbon nanotubes, coupling of quantum dots and carbon nanotubes [10]

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gold nanoparticles [11], silica nanoparticles [12], organic polymers [13], bi-or multilayer liposomes (14), magnetic and magnetofluorescent nanoparticles [15], silica nanoparticles [16]. These studies have highlighted the importance of the role of nanomaterial size, shape, material composition, surface chemistry; the choice of the cell type for the study; the effect of nanoma‐ terial-cell interactions, the fate of the nanomedicines and the resulting cellular responses [17, 18]. But each material has its own limitation in biological systems. The complexity of interaction between nanomaterials and cellular environment, biocompatibility, their progressive accu‐ mulation in live cells, inefficient bio-degradation and other pharmacokinetic properties including cell toxicity and immunogenicity presents a variety of obstacles for choosing the specific nanomaterials for testing. Considerable efforts have been directed towards surface modifications [19], multivalent attachment of small molecules [20] and coating for minimizing such effects. These measures also favour *in vivo* distribution through diversified biological organs and effective tissue specific targeting. Though use of nano-materials have been successful in *in vitro* cultured cells, its *in vivo* application by repeated injections is more challenging for shelf life, potential immunogenecity, biocompatibility and other physiological hurdles. An alternate approach for tracking the micro device through complex organs is via oral ingestion followed by better absorption and systemic spreading through body fluid instead of repeated intravenous injections.

PABA as the molecular building block with appropriate chemical saturated or unsaturated fatty acid substitution to build a library of PABA based nanomaterials for potential use as drug

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We have synthesized 4-N-pyridin-2-yl-benzamides from p-nitrobenzoic acid [22]. The synthesis involved amide formation with 2-aminopyridine followed by reduction of the nitro functionality utilizing a standard protocol using Pd/C under hydrogen atmosphere as the reducing agent. Subsequently, the free amine functionality present in benzamide was coupled with seven different acid chlorides (undecanoyl chloride, C=11; undec-10-enoyl chloride, C=11;1, Lauroyl chloride, C=12, Miristoyl Chloride, C=14, Palmetoyl chloride, C=16, Stearoyl chloride, C=18 and Oleoyl chloride C=18:1) to furnish the corresponding 4-alkylamido-Npyridin-2-yl benzamides respectively hereafter referred to as C11, C11U, C12, C14, C16, C18 and C18U based on the length of the side chains and unsaturated moieties coupled during synthesis. Synthesis of the Lauroyl chlrode and stearoyl chloride is shown in Figure 1.

The seven alkyl benzamides (1 mg) were added to methanol (2 ml) and heated to 60 ◦C till it dissolved completely. Deionized water (2 ml) was added slowly at the same temperature to obtain a milky white solution which, upon gradually cooling to room temperature, furnished cotton-like white aggregates. Three of the nanoaggregates exhibited intrinsic fluorescence (C11, C6 and C18) and to prepare rhodamine-B embedded Benzamide nanotubes (C11U, C12,C14, C18U) compounds, rhodamine B solution (0.1 ml, 1 mg of rhodamine B in 5.0 ml of deionized water) was added prior to the addition of deionized water (2 ml) which, on cooling,

delivery systems.

**Figure 1.** Synthesis of Lauroyl chlrode and stearoyl chloride

#### **2. Advantages of nanotubes**

Nanotubes, hollow cylindrical nanostructures are promising drug carriers offering many advantages over other drug delivery systems. Nanotubes, which have separated inner and outer surfaces, can be differentially functionalized either to load desired molecules inside or by suitably designing the chemical features of the outer surface allows for site-specific drug delivery. Relevant attachments include biologically active molecules, targeting sequences, intrinsic fluorescent or other imaging devices, biocompatible coatings, and others. Major focus in the development of nanotubes for biomaterial delivery relies on three important factors, chemical modification, biocompatibility and minimal damage of the harbouring environment. To date, the potential use of drug components for synthesizing the microstructure has not been realized primarily because of lack of methods for self-assembly to form a tubular structure and coupling them with tracking fluorescence markers.

#### **3. PABA nanotubes**

The core or the building block is an important component in biomaterial development. paminobenzoic acid (PABA) is frequently found as a structure moiety in drugs (in a database of 12111 commercial drugs, 1.5% (184 drugs) were found to contain the PABA moiety that have a wide range of therapeutic uses [21]. To minimize the problem of biocompatibility and cell toxicity, a more reliable choice is to select building blocks that can be used for developing a library of nanomaterials, which will have functional and structural diversity. We have chosen PABA as the molecular building block with appropriate chemical saturated or unsaturated fatty acid substitution to build a library of PABA based nanomaterials for potential use as drug delivery systems.

We have synthesized 4-N-pyridin-2-yl-benzamides from p-nitrobenzoic acid [22]. The synthesis involved amide formation with 2-aminopyridine followed by reduction of the nitro functionality utilizing a standard protocol using Pd/C under hydrogen atmosphere as the reducing agent. Subsequently, the free amine functionality present in benzamide was coupled with seven different acid chlorides (undecanoyl chloride, C=11; undec-10-enoyl chloride, C=11;1, Lauroyl chloride, C=12, Miristoyl Chloride, C=14, Palmetoyl chloride, C=16, Stearoyl chloride, C=18 and Oleoyl chloride C=18:1) to furnish the corresponding 4-alkylamido-Npyridin-2-yl benzamides respectively hereafter referred to as C11, C11U, C12, C14, C16, C18 and C18U based on the length of the side chains and unsaturated moieties coupled during synthesis. Synthesis of the Lauroyl chlrode and stearoyl chloride is shown in Figure 1.

**Figure 1.** Synthesis of Lauroyl chlrode and stearoyl chloride

gold nanoparticles [11], silica nanoparticles [12], organic polymers [13], bi-or multilayer liposomes (14), magnetic and magnetofluorescent nanoparticles [15], silica nanoparticles [16]. These studies have highlighted the importance of the role of nanomaterial size, shape, material composition, surface chemistry; the choice of the cell type for the study; the effect of nanoma‐ terial-cell interactions, the fate of the nanomedicines and the resulting cellular responses [17, 18]. But each material has its own limitation in biological systems. The complexity of interaction between nanomaterials and cellular environment, biocompatibility, their progressive accu‐ mulation in live cells, inefficient bio-degradation and other pharmacokinetic properties including cell toxicity and immunogenicity presents a variety of obstacles for choosing the specific nanomaterials for testing. Considerable efforts have been directed towards surface modifications [19], multivalent attachment of small molecules [20] and coating for minimizing such effects. These measures also favour *in vivo* distribution through diversified biological organs and effective tissue specific targeting. Though use of nano-materials have been successful in *in vitro* cultured cells, its *in vivo* application by repeated injections is more challenging for shelf life, potential immunogenecity, biocompatibility and other physiological hurdles. An alternate approach for tracking the micro device through complex organs is via oral ingestion followed by better absorption and systemic spreading through body fluid

Nanotubes, hollow cylindrical nanostructures are promising drug carriers offering many advantages over other drug delivery systems. Nanotubes, which have separated inner and outer surfaces, can be differentially functionalized either to load desired molecules inside or by suitably designing the chemical features of the outer surface allows for site-specific drug delivery. Relevant attachments include biologically active molecules, targeting sequences, intrinsic fluorescent or other imaging devices, biocompatible coatings, and others. Major focus in the development of nanotubes for biomaterial delivery relies on three important factors, chemical modification, biocompatibility and minimal damage of the harbouring environment. To date, the potential use of drug components for synthesizing the microstructure has not been realized primarily because of lack of methods for self-assembly to form a tubular structure and

The core or the building block is an important component in biomaterial development. paminobenzoic acid (PABA) is frequently found as a structure moiety in drugs (in a database of 12111 commercial drugs, 1.5% (184 drugs) were found to contain the PABA moiety that have a wide range of therapeutic uses [21]. To minimize the problem of biocompatibility and cell toxicity, a more reliable choice is to select building blocks that can be used for developing a library of nanomaterials, which will have functional and structural diversity. We have chosen

instead of repeated intravenous injections.

coupling them with tracking fluorescence markers.

**2. Advantages of nanotubes**

448 Application of Nanotechnology in Drug Delivery

**3. PABA nanotubes**

The seven alkyl benzamides (1 mg) were added to methanol (2 ml) and heated to 60 ◦C till it dissolved completely. Deionized water (2 ml) was added slowly at the same temperature to obtain a milky white solution which, upon gradually cooling to room temperature, furnished cotton-like white aggregates. Three of the nanoaggregates exhibited intrinsic fluorescence (C11, C6 and C18) and to prepare rhodamine-B embedded Benzamide nanotubes (C11U, C12,C14, C18U) compounds, rhodamine B solution (0.1 ml, 1 mg of rhodamine B in 5.0 ml of deionized water) was added prior to the addition of deionized water (2 ml) which, on cooling, produced pink-coloured aggregates. The aggregates were isolated under centrifuged condi‐ tions (4500 rpm for 20 min) followed by overnight drying at 60 ◦C to afford 0.5 mg of the final nanomaterials. All the PABA based nanomaterials C11, C11U, C12, C14. C16, c18 and C18U studied obtained by side chain variation are shown in Figure 2.

nanomaterials revealed that modifications and subsequent formation of PABA based bioma‐

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Scanning electron microscopy (SEM), transmission electronic microscopy (TEM) and atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to investigate the structural and morphological properties of the self-assembled nanomaterials. The TEM and SEM characterization suggest that the nanomaterials self-assembled using saturated acid side chains mainly form tubular structures with a hollow space inside, while those self-assembled from unsaturated side chains produced cube shaped particles (Figure 3). The mechanism of nanotubes formed by acid chlorides with saturated side chains is believed to that initially, they aggregate into sheets which later transform to curved structures and join to form nanotubes. This nanotube formation mechanism was also confirmed from a low-angle powder X-ray diffraction studies carried out on the of self-assembly of para-terphenylen-1,4′′-ylenebis (dodecanamide) and the results have shown that self-assembly results in layered sheets or rolled up nanotubes dependent on the experimental conditions [23]. Recently a frustrated aggregate internal rearrangement (FAIR) mechanism was also proposed for organic nanotube formation [24]. The authors suggested from density functional calculations that self-assembly takes place by forming sheet like structures driven by nonspecific and nondirectional inter‐ molecular interactions with weak intermolecular H-bonds providing additional stability to the structure. Instead of the fully formed H-bonded structure, the partially formed hydrogen bonded layers to avoid kinetic energy traps transform to curved structures. Mechanism by which the self-assembly of C11U and C18U results in the formation of cubic structures is also not known. It can be surmised however that like the tubular structure formation, the cubic structures also form by unidirectional growth of the sheets by the chemical subunits, followed by the folding of sheets into cubes by penalties that are compensated by favoured binding

To ascertain the size, a Dynamic Light-Scattering (DLS) study was carried out using different nanoparticles produced by side chain variation. In all cases, freshly prepared nanomaterials were mostly uniform in size with very few submicron sized aggregates, while materials examined after prolonged storage (after 3 days) contains more micron sized aggregates. DLS studies from fresh preparations estimated an average size in the range of 100 to 200 nm but prolonged storage leads to the formation of submicron-sized structures. The average height

Laser confocal microscopic images showed that three nanostructures, C-11, C-16 and C-18 emitted intrinsic green fluorescence, while remaining four nanomaterials (C-11U, C-12, C-14, C-18U) do not emit any intrinsic fluorescence (Figure 3). To verify the fluorescence enhance‐ ment, induced by self-assembly nanostructure, the fluorescence emission of the monomer and the self-assembled nanoparticles were compared using Nanodrop 3300 fluoro-spectrometer. The fluorescence intensity of the nanostructures (determined by a methanol/water solution)

of each nanoparticle as measured by 3 D reconstituted AFM images is 3-5 nm.

**4. Characterisation of nanoparticles**

terials did not result in change of properties.

energies [25].

**Figure 2.** Design and chemical synthesis of nanomaterials. (A) chemical structure of acid side chains, final self assem‐ bled product reaction condition, percentage of yield, fluorescent dyes summarized in a table. (B) Schematic diagram showing formation of two nanoparticles (C12 and C18) was drawn (C) Routine diagram and compatible SEM images showing rollover mechanism of two nanomaterial (C-14 and C16) formation.

Prior to the study of the cellular uptake of the seven nanomaterials which use the PABA template, a study was also undertaken to observe whether the biological properties of PABA changed due to the side chain substitution and self-assembly. The biological properties of PABA in self-assembled conjugates as monitored by the growth and viability of the wild type bacterial strains (E. coli K12) in cultured media in the presence of PABA or PABA containing nanostructures. A similar level of bacterial growth in culture media containing PABA or PABA nanomaterials revealed that modifications and subsequent formation of PABA based bioma‐ terials did not result in change of properties.

Scanning electron microscopy (SEM), transmission electronic microscopy (TEM) and atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to investigate the structural and morphological properties of the self-assembled nanomaterials. The TEM and SEM characterization suggest that the nanomaterials self-assembled using saturated acid side chains mainly form tubular structures with a hollow space inside, while those self-assembled from unsaturated side chains produced cube shaped particles (Figure 3). The mechanism of nanotubes formed by acid chlorides with saturated side chains is believed to that initially, they aggregate into sheets which later transform to curved structures and join to form nanotubes. This nanotube formation mechanism was also confirmed from a low-angle powder X-ray diffraction studies carried out on the of self-assembly of para-terphenylen-1,4′′-ylenebis (dodecanamide) and the results have shown that self-assembly results in layered sheets or rolled up nanotubes dependent on the experimental conditions [23]. Recently a frustrated aggregate internal rearrangement (FAIR) mechanism was also proposed for organic nanotube formation [24]. The authors suggested from density functional calculations that self-assembly takes place by forming sheet like structures driven by nonspecific and nondirectional inter‐ molecular interactions with weak intermolecular H-bonds providing additional stability to the structure. Instead of the fully formed H-bonded structure, the partially formed hydrogen bonded layers to avoid kinetic energy traps transform to curved structures. Mechanism by which the self-assembly of C11U and C18U results in the formation of cubic structures is also not known. It can be surmised however that like the tubular structure formation, the cubic structures also form by unidirectional growth of the sheets by the chemical subunits, followed by the folding of sheets into cubes by penalties that are compensated by favoured binding energies [25].

To ascertain the size, a Dynamic Light-Scattering (DLS) study was carried out using different nanoparticles produced by side chain variation. In all cases, freshly prepared nanomaterials were mostly uniform in size with very few submicron sized aggregates, while materials examined after prolonged storage (after 3 days) contains more micron sized aggregates. DLS studies from fresh preparations estimated an average size in the range of 100 to 200 nm but prolonged storage leads to the formation of submicron-sized structures. The average height of each nanoparticle as measured by 3 D reconstituted AFM images is 3-5 nm.
