Synthesis and characterisation of fluorescent FITC-insulin glulisine conjugates for insulin delivery

Drug development and its delivery have always been a pioneering area of research for the application and understanding of modern bioanalytical problems. The biodistribution of the drug, along with its stability and metabolism within biological systems, has been of prime importance in pharmaceutical drug development and research. The study carried out in this thesis aimed to evaluate novel fluorescent conjugates synthesised from commercially available rapid-acting insulin glulisine (Apidra®) and the fluorescent dye FITC (Fluorescein Isothiocyanate) for comparison with the biological activity of native insulin glulisine. The first objective of the study was to produce a single-labelled fluorescent-insulin conjugate (FITC-insulin glulisine), achieved without using any lengthy and expensive procedures involving protective groups for selective tagging and multi-step processes. The next objective was to study the stability of the insulin glulisine conjugate in several formulations at different temperatures (4oC, 20oC and 37oC) to assess appropriate storage temperature and shelf life without compromising its composition and biological activity. Thirdly, the biological activity of the FITC-insulin glulisine conjugate was studied and compared to that of native insulin glulisine. The formation of the desired mono-labelled conjugate was confirmed by MALDI-TOF mass spectroscopy, which identified the mass of the conjugate and the label position. Additional further conjugation analysis was conducted using Attenuated total reflectance Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. Further studies were conducted by assessing the lyophilic/hydrophilic nature of the synthesised FITC-insulin glulisine mono-labelled conjugate in octanol: water systems to ascertain its physical properties. A separate study examined the role of EDTA in chelating zinc from human insulin to assess what impact this has on producing FITC-human insulin conjugates labelled at B1. Experiments involved several parameters being examined with changing molar ratio altered from 1:1 to 3:1 and reaction time spanning from 18 hours to 24 hours with a pH of 7 to achieve the desired B1 mono-labelled FITC-insulin glulisine conjugate and this was successfully achieved at a reaction time of 18 hours at pH 7 with a molar ratio of 2:1. The B1 mono species was confirmed by MALDI-TOF mass spectrometry analysis with a molecular weight of 6207.7 Da for the mono labelled insulin glulisine conjugate. Mass spectroscopy fragmentation studies determined FITC was attached with the b-ion peak for FITC-Phenylalanine (N-terminus of Chain B) at m/z = 537.11 (FITC- MW- 389 g/mol and phenylalanine -MW- 165 g/mol) which confirms the position of conjugation at the first amino acid of the β-chain at position B1 (phenylalanine). The stability of the B1 labelled FITC-insulin glulisine conjugate was studied at temperatures of 4oC, 20oC and 37oC and extended stability of the B1 labelled conjugate was found in the Diluting Fluid (DF) stored at 4°C over the 56-day study period, suggesting excipients support the integrity of the FITC-insulin glulisine conjugate. On the other hand, Distilled Water (DW) at 37°C showed the least stability over the same study period. The biological activity experiments conducted on the FITC-insulin glulisine conjugate using western blot analysis confirmed pAKT and IRS tyrosine activity and the confocal microscopy analysis showed the GLUT4 translocation comparable to the native insulin glulisine. This biological activity study proved that the mono B1 conjugate showed similar biological activity and successfully translocated GLUT4 in the insulin signalling pathway, identical to native insulin glulisine, making it potentially useful in many biological and pharmaceutical applications. Native insulin glulisine, FITC and synthesised FITC-insulin glulisine conjugate were subjected to various analytical procedures for the -ATR -FTIR measurements to prove the successful conjugation of the fluorescent FITC to the native insulin glulisine without compromising the secondary structure of the insulin and the disappearance of the original isothiocyanate peak present for FITC at 2050 cm-1 was not found in the conjugate and formation of an additional peak for thiourea at 1395 cm-1 affirmed the conjugation. In other experiments human insulin (Actrapid®) was fluorescent-labelled with FITC at various EDTA concentrations ranging from 0mM to 2.4mM, which resulted in different chromatograms, reflecting varying percentages of the mono-labelled conjugate (A1 or B1) and di-labelled conjugate (A1B1) formation. At lower concentrations of EDTA, only the B1 mono-labelled FITC-insulin conjugate was produced, but beyond a crucial threshold of 0.2 mM EDTA, the di-labelled A1B1 conjugate began to form, with a 40% decrease in the mono-labelled conjugate at an EDTA concentration of 0.8 mM. Therefore, this EDTA study demonstrates that up to 0.2 mM EDTA enables optimal monoB1 labelling. A partitioning study on FITC-insulin glulisine conjugate compared to native insulin glulisine in an Octanol: water system demonstrated that the slow stirring method of mixing of the reaction mixture was favourable for the experiment and the logP values obtained for commercial insulin glulisine was -1.95 while the pure FITC logP value was determined to be 5.25, while the conjugated FITC-insulin glulisine had the logP value of -0.197 confirming the lipophilic influence of the fluorescein conjugation. This thesis demonstrates a streamlined and efficient protocol for FITC labelling of insulin glulisine established under mild conditions (pH 7, 2:1 FITC-to-insulin glulisine molar ratio, 18 h reaction time), achieving near complete yield without the need for protecting groups or multi-step synthesis. The resulting mono-labelled insulin glulisine (monoB1) retains full biological activity, including effective GLUT4 translocation via the insulin signalling pathway, comparable to native insulin. This approach offers a robust and simplified method for generating fluorescently conjugated insulin glulisine suitable for diverse biological and pharmaceutical applications, particularly in insulin delivery research.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2025

Author dc:creator

• Desai, Unmesh

Licence

• https://dora.dmu.ac.uk/bitstreams/f76f9360-a809-4a78-8b3b-abd167186b2d/download