This thesis describes the design and synthesis of functional hyperbranched polyamines and their potential applications. We successfully synthesized hyperbranched polyglycerols containing amino functionality using a novel Boc-protected amino ethanol glycidyl ether (BAG) monomer. A series of hyperbranched Boc-protected polyamino glycerols (PBAGs) were prepared through a one-pot ring-opening anionic multibranching polymerization to produce PBAGs with controlled molecular weights.
1H, 13C and 15N-NMR, GPC and MALDI-ToF measurements confirmed the successful polymerization of hyperbranched PAG polymers. Furthermore, we demonstrated that PAG can generate singlet oxygen species that can be used as a photodynamic therapeutic agent. To confirm the formation of singlet oxygen species, the -bi(methylene)dimalonic acid (ABDA) test and the UV-Vis experiment were used.
We expect that PAG with high biocompatibility and singlet oxygen generation ability will be widely used in biomedical fields. a) Schematic illustration of dendritic family. Synthetic scheme of (a) the BAG monomer and (b) the anionic ring-opening polymerization of PBAG and subsequent deprotection to give PAG. The distance between the signals corresponds to the mass of the respective monomers (AG: . 161.2 g/mol).
In vitro cell viability assay of polymers. polymer 4) determined by WST-1 assays using RAW264.7 cell lines. a) Potential singlet oxygen generation mechanism.
Hyperbranched polymers
The formula for calculating the degree of branching was devised by Frey and colleagues.4 This formula only applies to monomers of type ABm (m≥2). Moore and co-workers first reported hyperbranched polymers with narrow PDI values using the slow monomer addition method. Since this discovery, many scientists have synthesized polymers with narrow polydispersity index by the slow monomer addition method of ABm monomer.
However, due to the low molecular weight byproduct produced during the reaction, there was a limitation to the formation of high molecular weight polymers. On the other hand, the use of cyclic monomer eliminated the byproduct and allowed high molecular weight to be easily obtained. Frey and co-workers reported in 1999 a multi-branch anionic ring-opening polymerization of glycidol.5 Glycidol is a cyclic molecule and an AB2-type monomer.
Polyglycerols
Functionalized Polyglycerols
Biomedical application
PG is biocompatible due to its structural similarity to PEG, which is widely used in biochemistry, biomedicine, and industry.19 In 2006, Brooks et al. reported the biocompatibility of linear PG, hyperbranched PG, and PEG.12 All polymers showed high biocompatibility in vivo and in vitro tests. As a result of these experiments, high molecular weight PGs and their derivatives have been used for biochemistry,20,21 conjugation chemistry22 and drug delivery.23,24 Many scientists have observed biocompatible polymers as carriers of proteins or drugs.25 Combinations of anticancer drugs and biocompatible polymer result in increased accumulation of the drug in the cancer cell. The increase in effectiveness of these drugs is called the enhanced permeability and retention (EPR) effect.
With this conjugation, the drug can penetrate the tumor better. 26 , 27 Hyperbranched PG is a promising drug delivery material due to its hydroxyl functional groups, which make it easy to modify for biomedical applications. 28, 29 Kim and co-workers conjugated hyperbranched polyglycerols with doxorubicin, a chemotherapeutic agent, and enhanced efficacy in 2012. Haag and co-workers reported a new photoresponsive polycation that has a star-shaped amine shell and a biocompatible hyperbranched polyglycerol core.30,31 These polymers have positive charges. at a specific pH with a polyether core.
Polyamines
Polyamine functions
A 10 g/L solution of the polymer in acetonitrile and 10 g/L solution of the matrix solution were prepared separately. A 1.0 μL aliquot of the mixture was applied to a target plate and the solvent was evaporated before measurement. The synthesis of the BAG monomer was successfully identified via various spectroscopic and mass analyses, including 1H and 13C NMR (Figure 2) and ESI-MS.
The absorbance of the solution was recorded at a wavelength of 450 nm, using 600–650 nm as a reference. In addition, the number average molecular weight was calculated by comparing the peak integrals of methylene groups of the TMP initiator (peaks at 0.75 and 1.25 ppm, respectively) and polyether skeleton (peaks at 3.0–4.0 ppm). The deprotection of the Boc group of PBAG could be easily confirmed by the disappearance of the peak of the series of t-butyl groups at 1.34 ppm in the H NMR (Figure 3c).
In particular, the weight average molecular weight of the PBAG polymers was found to be g/mol with a polydispersity index (Mw/Mn) of 1.19–1.83 determined by GPC using PMMA as a standard due to the existence of the hydrophobic Boc - protection group. A similar phenomenon was observed in our previous report on the Boc-protected butanolamine glycidyl ether system.16. Under identical reaction conditions, approx. 5% of the Boc group deprotected, revealing a potential side reaction during the polymerization.
Moreover, we could identify the presence of tertiary amine group in the polymeric backbone as a result of the potential side reaction of the deprotected Boc group during the polymerization by using 15N NMR (Figure 5a). However, it should be noted that the fraction of the tertiary amine group is significantly lower than that of secondary amine groups in accordance with the model reaction performed. -ToF spectrometry was performed to identify the insertion of the TMP initiator and functional monomer segment into the PAG polymers.
As shown in Figure 5b, the distance between the signals corresponds to the mass of the respective monomers in the PAG polymer, which are present in varying degrees, which unequivocally shows the successful polymerization of PAG. For example, the mass peak at 1946.56 m/z corresponded to the polymer with TMP as initiator, 11 units of monomer and K + as one. a) Schematic representation of the potential side reaction of BAG monomer during the polymerization and its subsequent reaction with incoming new monomer. During the polymerization with potassium methoxide, a fraction of the monomer acts as an initiator, and the polymer formed as a side reaction may have a cyclic form, although the use of the slow monomer addition to keep the concentration of the monomers low during the reaction.
The DB of the selected polymer PBAG66 was determined to be approx. 0.41, which was slightly lower than the conventional hyperbranched polymers (0.4–0.6). The cell viability of the polymers was measured using the WST-1 assay, which is generally used for in vitro cytotoxicity assays of polymers and nanomaterials. This is because the amine groups in the PAG are not charged when the pH is high.
Based on all the results, we hypothesized that the amine groups of the polymer interact with light to generate singlet oxygen.
Hyperbrached polyamines based on novel amino glycidyl ether
Experimental
- Materials
- Analysis method
- Reaction to protect diethanolamine
- Reaction for synthesizing BAG
- Synthesis of PBAG
- Process of removing the Boc protecting group
- Cell viability assay
- Reactive oxygen species assay
- Characterization of hyperbranched polyamines
- Biocompatibility assay
- Singletoxygen generation assay
Differential scanning calorimetry (DSC) was performed using a DSC (model Q200, TA Instruments) in the temperature range of. The residue was purified by flash column chromatography with 17% hexane in ethyl acetate to give BAG monomer as a pale yellow viscous liquid (4.1 g, 32%). There is some error in the measurement of molecular weight by NMR measurement, we used 4500 g/mol as the value of Mn determined by NMR.
BAG monomer and PAG polymer were synthesized by the methods described in Figure 1. When the BAG monomer was prepared, we used an anionic multibranched ring-opening polymerization using a potassium alkoxide initiator, which was synthesized by reacting a solution of potassium methoxide and trimethylolpropane (TMP). As described in previous studies, slow monomeric addition of BAG monomer to deprotonated initiator TMP was performed and polymerized at 90 °C for 48 h to synthesize polymers in a controlled manner.
As shown in Figure 3, the characteristic proton peak of BAG monomer and PAG polymer was confirmed by 1 H NMR. As similarly determined in the structure of branched poly(ethyleneimine) (PEI), by distinguishing between the secondary and tertiary amine groups26, we could monitor the side reaction during the polymerization. We postulated that the longer spacer unit in the BAG monomer limited the branching of the terminal hydroxyl group compared to a glycidol monomer.
In the case of PAG50, which contains more amine groups, the study showed significant toxicity due to the toxic free amine groups; Therefore, cell viability decreased dramatically up to a concentration of 250 µg/ml. Among the various ROS, singlet oxygen can be easily generated by energy transfer or charge transfer. Because singlet oxygen is produced by energy transfer, it is widely used in photodynamic therapy.
We hypothesized that the lone electron pair of the amine functional group of the polyamine reacts with light to form singlet oxygen. The resulting superoxide radical reacts with the lone electron pair of the positively charged PAG to produce singlet oxygen. Therefore, the formation of singlet oxygen can be confirmed by studying the decrease in the amount of ABDA when photoirradiation is applied.
Conclusion
