Development of small peptide amphiphile based supramolecular hydrogels for various potential applications

Development of Small Peptide Amphiphile Based Supramolecular Hydrogels

for Various Potential Applications

by

Department of Chemistry

Indian Institute of Technology Guwahati Guwahati, Assam - 781039

India

A Thesis Submitted in Partial Fulfillment of the Requirements for the degree of

IIT Guwahati, April 2019

Dedicated To

My Parents

Declaration

I hereby declare that the matter embodied in this thesis is the result of investigations carried out by me in the Department of Chemistry, Indian Institute of Technology Guwahati, India, under the guidance of Dr. Debapratim Das. In keeping with the general practice of reporting scientific observations, due acknowledgments have been made wherever the work described is based on the findings of other investigators.

(Nilotpal Singha)

INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI Dr. Debapratim Das Associate Professor Department of Chemistry Contact: + 91 361 258 3301 Fax: + 91 361 258 2349 E-mail: ddas@iitg.ac.in

3^rd April, 2019

To whom it may concern

This is to certify that the thesis entitled “Development of small peptide amphiphile based supramolecular hydrogels for various potential applications” submitted by Nilotpal Singha (Roll No.

146122001) for the award of Ph.D. degree to IIT Guwahati, is absolutely based on his own research work and that neither this thesis nor any part of it has been submitted for any degree/diploma or any academic award anywhere before.

(Dr. Debapratim Das)

Acknowledgments

This thesis is the last milestone of my journey in acquiring my Ph.D. and has been kept on track which is also governed by the support and encouragement of numerous people including my lab mates, well-wishers, friends, and various institutions. I would like to thank all those people who made this thesis successful and a remarkable experience for me. Also, it is a great opportunity to express my gratitude to all those who contributed in several ways to the victory of this study.

Prior to all, I would like to express my sincere gratefulness to my supervisor Dr. Debapratim Das. This work would not have been accomplished in the absence of his guidance, scholarly inputs, consistent encouragement and unconditional support that I received throughout the research work. I am truly honored and blessed to have been a part of his research team and I fruitfully overcame many obstacles which assisted me to learn a lot from his research expertise. His unflinching courage and conviction always inspire me, and I anticipate to continue further research with his gallant thoughts. He has been by my side always whenever and wherever I needed. He is known for his amicability and positive disposition. Thank you, Sir, for all your immense support, enthusiasm, and dedication.

Besides my supervisor, I would like to give special thanks to my doctoral committee members:

Dr. Sunanda Chatterjee, Dr. Debasis Manna, and Dr. Dipankar Srimani for their encouragement, insightful comments, and suggestions.

I am grateful to the collaborators for deploying me their expertise and instinct to my scientific and technical problems: Dr. Antara Dasgupta, Dr. Anindita Ukil (University of Calcutta, India), Dr.

Ranajit Ghosh (CMERI, India), Dr. Ananya Debnath (IIT Jodhpur, India).

I would like to thank the Department of Chemistry, IIT Guwahati for availing me the innumerable facilities. Also, I am thankful to IIT Guwahati for financial support.

I would also like to extend huge, warm thanks to my past and present lab mates Dr. Julfikar Hassan Mondal, Dr. Sahnawaz Ahmed, Bapan Pramanik, Payel Dowari, Dr. Subhajit Ghosh, Basab Kanti Das, Dr. Tanur Sinha, Sumit Chowdhuri, Supriya for providing an amusing and fun-filled environment. I am especially grateful to Dr. Julfikar Hassan Mondal and Dr. Sahnawaz Ahmed for their initial guidance, support, and motivation in the lab. My heartfelt thanks to Payel, Bapan, and Basab for their cooperation during the course of my work.

My special acknowledgments go to my friends, Ayan, Rajat, Utsab, Mostakim, Rana, Subas, for their support during my research endeavor. I am indebted to Milan Mahadani for his valuable help with TEM analysis.

I sincerely thank all the research scholars and the technical staffs of the Department of Chemistry for their help and friendship. I would like to express my gratitude to all others who are associated with my work directly or indirectly at IIT Guwahati.

Abstract

The thesis “Development of small peptide amphiphile based supramolecular hydrogels for various potential applications” deals with the development of new short peptide amphiphile based hydrogels with the help of different aromatic functional groups.

Chapter 1 is a brief introduction of peptide-based hydrogels with up to date literature review.

Chapter 2 describes the hydrogelation mechanism of a peptide amphiphile (PA-1) conjugated to naphthalene diimide (NDI) and its application in both cell imaging as well as intracellular pH sensing.

Chapter 3 deals with another naphthalene diimide (NDI) appended peptide amphiphile (PA-2) and its application in sensing the VOC with the help of carbon quantum dots.

Chapter 4 describes the untraditional unique behaviors of a pyrene containing short tri-peptide (PyKC) based hydrogel.

Chapter 5 contains the potential application of PyKC hydrogel in the protection and retention of

enzymes’ geometrical conformation and activity respectively from different extreme conditions like

denaturants, temperature, acid, and base.

List of Abbreviations

Abbreviations

ADI Arylene Diimide NDI Naphthalene Diimide NMR Nuclear Magnetic Resonance

ESI-Ms Electrospray Ionization Mass Spectrometry UV-Vis Ultraviolet–Visible Spectroscopy

FL Fluorescence



max

The wavelength in the absorption spectrum where the absorbance is maximum MAC Minimum Aggregation Concentration

MeOH Methanol EtOH Ethanol

ACN Acetonitrile DMSO Dimethyl Sulfoxide

DMF N,N-Dimethylformamide THF Tetrahydrofuran

DIPEA N,N-Diisopropylethylamine TEA Triethylamine

NaOH Sodium Hydroxide

FESEM Field Emission Scanning Electron Microscopy FETEM Field Emission Transmission Electron Microscopy

AFM Atomic force microscopy

FTIR Fourier-transform Infrared Spectroscopy DLS Dynamic Light Scattering

ζ Zeta potential PBS Phosphate Buffer

Tris Tris(hydroxymethyl) aminomethane CD Circular Dichroism

ICD Induced circular Dichroism PA Peptide Amphiphile G’ Storage Modulus G” Loss Modulus

MGC Minimum Gelation Concentration T

_g

Gel Melting Temperature

D

₂

O Deuterium Oxide

d

₆

-DMSO Deuterated Dimethyl Sulfoxide

nM Nanomolar

M Micromolar mM Millimolar ppm Parts Per Million

g/mL Microgram Per Milliliter

HRTEM High Resolution Transmission Electron Microscopy DFT Density Functional Theory

Phe Phenylalanine Trp Tryptophan Tyr Tyrosine

ECM Extra Cellular Matrix

MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide VOC Volatile Organic Compound

SMHG Supramolecular Hydrogel PHG Polymeric Hydrogel CQD Carbon Quantum Dot

PVA Polyvinylalcohol

CV-lipase Lipase from Chromobacterium viscosum CR-lipase Lipase from Candida rugosa

PXRD Powder X-ray Diffraction

TCEP Tris(2-carboxyethyl)phosphine hydrochloride GSH Glutathione

HPLC High Performance Liquid Chromatography I-V Current-Voltage

MD Molecular Dynamic

HBTU

Hexafluorophosphate Benzotriazole Tetramethyl Uronium

HOBT

Hydroxybenzotriazole

TFA Trifluoroacetic acid Py 1-Pyrenebutyric acid

Wt% 10 mg/mL

Contents

Chapter-1

¹⁹

1.1 Prelude

²⁰

1.2 Hydrogels

²⁰

1.2.1 Polymeric Hydrogels 21

1.2.2 Supramolecular Hydrogels 21

1.2.3 Supramolecular Polymeric Hydrogels 22

1.3 Peptide-Based Hydrogels

²³

1.3.1 Hydrogels Based on Secondary Structural Motifs 24

1.3.2 Short Peptide-Based Hydrogels 27

1.3.3 Peptide Amphiphile Based Hydrogels 29

1.3.3.1 Amphiphilic Peptides 30

1.3.3.2 Lipidated Peptide Amphiphiles 31

1.3.4 Peptide Amphiphiles With Different Substitutions 33

1.4 Applications of PA Hydrogels

³⁴

1.4.1 Tissue Scaffolds 34

1.4.2 Drug Delivery Vehicles 35

1.4.3 Antimicrobial Hydrogels 36

1.4.4 Template for Nanofabrication 37

1.4.5 Intracellular Localization 38

1.4.6 Probable Future Applications 38

1.5 The Present Thesis

³⁹

Chapter-2

⁴¹

2.1 Introduction

⁴²

2.2 Results and Discussion

⁴⁴

2.2.1 Design, Synthesis, and Gelation 44

2.2.2 Stepwise Aggregation 44

2.2.3 Supramolecular Interaction Involved In The Aggregation 49

2.2.4 pH Sensitivity 50

2.2.5 Rheology 52

2.2.6 Cytotoxicity 52

2.2.7 Intracellular Localization 52

2.3 Conclusion

⁵⁵

2.4 Experimental Section

⁵⁶

2.4.1 Materials 56

2.4.2 Synthesis and Characterization 56

2.4.2.1 Synthesis of 1a 57

2.4.2.2 Synthesis of 1b 57

2.4.2.3 Synthesis of PA-1 58

2.4.3 Sample Preparation 58

2.4.4 Preparation of Hydrogel 59

2.4.5 DLS and Zeta Potential 59

2.4.6 Field Emission Scanning Electron Microscope (FESEM) 59

2.4.7 Transmission Electron Microscope (TEM) 59

2.4.8 Circular Dichroism (CD) 59

2.4.9 Surface Tension 60

2.4.10 Rheology 60

2.4.11 Cell Culture 60

2.4.12 MTT Assay 60

2.4.13 Cellular and Subcellular Localization 61

2.4.14 Lambda Scanning 61

Chapter-3

⁶³

3.1 Introduction

⁶⁴

3.2 Results and Discussions

⁶⁵

3.2.1 Gas Sensing 71

3.3 Conclusion

⁷⁷

3.4 Experimental Section

⁷⁸

3.4.1 Materials 78

3.4.2 Synthesis of PA-2 78

3.4.3 Characterization of PA-2 79

3.4.4 Synthesis of Carbon Quantum Dot (CQD) 79

3.4.5 NMR Studies 80

3.4.6 FETEM 80

3.4.7 Preparation of Hydrogel 80

3.4.8 Preparation of Composite Devices 80

3.4.9 Rheology 81

3.4.10 Fourier Transformed Infrared Spectroscopy 81

3.4.11 Density Functional Theory 81

3.4.12 Gas Sensing Measurement 81

Chapter-4

⁸³

4.1 Introduction

⁸⁴

4.2 Results and Discussions

⁸⁵

4.2.1 PyKC-Dimer Configuration from Electronic Structure Calculation 94 4.2.2 PyKC-Dimers in Water from Molecular Dynamics Simulation 97

4.3 Conclusion

¹⁰¹

4.4 Experimental Section

¹⁰¹

4.4.1 Materials 101

4.4.2 Synthesis of Peptides 102

4.4.3 Characterization of Synthesized Peptides 103

4.4.3.1 Characterization of PyKC 103

4.4.3.2 Characterization of Pep-2 103

4.4.3.3 Characterization of Pep-3 103

4.4.3.4 Characterization of Pep-4 103

4.4.3.5 Characterization of Pep-5 104

4.4.3.6 Characterization of Pep-6 104

4.4.3.7 Characterization of Pep-7 104

4.4.9 Powder X-Ray Diffraction (PXRD) 106

4.4.10 Dissolution Studies 106

4.4.11 Simulation Details 107

Chapter-5

¹⁰⁹

5.1 Introduction

¹¹⁰

5.2 Results and Discussions

¹¹¹

5.2.1 Stability and retention of structural conformation of the lipase 112

5.2.2 Retention of activity of lipase 113

5.3 Conclusion

¹¹⁶

5.4 Experimental Section

¹¹⁶

5.4.1 Materials 116

5.4.2 Synthesis and Characterization 117

5.4.2.1 Bodipy-butyric acid 117

5.4.2.2 Bodipy-NHS ester 117

5.4.3 Preparation of enzyme incorporated hydrogel 118

5.4.4 Field Emission Transmission Electron Microscopy (FETEM) 118

5.4.5 Circular Dichroism (CD) 118

5.4.6 Enzyme activity

NMR, MASS, and HPLC of the Synthesized Compounds

¹²¹

References

¹³⁹

Publications

¹⁶¹

Chapter - 1

Introduction

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Chapter - 2

Hydrogelation from a Peptide Amphiphile (PA-1) Conjugated to Naphthalene Diimide and Its Application in Both Cell Imaging and

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Carbon Quantum Dot/Peptide Composite Xerogel Based Sensor:

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Chapter – 4

A Water Insoluble Supramolecular Hydrogel with Unique

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Chapter – 5

Controlled encapsulation, long time protection, and instant triggered release with a minimum damage of biomolecules by a

smart tri-peptide hydrogel

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NMR, MASS, and HPLC of the Synthesized Compounds

References

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