Development of a PEG Derivative Containing Hydrolytically Degradable Hemiacetals.

Synthetic polymers are ubiquitous in the biomedical sciences, with applications in drug delivery, medical devices, and artificial matrices for tissue engineering.1-4 Since the success of the first synthetic poly(glycolic acid)-based suture in the 1960’s, vast effort has been devoted to designing synthetic biodegradable polymers.5 Given the complexity of regenerative medicine, there is a need to tailor biomaterials for specific applications.6 Among the hydrophilic synthetic polymers, poly(ethylene glycol) (PEG) is a widely used material due to its resistance to protein adsorption and its biocompatiblity.7 PEG and PEG-copolymers play critical roles ranging from PEGylation therapeutics to hydrogel scaffolds mimicking the natural extracellular matrix for cell culture and tissue regeneration. PEG, or poly(ethylene oxide) PEO with molecular weight exceeding 20 kDA, is a hydrolytically non-degradable polymer with excellent solubility in water and various organic solvents.8 As a result of PEG’s non-degradability, the entire polymer chain is excreted through the kidneys ( 30 kDa). 9 Therefore, only PEG of molecular weight less than 50 kDa is typically used in biomedical applications to ensure elimination from the body.10 To overcome this limitation, efforts have been made to introduce biodegradability into PEG or to make copolymers of PEG and biodegradable polymer moieties such as esters.11, 12 Polyesters, as well as other similarly structured polymers, degrade via hydrolysis and give rise to products with carboxylic acid terminal groups.13 As a result, their degradation may create an acidic environment that can induce tissue toxicity.14 In this Communication, we report a synthetic scheme introducing hemiacetals randomly into the backbone of PEG which we will refer to as ROPEG, randomly oxidized PEG. This simple synthetic scheme of incorporating minimal random hemiacetals within the PEG backbone will retain beneficial characteristics of PEG while allowing hydrolytic degradation to non-acidic by-products.15, 16 Utilizing the Fenton reaction of hydrogen peroxide and ferric chloride at a neutral pH we developed a simple method of introducing hemiacetals into the PEG backbone. Previous reports have shown the Fenton reaction to depolymerize PEG but particularly requiring highly acidic conditions to drive the reaction to completion.17, 18 Almkvist et al. oxidized PEG via hydroxyl radicals generated by Fenton’s reagent (Fe(II)/H2O2) in aqueous solution and demonstrated PEG degradation products of alcohols, aldehydes and formate esters using 1H NMR.17 This degradation pathway suggests that there are multiple steps of PEG oxidation and degradation into a variety of PEG oligomers, including PEG hemiacetals, an intermediate formed prior to complete depolymerization of the backbone.17 We found that keeping the pH neutral during the Fenton reaction can oxidize the PEG backbone to ROPEG, a degradable polymer retaining the many advantages of PEG, while retaining higher molecular weight elements. The Fenton reagent used in our studies consisted of 35% (v/v) hydrogen peroxide and 2.5mM FeCl3 buffered in 1X PBS, and the pH was adjusted to 7 with 5M NaOH. PEG 3400 Da was added to the reagent at 10 % (w/v) and oxidation was performed at either 4°C or room temperature. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) of PEG treated with the Fenton reaction at 4°C and at neutral pH before and after dialysis with 1000 Da molecular weight cutoff membrane is provided in the Supporting Information. We demonstrate that some degradation of PEG does take place; however, 1H NMR spectra show hemiacetal formation in the backbone of non-degraded PEG at 4°C and room temperature (Figure 1). Resonance at 4.9 ppm and 3.5-4 ppm corresponds to the hemiacetal protons in the ROPEG and all methylene protons respectively. The peak ratio of hemiacetal protons to methylene protons for synthesis at 4°C for 3 days, 4°C for 5 days, and room temperature for 24 hours was 1:557, 1:372, and 1:135, respectively; this corresponds to approximately 0.7%, 1%, and 3% oxidation of methylene subunits to hemiacetals. This suggests the possibility of tuning the number of hemiacetals in the PEG backbone by modifying reaction time and temperature, and as a result tuning the rate of hydrolytic degradation. Difficulties arise, however, with elevated oxidation temperatures leading to increased degradation rate. Consequently, higher oxidation temperatures produce degradation products that are difficult to separate with dialysis or column purification. 13C NMR spectra of the ROPEG (not purified) synthesized at room temperature for 24 hours corroborates this, demonstrating hemiacetal formation in ROPEG as indicated with resonance at 100 ppm, but also presence of what is most likely PEG-formate ester degradation products, demonstrated by resonance at 170 ppm (Figure 2). Figure 1 1H NMR spectra of PEG (A), ROPEG synthesis at 4°C for 3 days (B), ROPEG synthesis at 4°C for 5 days (C), and ROPEG synthesis at room temperature for 24 hours (D). Figure 2 13C NMR spectra of PEG (A), ROPEG synthesized at room temperature for 24 hours depicting hemiacetal formation in ROPEG with resonance at 100 ppm (B). Z most likely represents resonance corresponding to degradation products of PEG-formate ester and Y represents . As previously mentioned, hemiacetals can be hydrolyzed to aldehydes and alcohols, a process that can be catalyzed via acidic environments.19 Thus, we sought to provide further evidence of hemiacetal formation within the backbone of ROPEG by treating dialyzed ROPEG synthesized at 4°C and a control group of unmodified PEG with 0.5M hydrochloric acid for 24 hours (Figure 3). Analysis by MALDI-TOF MS shows a bimodal molecular weight distribution due to hydrolysis of the hemiacetals in ROPEG synthesized at 4°C, while the molecular weight distribution of the unmodified PEG demonstrates that no degradation occurred. Figure 3 MALDI-TOF MS of three day oxidized ROPEG dialyzed with 1000Da cutoff in order to remove most of the degraded PEG products (A), three day oxidized ROPEG treated with 0.5 M hydrochloric acid to verify hemiacetals in the backbone (B), PEG 3400 Da (C), PEG . Further analysis of hemiacetal formation with the Fenton reaction at a neutral pH was evaluated with a smaller molecule, tri(ethylene glycol) dimethyl ether (TEGDME). Similarly to 3400 Da PEG, hemiacetals were formed in the backbone of TEGDME (178 Da) at room temperature as depicted in 1H NMR spectra of Supplemental figure 2. In conclusion, PEG was randomly functionalized with hemiacetals (ROPEG) via the Fenton reaction. We have demonstrated a simple method of synthesizing a degradable PEG polymer capable of hydrolyzing into non-acidic by-products. This new technique of ROPEG synthesis can be utilized in a variety of areas such as PEGylation therapeutics and tissue engineering scaffolds. PEGylation has emerged as an effective strategy to alter the pharmacokinetic profiles of a variety of drugs.20 Unfortunately, this technology has been limited to lower molecular weights of PEG which can be excreted by the kidneys, given that high molecular weights will accumulate in the liver.9 We have demonstrated synthesis of degradable hemiacetal moieties in the backbone of PEG within a small molecule of 3 repeat units (TEGDME) as well as a larger molecule (PEG 3400 Da) of approximately 77 repeat units. Therefore, ROPEG synthesis can be employed to improve PEGylation therapeutics. In tissue engineering, biomaterials are designed to be degradable to allow growing tissue to replace the scaffold. This normally requires using degradable polymers such as PLA and PGA, which have been previously observed to cause inflammatory responses.21 ROPEG will provide a simple method of designing a degradable scaffold that will have the biocompatible properties of PEG while also allowing degradation. Further investigation of tuning hemiacetal formation for injectable hydrogel scaffolds will be studied and reported in forthcoming papers.