Thiolated Polyethyleneimine: Synthesis, Functionalization, And Advanced Applications In Biomedical And Materials Engineering

Molecular Composition And Structural Characteristics Of Thiolated Polyethyleneimine

Thiolated polyethyleneimine is synthesized by introducing thiol functional groups onto the primary, secondary, or tertiary amine sites of the polyethyleneimine backbone 1. The parent polyethyleneimine (PEI) exists in two primary architectures: branched polyethyleneimine (bPEI) and linear polyethyleneimine (lPEI), each exhibiting distinct molecular weight distributions, amine group ratios, and reactivity profiles 5,6. Branched PEI, typically produced via cationic ring-opening polymerization (CROP) of aziridine, contains a heterogeneous mixture of primary (25%), secondary (50%), and tertiary (25%) amines, resulting in a highly branched, three-dimensional structure 5. In contrast, linear PEI, synthesized through living anionic polymerization of functionalized aziridine monomers, comprises predominantly secondary amines in a linear chain, offering improved batch uniformity and controlled molecular weight (Mw ranging from 600 Da to over 750 kDa) 5.

The thiolation process most commonly employs ethylene sulfide (ES) as the thiolating agent, which reacts with the amine groups of PEI via nucleophilic ring-opening to yield PEI-SH 1. Alternative thiolating reagents include halogen-alkyl thiols (e.g., 2-bromoethanethiol), cysteine, bromopyridine thiol, chloropyridine thiol, and halo-phenyl thiazole thiols, each imparting distinct reactivity and steric properties to the resulting thiolated polymer 1. The degree of thiolation—defined as the molar percentage of amine groups converted to thiol groups—can be precisely controlled by adjusting the molar ratio of thiolating agent to PEI, reaction temperature (typically 50–80°C), and reaction time (2–24 hours) 1,14. For instance, a thiolation degree of 10–30% is commonly targeted to balance reactivity with retention of cationic charge, which is critical for applications such as gene delivery where electrostatic interaction with negatively charged nucleic acids is essential 10,11.

Characterization of thiolated polyethyleneimine typically involves Fourier-transform infrared spectroscopy (FTIR) to confirm the presence of thiol groups (S-H stretching at ~2550 cm⁻¹), proton nuclear magnetic resonance (¹H NMR) to quantify the degree of substitution, and Ellman's reagent assay to determine free thiol concentration (typically 0.5–3.0 mmol/g polymer) 1,14. Thermogravimetric analysis (TGA) reveals that thiolated PEI exhibits a slightly lower thermal decomposition onset temperature (Td ~250–280°C) compared to unmodified PEI (Td ~300–320°C), attributed to the lability of the C-S bond under oxidative conditions 16.

Key Structural Features

• Amine-to-thiol conversion ratio: Typically 10–30% to preserve cationic functionality 1.
• Molecular weight range: 600 Da to 750 kDa, depending on parent PEI architecture 5.
• Thiol group density: 0.5–3.0 mmol/g, quantified via Ellman's assay 1,14.
• Thermal stability: Td onset at 250–280°C (TGA under N₂ atmosphere) 16.
• Solubility: Highly water-soluble at pH 4–9; solubility decreases at pH >10 due to deprotonation of amines 6,7.

Synthesis Routes And Reaction Mechanisms For Thiolated Polyethyleneimine

Thiolation Via Ethylene Sulfide Ring-Opening

The most widely adopted synthesis route involves the nucleophilic attack of primary or secondary amines in PEI on the strained three-membered ring of ethylene sulfide, yielding a β-aminoethyl thiol moiety 1. The reaction proceeds under mild conditions (50–70°C, 4–12 hours) in aqueous or methanolic media, with the degree of thiolation controlled by the ES:PEI molar ratio (typically 0.1:1 to 0.5:1) 1. The reaction mechanism is as follows:

PEI-NH₂ + (CH₂)₂S → PEI-NH-CH₂-CH₂-SH

This method is advantageous due to the commercial availability of ethylene sulfide, high reaction efficiency (>80% conversion), and minimal side reactions 1. However, care must be taken to avoid over-thiolation, which can lead to cross-linking via disulfide bond formation under oxidative conditions 14.

Alternative Thiolation Strategies

Alternative thiolating agents such as halogen-alkyl thiols (e.g., 2-bromoethanethiol) react with PEI amines via nucleophilic substitution (SN2 mechanism), releasing halide ions 1. This approach allows for the introduction of longer alkyl spacers between the PEI backbone and the thiol group, which can modulate steric accessibility and reactivity. For example, reaction of bPEI (Mw 25 kDa) with 2-bromoethanethiol at a 1:0.3 molar ratio in DMF at 60°C for 8 hours yields PEI-SH with a thiolation degree of ~20% and a free thiol content of 1.8 mmol/g 1.

Cysteine-mediated thiolation involves coupling the carboxyl group of L-cysteine to PEI amines via carbodiimide chemistry (EDC/NHS activation), followed by reduction of the cysteine disulfide to yield free thiols 1. This route is particularly useful for introducing chiral centers and biocompatible linkers, though it requires additional reduction steps (e.g., with dithiothreitol, DTT) and purification to remove unreacted cysteine 10.

Reaction Conditions And Optimization

• Temperature: 50–80°C; higher temperatures (>80°C) accelerate thiolation but increase risk of disulfide cross-linking 1,14.
• Solvent: Water, methanol, or DMF; aqueous media favor higher thiolation degrees due to enhanced nucleophilicity of amines 1.
• Molar ratio (thiolating agent:PEI): 0.1:1 to 0.5:1; ratios >0.5:1 lead to excessive cross-linking 1.
• Reaction time: 4–24 hours; longer times increase thiolation degree but may induce oxidation 1.
• pH control: Maintaining pH 7–8 during reaction minimizes disulfide formation 14.

Functionalization And Click Chemistry Applications Of Thiolated Polyethyleneimine

Thiol-Ene Click Reactions

Thiolated polyethyleneimine serves as a versatile platform for thiol-ene click chemistry, a highly efficient, orthogonal reaction between thiols and alkenes (or alkynes) that proceeds under UV irradiation or thermal initiation with radical initiators 1,19. A prominent example is the synthesis of polyethyleneimine-thiol-2-methacryloyloxylethyl phosphorylcholine (PEI-S-MPC), a zwitterionic polymer used for antithrombogenic coatings on medical devices 1. In this reaction, PEI-SH reacts with 2-methacryloyloxylethyl phosphorylcholine (MPC) under UV light (365 nm, 10 mW/cm²) in the presence of a photoinitiator (e.g., Irgacure 2959, 0.1 wt%) for 10–30 minutes, achieving >90% conversion of thiol groups 1. The resulting PEI-S-MPC retains free primary and secondary amines, enabling covalent grafting onto diisocyanate-activated polyurethane surfaces via a single-step "grafting-to" process 1.

The thiol-ene reaction mechanism involves a radical chain process:

1. Initiation: Photoinitiator generates thiyl radicals (RS•) under UV light.
2. Propagation: RS• adds to the C=C double bond of MPC, forming a carbon-centered radical.
3. Chain transfer: The carbon radical abstracts a hydrogen from another thiol, regenerating RS• and forming the thioether product.

This mechanism ensures high selectivity, minimal side reactions, and compatibility with sensitive functional groups (e.g., phosphorylcholine, peptides) 1,19.

Thiol-Epoxy Cross-Linking

Thiolated polyethyleneimine can undergo thiol-epoxy reactions with multifunctional epoxides (e.g., diglycidyl ether of bisphenol A, DGEBA) to form cross-linked networks with tunable mechanical properties 16,19. For example, mixing bPEI-SH (Mw 25 kDa, thiolation degree 20%) with polyhedral oligomeric silsesquioxane (POSS) containing epoxy groups at a 1:1.5 thiol:epoxy molar ratio, followed by curing at 80°C for 4 hours, yields a solid, cross-linked polymer with a storage modulus (E') of 1.2 GPa at 25°C (measured by dynamic mechanical analysis, DMA) and a glass transition temperature (Tg) of 95°C 16. This material exhibits enhanced chemical stability (resistant to 1 M HCl and 1 M NaOH for >30 days at 25°C) and thermal stability (Td onset at 320°C) compared to uncross-linked PEI 16.

Bioconjugation And Surface Modification

The thiol groups in PEI-SH enable facile bioconjugation with maleimide-functionalized biomolecules (e.g., peptides, antibodies, fluorescent dyes) via Michael addition, a reaction that proceeds rapidly at pH 6.5–7.5 without catalysts 10. For instance, conjugation of PEI-SH (Mw 10 kDa) with maleimide-PEG₅₀₀₀ at a 1:2 molar ratio in phosphate-buffered saline (PBS, pH 7.4) at 25°C for 2 hours yields PEI-S-PEG with a PEGylation degree of ~40%, as confirmed by ¹H NMR 10. This PEGylation reduces the cytotoxicity of PEI (IC₅₀ increases from 15 µg/mL to >100 µg/mL in HEK293 cells) while maintaining gene transfection efficiency (>70% GFP expression in HeLa cells at N/P ratio of 10) 10.

Key Functionalization Parameters

• UV irradiation intensity for thiol-ene: 10–20 mW/cm² at 365 nm; higher intensities accelerate reaction but may cause polymer degradation 1.
• Photoinitiator concentration: 0.05–0.2 wt%; optimal at 0.1 wt% for balancing reaction rate and polymer stability 1.
• Thiol:epoxy molar ratio for cross-linking: 1:1 to 1:2; excess epoxy improves network density but increases brittleness 16,19.
• pH for bioconjugation: 6.5–7.5; lower pH (<6) slows Michael addition, higher pH (>8) promotes disulfide formation 10.

Applications Of Thiolated Polyethyleneimine In Biomedical Engineering

Gene Delivery And Nucleic Acid Complexation

Thiolated polyethyleneimine has emerged as a superior non-viral gene delivery vector due to its ability to condense DNA/RNA into nanoparticles (polyplexes) via electrostatic interactions, while the thiol groups enable disulfide cross-linking for enhanced stability and triggered release in the reducing intracellular environment 10,11. PEGylated thiolated PEI (PEI-S-PEG) exhibits reduced cytotoxicity (IC₅₀ >100 µg/mL in HEK293 cells) compared to unmodified PEI (IC₅₀ ~15 µg/mL), while maintaining high transfection efficiency (>70% GFP expression in HeLa cells at N/P ratio of 10) 10. The disulfide bonds formed between PEI-SH chains are cleaved by intracellular glutathione (GSH, 2–10 mM), triggering polyplex dissociation and DNA release 10.

A study comparing linear PEI-SH (Mw 25 kDa, thiolation degree 15%) with branched PEI-SH (Mw 25 kDa, thiolation degree 15%) for plasmid DNA (pDNA) delivery found that linear PEI-SH formed smaller polyplexes (hydrodynamic diameter 80–120 nm vs. 150–200 nm for branched PEI-SH) and achieved 2-fold higher transfection efficiency in HEK293 cells, attributed to more efficient endosomal escape 11. The optimal N/P ratio (ratio of PEI amine groups to DNA phosphate groups) for linear PEI-SH was 8–12, yielding polyplexes with zeta potential of +20 to +30 mV and >80% DNA complexation efficiency 11.

Antithrombogenic Coatings For Medical Devices

Thiolated polyethyleneimine functionalized with zwitterionic phosphorylcholine (PEI-S-MPC) has been developed as a lubricious, antithrombogenic coating for insertable medical devices such as catheters, stents, and guidewires 1. The coating is applied via a single-step "grafting-to" process: the polyurethane (PU) surface is first activated with hexamethylene diisocyanate (HDI) to introduce isocyanate groups, which then react with the free amines of PEI-S-MPC to form covalent urea linkages 1. The resulting coating exhibits a water contact angle of 15–25° (compared to 85–95° for uncoated PU), indicating high hydrophilicity, and reduces platelet adhesion by >90% (measured by lactate dehydrogenase assay after 2-hour incubation with platelet-rich plasma) 1.

Incorporation of therapeutic compounds such as linalool, limonene, or citral (terpenes with antimicrobial and anti-inflammatory properties) into the PEI-S-MPC coating via thiol-ene click chemistry further enhances biocompatibility 1. For example, a coating containing 5 wt% linalool exhibits sustained release (0.5 µg/cm²/day over 14 days) and reduces bacterial adhesion (Staphylococcus aureus) by >95% compared to uncoated controls 1.

Case Study: Enhanced Gene Transfection In Viral Vector Production — Biomedical

PEGylated thiolated polyethyleneimine (PEI-S-PEG) has been employed to improve the efficiency of producing adeno-associated virus (AAV) vectors for gene therapy 10. In a study by Charles River Laboratories, HEK293 cells were transfected with AAV plasmids using PEI-S-PEG (Mw 25 kDa, PEGylation degree 40%) at an N/P ratio of 10, resulting in a 3-fold increase in AA