Influence of Lysine and TRITC Conjugation on the Size and Structure of Dextran Nanoconjugates with Potential for Biomolecule Delivery to Neurons.

Darya Zeini,Bo Nyström,Joel C Glover,Kenneth D Knudsen

doi:10.1021/acsabm.1c00544

Abstract

As a potent nonviral system for biomolecular delivery to neurons via their axons, we have studied molecular characteristics of lysinated fluorescent dextran nanoconjugates with degrees of conjugation of 0.54-15.2 mol lysine and 0.25-7.27 mol tetramethyl rhodamine isothiocyanate (TRITC) per mol dextran. We studied the influence of conjugation with lysine and TRITC on the size and structure of different molecular weight dextrans and their mobility within axons. Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) experiments revealed significant differences in the size and structure of unmodified and modified dextrans. Unexpectedly, lower-molecular-weight conjugated dextrans exhibited higher molecular volumes, which we propose is due to fewer intramolecular interactions than in higher-molecular-weight conjugated dextrans. Assessment of retrograde and anterograde movement of lysine- and TRITC-conjugated dextrans in axons in the lumbar spinal cord of chicken embryos showed that lower-molecular-weight dextrans translocate more efficiently than higher-molecular-weight dextrans, despite having larger molecular volumes. This comparative characterization of different molecular weight dextrans will help define optimal features for intracellular delivery.

Highlights

The degree of lysine conjugation was tuned by the amount of cyanogen bromide and lysine in the solution. 1H NMR (Bruker 400 MHz, D2O, δ, ppm) spectra of lysine-conjugated dextran in D2O exhibited the characteristic signal of protons from lysine (Figure 1)
The chemical shift values of the signals in 1H NMR spectra of the dextran and lysine moiety of this product were in accordance with literature values.[44−46] The degree of substitution (DS) of lysine per each glucose unit was calculated by dividing the integral of the peak at 3.01 ppm, corresponding to 1 proton of lysine, with that of the peak at 4.99 ppm, corresponding to 1 proton from a single unit of glucose
Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) analyses showed that lysine-conjugated dextrans form association complexes, probably through intermolecular hydrogenbonding interactions, and that the volume of association complexes decreases with an increasing degree of conjugation

Summary

Introduction

Dextran-based polymers have received great interest for controlled drug release and gene transfection applications.[1−7]Targeted and localized delivery systems based on dextran polymers have several benefits such as high aqueous solubility, high chemical stability, target site specificity, few deleterious effects on cellular function, suitability for programmed release applications, good patient compliance, and high overall efficiency.[8−11] For example, one limitation to the use of nucleic acids for studies of normal and pathophysiological processes and for the potential treatment of neurological disease is the specificity, with which they can be delivered to selected neuron populations.[11,12] This problem can be solved while simultaneously avoiding the risks inherent in using viral carriers using a suitable nonviral polymeric nanocarrier.[12,13]Selectively localized injection of a suitable polymeric nanocarrier can restrict intracellular accumulation to neurons that project to a specific target. Dextran-based polymers have received great interest for controlled drug release and gene transfection applications.[1−7]. Targeted and localized delivery systems based on dextran polymers have several benefits such as high aqueous solubility, high chemical stability, target site specificity, few deleterious effects on cellular function, suitability for programmed release applications, good patient compliance, and high overall efficiency.[8−11] For example, one limitation to the use of nucleic acids for studies of normal and pathophysiological processes and for the potential treatment of neurological disease is the specificity, with which they can be delivered to selected neuron populations.[11,12] This problem can be solved while simultaneously avoiding the risks inherent in using viral carriers using a suitable nonviral polymeric nanocarrier.[12,13]. Biodegradable, and biocompatible nonionic polysaccharide, widely employed for biomedical applications.[15−22] The chemical structure of dextran consists mainly of α-(1 → 6) glycosidic bonds with branching at positions α-(1 → 2), α-(1 → 3), and α-(1 → 4).[9,13,23] The degree of branching in dextran polymers is related to the molecular weight of dextran[24] and is usually pronounced for high molecular weights

Objectives

Results

Conclusion