1 SYNTHESIS OF CARBON NANODOTS-PEPTIDE CONJUGATES DECORATED WITH GERMANIUM FOR BIOIMAGING KHANANI MACHUMELE SUPERVISED BY: DR MAYA MAKATINI DR MANOKO S. MAUBANE-NKADIMENG ii 31-10-2023 Declaration of originality I, at this moment, state that I conducted the work recorded in this document. This thesis was wholly conducted and written by me under the supervision of Dr Maya Makatini and Dr Manoko Maubane-Nkadimeng. I have rightfully credited the sources used in the thesis. This thesis has not been submitted before for any degree. --------------------------` ---------------------- iii Acknowledgements First and foremost, I express my gratitude to God for granting me the opportunity to use my work as a means to glorify His name. I extend my appreciation to the School of Chemistry for providing me with the chance to advance in my field, as well as to the National Research Foundation for their financial support. A special acknowledgment goes to Dr. Maya Makatini and Dr Manoko S. Maubane- Nkadimeng, my supervisors, for entrusting me with this project and generously sharing their extensive knowledge. I am immensely grateful for the encouragement they provided me during times when hope seemed scarce. Their unwavering dedication to their work and their efforts to promote women in science serve as a true inspiration. To my parents, Nomalungelo Felicia Machumele and Nkateko Jones Machumele, I owe a profound debt of gratitude for their unwavering support throughout my postgraduate studies. They have been my unwavering sources of strength, especially when faced with discouragement in pursuing an academic career. I cannot adequately express the depth of my gratitude as they have made countless sacrifices to shape me into the person I am today. Thank you for being amazing parents. I extend my heartfelt appreciation to my brother, Nkateko Machumele, for his constant motivation and daily encouragement. A special thank you goes to my sister, Gauta Maseko, for her regular calls that uplift my spirits. I would also like to thank Nombulelo Kwayiba, Tsebo Utlwa, Nicole Baloyi, Khensi Baloyi, Irene Machumele, Maggy Machumele, Khongelani Machumele, and all other family members for their support. I am grateful to my colleagues: Thapelo Mbele, Refilwe Moepya, Ndivhuwo Shumbula, Thabo Peme, Ntombizanele Ngqinayo, Mudzuli Maphupha, Sinazo Cobongela, Kamogelo Butsi, Edward Chavalala and Nompumelelo Mathebula. I am also grateful to Mary-Ann Xaba, Khwezi Ndawonde, Elias Baloyi and David Moloto for their support. Special thanks go to my friends: Tshwarela Kolokoto, Mulweli Nethathe, Lindelwa Didiza, Linda Didiza, Dineo Tiro, Oreneile Tshetlo, Lerato Mokoloko, Lindelo Mguni, Mufaro buhlebenkosi Mugari and Nthabiseng Khanye. Thank you to my partner Thabiso Matome Mashatola for making sure I complete my work. Lastly, I express my gratitude to the iv National Research Foundation (NRF) and the South African Medical Research Council (SAMRC) for their funding support. Awards National Young Chemists’ Symposium (Oct 3, 2022)- Award Won -1st Place. National Young Chemists’ Symposium (Jul 8, 2021) - Award Won -2nd Place. 12th Wits Cross-Faculty Postgraduate Symposium (July 26-28, 2021)- Award Won -3rd Place. Scholarship awards NRF Scholarship: 2019 – 2022 Tshipi mine bursary: 2016 – 2017 Rand Refinery bursary: 2012 – 2016 Publications 1. Machumele, K., Mokoloko, L.L., Makatini, M. and Maubane-Nkadimeng, M.S., 2023. Physicochemical properties of in situ microwave-assisted germanium oxide modified nitrogen-doped carbon dots. Diamond and Related Materials, p.110195. 2. Machumele, KP., Ntombela, T., Peme, T., Maubane-Nkadimeng, M.S. and Makatini, M. 2023. The total structure of the penetration accelerating sequences (Pas) by two- dimensional nuclear magnetic resonance spectroscopy. Manuscript ID: MRC-23- 0073. (Submitted-28 July 2023) v Conferences ● Chemistry And Biology of Peptides Gordon Research Conference (Oct 30 - Nov 4, 2022) Physical, International Conference, USA Poster Presentation Title: Carbon nanodots-peptide conjugates for drug delivery in cancer therapy ● Chemistry And Biology of Peptides Gordon Research Seminar (Oct 28–30, 2022) Physical, International Conference, USA Oral Presentation Title: Carbon nanodots-peptide conjugates for drug delivery in cancer therapy ● National Young Chemists’ Symposium (Oct 3, 2022) Poster Presentation Title: Carbon nanodots-peptide conjugates for drug delivery in cancer therapy ● The Wits PhD Seminar (Sep 12-13, 2022) Oral Presentation Title: carbon nanodots-peptide conjugates for drug delivery in cancer therapy ● 13th Wits Cross-Faculty Postgraduate Symposium (Jul 11-13, 2022) Grad Flash Video Title: Carbon nanodots-peptide conjugates for drug delivery in cancer therapy ● 7th Nano Today Conference 2021 (Nov 15-18, 2021) Virtual, International Conference, China Poster Pitch Video Title: Synthesis and characterization of carbon dots decorated with germanium, conjugated to iRGD peptides for tumor-targeting fluorescence bio-imaging and drug delivery. ● Wits EIE Women's Conference 2021 (Nov 5, 2021) vi Grad flash video title: Nano-technological innovation of using carbon nanodots for drug delivery in cancer therapy. ● 12th Wits Cross-Faculty Postgraduate Symposium (Jul 26-28, 2021) Grad Flash Video Title: Nano-technological innovation of using carbon nanodots for drug delivery in cancer therapy. ● South African Nanotechnology Initiative - Nanosciences Young Researcher’s Symposium 2021 (Oct 7-8, 2021) Poster Pitch Video Title: New advances in using carbon nanodots for drug delivery in cancer therapy. ● The Wits PhD Seminar (Aug 30-31, 2021) Oral Presentation Title: Synthesis and characterization of carbon dots decorated with germanium, conjugated to iRGD peptides for tumor-targeting fluorescence bio-imaging and drug delivery. ● National Young Chemists’ Symposium (Jul 8, 2021) Poster Presentation Title: Synthesis and Characterization of carbon dots decorated with germanium, conjugated to iRGD peptides for tumor-targeting fluorescence bio-imaging and drug delivery. ● 11th Wits Cross-Faculty Postgraduate Symposium (Oct 12-14, 2020) Grad Flash Video Title: Synthesis and characterization of carbon dots decorated with germanium, conjugated to iRGD peptides for tumor targeting fluorescence bio-imaging and drug delivery. vii Abstract The World Health Organization Global Cancer Observatory estimates that cancer caused 9.96 million deaths worldwide in 2020, making early detection crucial for diagnosis and treatment. Accurate identification of cancer plays a crucial role in the diagnosis and treatment process. It allows for customized and efficient therapies, minimizes unnecessary procedures and adverse effects, and improves the prognostic insights for patients and healthcare providers alike. The challenges in diagnosis include overdiagnosis, false positives/negative outcomes, and limited sensitivity. Advanced technologies are needed for better imaging accuracy and minimizing harm. This study aims to fabricate carbon dot-peptide conjugates to enhance bio-imaging capacity and selectivity. The peptides used are derived from the GKPILFF cell-penetrating peptide sequence and the RLRLRIGRR peptide, which is selective to cancerous cells. The Carbon dots were used to provide the photoluminescent properties required for bio-imaging of cancerous cells. Carbon dots (CDs) were synthesized using iso-ascorbic acid as the source of carbon using a microwave-assisted method. The nitrogen and germanium-modified carbon dots (Iso-N-Ge- CDs) demonstrated the highest photoluminescent properties compared to the unmodified CDs (Iso-CDs) and those with either N (Iso-N-CDs) or Ge (Iso-Ge-CDs). Photoluminescence emissions of longer wavelengths suitable for cell imaging were observed for the CDs, and the Iso-N-Ge-CDs demonstrated excitation-dependent emission wavelength behavior, pH sensitivity, and Fe3+ sensitivity. The 13 peptides derived from the peptide accelerating sequence GKPILFF and the cancer- selective peptide RLRLRIGRR were successfully synthesized. The peptides were characterized using Liquid Chromatography Mass Spectrometry (LCMS) and purified using preparative High-Pressure Liquid Chromatography (prep-HPLC). The secondary structure of the L-GKPILFF penetration acceleration peptide sequence (Pas) adopted a helical secondary structure. The D-GKPILFF derivative was found to adopt a random coil structure. These were confirmed using Nuclear Magnetic Resonance (NMR) techniques such as Total Correlation Spectroscopy (TOCSY) and Rotating Frame Overhauser Enhancement Spectroscopy (ROESY) NMR. The CDs-peptide conjugates were successfully synthesized, and the confirmation of conjugation involved multiple methods, including UV-Vis and PL techniques. To the best of viii our knowledge, the thesis incorporates the first study to demonstrate long-range interactions through ROESY NMR. The NMR analysis indicated that the helical structure of the peptide could be affected after conjugation, leading to notable peak shifts. Since the helical structure is crucial for the peptide's bioactivity and stability enhancement, NMR spectra with fewer structural changes in the peptide region may improve its biological properties. The research contained valuable information for scientists aiming to design and characterize Carbon dot- peptide conjugates with enhanced permeability and selectivity that can effectively deliver materials into cytosolic space. ix Table of Contents Contents Declaration of originality ........................................................................................................... ii Acknowledgements ................................................................................................................... iii Scholarship awards ................................................................................................................... iv Publications ............................................................................................................................... iv Conferences................................................................................................................................ v List of figures .......................................................................................................................... xiv List of tables .......................................................................................................................... xviii List of schemes ....................................................................................................................... xix List of abbreviations ................................................................................................................ xx 1. Introduction ........................................................................................................................ 1 1.1 Background and motivation ........................................................................................ 1 1.2 Problem statement ....................................................................................................... 1 1.3 Carbon Dots as Potential Cancer Diagnostic and Therapeutic Agents ....................... 2 1.4 Limitations of current technologies............................................................................. 3 1.5 Conjugation of carbon dots to peptides for bio-imaging............................................. 3 1.6 Aim and objectives ...................................................................................................... 4 1.7 The study's novelty and prospective applications in the context of South Africa ...... 5 1.8 Outline of the thesis..................................................................................................... 7 1.9 References ................................................................................................................... 9 2. Literature review ............................................................................................................... 12 2.1 Introduction ............................................................................................................... 12 2.2 Global and local impact of diseases caused by cancer and tumors ........................... 12 2.2.1 Treatment and monitoring.................................................................................. 13 2.3 Carbon dots ............................................................................................................... 14 x 2.3.1 Carbon dots – what are they? ............................................................................. 14 2.3.2 The potential of carbon dots for bio-application................................................ 15 2.3.3 In vivo and in vitro studies using fluorescent carbon dots ................................. 16 2.3.4 N-doped carbon dots decorated with Ge ............................................................ 17 2.4 Peptides and their bio-application ............................................................................. 18 2.4.1 Cell-penetrating peptides ................................................................................... 19 2.4.2 Cell homing peptides ......................................................................................... 20 2.4.3 Cell homing and penetrating peptides................................................................ 21 2.5 Peptide-Carbon dot conjugates.................................................................................. 22 2.6 Derivatization of carbon dots and peptide and their effects ...................................... 24 2.6.1 Functionalization of carbon dots ........................................................................ 24 2.6.2 Different types of interaction for the functionalization of carbon dots ............. 25 2.6.3 Manipulation of the Amino Acid Sequence....................................................... 27 2.6.4 C-Terminal Amidation ....................................................................................... 29 2.6.5 N-Terminal modification ................................................................................... 29 2.6.6 Structure-Activity Relationships (SAR) ............................................................ 31 2.7 References ................................................................................................................. 33 3. Physicochemical properties of in situ microwave-assisted germanium oxide modified nitrogen-doped carbon dots...................................................................................................... 47 3.1 Abstract ..................................................................................................................... 47 3.2 Introduction ............................................................................................................... 48 3.3 Experimental ............................................................................................................. 51 3.3.1 Materials ............................................................................................................ 51 3.3.2 Synthesis of various carbon dots........................................................................ 51 3.4 Characterization techniques ...................................................................................... 53 3.5 Results and discussion ............................................................................................... 53 3.5.1 Morphological characterization ......................................................................... 53 xi 3.5.2 Chemical properties ........................................................................................... 55 3.5.3 XRD analysis ..................................................................................................... 58 3.5.4 Photophysical properties .................................................................................... 59 3.6 Conclusion ................................................................................................................. 66 3.7 References ................................................................................................................. 68 4. Synthesis and characterization of enhanced cell-penetrating peptides derivatives .......... 74 4.1 Abstract ..................................................................................................................... 74 4.2 Introduction ............................................................................................................... 74 4.3 Design of the short peptide derivatives ..................................................................... 78 4.3.1 L- and D-amino acids......................................................................................... 78 4.3.2 Adamantane and Palmitic acid........................................................................... 79 4.3.3 RGD homing moiety .......................................................................................... 80 4.4 Methods and Procedures for Peptide Synthesis ........................................................ 83 4.4.1 General peptides synthesis ................................................................................. 83 4.4.2 Peptide purification ............................................................................................ 86 4.4.3 Modifications for optimization .......................................................................... 87 4.5 Synthesis of the GKP peptide derivatives ................................................................. 92 4.5.2 Synthesis of the BP peptide derivatives ............................................................. 97 4.5.3 Summary of all synthesized analogues .............................................................. 99 4.6 Conclusion ............................................................................................................... 101 4.7 References ............................................................................................................... 102 5. The total structure of the penetration accelerating sequences (Pas) by two-dimensional nuclear magnetic resonance spectroscopy. ............................................................................ 105 5.1 Abstract ................................................................................................................... 105 5.2 Introduction ............................................................................................................. 106 5.3 Materials .................................................................................................................. 107 5.4 Synthesis of peptides ............................................................................................... 107 xii 5.5 Results and discussions ........................................................................................... 108 5.5.1 NMR elucidation of L-GKPILFF peptide........................................................ 108 5.5.2 Full structural elucidation using HSQC NMR ................................................. 115 5.5.3 Secondary structure of GKPILFF peptide ....................................................... 118 5.5.4 Structural elucidation using computational techniques ................................... 121 5.6 Conclusion ............................................................................................................... 125 5.7 References ............................................................................................................... 126 6. The use of NMR techniques for Carbon dots- GKP peptide conjugates ........................ 130 6.1 Abstract ................................................................................................................... 130 6.2 Introduction ............................................................................................................. 130 6.3 Synthesis of carbon nanodot-GKP peptide conjugates ........................................... 131 6.4 Results and discussion ............................................................................................. 134 6.4.1 UV-Vis spectroscopy ....................................................................................... 134 6.4.2 Photoluminescence spectroscopy..................................................................... 140 6.4.3 NMR of CDs conjugates .................................................................................. 150 6.4.4 NMR of CDs before and after dialysis ............................................................ 150 6.5 Conclusion ............................................................................................................... 166 6.6 References ............................................................................................................... 168 7. Final conclusions and recommendations ........................................................................ 174 8. Experimental procedures ................................................................................................ 177 8.1 Reagents and chemicals .......................................................................................... 177 8.1.1 Reagents for peptides ....................................................................................... 177 8.1.2 Reagents for carbon dots .................................................................................. 177 8.2 Instrumentation........................................................................................................ 177 8.2.1 Instrumentation used for peptides and amino acids ......................................... 177 8.2.2 Instrumentation used for carbon dots and their conjugates. ............................ 179 8.2.3 General procedure for synthesizing carbon dots .............................................. 181 xiii 8.2.4 General procedure for conjugating carbon dots to peptides ............................ 182 8.2.5 General procedure for the swelling, activation, and coupling of the first amino acid to rink amide resin .................................................................................................. 182 8.2.6 General procedure for analysing the substitution of the first amino acid ........ 182 8.2.7 General procedure for the synthesis of peptides .............................................. 183 8.2.8 General procedure for the synthesis of peptides containing adamantane and palmitic acid on the N-terminal ...................................................................................... 184 8.2.9 General procedure for the cleavage of the peptides from the resin ................. 185 9. Appendix ........................................................................................................................ 186 9.1 Chapter 3 ................................................................................................................. 186 9.2 Chapter 4 ................................................................................................................. 202 9.3 Chapter 5 ................................................................................................................. 219 xiv List of figures Figure 2.1 Stats as per The ASCO Post, in partnership with the American Society of Clinical Oncology 2021. 8 ...................................................................................................................... 13 Figure 2.2 This is an illustration of a carbon dot with functional groups. ............................... 15 Figure 2.3 Illustration of how amino acids are building blocks of peptides and how proteins are much larger forms of peptides. .......................................................................................... 19 Figure 2.4 Multipurpose functions in one individual nanostructure ........................................ 24 Figure 2.5 Covalent modification of carbon dots 119 ............................................................... 26 Figure 2.6 Illustration of L-amino acids versus D-amino acids ............................................... 28 Figure 2.7 Shows a schematic diagram with essential contacts for the development of a complex between PMB and the lipid A component of the LPS colour-coded fatty acyl chain at the end, or FA. 154................................................................................................................. 32 Figure 3.1 TEM micrographs and (inserted) size distribution curves of (a) Iso-CDs, (b) Iso- Ge-CDs, (c) Iso-N-CDs and (d) Iso-N-Ge-CDs. ..................................................................... 55 Figure 3.2 FT-IR spectra of (a) Iso-CDs, (b) Iso-N-CDs, (c) Iso-N-CDs and (d) Iso-N-Ge- CDs. ......................................................................................................................................... 56 Figure 3.3 (a) Survey scans of Iso-CDs and Iso-N-Ge-CDs showing four major peaks of carbon, oxygen, nitrogen and germanium, and the expanded survey scan of (b) C1s (c) N1s and (d) Ge2p3. ......................................................................................................................... 58 Figure 3.4 XRD spectra of the various CDs. ........................................................................... 59 Figure 3.5 Photoluminescence (PL) of (a) Iso-CDs (b) Iso-Ge-CDs (c) Iso-N-CDs d) Iso-N- Ge-CDs .................................................................................................................................... 60 Figure 3.6 Photoluminescence spectra of the Iso-N-Ge-CDs at pH ranging from 1 to 11. ..... 62 Figure 3.7 (a) The emission peak intensity ratio of Iso-N-Ge-CDs with Cu2+, Fe3+, Co3+, Zn3+, Ni2+, Cd2+, Ca2+, Na+, Al3+, K+, As3+and As2+ ions, (b) Photoluminescence (PL) of carbon dots of Iso-N-Ge-CDs with different metal ions, (c) fluorescent properties of t ............................. 64 Figure 3.8 UV-Vis absorption spectra of Iso-CDs, Iso-Ge-CDs, Iso-N-CDs and Iso-N-Ge- CDs .......................................................................................................................................... 65 Figure 3.9 Fluorescent Microscopy of Iso-N-Ge-CDs under blue (a) and red (b) wavelength66 Figure 4.1 Using an information-theoretic technique based on Jensen-Shannon divergence, active sites were predicted at D amino acid 81 (D, Aspartic Acid), 84αα (D, Aspartic Acid), and 438 (E, Glutamic Acid).12 ................................................................................................. 77 xv Figure 4.2 D-amino acid ((R)-alanine) and L- amino acid ((S)-alanine) ................................. 78 Figure 4.3 Peptide sequence of GKP peptide. ......................................................................... 78 Figure 4.4 Peptide sequence of the L and D- GKP peptides with the adamantane moiety on the N terminus .......................................................................................................................... 79 Figure 4.5 Peptide sequence of the GKP peptide with the palmitic acid moiety on the N terminus.................................................................................................................................... 80 Figure 4.6 Peptide sequence of the GKP peptide with the RGD moiety on the N terminus ... 81 Figure 4.7 UHPLC-MS spectrum of crude GKPILFF peptide on the first attempt ................. 87 Figure 4.8 Side-by-side comparison of HBTU and HATU ..................................................... 89 Figure 4.9 Side-by-side comparison of DIPEA and NMM ..................................................... 91 Figure 4.10 LCMS spectrum of the crude sample after optimization of the synthesis ............ 92 Figure 4.11 LCMS of purified GKPILFF ................................................................................ 94 Figure 4.12 LCMS of crude GKPILFF-ada peptide ................................................................ 95 Figure 4.13 LCMS of purified RGDGKPILFF peptide ........................................................... 97 Figure 4.14 LCMS of purified RLRLRIGRR peptide sequence ............................................. 98 Figure 5.1 An illustration of the primary structure of the GKPILFF penetration acceleration sequence ................................................................................................................................. 108 Figure 5.2 1H NMR spectrum of the L-GKPILFF peptide. ................................................... 109 Figure 5.3 An amide region in the 1H NMR spectra of the GKPILFF peptides a) L peptide and b) D peptide. .................................................................................................................... 110 Figure 5.4 A TOCSY NMR spectra of the GKPILFF peptide in the amide region with dashed lines representing each spin system between 7.75 ppm and 8.70 ppm for a) the L peptide and b) D-peptide. .......................................................................................................................... 111 Figure 5.5 A Sequential walk of the ROESY spectra in the region between 8.85 and 7.55 ppm connecting Gly-1, Lys-2, and Pro-3. ...................................................................................... 112 Figure 5.6 A magnification of the sequential walk on the ROESY spectra in the region between 8.02 and 7.85 ppm connecting Ile-4, leu-5, Phe-6, and Phe-7................................. 113 Figure 5.7 A ROESY overlapping spectra of the L- GKPILFF peptide in red and blue and D- GKPILFF peptide in yellow and turquoise in the region between 8.70 and 7.65 ppm. ........ 115 Figure 5.8 A magnification of the HSQC NMR spectra in the region between 7.31 and 7.04 ppm in the F2(X) axis and 132.5 and 124.5 ppm in the F1(Y) axis for the L-peptide .......... 116 xvi Figure 5.9 The CD spectra of α-helical peptides and the secondary structure of the GKPILFF penetration acceleration sequence for a) the L peptide in orange and b) the D-peptide in blue. ................................................................................................................................................ 118 Figure 5.10 The helical wheel projection of the GKPILFF penetration acceleration sequence for a) the L peptide and b) the D-peptide ............................................................................... 120 Figure 5.11 A magnification of the ROESY spectra in the region between 3.10 and 2.60 ppm in the F2(X) axis and 5.50 and 4.10 ppm in the F1(Y) axis for a) the L peptide and b) D- peptide showing the difference in the long-range interactions. ............................................. 121 Figure 5.12 The structure of the linear L-GKPILFF peptide and its folded state after MD simulations at 325 K. ............................................................................................................. 122 Figure 5.13 The structure of the linear D-GKPILFF peptide and its folded state after MD simulations at 325 K. ............................................................................................................. 122 Figure 5.14 The potential energy and root-mean-square deviation (RSMD) analyses of the L- and D- GKPILFF peptide....................................................................................................... 123 Figure 5.15 Figure 5.15 The Ramachandran plots show the stereochemical analysis of the peptides’ psi (ɸ) – phi (Ψ) dihedral angles. (a) L-peptide and (b) D-peptide. The red regions represent the favoured region, the yellow represents the allowed regions, and the white rep................................................................................................................................. 123 Figure 6.1 Illustration of the synthesized carbon dots conjugated to the peptide .................. 134 Figure 6.2 Absorption spectra of GKP, GKP-Iso-N-Ge-CDs, and Iso-N-Ge-CDs ............... 136 Figure 6.3 Side by side comparison of all the GKP peptide analogue-conjugates against the Iso-N-Ge-CDs. Black line represents the GKP peptide derivative, blue line represents the Iso- N-Ge-CDs and the green/lime line Conjugate. ...................................................................... 137 Figure 6.4 Side by side comparison of all the BP peptide analougue-conjugates against the Iso-N-Ge-CDs. Black line represents the BP peptide derivative, blue line represents the Iso- N-Ge-CDs and the green/lime line Conjugate. ...................................................................... 139 Figure 6.5 PL data for GKP in black, Iso-N-Ge-CDs in blue, and GKP-Iso-N-Ge-CDs in green ....................................................................................................................................... 141 Figure 6.6 PL study on the effects of conjugation by different CDs. Black line represents the GKP peptide derivative, blue line represents the Iso-N-Ge carbon dots -and the green/lime line Conjugate. ....................................................................................................................... 144 xvii Figure 6.7 PL study on the effects of conjugation by different CDs. Black line represents the BP peptide derivative, blue line represents the Iso N Ge carbon dots -and the green/lime line Conjugate. .............................................................................................................................. 146 Figure 6.8 PL spectra of the GKP-Iso-N-Ge-CDs conjugate at various excitations ............. 148 Figure 6.9 pH study on the GKP-Iso-N-Ge-CDs ................................................................... 149 Figure 6.10 Stacked 1H NMR spectra of urea (pink), CDs before (red), and after dialysis (brown) in deuterated DMSO. ............................................................................................... 151 Figure 6.11 Overlapping 1H NMR spectra of urea (pink), CDs before (red), and after dialysis (brown) in deuterated DMSO. ............................................................................................... 152 Figure 6.12 The COSY NMR of the CDs after dialysis and an insert of the allylic region between 2.5 and 0.4 ppm a) COSY and HSQC NMR. .......................................................... 153 Figure 6.13 The HMBC NMR of the a) CDs after dialysis and b) the allylic region between 2.0 and 0.4 ppm ...................................................................................................................... 154 Figure 6.14 The 13C NMR of the a) GKP-Iso-N-Ge-CDs conjugate and the b) Iso-N-Ge-CDs ................................................................................................................................................ 155 Figure 6.15 The overlapping 1H NMR spectra. Indicated in red and blue/green, are the spectra of the GKP-Iso-NGe-CDs conjugate and GKP peptide ......................................................... 156 Figure 6.16 The ROESY NMR of the a) Iso-N-Ge-CDs, b) GKP conjugate and of c) GKP- Iso-N-Ge-CDs ........................................................................................................................ 157 Figure 6.17 Peptide wrapping around the CDs ...................................................................... 158 Figure 6.18 The mechanisms of the CDs formed as well as the possible structure of the final conjugates. ............................................................................................................................. 159 Figure 6.19 The COSY NMR spectra show the 1H-1H correlations within the a) GKP-Iso-N- Ge-CDs conjugate and b) GKP. ............................................................................................. 160 Figure 6.20 The overlapping of the peptide and the conjugate COSY spectra, with the peptide in red and the conjugate in green. .......................................................................................... 160 Figure 6.21 COSY NMR of four overlapping sections with the peptide in red and the conjugate in green. ................................................................................................................. 162 Figure 6.22 This shows the HSQC NMR of the peptide in red and the conjugate in green. . 163 Figure 6.23 This illustrates the HSQC NMR of the a) aromatic region and b) methyl region of the peptide in red and the conjugate in green. ....................................................................... 164 xviii Figure 6.24 This shows the 1H NMR of the a) GKP-Iso-N-Ge-CDs for b) GKP-ada-Iso-N- Ge-CDs c) GKP-pal-Iso-N-Ge-CDs d) D-GKP-Iso-N-Ge-CDs and e) D-GKP-ada-Iso-N-Ge- CDs f) D-GKP pal-Iso-N-Ge-CDs of the conjugate in red and the peptides in blue. ............ 165 Figure 6.25 The 1H NMR of the a) BP-Iso-N-Ge-CDs for b) BP-ada-Iso-N-Ge-CDs c) BP- pal-Iso-N-Ge-CDs, d) D-BP-Iso-N-Ge-CDs, and e) D-BP-ada-Iso-N-Ge-CDs, f) D-BP-pal- Iso-N-Ge-CDs of the conjugate in red and the peptides in blue. ........................................... 166 List of tables Table 3.1 CDs microwave-assisted synthesis conditions ........................................................ 52 Table 3.2 Data derived from the Photoluminescence data. ..................................................... 61 Table 4.1 K4G7W peptide analogues investigated by Dorit Avrahami et al. conjugated to Palmitic Acid and D-amino acids ............................................................................................ 76 Table 4.2 Peptide analogues and the modification .................................................................. 82 Table 4.3 Modification of the synthesis procedure of GKP peptide for optimization ............. 91 Table 4.4 Summary of peptides derived from GKPILFF and RLRLRIGRR sequences. ........ 99 Table 5.1 Assignment of NMR chemical shifts (ppm) of long-range interaction in L- GKPILFF ............................................................................................................................... 114 Table 5.2 Full assignment of all the protons and carbons in the L-peptide. .......................... 117 Table 5.3 Peptide fold occurrence in the dataset ................................................................... 119 Table 5.4 Stereochemical analysis of the peptides’ backbone psi (ɸ) – phi (Ψ) parameters of each amino acid residue ......................................................................................................... 124 Table 6.1 List of GKP and BP peptide analogues and their respective sequences, which were conjugated to Iso-N-Ge-CDs ................................................................................................. 132 Table 6.2 Summary of the peaks observed in the UV-Vis spectrometer of GKP conjugates. ................................................................................................................................................ 138 Table 6.3 Summary of the peaks observed by the UV-Vis spectrometer of the BP conjugates. ................................................................................................................................................ 140 Table 6.4 Summary of the peaks observed by the Photoluminescent spectrometer for GKP peptide derivatives and the conjugates. ................................................................................. 145 Table 6.5 Summary of the peaks observed by the Photoluminescent spectrometer for BP peptide derivatives and the conjugates. ................................................................................. 147 xix Table 6.6 The summarized NMR data of the short chain confirming the pentanal structure. ................................................................................................................................................ 154 Table 6.7 The summarized NMR data of the carbon dots ..................................................... 155 Table 8.1 List of the precursors for the synthesis of the carbon dots .................................... 181 Table 8.2 List of the amino acid (0.2 M) added to the resin together with NMM (1.0 M) and HBTU (0.19 M) during synthesis. ......................................................................................... 184 List of schemes Scheme 3.1 A simple illustration of the fabrication process for the various CDs and Ge-CDs composites presented in this work. .......................................................................................... 52 Scheme 4.1 General synthetic scheme for peptides ................................................................ 84 Scheme 4.2 Mechanism of deprotonation with DIPEA and activation with HBTU .............. 89 Scheme 4.3 Mechanism of piperidine ..................................................................................... 90 Scheme 4.4 Optimized procedure for the synthesis of peptides ............................................. 93 Scheme 5.1 Synthetic scheme of peptide formation ............................................................. 107 xx List of abbreviations AA Amino Acid Ada adamantane carboxylic acid ACN Acetonitrile CDs Carbon dots CD circular dichroism CPPs cell-penetrating peptides AlCl3 Aluminium chloride Boc tert-butyloxycarbonyl CDCl3 deuterated chloroform COSY correlated spectroscopy (CD3)2SO2 Deuterated Dimethylsulfoxide DCM Dichloromethane DIC 1, 3-diisopropyl carbodiimide DIPEA N, N-diisopropylethylamine DMF N, N-dimethylformamide DMSO Dimethylsulfoxide EDT 1,2-Ethanedithiol EtOAc Ethyl acetate FDA Food and Drug Administration Fmoc 9-Fluorenylmethoxycarbonyl FWHM Full width at half maxima xxi HATU Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium HBTU O-Benzotriazole- N,N,N',N'-tetramethyl-uronium hexafluorophosphate HOBt 1-Hydroxybenzotriazole Hz Hertz UHPLC Ultra High-Performance Liquid Chromatography Prep-HPLC Preparative High-Performance Liquid Chromatography Ppm parts per million HRMS High-resolution mass spectroscopy Mg Milligram MIC Minimum Inhibitory Concentration Min Minute ml Milliliter mM Millimolar MS Mass spectrometry m.p Melting point m Multiplet m/z mass to charge ratio nm Nanometres NMR Nuclear Magnetic Resonance NMM N-Methylmorpholine NOESY Nuclear Overhauser Effect Spectroscopy PDCs Peptide–drug conjugates xxii ROESY Rotating Frame Overhauser Enhancement Spectroscopy SPPS Solid phase peptide synthesis SPR Surface plasmon resonance TEM Transmission electron microscopy TFA Trifluoroacetic acid TIPS Triisopropyl silane TIC Total ion chromatograms TOCSY total related spectroscopy μg Microgram μL Microliter TLC Thin Layer Chromatography UV-VIS Ultraviolet-visible spectroscopy 1 1. Introduction 1.1 Background and motivation Carbon dots (CDs) are non-toxic and extremely biocompatible nanoparticles, making them suitable for use in biological applications.1 They are highly photostable, which means they may be exposed to light repeatedly without deteriorating or losing their luminous qualities.2 CDs are also known to have a high fluorescence quantum yield, which indicates that when activated by light, they create intense fluorescence signals.3 These properties of CDs make them extremely sensitive to bioimaging and suitable for long-term bioimaging research. CDs may be targeted to cancer cells with remarkable selectivity by conjugating them with certain peptides.4,5 This allows for the identification of cancer cells while ignoring healthy ones. Therefore, carbon dot-peptide conjugates have the potential to increase bioimaging sensitivity and specificity for cancer detection and surveillance, as well as to enable multimodal imaging for more thorough cancer diagnosis. 1.2 Problem statement According to The World Health Organization, cancer is the leading cause of death globally and was responsible for almost 10 million deaths and 19.3 million new cancer cases in 2020.5 Current technologies for the treatment of cancer include surgery, radiation therapy, and chemotherapy which are toxic to the body.6 Remission of cancer might last months, years, or one’s entire life with current treatment. However, it does not always imply that one is cancer- free or cured.7 Additionally, overdiagnosis, false positive/negative results, and low sensitivity of instruments are one of the challenges faced when diagnosing cancer. Patients labeled as ‘cancer-free’ sometime develop cancer again even after years.8 This calls for new technologies for diagnosing cancer that have high imaging efficiency and is less harmful to the body. This work proposes the use of nanotechnology for bio-imaging and monitoring methods using nitrogen-doped carbon dots (N-CDs) with germanium to be conjugated to tumour-homing peptides. This conjugate should be able to target cancer for imaging. Germanium, which is known to have anticancer properties, was successfully incorporated into the as-synthesized carbon dots. Optical and photoluminescence properties of GeO2 have 2 been well documented and which was incorporated into the carbon dots. However, Germanium, which is known to have anticancer properties, was successfully incorporated into the as-synthesized carbon dots.9 Spiro-germanium Compounds, 2-Carboxyethyl Germanium Sesquioxide (Ge-132), Amino Acid Germanium, and Lactic Acid-Citrate Germanium are organogermanium compounds that have been proven to have activity against tumors and have been incorporated into drugs. A study conducted by Mark G. Mainwaring and colleagues at the University of Florida College of Medicine and Veterans Affairs Medical Center, Gainesville, demonstrated that treatment of Spindle cell carcinoma with orally administered germanium sesquioxide could cause complete remission.10 Herein, cancer-selective bio-imaging carbon dot-peptide conjugate was explored. The incorporation of germanium to the N-doped carbon dots is an area that has scarce information and we hypothesized that it may exhibit superior fluorescence. Additionally, the peptides used in this study are known for their enhanced permeability and selectivity which are crucial properties for conjugates. 1.3 Carbon Dots as Potential Cancer Diagnostic and Therapeutic Agents The increasing demand for low-cost and highly efficient diagnostic and therapeutic agents is increasing exponentially globally, which requires research and development for creating new and innovative drugs and diagnostics. Numerous well-known and effective drugs should be reformulated to provide enhanced efficiency or more beneficial therapy in efforts to meet this demand. A nanotherapeutic delivery and diagnostic system are drug delivery concepts in nanotechnology, which generally refers to a technique that develops nanoscale devices (1 to 100 nm) for enhanced delivery of small drug molecules to the cells of interest for treatment or detection.11, 12 In 2005, the Food and Drug Administration (FDA) approved Abraxane as the first commercially available nanomedicine for metastatic breast cancer, which encapsulates the anticancer drug paclitaxel within protein (albumin) nanoparticles.13 Other nanomedicines encapsulated with albumin include 5-fluorouracil, doxorubicin, 10-hydroxy camptothecin, and methotrexate, where chemotherapy drugs were utilized.14 This shows that conjugates 3 have great prospects in the medical field, and selective bio-imaging conjugates may address the limitations of current technologies for bioimaging. 1.4 Limitations of current technologies Imaging tools such as Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and Computed Tompography (CT) scans can assist to diagnose cancer and identify the amount of its spread, a process known as staging.15 This information is essential for selecting the best treatment and predicting prognosis.16 Despite developments in imaging technology, visualizing malignancies or tumours still poses various problems. Among the current challenges are sensitivity, selectivity towards cancer cells and deep penetration of the cells.17,18 This means that emerging imaging technology should be able to identify even tiny tumours while also discriminating between diseased and non-cancerous cells with accuracy. Although imaging is crucial in cancer diagnosis and therapy, more research and innovation are still needed to overcome these problems and enhance accuracy and sensitivity. In this case, cancer-selective carbon-peptide conjugates will be used to overcome these challenges. 1.5 Conjugation of carbon dots to peptides for bio-imaging There are several conjugate systems with promising results such as antibody-dye conjugates which are agents that can detect and bind to particular targets, such as proteins or cells by labelling antibodies with fluorescent but used dyes with dose-limiting toxicities.19 Other bio- imaging conjugates include quantum dot-protein conjugates, green fluorescent protein (GFP) and magnetic nanoparticle-antibody conjugates.20, 21, 22 The drawback with these conjugates is that they often make use of chemical dyes which can be toxic if used inappropriately.23 Although they can be highly selective, the cost of producing synthetic antibodies or proteins can be high depending on several factors, including the complexity of the molecule, the type and scale of production method used, and the purification and characterization requirements.24 Carbon dots are metal free, biocompatible and cheap to synthesize because waste can be used as a carbon source. Additionally, short peptides significantly reduce the cost of synthesis because fewer reagents are used and purification is more facile.25 4 1.6 Aim and objectives Aim The purpose of this research work is to synthesize carbon dot-peptide conjugates to enhance the bio-imaging capacity and selectivity of the conjugates. Factors that affect permeability include bioavailability, potency, stability, and half-life of the drug in the body. Theragnostic is a rising area of study which is a combination of therapeutics and diagnostics. To achieve the aims of this thesis, the following objectives were outlined: • Synthesize N-doped carbon dots to enhance fluorescence, using a microwave assisted synthesis method. • Synthesize Ge-modified carbon dots to add anticancer properties and investigate other defects that may introduce enhanced fluorescent properties to the carbon dots. • Synthesize L and D amino acid derivatives of cell-penetrating and/or cancer-homing peptides for structure-activity studies. • Synthesize peptides with adamantane and palmitoyl moieties to enhance cell permeability. • To fully elucidate the synthesized peptide analogues using NMR for insight into the structure vs. activity study. • To determine the optical properties of the nanoparticles and the conjugates by using ultraviolet-visible spectroscopy and photoluminescence spectroscopy. • To investigate the bio-imaging capacity of the nanoparticles and the conjugates by using optical fluorescent microscopy 5 1.7 The study's novelty and prospective applications in the context of South Africa The preparation of carbon dots (CDs) and nitrogen-doped carbon dots (NCDs) with germanium oxide (GeO2) using a facile one-step microwave-assisted synthesis procedure was explored. Studies have shown that the CDs, NCDs, and GeO2 nanoparticles individually have excellent qualities for bio-imaging applications, including photoluminescence, biocompatibility, good stability, and low toxicity.26 ,27 While CDs have been thoroughly explored in literature, CDs with germanium oxide on their surface are an avenue that has scarce information. To enlarge the scope of these CDs-Ge/GeO2 composites in practical applications, particularly bioimaging applications, this study reports on a simple microwave reaction for the synthesis of germanium oxide-modified NCDs. Iso-ascorbic acid and urea were utilized to evaluate the response of Ge-132. Ge-NCDs produced were fluorescent, water-soluble, and emitted different colors due to the different sizes. To the best of our knowledge, the effect of both germanium and nitrogen on the photoluminescent traits of CDs has not been reported. We found that the CDs modified with germanium oxide have excellent photoluminescent traits, which are crucial for bio-imaging. The short penetration accelerating sequence (PAS) has gained increasing attention from researchers due to its ability to enhance cellular uptake.28 The seven-amino acid sequence recognized by lysosomal protease cathepsin D promotes the endosomal escape of cell- penetrating peptides (CPPs) and improves the cytosolic release of the delivered cargo.29 To date, the peptide's mechanism is still unknown, and there is no available NMR data or other structural information. This is a major limitation in understanding the mechanism of penetration and how the peptide interacts with the target site.30 In short, carbon dot-peptide conjugates offer a lot of potential in bioimaging.31 It is feasible to generate fluorescent probes that may selectively target certain cells or tissues by conjugating peptides to carbon dots, giving a potent tool for observing biological processes. Carbon dots' strong photostability makes them useful for long-term imaging research. Many studies have shown that carbon dot-peptide conjugates may be used for bioimaging applications such as in 6 vivo imaging of malignancies, imaging of cellular uptake, and imaging of protein-protein interactions and this study expands the scope of carbon dot-peptide conjugates. 7 1.8 Outline of the thesis Chapter 1 This chapter serves as a layout of the context and rationale for drug delivery for anticancer drugs and emphasizes the need for a new class of drugs that are multifaceted to overcome the current limitations and enhance pharmacokinetic properties. Moreover, the main aim and objectives of the study are also discussed. Chapter 2 First, the impact of tumours and cancerous diseases were reviewed. Subsequently, carbon dots and peptides are thoroughly explored to fully understand the synergetic effects of the Peptide-Carbon dot conjugates for treatment and drug delivery. Furthermore, the target sites and receptors involved in enhanced drug delivery were examined. Chapter 3 The synthesis and characterization of carbon dots were investigated, where we evaluated the effects of introducing germanium into the CDs as well as doping with nitrogen. The nitrogen doped CDs containing germanium were found to have the highest photoluminescent properties. Chapter 4 Herein, the synthesis and characterization of peptide derivatives were analysed following the conjugation of the peptides to the carbon dots. There were 13 peptides which were successfully synthesized, and the different retention times were indicative of different structures for each peptide derivative. These peptides were variations of the penetration accelerating peptide (sequence: GKPILFF) and cancer targeting peptide (sequence: RLRLRLIGRR). Chapter 5 In this section, the full elucidation of the penetration accelerating GKPILFF peptide derivatives was examined to provide further insight into drug delivery and bioactivity. The peptide was found to have a helical structure known to enhance stability in bioactive peptides. 8 Chapter 6 In this part, the full elucidation of the conjugates was examined to provide further insight into drug delivery and bioactivity. Long-range interactions between the peptide and the CDs were confirmed in the ROESY NMR spectra of the conjugates. Chapter 7 Finally, this chapter will supply overall conclusions and recommendations for possible future work on the synthesis of carbon dot Peptide conjugates for drug delivery and treatment of cancer. The NMR results of the conjugates indicate that the helical structure may be impacted due to noticeable peak shifts. Considering the potential influence of the helical structure on its activity, conjugates with NMR spectra showing less structural deviation from the peptide may possess more favourable biological properties. These promising options include GKP-Iso-N Ge, GKP-ada-Iso-N-Ge, BP-pal-Iso-N-Ge, and DBP-ada-Iso-N-Ge, which still exhibit distinguishable peaks compared to the original peptide. Chapter 8 The detailed procedures for synthesizing the carbon dots and peptides are fully explained in this chapter. Additionally, the instruments used for characterization and calculations used was presented. 9 1.9 References 1. Wang, Y., Zhu, Y., Yu, S. & Jiang, C. Fluorescent carbon dots: rational synthesis, tunable optical properties and analytical applications. RSC Adv. 7, 40973–40989 (2017). 2. Peng, Z., Miyanji, E. H., Zhou, Y., Pardo, J., Hettiarachchi, S. D., Li, S., Blackwelder, P. L., Skromne, I. & Leblanc, R. M. Carbon dots: promising biomaterials for bone-specific imaging and drug delivery. Nanoscale 9, 17533–17543 (2017). 3. Zhang, Y., Liu, X., Fan, Y., Guo, X., Zhou, L., Lv, Y. & Lin, J. One-step microwave synthesis of N-doped hydroxyl-functionalized carbon dots with ultra-high fluorescence quantum yields. Nanoscale 8, 15281–15287 (2016). 4. Zhang, Q., Zhang, Y., Li, K., Wang, H., Li, H. & Zheng, J. A Novel Strategy to Improve the Therapeutic Efficacy of Gemcitabine for Non-Small Cell Lung Cancer by the Tumor- Penetrating Peptide iRGD. PLoS ONE 10, e0129865 (2015). 5. Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A. & Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA. Cancer J. Clin. 71, 209–249 (2021). 6. Abola, M. V., Prasad, V. & Jena, A. B. Association between treatment toxicity and outcomes in oncology clinical trials. Ann. Oncol. 25, 2284–2289 (2014). 7. Imura, M., Katada, J. & Shiga, T. Epidemiological Study Regarding the Incidence of Venous Thromboembolism in Patients After Cancer Remission. Cardiol. Ther. 11, 611– 623 (2022). 8. Understanding Cancer Prognosis - NCI. (2014). at (Last accessed: 21 Oct. 2023) 9. Schauss, A. G. Nephrotoxicity and neurotoxicity in humans from organogermanium compounds and germanium dioxide. Biol. Trace Elem. Res. 29, 267–280 (1991). 10. Li, L., Ruan, T., Lyu, Y. & Wu, B. Advances in Effect of Germanium or Germanium Compounds on Animals—A Review. J. Biosci. Med. 5, 56–73 (2017). 11. Sinha, R., Kim, G. J., Nie, S. & Shin, D. M. Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery. Mol. Cancer Ther. 5, 1909–1917 (2006). 12. Werengowska-Ciećwierz, K., Wiśniewski, M., Terzyk, A. P. & Furmaniak, S. The Chemistry of Bioconjugation in Nanoparticles-Based Drug Delivery System. Adv. Condens. Matter Phys. 2015, e198175 (2015). 10 13. Zhong, H., Chan, G., Hu, Y., Hu, H. & Ouyang, D. A Comprehensive Map of FDA- Approved Pharmaceutical Products. Pharmaceutics 10, 263 (2018). 14. Lee, S., Trinh, T. H. T., Yoo, M., Shin, J., Lee, H., Kim, J., Hwang, E., Lim, Y. & Ryou, C. Self-Assembling Peptides and Their Application in the Treatment of Diseases. Int. J. Mol. Sci. 20, 5850 (2019). 15. Bagci, U., Udupa, J. K., Mendhiratta, N., Foster, B., Xu, Z., Yao, J., Chen, X. & Mollura, D. J. Joint segmentation of anatomical and functional images: Applications in quantification of lesions from PET, PET-CT, MRI-PET, and MRI-PET-CT images. Med. Image Anal. 17, 929–945 (2013). 16. Wang, Z. J., Reddy, G. P., Gotway, M. B., Higgins, C. B., Jablons, D. M., Ramaswamy, M., Hawkins, R. A. & Webb, W. R. Malignant Pleural Mesothelioma: Evaluation with CT, MR Imaging, and PET. RadioGraphics 24, 105–119 (2004). 17. O’Farrell, A., Shnyder, S., Marston, G., Coletta, P. & Gill, J. Non-invasive molecular imaging for preclinical cancer therapeutic development. Br. J. Pharmacol. 169, 719–735 (2013). 18. Heidenreich - 2011 - Consensus Criteria for the Use of Magnetic Resonan.pdf. at (Last accessed: 21 Oct. 2023) 19. Lu, G., Nishio, N., van den Berg, N. S., Martin, B. A., Fakurnejad, S., van Keulen, S., Colevas, A. D., Thurber, G. M. & Rosenthal, E. L. Co-administered antibody improves penetration of antibody–dye conjugate into human cancers with implications for antibody–drug conjugates. Nat. Commun. 11, 5667 (2020). 20. Bilan, R., Nabiev, I. & Sukhanova, A. Quantum Dot-Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery. ChemBioChem 17, 2103–2114 (2016). 21. Miyawaki, A. & Niino, Y. Molecular Spies for Bioimaging—Fluorescent Protein-Based Probes. Mol. Cell 58, 632–643 (2015). 22. Kale, A., Kale, S., Yadav, P., Gholap, H., Pasricha, R., Jog, J. P., Lefez, B., Hannoyer, B., Shastry, P. & Ogale, S. Magnetite/CdTe magnetic–fluorescent composite nanosystem for magnetic separation and bio-imaging. Nanotechnology 22, 225101 (2011). 23. Wang, Y., Hu, R., Lin, G., Roy, I. & Yong, K.-T. Functionalized Quantum Dots for Biosensing and Bioimaging and Concerns on Toxicity. ACS Appl. Mater. Interfaces 5, 2786–2799 (2013). 11 24. Roque, A. C. A., Lowe, C. R. & Taipa, M. Â. Antibodies and Genetically Engineered Related Molecules: Production and Purification. Biotechnol. Prog. 20, 639–654 (2004). 25. B. Essner, J., H. Laber, C., Ravula, S., Polo-Parada, L. & A. Baker, G. Pee-dots: biocompatible fluorescent carbon dots derived from the upcycling of urine. Green Chem. 18, 243–250 (2016). 26. Edison, T. N. J. I., Atchudan, R., Sethuraman, M. G., Shim, J.-J. & Lee, Y. R. Microwave assisted green synthesis of fluorescent N-doped carbon dots: Cytotoxicity and bio- imaging applications. J. Photochem. Photobiol. B 161, 154–161 (2016). 27. Karatutlu, A., Song, M., P. Wheeler, A., Ersoy, O., R. Little, W., Zhang, Y., Puech, P., S. Boi, F., Luklinska, Z. & V. Sapelkin, A. Synthesis and structure of free-standing germanium quantum dots and their application in live cell imaging. RSC Adv. 5, 20566– 20573 (2015). 28. Takayama, K., Hirose, H., Tanaka, G., Pujals, S., Katayama, S., Nakase, I. & Futaki, S. Effect of the Attachment of a Penetration Accelerating Sequence and the Influence of Hydrophobicity on Octaarginine-Mediated Intracellular Delivery. Mol. Pharm. 9, 1222– 1230 (2012). 29. Woldetsadik, A. D., Vogel, M. C., Rabeh, W. M. & Magzoub, M. Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells. FASEB J. 31, 2168–2184 (2017). 30. Majumdar, S. and Siahaan, T.J. Peptide‐mediated targeted drug delivery. Medicinal research reviews, 32, 637-658 (2012). 31. Li, J., Wu, D., Miao, Z. and Zhang, Y. Preparation of quantum dot bioconjugates and their applications in bio-imaging. Current Pharmaceutical Biotechnology, 11, pp.662-671 (2010). 12 2. Literature review 2.1 Introduction This Chapter investigates the prevalence of cancer-related mortality internationally and nationally. The imaging of tumors is important for prognosis and treatment; this is an area where the development of nanotechnology could assist. Carbon dots conjugated to peptides have been studied for their potential use in the bio-imaging of cancer cells, and this is explored in this chapter. Furthermore, by conjugating peptides to the surface of carbon dots, they can selectively target cancer cells or biomolecules that are overexpressed on cancer cells, allowing for more precise imaging of the tumor. The effects of doping carbon dots and manipulating peptides were discussed to further understand the structure-activity relationship. 2.2 Global and local impact of diseases caused by cancer and tumors At the end of 2020, the Global Cancer Observatory (GLOBOCAN) 2020 supplied an updated approximation of cancer-related mortality and prevalence which was estimated at 19.3 million new cancer cases globally and nearly 10.0 million people deaths in the year 2020.1 It is caused by damaged cells due to malfunctions during cell division induced by harmful substances or inherited DNA genes.2 When a patient develops a tumor that leads to cancer, it can be due to the environment that the patient was exposed to or their lifestyle habits such as smoking, which accounts for 90-95% of reported tumor cases; moreover, only 5% and less of the cases are due to inheritable defected genes from other family members.3 In South Africa, 56 802 people died of cancer in 2021, as seen in Figure 2.1, according to the American Society of Clinical Oncology (ASCO) Post, and the deadliest forms of cancer were found to be lung cancer, oesophagal cancer, and cervical cancer.4 Mine workers are often exposed to excessive chemicals underground, which cause them to develop cancer. A clinical study of South African mine workers by Ndlovu and colleagues at the School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, assessed the asbestos-related diseases in mine workers and demonstrated that the chemical exposure underground caused asbestosis, mesothelioma, and lung cancer.5,6 Exposure to UV rays from the sun and strong air pollution also cause cancer. Studies show 13 that an unhealthy lifestyle can also induce cancer, such as smoking, passive smoking, drinking alcohol, and obesity.7 Figure 2.1 Stats as per The ASCO Post, in partnership with the American Society of Clinical Oncology 2021. 8 Any kind of swelling caused by abnormal growth of tissue is regarded as a tumor, but not all tumors are cancerous or malignant.9 Normally, dead cells that have gone through apoptosis are replaced by new cells, but tumors form when the dead cells remain to form a growth. Life-threatening tumors are classified as malignant tumors because they can spread throughout the body and also limit the function of other organs. Malignant tumors can be surgically removed, but regrowth is possible.10 On the other hand, benign tumors pose a lower health risk because they do not spread in the body and can often be left untreated.11 2.2.1 Treatment and monitoring Imaging procedures play a crucial role in determining the appropriate treatment and location of malignant tumors, as well as determining the stage of development.12 More research has gone into diagnosing and treating malignant tumors because they are fatal and aggressive. 13 One of the most important aspects of treating malignant tumors is monitoring their size and growth. This can assist in determining the correct treatment and help monitor the response of the tumor toward certain treatments. One way to image and monitor the size and growth of the tumor is through the use of nanotechnology. Nanomaterials such as gold nanoparticles, iron oxide nanoparticles, and quantum dots have been explored extensively for tumor monitoring due to their unique optical properties and biocompatibility.14 These nanomaterials can be functionalized with targeting molecules such as antibodies or peptides to specifically bind to cancer cells, making them useful for early cancer diagnosis and targeted therapy.15 14 Among the carbon nanomaterial family, carbon dots (CDs) have the potential to be used in tumor monitoring due to their unique optical properties and biocompatibility.16 CDs are small carbon-based nanoparticles (diameters less than 10 nm) that exhibit strong fluorescence, making them ideal for use in biological imaging.17 In addition, CDs have low toxicity and can be easily modified to target specific cells or tissues. Moreover, the competitive advantage CDs possess over other nanomaterials is that they can be made from waste materials such as agricultural waste, food waste, and industrial waste, which makes their production cheap and sustainable.18 Several studies have demonstrated the potential of CDs for tumor imaging and monitoring.19 For example, CDs have been used to label cancer cells and track their migration and proliferation in vivo.20 Additionally, they have been functionalized with targeting molecules such as antibodies or peptides to specifically bind to cancer cells, making them useful for early cancer diagnosis and targeted therapy.21 2.3 Carbon dots 2.3.1 Carbon dots – what are they? CDs are nanoparticles that are less than 10 nm in sizes and predominantly contain carbon atoms, as seen in Figure 2.2.22 These nanoparticles are biocompatible, which means that they are not harmful to living tissue and maintain their efficiency in vitro and in vivo. 23,24 These nanoparticles have strong luminescent properties and give off bright colors under UV light, which allows for many bio-applications to emerge, such as fluorescence bio-imaging, bio- analysis, and micro-arrays. CDs have both excellent antimicrobial properties and anti-cancer properties. 25 15 Figure 2.2 This is an illustration of a carbon dot with functional groups. 2.3.2 The potential of carbon dots for bio-application Several studies have been conducted with CDs which explore the potential of using them in the medical industry, especially for cancer.26 Below is a list of some of the fluorescent bio- applications of carbon nanomaterials that have been used in biology or medicine: ● Fluorescent biological labels CDs have been employed in a variety of biological applications, including cellular imaging, bioimaging, and biosensing. 21 They are useful in both in vitro and in vivo labelling of diverse biological substances such as proteins, nucleic acids, and lipids.22 One advantage of utilizing CDs as fluorescent biological identifiers is their high photostability, which means they do not easily bleach or fade, which is useful for long-term imaging.29 They are also less susceptible to photobleaching and photodamage than standard fluorescent dyes, making them excellent for live cell imaging.30 ● Drug and gene delivery The fluorescence of CDs can be used to trace the distribution of medicines and genes.31 CDs, for example, can be functionalized with specific targeting ligands like antibodies or peptides, allowing them to bind to specific cells or tissues.32 The fluorescence of the CDs can be monitored once they have been picked up by the target cells to follow the intracellular delivery of the medication or gene.33-35 ● Bio-detection of pathogens 16 The fluorescence features of the CDs can be used to detect the presence of the pathogen or biomarker once they have been functionalized and exposed to the target pathogen or biomarker.36 This can be accomplished through the use of several detection techniques like fluorescence microscopy, flow cytometry, and spectroscopy. ● Magnetic resonance imaging (MRI) contrast enhancement The fluorescence and magnetic resonance imaging (MRI) are separate imaging modalities, therefore, the fluorescence properties of CDs are not directly applicable to MRI contrast enhancement.32 However, CDs can be functionalized with magnetic materials such as iron oxide nanoparticles to improve their magnetic characteristics and allow them to be used as MRI contrast agents. 33 Magnetic CDs can also be functionalized with specific targeting ligands like antibodies or peptides to allow them to bind to certain cells or tissues.39 This can improve the sensitivity and specificity of MRI imaging and allow for earlier disease identification. Moreover, the fluorescence of CDs is used for probing DNA structures and bio-sensing, which demonstrates the prospects of fluorescent CDs in the medical field.40, 41 2.3.3 In vivo and in vitro studies using fluorescent carbon dots Carbon nanoparticles for bio-labelling studies are gaining popularity because of their low toxicity and efficiency. One of the advantages of CDs is that they can be manipulated to exhibit bright luminescent properties for bio-imaging experiments.42 CDs are a buildup of organic molecules and are generally made up of carbon, oxygen, and hydrogen.43 They have defected 𝑠𝑝2 domains which cause the surface states to be flawed, inducing the low energy luminescent emission.44 The excitation wavelength can be altered because CDs have excitation-dependent emission properties, which can be used for fluorescence microscopy experiments.45 These experiments are often conducted on living cells or even mice for bio- labelling, bio-sensing, and probing of cells. Bio-labelling is the labelling of a living cell using nanoparticles for organelle and even tumours.42, 43 This can help confirm the capability of the cell uptake of CDs.48 It can also be used to differentiate between various parts of a cell or organelle. A study conducted by Edward Jan and Nicholas A. Kotov 49 proved that it is possible to monitor an embryonic 17 neural stem cell of a mouse as it differentiates into various cells such as neurons, astrocytes, and oligodendrocytes using carbon nanotubes. Many diseases, including tumours, go undetected because the instruments available are not sensitive enough to detect them at an early stage and subsequently detect the disease once fully developed.50 This gives rise to the need for biosensors. Studies have shown that CDs are easily functionalized, which makes it possible to target bio-makers which are present when certain diseases affect the body.51, 52 Additionally, CDs have electrical properties, which will make it easier to produce devices that detect bio-makers at high sensitivity.47 For instance, CA-125 is a protein in the body encoded by the MUC16 gene, which is routinely used as a biomarker for ovarian cancer because the presence of the protein is increased in the blood when cancer is present.47,55 The use of CDs as a biosensor for cancer has shown to have high sensitivity, selectivity, and stability.56 The use of CDs in drug delivery for bio-imaging is very popular because the drug can be used on a confocal microscope to monitor the drug released. Doxorubicin is a strong chemotherapeutic drug that diminishes cancer cells or ceases the growth of cancer cells.57 CDs have a structure composed of carbon and functional groups found on their surface. CDs’ luminous characteristics can be improved via doping. This involves the deliberate insertion of impurities into a substance to change its qualities. Doping CDs can introduce atoms or molecules that act as traps for excited electrons, enhancing the dots’ fluorescence quantum yield. Nitrogen, sulfur, phosphorus, and metal ions such as copper, silver, and gold are common dopants used to improve their luminous properties. The specific dopant utilized will be determined by the desired attributes and the synthesis method used to create the CDs. 2.3.4 N-doped carbon dots decorated with Ge The properties of CDs can be enhanced by doping with various elements, such as nitrogen. For instance, nitrogen doping of carbon is one of the ways of increasing the luminescent properties of CDs which play an important role in bio-imaging.58 CDs have incredible properties, and the use of dopants and co-doping assists in tuning their properties to suit a particular application.59 For example, Fatemeh Khodadadei, and co-workers fabricated 10 nm nitrogen-doped graphene quantum dots (N-GQDs) containing ten graphitic layers.60 18 Additionally, the N-GQDs were loaded with the Methotrexate (MTX) cargo by strong π-π stacking interaction. The layered structure of N-GQDs was exploited to render a high specific area to intercalate the MTX between the layers generating a comprehensive anti-cancer drug delivery system. On the other hand, the properties of CDs have also been modified using additives such as germanium. Germanium is a group 4 metalloid and is prone to have 𝑠𝑝3 like bonding which alters the properties of CDs differently compared to nitrogen.61 Studies have shown that Ge- CD (carbon dots doped with germanium) are biocompatible with exceptional emission properties and can be used for bio-application.62 Organogermanium compounds have been shown to have anti-cancer properties.63 Spirogermanium compounds, 2-carboxyethyl germanium sesquioxide (Ge-132), amino acid germanium, and lactic acid-Citrate germanium are organogermanium compounds that have been proven to have activity against tumours and have been incorporated in drugs.63 A study conducted by Mark G. Mainwaring and colleagues demonstrated that treatment of Spindle cell carcinoma with orally administered germanium sesquioxide could cause complete remission.64 This implies that it can be hypothesized that the N-doped CDs decorated with germanium can have anti-cancer properties.65 When germanium is introduced into the N- doped carbon dot, then germanium oxide, amino acid germanium, and other organogermanium compounds will be present in the CDs, which can cause the CDs to have antitumor properties.66 2.4 Peptides and their bio-application Peptides are molecules containing two or more amino acids and are the building blocks of proteins, as demonstrated in Figure 2.3.67 Attaching a natural peptide ligand or peptidomimetic to an active therapeutic agent can be a direct targeting strategy achieved by binding a receptor that is expressed on the target organ or tumour and undergoes endocytosis.68 19 Figure 2.3 Illustration of how amino acids are building blocks of peptides and how proteins are much larger forms of peptides. On the other hand, stereoisomers can be evaluated as potential ligands for receptors utilizing computation and analysis of peptide libraries. These are employed to elucidate the arrangement of atoms of the peptide in crystalline form, with the prediction of ligand conformation and orientation of the peptides.69 This kind of approach bypasses the necessity for the manual synthesis of numerous peptide libraries and screening. However, the disadvantage is that they primarily hinge on data about the receptor targets, and unknown targets that may exist cannot be screened.70, 71 Moreover, peptides that demonstrate biological activity in silico don’t necessarily have activity in vivo.72 Nonetheless, these strategies can supply an excellent foundation for constructing targeting peptide libraries.73 2.4.1 Cell-penetrating peptides There are several obstacles that most therapeutic agents need to overcome before reaching the intended site to achieve their physiological function. The ability to permeate membranes is a crucial feature that most therapeutic agents require to reach the targeted site. Therefore, molecules or biomolecules that enhance the cell-penetrating properties must be incorporated into active compounds. For example, the GKPILFF fragment as well as the synthetic retro peptide (FFLIPKG) both originate from a cathepsin D enzyme, a lysosomal aspartyl protease and are penetration accelerating sequences (Pas).74 Literature has shown that they were able to substantially enhance cellular uptake when bonded to a bioactive agent.75 20 In a study by Woldetsadik and co-workers,76 the selectivity of a peptide correlating to the mitochondrial membrane–binding N-terminus sequence of HKII (pHK) was investigated by evaluating the apoptotic selective detachment of HKII from mitochondria in cancerous cells. To further improve the properties of the peptide, the penetration-accelerating GKPILFF peptide was covalently coupled to the pHK peptide resulting in enhanced cellular uptake and cell-death efficacy. In another study by Kristensen and co-workers,77 the peptides NR2B9c and N-dimer were fashioned to hinder binding forces between the N-methylated D-aspartate and the postsynaptic density-95 (PSD-95)/disc large/ZO-1 (PDZ) domains of PSD-95 for relieving neuronal damage. Each of these peptides was covalently incorporated into the cell- penetrating peptide TAT to promote permeation and neuro-cell absorption in the highly selective semipermeable barrier of the brain. The peptide conjugates not only effectively penetrated the vascular system of the mouse brain's central nervous system but also exhibited a similar level of accumulation in brain tissue as other significant polymeric nanomedicines.78 2.4.2 Cell homing peptides Targeted delivery increases the efficacy and decreases the side effects caused by the overall toxicity of the corresponding therapeutic compound, and can function either by direct conjugation or via promoted co-administrative effects.79 Most therapeutic agents rely on passive diffusion as transportation across cell membranes; however, one of the main limitations of most pharmaceutical agents, especially highly toxic substances such as chemo drugs, is the inability to target the diseased site without affecting the normal tissue.80 This has led to a need for a new class of drugs that incorporate targeting molecules or biomolecules. Peptides that possess cell-targeting properties often bind to receptors and other molecules present in the cell membrane, which makes them attractive candidates for targeted drug delivery systems for bio-imaging.80 One of the notable tumour-homing peptides is the Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence which has been shown to bind to clotted plasmas proteins present in tumour vessels.81 For instance Zhang, Y. and co-workers designed a nano-vehicle containing the liposome conjugated to the CREKA peptide and loaded with a reversible platelet inhibitor ticagrelor (T). Primarily they showed that platelets could induce the transition of tumour cells, into 21 invasive phenotypes in vitro and thereafter demonstrated that CREKA-Lipo-T can eliminate this effect. These findings showed that CREKA-Lipo-T could efficiently block tumour cell acquisition of an invasive phenotype. 82 Other well-known tumour-homing molecules include folic acid and arginine-glycine- aspartate, which strongly target the folate receptor and integrin αvβ3, respectively.83,84 To harness the properties of both previously mentioned tumour targeting molecules, Yan, H. and co-workers designed the PTX@MSNs-NH2-FA-RGD conjugate to assess its potential to kill MCF-7 cells, and was found to be 1.6 times effective in contrast to the pristine PTX compound, indicating that PTX@MSNs-NH2-FA-RGD had enhanced tumour selective cytotoxicity.85 2.4.3 Cell homing and penetrating peptides Cell homing and penetrating peptides are peptides that have a high affinity to certain receptors or sites found in the cell. Additionally, they also can permeate the cell so that they can perform their physiological function.86 Cell homing and penetrating peptides can also be designed by combing peptides or molecules that can facilitate each function, respectively. This strategy can be accomplished in several ways by utilizing specific moieties such as carboxylic groups, amines, and thiols to name a few.87 Iwasaki, T., and co-workers designed enantiomeric antimicrobial peptide (AMP) analogues derived from beetle defensins and observed their anti-cancer properties in several cancer cell lines. These results demonstrated a negative charge-dependent selectivity in cancer cells attributed to the d-9-mer peptides. Therefore, they have selective cytotoxicity and target anionic phosphatidylserine on the cell surface in the cancer cell membrane. Furthermore, combinations of d-peptide B (RLRLRIGRR-NH2) and the anti-cancer agent dexamethasone demonstrated synergic growth inhibitory activity against mouse myeloma.88 For example, Myrberg, H constructed a cell-homing and penetrating peptide that contained the cyclized peptide sequence, which is a targeting peptide, and the cell-penetrating peptide pVEC. Although the anti-cancer cCPGPEGAGC (PEGA) sequence is unable to penetrate the plasma membrane, it can enter many breast cancer cells in vitro when coupled to the peptide pVEC. Additionally, the conjugate accumulates mostly in the blood veins of tumorous breast 22 tissue, where it is subsequently taken up, and the PEGA-in pVEC’s in vivo cell-directing ability is maintained.89 2.5 Peptide-Carbon dot conjugates Nanostructured delivery systems can be used as a delivery system for peptides.90 Research from over the years has demonstrated that this strategy is capable of improving the absorption of the peptide and half-life.91 Different types of nanoparticles (NPs), such as carbon nanoparticles, micelles, and dendrimers, to name a few, can be used for enhanced selective targeted therapy of peptides facilitated by several chemical interactions. Polymetric and mesoporous silica nanoparticles are the most popular class of nanoparticles utilized for active agents and peptide drugs. Moreover, the exterior of nanoparticles can be modified with specific biomolecules, such as carbohydrates, polypeptides, synthetic polymers, enzymes, glycans, antibodies, nucleic acids, and oligonucleotides (ONTs), to name a few, which can feasibly be employed as targeted delivery moieties or cargo.92, 93 While bioconjugation involves the fusion of two molecules, of which one of them is a biomolecule, normal conjugation occurs when two molecules are bonded together. Both strategies can be facilitated by covalent and non-covalent bonds. Fabrication of bioconjugates often involves straightforward, easy, and normally a one-step reaction which is generally confirmed using characterization techniques such as zeta potential, UV-vis, NMR, and FTIR.94 This technique is also used for the incorporation of synthetic labels, for instance, isotope labels, fluorescent dyes, affinity tags, and biotin, to biological molecules for monitoring the internalization and bioavailability.95, 96 For instance, Lei Yang and co-workers designed conjugates that comprised CDs that were functionalized with polyethylene glycol (PEG),97 which facilitated the covalent conjugation of a nuclear localization signal peptide. Their research showed that the conjugate was biocompatible, and the quantum yield was found to be 75.8%. Furthermore, intracellular localization of the NLS-CDs conjugate in MCF7 and A549 cells was confirmed using confocal laser scanning microscopy images. In a similar study by Yu Cao and co-workers,98 polyethene glycol (PEG) was also used to provide covalent interactions between the graphene quantum dots; however, rather than conjugating a peptide, an aptamer was used to promote internalization and targeted cell- 23 specific therapeutic effects. The GQD-PEG-P conjugate demonstrated effective discrimination of cancer cells from somatic cells and the capacity to detect cancer-related miRNA. In recent years, the integration of diagnostic and therapeutic agents that provide multipurpose functions in one individual nanostructure has proven to be critical to developing a new class of theranostic (a term used to describe the combination of diagnostic and therapeutic).99 These platforms have received profound attention, especially in the area of personalized nanomedicine and clinical application. In another study where the cRGD peptide was coupled to nanoparticles, the cRGD@TAT- DINPs showed increased cytotoxicity on both MDA-MB-231 and A549 cells, according to an in vitro investigation. In particular, combination chemo/photothermal/photodynamic therapy significantly increased the therapeutic efficacy of the produced nanoparticles under NIR light.100 Intricate and versatile platforms can be facilitated by these multifunctional nanostructures, which can open a new gate towards a diagnostic, site-homing, and active treatment using a single multifunctional system. Unfortunately, some of the limitations of such technologies include a limited stability profile, high cost, and short storage life. Additionally, they faced major intrinsic hurdles caused by production batches which led to harsh bench-to-bedside transformation and poor reproducibility. To address these limitations, researchers are finding various ways to improve on these limitations by using cheap resources such as wasted CDs synthesis and facile one-step synthesis to enhance reproducibility.101 At present, another alternative to nanoparticulate delivery systems is bioconjugates. Reports show that medicinal business sector and academic scholars are investing heavily in bioconjugate structures due to the appealing potential features. Bioconjugation is a pervasive technique that finds a multitude of applications in different branches of life sciences, including selective targeted therapy, biosensing biological assays, imaging, and gene delivery applications. Therefore, the intracellular concentrations for each construct must be determined separately before using cell-permeable peptide/cargo constructs for intracellular investigations of structure-function connections. This will ensure that the improved biological activity solely relies on the conjugation of each component. The conjugation to homing peptides is known to 24 bind to the specific receptors present in the cells; however, it may not necessarily penetrate the cells, as seen in Figure 2.4.102 To address this potential challenge, conjugation to a cell- penetrating peptide may be necessary. Another method would be to use a peptide that has both the targeting properties and cell-penetrating properties to deliver the drug cargo. Figure 2.4 Multipurpose functions in one individual nanostructure 2.6 Derivatization of carbon dots and peptide and their effects 2.6.1 Functionalization of carbon dots To facilitate CD modification, molecules with unique chemical characteristics and reducing groups on the surface of CDs can be employed.103 Luminescence properties can be enhanced using synthetic approaches based on known organic chemistry reactions desirable functional groups that can be covalently conjugated to CDs. External functional groups, including carbonyl and carboxyl groups, can facilitate trapping states with different energy levels by treatment of CDs utilizing polymers and organic compounds, producing luminescent CDs that emit light with various energies.104 Additionally, covalent modifications can accomplish enhanced control of the size, stabilization of surface energy traps, and physical features of CDs.105 On the other hand, non-covalent modifications rely on interactions between CDs and the substance being added, known as the London force or the -dispersion force. Non-covalent alterations also have a less detrimental impact on the CDs’ structure than covalent modifications, making it possible to tailor the photoluminescence and physicochemical characteristics.106, 107 Significantly improved biocompatibility and optical properties can be 25 observed after modification with these molecules, and typically the synthesis is straightforward. For instance, if the surface of the CDs readily contains moieties such as amino groups, then generally non-local π orbital and molecular orbital resonance structures are found, which is associated with the enhanced QY and adjustable photoluminescence.108 Furthermore, normal CDs, which have been subsequently modified with amino-containing compounds, are known to possess distinctly improved QY.109 As an alternative, oxygenated moieties on the surface of CDs are affiliated with the photoluminescence properties usually caused by electron-hole recombination and surface energy traps.110 The relationship between the surface functionality of CDs and the different properties produced, oxidation on the surface of CDs and photoluminescence can be customized by fine-tuning the amount of oxygenated and amino groups to achieve specific properties. Moreover, the main feature of CDs that makes them widely applicable in selectively targeted therapy applications is functionalizing with bioactive molecules.111 Pharmaceutical agent and receptor targeting molecules can also be functionalized together to the surface of CDs, resulting in a nanohybrid that can recognize the ailing sites and discharge medicinal agents. 112 CDs and similar nanostructures have been utilized as drug delivery systems (DDS) with a focus on promoting properties such as drug solubility, half-life, and accumulation.113 Targeting and controlling drug release at the tumorous area results in diminishing the drugs’ side effects and improving their bioavailability and tolerance. 114 2.6.2 Different types of interaction for the functionalization of carbon dots The covalent and non-covalent interaction methods of functionalizing CDs are both readily used and have different advantages, which are high on the application of the material.115 Both techniques can improve selective targeted therapy and control; however, covalent bonds are known to be very strong, and the integrity of the conjugates may be superior.116 On the other hand, in applications where the attached molecule is required to release and bind to receptors, non-covalent interactions may potentially allow better drug release of the cargo drug attributed to the high affinity to the target site. 117 One of the most popular methods of covalently coupling peptides to CDs is EDC/NHS coupling owing to its high efficiency and stability.118 This technique achieves conjugation 26 through activation of the carboxylic acid groups, which allows it to react with the free amine groups present on the peptide, as seen in Figure 2.5. 119 Figure 2.5 Covalent modification of carbon dots 119 For example, Dada, S. N. and co-workers120 fabricated folic acid-modified carbon dots (FA- CDs) conjugated to doxorubicin (Dox) using the EDC/NHS strategy and subsequently compared the covalently and noncovalently bonded nanohybrids as cancer-selective targeted therapy system. For targeting cancer cells, folic acid was integrated into the surface of CDs to attach to the overexpressing folate receptors. The data demonstrated that the non-covalent FA-CD-Dox displayed superior efficiency against MDA-MB-231 in contrast to the CD-Dox and covalent FA-CD-Dox.120 Similar work was conducted by Qinghui Zeng and co-workers, and their data demonstrated successful delivery of CD–DOX conjugates to HepG2 cancer cells and HL-7702 normal.114 The various non-covalent modification of CDs includes dipole-dipole interactions, complex formation, or π-π forces between CDs, to name a few.106 One of the benefits of these non- covalent interactions include maintaining the crucial properties of the CDs while introducing a new feature that enhances or adds new properties.103 Furthermore, non-covalent modifications present minimal adverse influence on the pristine framework of CDs and also help provide new moieties, targeting molecules, or metal nanoparticles on the exterior of the CD.121 Consequently, these new features can establish a supplementary dimension in controlling the interfacial properties and render a powerful link to connect nanoparticles to biological molecules. The integrity of the nanostructure is the supreme virtue of non-covalent interaction, which can be fabricated based on inherent functional groups and dispersion force from van der Waals forces present on the CDs. For instance, in a study conducted by BR. Liu, 27 successful delivery of CdSe/ZnS quantum dots by cell penetration peptides (PR9) was reported. The intracellular trafficking data and functional assay evidence revealed that the ingestion of the quantum dots was predominantly through endocytosis.122 The electrostatic interaction between the peptides and the quantum dots was found to play a key role in the complex formation during conjugation and these interactions were analyzed using Zeta potential. One of the toughest membranes in the body is the blood-brain barrier (BBB) which is a protective layer separating the brain’s blood vessels and the cells and other components that makeup brain tissue.123 This barrier provides a defense mechanism against bacteria, viruses, and toxins that may exist in our blood. The selective permeability of this membrane causes major medical drawbacks for drug-delivery systems that target the brain, which has led to high demand for drug-delivery systems that can penetrate the brain. Shanghai Li, and co- workers observed the penetration of transferrin-conjugated C-Dots in the central nervous system (CNS) by crossing the BBB in vivo using a zebrafish model study.124 This in vivo study revealed that the CDs alone could not pass through the blood vessels that vascularize the central nervous system and that the conjugation to transferrin glycoprotein is crucial for permeability. The diameter of CDs was approximately 5 nm, with plenty of carboxylic groups dispersed on the surface for feasible chemical interaction with the biological molecule. In another study by Ullah, Z. and co-workers, a comprehensive theoretical study on adsorption performance investigated the Pyrimidin-2-amine drug molecule on graphene quantum dots (GQD).125 The computational simulations investigated non-covalent interactions, density functional theory (DFT), and time-dependent density functional theory (TD-DFT), and the data revealed that the enhanced interaction with doping (N, B) quantum dot complexes with a ΔG value of −13.38 kcal/more, ΔE of –23.49, and ΔH of 22.60 kcal/mol. This