Self-assembled peptide microspheres for sustainable release of sulfamethoxazole

Abstract

The self-assembly of two peptides Boc-Phe-Aib-Gabu-OMe 1 and Boc-Tyr-Aib-Gabu-OMe 2 (Aib = α-aminoisobutyric acid, Gabu = γ-aminobutyric acid) containing a core of a conformationally rigid achiral amino acid and a conformationally flexible achiral amino acid at the C-terminus was studied to fabricate a sustainable delivery vehicle. From X-ray crystallography, peptide 1 with the Phe residue adopts a type II turn-like structure whereas the Tyr analogue 2 adopts a type II′ turn-like structure. However, peptide 1 self-associates to form four membered ring-like porous structures through multiple intermolecular hydrogen bonds but peptide 2 self-associates to form five membered ring-like porous structures by intermolecular hydrogen bonds. Field emission scanning electron microscopy (FE-SEM) revealed that both the peptides exhibit microsphere morphologies. Further, these microspheres were loaded with the bacteriostatic antibiotic sulfamethoxazole. The growth inhibition of E. coli is greater for the peptide 1-sulfamethoxazole formulation than for the peptide 2-sulfamethoxazole formulation which indicates that the formulation leads to sustained release of the encapsulated drug. The peptide 2 microspheres slowly release the encapsulated sulfamethoxazole, more so than the peptide 1 microspheres.

---

Introduction

Therapeutic agents often cause clinical issues like unwanted side effects and systemic toxicity which are mainly attributed to their pharmacokinetics and pharmacodynamics.¹ Hence, suitable carrier materials are needed to overcome the undesirable functions of drugs.² The primary goal of a drug delivery vehicle is to deliver the necessary amount of drug to the targeted site with sustained release of the drug for a necessary period of time under physiological conditions.³ In recent years, various materials including organic-based nanomaterials such as dendrimer nanoparticles,⁴ polymers and polymer-based micelles,⁵ carbon nanotubes,⁶ iron oxide nanoparticles,⁷ metal nanoparticles,⁸ silica,⁹ and metal–organic frameworks¹⁰ have been widely used for the fabrication of efficient drug delivery vehicles. But most of these nanomaterials are known to cause undesired effects due to their intrinsic toxicity¹¹ or immunogenicity¹² caused by surface modification. In this regard self-assembled peptide-based microspheres and vesicles are interesting and highly promising as delivery vehicles.¹³ Moreover, many anti-tumour and antidepressant drugs, and statins are highly hydrophobic and therefore they need a formulation for solubilisation and targeted delivery.¹⁴ So peptide-based delivery vehicles will be useful due to their hydrophobic–hydrophilic balance, folded structure,¹⁵ recognition properties,¹⁶ biocompatibility¹⁷ and biodegradability.¹⁸ Peptides containing non coded amino acids in particular are very important because of their immunity towards enzymatic degradation.¹⁹

We are developing self-assembled peptide-based delivery vehicles.²⁰ Recently we have reported the hydrogen peroxide responsive release of an anti cancer drug from peptidomimetic microspheres.²¹ Herein we report the formation of microspheres via the self-assembly of tripeptides containing an aromatic L-amino acid at the N-terminus, a core of a conformationally rigid achiral amino acid and a conformationally flexible achiral amino acid at the C-terminus. We have synthesized Boc-Phe-Aib-Gabu-OMe 1 and Boc-Tyr-Aib-Gabu-OMe 2. Field emission scanning electron microscopy (FE-SEM) of the peptides reveals their microsphere morphologies. Single crystal X-ray diffraction reveals that peptide 1 with the Phe residue adopts a type II turn-like structure and self-associates to form four membered ring-like porous structures through multiple intermolecular hydrogen bonds. But the Tyr analogue 2 adopts a type II′ turn-like structure and self-associates to form five membered ring-like porous structures by intermolecular hydrogen bonds. We have exploited these porous microspheres to load the bacteriostatic antibiotic sulfamethoxazole. The formulation leads to sustained release of the encapsulated drug. The drug release profile shows that the peptide 2 microspheres are slowly releasing the encapsulated drug, more so than the peptide 1 microspheres. Hence, the growth inhibition of E. coli is greater for the peptide 1-sulfamethoxazole formulation than for the peptide 2-sulfamethoxazole formulation.

Results and discussion

For tripeptides 1 and 2 the design principle explored the effect of the conformationally rigid Aib²² residue at the central position and flexible achiral γ-aminobutyric acid²³ at the C-terminus, and the folding propensities were studied (Scheme 1). The Tyr OH in peptide 2 will incorporate additional hydrogen bonds.²⁴ The peptides contain natural coded amino acids like phenylalanine or tyrosine, and α-aminoisobutyric acid, available in nature from alamethicin and γ-aminobutyric acid, is the chief inhibitory neurotransmitter in the mammalian central nervous system. The reported tripeptides Boc-Phe-Aib-Gabu-OMe 1 and Boc-Tyr-Aib-Gabu-OMe 2 were synthesized by conventional solution-phase methodology and purified and characterized by ¹H-NMR, ¹³C-NMR, FT-IR and mass spectrometry (MS) analysis.

Scheme 1 The schematic presentation of peptides 1 and 2.

To investigate the conformational preference of peptides 1 and 2, solid state FT-IR spectroscopy was performed. The frequency range 3500–3200 cm⁻¹ corresponds to the N–H stretching vibrations and 1800–1500 cm⁻¹ is important for the stretching band of amide I and the bending peak of amide II. From the FT-IR spectra, the bands at 3313 cm⁻¹ for peptide 1 and 3322 cm⁻¹ for peptide 2 indicate that the NH groups are hydrogen bonded (Fig. 1).²⁵ There are no peaks around 3400 cm⁻¹ for non hydrogen bonded NH protons. The amide I and amide II bands appear at 1678 cm⁻¹ and 1542 cm⁻¹ for peptide 1 and at 1678 cm⁻¹ and 1538 cm⁻¹ for peptide 2 indicating turn-like structures for both peptides (Fig. 1).²⁵ This also suggests that the peptides have an extensively hydrogen-bonded network structure.

Fig. 1 The solid state FT-IR spectra of (a) peptide 1 and (b) peptide 2.

The conformation in the solid state and the packing of the reported peptides 1 and 2 at the atomic level were studied by single crystal X-ray diffraction analysis. A colourless orthorhombic crystal of compound 1 was obtained from methanol–water solution by slow evaporation. The X-ray crystallography data shows that the asymmetric unit contains one molecule of peptide 1.²⁶ The ORTEP diagram of peptide 1 shows that the peptide backbone adopts a type II′ turn-like conformation (Fig. 2a). The C-terminal γ-aminobutyric acid adopts gauche–anti conformation.^20b There is an intramolecular N–H⋯O hydrogen bond between Boc C=O and Gabu NH. There is also an N–H⋯N hydrogen bond between Aib NH and Gabu N resulting in a rigid conformation of peptide 1 in the solid state (Fig. 2a). The colourless orthorhombic crystal of compound 2 was obtained from methanol–water solution by slow evaporation. The asymmetric unit contains two molecules of peptide 2.²⁶ The solid state conformation of compound 2 revealed that it adopted a type II turn-like structure through intramolecular N–H⋯O hydrogen bonding interactions between Boc C=O and Gabu NH. The C-terminal γ-aminobutyric acid adopts gauche–anti conformation in molecule A and anti–anti conformation in molecule B. There are also two N–H⋯N hydrogen bonds (N13–H13⋯N10 and N46–H46⋯N43). From Fig. 2b, it is evident that there exists an intermolecular N–H⋯O hydrogen bond between Tyr–OH of molecule A and Aib NH of molecule B. The important backbone torsion angles are listed in Table 1.

Fig. 2 The ORTEP diagrams of (a) peptide 1 and (b) peptide 2. Ellipsoids are drawn at the 50% probability level. The hydrogen bonds are shown as dotted lines.

Table 1 Selected backbone torsion angles (deg) for peptides 1 and 2

	ϕ1/°	ψ1/°	ϕ2/°	ψ2/°	ϕ3/°	ψ3/°
Peptide 1	48.65	−131.54	−69.80	−13.36	131.05	179.05
Peptide 2, A	−61.56	121.65	60.61	18.48	76.56	173.26
Peptide 2, B	−58.39	128.01	65.71	12.53	−106.29	−23.08

---

In higher order packing, the individual subunits of peptide 1 are themselves regularly interlinked through multiple intermolecular hydrogen-bonding interactions (N1–H10⋯O4 and N2–H19⋯O3) and thereby form a supramolecular four membered ring-like porous structure along the crystallographic a direction (Fig. 3a) where the Phe rings are separated by hydrophobic alkyl chains and there are no π–π interactions.²⁷ The average internal diameter of the pore is 5.9 Å. Moreover, the individual turn-like molecules A and B of compound 2 are themselves regularly inter-linked through cooperative multiple intermolecular hydrogen bonding interactions (N55–H55⋯O65, N43–H43⋯O33 and O66–H66–O21), thereby forming a supramolecular five membered ring like porous structure along the crystallographic c direction (Fig. 3b). Here also the Tyr rings are separated by hydrophobic alkyl chains and there are no π–π interactions. The internal diameter of the pore is ca. 6.3 Å. The hydrogen bonding parameters for peptides 1 and 2 are listed in Table 2.

Fig. 3 Ball and stick models showing (a) the intermolecular hydrogen bond mediated four membered porous packing of peptide 1 and (b) the intermolecular hydrogen bond mediated packing of peptide 2.

Table 2 Hydrogen bonding parameters of tripeptides 1 and 2^a

	D–H⋯A	D⋯H (Å)	H⋯A (Å)	D⋯A (Å)	D–H⋯A (°)
a Symmetry equivalent directions a = −1 + x, y, z, b = 1 − x, −1/2 + y, 1/2 − z, c = 1 − x, −1/2 + y, −1/2 − z, d = 1 + x, y, z, e = 1/2 + x, −1/2 − y, −1 − z, f = −2 − x, 1/2 + y, −1/2 − z.
1	N1–H10⋯O4	0.86	2.14	2.976(3)	164^a
	N2–H19⋯O3	0.86	2.22	3.051(3)	163^b
	N3–H26⋯O2	0.86	2.22	2.934(3)	140
	N3–H26⋯N2	0.86	2.33	2.762(3)	108
2	N10–H10⋯O66	0.86	2.15	2.990(9)	168^c
	N13–H13⋯O29	0.86	2.20	2.887(9)	152
	N22–H22⋯O20	0.86	2.37	2.943(2)	125^d
	N13–H13⋯N10	0.86	2.36	2.731(2)	106
	O33–H33⋯O54	0.82	1.85	2.666(7)	171^d
	N43–H43⋯O33	0.86	2.15	2.982(8)	163
	N46–H46⋯O62	0.86	2.11	2.934(9)	159
	N46–H46⋯N43	0.86	2.32	2.739(2)	110
	N55–H55⋯O65	0.86	2.09	2.842(2)	146^e
	O66–H66⋯O21	0.82	1.82	2.640(8)	174^f

---

The self-assembly of the tripeptides 1 and 2 was studied by field emission scanning electron microscopy (FE-SEM). The solutions of the reported peptides in methanol–water (0.5 mg mL⁻¹) were drop-casted on a microscopic glass cover slip, dried under vacuum at 30 °C for two days and investigated by FE-SEM. From FE-SEM, peptide 1 in methanol exhibits a polydisperse microsphere morphology (Fig. 4a). The microspheres have a diameter of ca. 0.75 μm. The inset of Fig. 4a shows the holes on the sphere. Fig. 4c shows the polydisperse spherical structure obtained from peptide 2. The inset of Fig. 4c shows the porous surface of the microsphere. The microspheres are very stable and insoluble in water. From previous reports, the peptide based microspheres have advantages as drug delivery vehicles due to their biocompatibility, responsive release and biodegradability.¹⁷ We have tried to encapsulate the bacteriostatic antibiotic sulfamethoxazole in the peptide microspheres.¹⁸ To carry out a systematic inquiry into the morphological changes of the peptide microspheres by drug sulfamethoxazole loading, FE-SEM experiments were performed. Fig. 4b and d show the FE-SEM images of the drug loaded peptides 1 and 2 respectively. From the FE-SEM images it is clear that after sulfamethoxazole loading there is almost no change in the size or morphology of the microspheres. We have also carried out a FE-SEM study of only sulfamethoxazole under the same conditions. Fig. 4e shows the branched fibre like morphology of sulfamethoxazole. The fibres have a diameter of ca. 100 nm and are several micrometers in length. The confocal microscope images show that the sulfamethoxazole appended peptide 1 microspheres exhibit blue fluorescence upon excitation at 360 nm (Fig. 4f).

Fig. 4 (a) FE-SEM image of peptide 1 polydisperse microspheres. Inset shows the holes on the sphere. (b) FE-SEM image of peptide 1 microspheres after sulfamethoxazole loading. (c) Polydisperse microspheres of peptide 2. (d) FE-SEM image of peptide 2 microspheres after drug loading. (e) FE-SEM image showing branched fibre like morphology of sulfamethoxazole. (f) Confocal microscope image of peptide 1 and sulfamethoxazole showing blue fluorescent microspheres upon excitation at 360 nm.

For further investigation of the peptide–drug interaction, UV-vis and fluorescence experiments were performed as they are very sensitive techniques to study the changes in microenvironments. Fig. 5a shows the UV-vis titration spectra of peptide 1 with the gradual addition of sulfamethoxazole. The fluorescence intensity at 305 nm, from the phenyl groups, decreases with increasing sulfamethoxazole concentration and is also red shifted. A new emission peak appears at 340 nm which indicates an interaction between the drug molecules and peptide 1 (Fig. 5b). The spectroscopic study of sulfamethoxazole and peptide 2 show similar results (ESI Fig. S1†).

Fig. 5 (a) UV-vis spectra and (b) fluorescence spectra of peptide 1 with increasing concentration of sulfamethoxazole. The excitation wavelength is 275 nm. (Peptide 1, conc. = 1.00 × 10⁻⁵ M).

The concentration of the drug molecules has been quantified by UV-vis spectroscopy and the results are expressed as encapsulation efficiency (%). To 3 mL of a 0.50 × 10⁻³ M drug solution in methanol, 5 mg of peptide 1 or 2 was added and stirred overnight, the solvent was evaporated and the microspheres were washed with 5 mL water. The residue was dissolved in 3 mL methanol and absorption spectra were taken. The encapsulation efficiency was calculated as (amount of drug added – amount of free drug)/amount of drug added. The encapsulation efficiencies of the peptide 1 and peptide 2 microspheres are 78.39% and 52.46% respectively, and the drug loading content (i.e. (weight of the encapsulated drug in the microspheres/weight of microspheres used)/100) for the peptide 1 vesicles is 6.24% and for the peptide 2 vesicles is 4.17%. We have also studied the release of the encapsulated sulfamethoxazole from the peptide 1 and 2 microspheres. Sulfamethoxazole loaded peptide 1 or 2 microspheres were immersed into 5 mL sodium phosphate buffer (pH = 6.2) in a 15 mL centrifuge tube, centrifuged at 4000 rpm for 8 min, and monitored by UV-Visible spectroscopy at different time intervals. Under slightly acidic conditions (sodium phosphate buffer of pH = 6.2), the protonation of sulfamethoxazole NH₂ increases the hydrophilicity of the drug molecules and governs the slow release from the vesicles. Fig. 6 shows that the peptide 1 and 2 microspheres are slowly releasing the encapsulated drug with complete release by 28 h and 22 h respectively. The pH of 6.2 is very close to that of in vivo conditions. At pH = 5.8, total release of the drug takes place within 15–18 h for peptides 1 and 2 (ESI Fig. S2†). At higher acidic and basic pH values (i.e. pH = 2, 10) deprotection of the Boc and –OMe groups affect the self-assembly of the peptides and the encapsulated drugs are completely released in the time period 3–5 h (ESI Fig. S2†). But no hydrolysis of the Boc or –OMe groups occurs at pH = 6.2 (ESI Fig. S3†).

Fig. 6 Drug release profile of sulfamethoxazole loaded peptide 1 and 2 microspheres in sodium phosphate buffer (pH = 6.2) obtained from UV-vis spectroscopy.

The sustainable release and in vitro antibacterial activity of sulfamethoxazole in water and DMSO (10%) on E. coli was evaluated by optical density (OD) measurement.²⁸ It was found that E. coli was more susceptible to the peptide microsphere encapsulated sulfamethoxazole solutions than the naked sulfamethoxazole solution (control). Fig. 7 shows the growth inhibition plot of E. coli against the sulfamethoxazole solutions in water–DMSO (10%). From Fig. 7, up to eight hours, the growth inhibition of E. coli is greater for the peptide 1-sulfamethoxazole formulation than for the peptide 2-sulfamethoxazole formulation. This indicates that the peptide 2 microspheres are slowly releasing the encapsulated drug, more so than the peptide 1 microspheres, which is also supported by the drug release profile obtained from UV-vis spectroscopy. The E. coli is more susceptible to the encapsulated sulfamethoxazole solutions even at low concentrations.

Fig. 7 The growth inhibition plot of E. coli.

The in vitro antibacterial activity of the released sulfamethoxazole in water–DMSO (10%) on E. coli was also evaluated by the measurement of the mean diameter of growth inhibition zones in centimetres.²⁹ Fig. 8 shows the growth inhibition zones of E. coli bacteria against the sulfamethoxazole encapsulated peptide 1 and 2 microspheres in water–DMSO (10%). It was found that the diameter of the growth inhibition zones increased with increasing released sulfamethoxazole concentration. The solvent used, water–DMSO (10%), showed no antibacterial effect on the tested bacteria (ESI Fig. S4†). The control experiments show that peptides 1 and 2, and the pH = 6.2 buffer have no antibacterial effects (ESI Fig. S5†). The control experiments also show that protonation has no affect on the antibacterial activity of the drug (ESI Fig. S6†).

Fig. 8 The growth inhibition zone of E. coli bacteria against sulfamethoxazole (drug), the encapsulated peptide 1-sulfamethoxazole formulation (PAG) and the peptide 2-sulfamethoxazole formulation (TAG) in water–DMSO (10%).

Conclusions

In conclusion, the self-assembly propensities of tripeptides containing L-phenylalanine, L-tyrosine, α-aminoisobutyric acid and γ-aminobutyric acid have been reported. Peptide 1, with the Phe residue, adopts a type II turn structure whereas the Tyr analogue 2 adopts a type II′ turn structure. X-ray crystallography reveals that peptide 1 self-associates to form four membered ring-like porous structures through multiple intermolecular hydrogen bonds however peptide 2 self-associates to form five membered ring-like porous structures. Field emission scanning electron microscopy (FE-SEM) revealed that the peptides exhibit microsphere morphologies. Further, these microspheres were loaded with the bacteriostatic antibiotic sulfamethoxazole. The formulation led to sustained release of the encapsulated drug sulfamethoxazole. The growth inhibition of E. coli is greater for the peptide 1-sulfamethoxazole formulation than for the peptide 2-sulfamethoxazole formulation which indicates that the peptide 2 microspheres are slowly releasing the encapsulated drug, more so than the peptide 1 microspheres. The results presented here may foster new formulations for sustainable drug release with advanced applications and reduced side effects including nausea, loss of appetite and vomiting.

Experimental

General

All L-amino acids were purchased from Sigma chemicals. HOBt (1-hydroxybenzotriazole) and DCC (dicyclohexylcarbodiimide) were purchased from SRL.

Peptide synthesis

The peptides were synthesized by conventional solution-phase methods using a racemization free fragment condensation strategy. The Boc group was used for N-terminal protection, and the C-terminus was protected by a methyl ester. Coupling was mediated by dicyclohexylcarbodiimide/1-hydroxyl benzotriazole (DCC/HOBt). The products were purified by column chromatography using silica (100–200 mesh size) gel as a stationary phase and an n-hexane–ethyl acetate mixture as an eluent. The intermediates and final compounds were fully characterized by 500 MHz and 400 MHz ¹H NMR spectroscopy, 125 MHz ¹³C NMR spectroscopy, FT-IR spectroscopy and mass spectrometry. The tripeptides 1 and 2 were characterized by X-ray crystallography.

NMR experiments

All NMR studies were carried out on a Brüker AVANCE 500 MHz and Jeol 400 MHz spectrometer at 278 K. Compound concentrations were in the range 1–10 mM in CDCl₃ and (CD₃)₂SO.

FT-IR spectroscopy

All reported solid-state FT-IR spectra were obtained with a Perkin Elmer Spectrum RX1 spectrophotometer using the KBr disk technique.

Mass spectrometry

Mass spectra were recorded on a Q-Tof Micro YA263 high-resolution (Waters Corporation) mass spectrometer by positive-mode electrospray ionization.

UV/vis spectroscopy

UV/Vis absorption spectra were recorded on a Perkin Elmer UV/Vis spectrophotometer.

Fluorescence spectroscopy

Fluorescence spectra were recorded on a Horiba Jobin Yvon fluorescence spectrometer.

Confocal microscopy

Mixtures of the reported peptides and drug sulfamethoxazole were placed on glass slides, and then the slides were washed with fresh buffer solution repeatedly. Finally, the slides were dried under vacuum, and images were taken by a Zeiss LSM 710 confocal microscope.

Field emission scanning electron microscopy

The morphologies of the reported peptides were investigated using field emission-scanning electron microscopy (FE-SEM). A small amount of a solution of the peptide was placed on a clean silicon wafer and was then dried by slow evaporation. The material was then allowed to dry under vacuum at 30 °C for two days. The material was gold-coated, and the micrographs were taken using FE-SEM apparatus (Jeol Scanning Microscope-JSM-6700F).

Single crystal X-ray diffraction study

Intensity data of peptides 1 and 2 were collected with MoKα radiation using a Bruker APEX-2 CCD diffractometer. Data were processed using the Bruker SAINT package and the structure solution and refinement procedures were performed using SHELX97. CCDC 1447358 and 1447364 contain the supplementary crystallographic data for peptides 1 and 2 respectively.†

Loading procedure

15 mg of peptide was dissolved in 1 mL methanol containing 1.5 mg of sulfamethoxazole. The solution was stirred and drop-casted on a Petri dish and dried. Finally, the drug loaded microspheres were dried under vacuum and washed with phosphate buffer solution (several times) to remove unbound drug molecules.

Acknowledgements

We acknowledge the CSIR, India, for financial assistance (Project No. 02(0206)/14/EMR-II). A. Pramanik, T. Das and K. Maji thanks CSIR, India for research fellowship. A. Paikar acknowledges the UGC, India for fellowship.

Notes and references

1. (a) P. T. Wong and S. K. Choi, Chem. Rev., 2015, 115, 3388–3432; (b) T. Liu and R. B. Altman, J. Chem. Inf. Model., 2015, 55, 1483–1494; (c) P. Vitovič, J.-M. Alakoskela and P. K. J. Kinnunen, J. Med. Chem., 2008, 51, 1842–1848; (d) W. G. H. Steve and E. Unger, Chem. Res. Toxicol., 2006, 19, 1564–1569; (e) Y. Yamanishi, E. Pauwels and M. Kotera, J. Chem. Inf. Model., 2012, 52, 3284–3292.
2. (a) K. Uekama, F. Hirayama and T. Irie, Chem. Rev., 1998, 98, 2045–2076; (b) M. Karimi, A. Ghasemi, P. S. Zangabad, R. Rahighi, S. M. M. Basri, H. Mirshekari, M. Amiri, Z. S. Pishabad, A. Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A. Vaseghi, A. R. Aref, L. Haghani, S. Bahramia and M. R. Hamblin, Chem. Soc. Rev., 2016, 45, 1457–1501; (c) R. Xing, T. Jiao, L. Yan, G. Ma, L. Liu, L. Dai, J. Li, H. Möhwald and X. Yan, ACS Appl. Mater. Interfaces, 2015, 7, 24733–24740; (d) X. Yan, P. Zhu, J. Fei and J. Li, Adv. Mater., 2010, 22, 1283–1287; (e) Q. Zou, L. Zhang, X. Yan, A. Wang, G. Ma, J. Li, H. Möhwald and S. Mann, Angew. Chem., Int. Ed., 2014, 53, 2366–2370.
3. (a) V. R. Devadasu, V. Bhardwaj and M. N. V. R. Kumar, Chem. Rev., 2013, 113, 1686–1735; (b) E.-K. Lim, T. Kim, S. Paik, S. Haam, Y.-M. Huh and K. Lee, Chem. Rev., 2015, 115, 327–394; (c) H. Zhang, J. Fei, X. Yan, A. Wang and J. Li, Adv. Funct. Mater., 2015, 25, 1193–1204.
4. (a) S. C. Zimmerman, J. R. Quinn, E. Burakowska and R. Haag, Angew. Chem., Int. Ed., 2007, 46, 8164–8167; (b) Z. Zhou, Y. Shen, J. Tang, E. Jin, X. Ma, Q. Sun, B. Zhang, E. A. Van Kirk and W. J. Murdoch, J. Mater. Chem., 2011, 21, 19114–19123; (c) M. Fischer and F. Vogtle, Angew. Chem., Int. Ed., 1999, 38, 884–905; (d) Z. Sideratou, C. Kontoyianni, G. I. Drossopoulou and C. M. Paleos, Bioorg. Med. Chem. Lett., 2010, 20, 6513–6517; (e) L. M. Kaminskas, B. D. Kelly, V. M. McLeod, G. Sberna, D. J. Owen, B. J. Boyd and C. J. H. Porter, J. Controlled Release, 2011, 152, 241–248; (f) H. T. Chen, M. F. Neerman, A. R. Parrish and E. E. Simanek, J. Am. Chem. Soc., 2004, 126, 10044–10048; (g) S. Svenson, Chem. Soc. Rev., 2015, 44, 4131–4144.
5. (a) S. Dhar, F. X. Gu, R. Langer, O. C. Farokhzad and S. J. Lippard, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 17356–17361; (b) A. O. Eniola and D. A. Hammer, J. Controlled Release, 2003, 87, 15–22; (c) F. Gu, L. Zhang, B. A. Teply, N. Mann, A. Wang, A. F. Radovic-Moreno, R. Langer and O. C. Farokhzad, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 2586–2591; (d) S. S. Chandran, A. Nan, D. M. Rosen, H. Ghandehari and S. R. Denmeade, Mol. Cancer Ther., 2007, 6, 2928–2937.
6. (a) Z. Liu, S. M. Tabakman, Z. Chen and H. Dai, Nat. Protoc., 2009, 4, 1372–1381; (b) S. Dhar, Z. Liu, J. Thomale, H. Dai and S. J. Lippard, J. Am. Chem. Soc., 2008, 130, 11467–11476; (c) Z. Liu, K. Chen, C. Davis, S. Sherlock, Q. Cao, X. Chen and H. Dai, Cancer Res., 2008, 68, 6652–6660; (d) W. Wu, S. Wieckowski, G. Pastorin, M. Benincasa, C. Klumpp, J. P. Briand, R. Gennaro, M. Prato and A. Bianco, Angew. Chem., Int. Ed., 2005, 44, 6358–6362.
7. (a) A. K. Gupta and M. Gupta, Biomaterials, 2005, 26, 3995–4021; (b) S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst and R. N. Muller, Chem. Rev., 2008, 108, 2064–2110.
8. (a) E. C. Dreaden, A. M. Alkilany, X. Huang, C. J. Murphy and M. A. E. Sayed, Chem. Soc. Rev., 2012, 41, 2740–2779; (b) X. Wang and Z. Guo, Chem. Soc. Rev., 2013, 42, 202–224; (c) C. R. Patra, R. Bhattacharya, D. Mukhopadhyay and P. Mukherjee, Adv. Drug Delivery Rev., 2010, 62, 346–361.
9. Y. Du, L. E. Luna, W. S. Tan, M. F. Rubner and R. E. Cohen, ACS Nano, 2010, 4, 4308–4316.
10. (a) K. T. Holman, Angew. Chem., Int. Ed., 2011, 50, 1228–1230; (b) S. Sengupta, A. Goswami and R. Mondal, New J. Chem., 2014, 38, 2470–2479; (c) J. Zhuang, C.-H. Kuo, L.-Y. Chou, D.-Y. Liu, E. Weerapana and C.-K. Tsung, ACS Nano, 2014, 8, 2812–2819.
11. L. A. Mitchell, F. T. Lauer, S. W. Burchiel and J. D. McDonald, Nat. Nanotechnol., 2009, 4, 451–456.
12. B. S. Zolnik, A. G. -Fernandez, N. Sadrieh and M. A. Dobrovolskaia, Endocrinology, 2010, 151, 458–465.
13. X. Yan, P. Zhu and J. Li, Chem. Soc. Rev., 2010, 39, 1877–1890.
14. H. Gelderblom, J. Verweij, K. Nooter and A. Sparreboom, Eur. J. Cancer, 2001, 37, 1590–1598.
15. (a) A. Baral, S. Roy, A. Dehsorkhi, I. W. Hamley, S. Mohapatra, S. Ghosh and A. Banerjee, Langmuir, 2014, 30, 929–936; (b) T. Rehm, V. Stepanenko, X. Zhang, F. Würthner, F. Gröhn, K. Klein and C. Schmuck, Org. Lett., 2008, 10, 1469–1472; (c) M. Gupta, A. Bagaria, A. Mishra, P. Mathur, A. Basu, S. Ramakumar and V. S. Chauhan, Adv. Mater., 2007, 19, 858–861; (d) S. Ghosh, M. Raches, E. Gazit and S. Verma, Angew. Chem., Int. Ed., 2007, 46, 2002–2004.
16. (a) D. T. Bong, T. D. Clark, J. R. Granja and M. R. Ghadiri, Angew. Chem., Int. Ed., 2001, 40, 988–1011; (b) C. H. Gorbitz, M. Nilsen, K. Szeto and L. W. Tangen, Chem. Commun., 2005, 4288–4290.
17. (a) C. Yan and D. J. Pochan, Chem. Soc. Rev., 2010, 39, 3528–3540; (b) M. Reches and E. Gazit, Science, 2003, 300, 625–627; (c) J. D. Hartgerink, E. Beniash and S. I. Stupp, Science, 2001, 294, 1684–1688; (d) S. Santoso, W. Hwang, H. Hartman and S. Zhang, Nano Lett., 2002, 2, 687–691; (e) S. Cavalli, F. Albericio and A. Kros, Chem. Soc. Rev., 2010, 39, 241–263; (f) A. J. V. Hell, M. M. Fretz, D. J. A. Crommelin, W. E. Hennink and E. Mastrobattista, J. Controlled Release, 2010, 141, 347–353; (g) A. J. V. Hell, C. I. C. A. Costa, F. M. Flesch, M. Sutter, W. Jiskoot, D. J. A. Crommelin, W. E. Hennink and E. Mastrobattista, Biomacromolecules, 2007, 8, 2753–2761.
18. Z. S. Mehdi, Journal of Chemistry and Biochemistry, 2015, 3, 63–74.
19. T. T. Tran, H. Treutlein and A. W. Burgess, Protein Eng., Des. Sel., 2006, 19, 401–408.
20. (a) A. Pramanik, A. Paikar and D. Haldar, RSC Adv., 2015, 5, 53886–53892; (b) S. K. Maity, S. Bera, A. Paikar, A. Pramanik and D. Haldar, CrystEngComm, 2013, 15, 5860–5866; (c) S. Maity, P. Jana, S. K. Maity and D. Haldar, Soft Matter, 2011, 7, 10174–10181; (d) S. Maity, P. Jana, S. K. Maity and D. Haldar, Langmuir, 2011, 27, 3835–3841.
21. S. K. Maity, S. Bera, A. Paikar, A. Pramanik and D. Haldar, CrystEngComm, 2014, 16, 2527–2534.
22. (a) S. K. Maji, A. Banerjee, M. G. B. Drew, D. Haldar and A. Banerjee, Tetrahedron Lett., 2002, 43, 6759–6762; (b) D. Haldar, M. G. B. Drew and A. Banerjee, Tetrahedron, 2006, 62, 6370–6378.
23. D. Haldar, Tetrahedron, 2008, 64, 186–190.
24. P. Jana, S. Maity, S. K. Maity and D. Haldar, Chem. Commun., 2011, 47, 2092–2094.
25. (a) C. Toniolo and M. Palumbo, Biopolymers, 1977, 16, 219–224; (b) V. Moretto, M. Crisma, G. M. Bonora, C. Toniolo, H. Balaram and P. Balaram, Macromolecules, 1989, 22, 2939–2944; (c) D. Haldar and A. Banerjee, Int. J. Pept. Res. Ther., 2006, 12, 341–348.
26. Crystallographic data of peptide 1: C₂₃H₃₅N₃O₆, M_w = 449.54, orthorhombic, space group P 2₁ 2₁ 2₁, a = 9.152(3), b = 9.804(4), c = 27.360(1) Å, α = β = γ = 90°, V = 2454.8(16) ų, Z = 4, d_m = 1.216 Mg m⁻³, T = 296 K, R₁ 0.0534 and wR₂ 0.1232 for 5001 data with I > 2σ(I). Peptide 2: C₄₆H₇₀N₆O₁₄, M_w = 931.09, orthorhombic, space group P 2₁ 2₁ 2₁, a = 8.31575(11), b = 20.7760(2), c = 29.5002(4) Å, α = β = γ = 90°, V = 5096.7(11) ų, Z = 4, d_m = 1.213 Mg m⁻³, T = 100 K, R₁ 0.0341 and wR₂ 0.0880 for 8699 data with I > 2σ(I). Intensity data were collected with MoKα radiation using Bruker APEX-2 CCD diffractometer. Data were processed using the Bruker SAINT package and the structure solution and refinement procedures were performed using SHELX97.³⁰ The data for peptides 1 and 2 have been deposited at the Cambridge Crystallographic Data Centre with reference number CCDC 1447358 and 1447364 respectively†.
27. P. Jana, S. Maity and D. Haldar, CrystEngComm, 2011, 13, 973–978.
28. E. D. Brown and G. D. Wright, Chem. Rev., 2005, 105, 759–774.
29. M. Sangra, S. Jaiswal and G. D. Gupta, Indo American Journal of Pharmaceutical Research, 2014, 4, 2943–2950.
30. G. M. Sheldrick, SHELX 97, University of Göttingen, Germany, 1997.

---

Footnote

† Electronic supplementary information (ESI) available: Synthesis and characterization of trisamides, ¹H NMR, ¹³C NMR, Fig. S1–S3, Fig. S3–S15. CCDC 1447358 and 1447364. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra07095g

---