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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Macromol Rapid Commun. 2017 Dec 18;39(5):10.1002/marc.201700652. doi: 10.1002/marc.201700652

Synthesis and Biological Evaluation of a Degradable Trehalose Glycopolymer Prepared by RAFT Polymerizationa

Uland Y Lau 1, Emma M Pelegri-O’Day 2, Heather D Maynard 3,✉
PMCID: PMC5986558  NIHMSID: NIHMS970009  PMID: 29251372

Abstract

There is a significant need for new biodegradable protein stabilizing polymers. Herein, we describe the synthesis of a polymer with trehalose side chains and hydrolytically degradable backbone esters and its evaluation for protein stabilization and cytotoxicity. Specifically, an alkene-containing parent polymer was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization and thiolated trehalose was installed using a radical initiated thiol-ene reaction. The stabilizing properties of the polymer were investigated by thermally stressing granulocyte colony-stimulating factor (G-CSF), which was expressed and purified using a custom designed G-CSF fusion protein with a polyhistidine-tagged maltose binding protein. The degradable polymer was shown to stabilize G-CSF to 66% after heating at 40 °C. P(BMDO-co-BMA-trehalose) was degraded and its cellular compatibility was investigated. While the polymer was noncytotoxic, cytotoxic effects were observed from the degraded products in fibroblasts and murine myeloblasts. These data provide important information for future use of BMDO-containing trehalose glycopolymers for biomedical applications.

Keywords: biomimetic, degradation, proteins, radical polymerization, synthesis

Graphical Abstract

A novel degradable trehalose polymer containing BMDO is prepared using post-polymerization thiol-ene chemistry. The therapeutic protein GCSF is expressed and purified, and the degradable trehalose polymer is shown to stabilize GCSF against heat stress. Although the parent polymer is cytocompatible, the products of hydrolytic degradation demonstrated cytotoxicity at high concentrations providing the working concentrations for the polymer.

graphic file with name nihms970009u1.jpg

1. Introduction

Poly(ethylene glycol) (PEG) is widely used in polymer-based therapeutics to improve the pharmacological properties of protein drugs. Decades of clinical experience and product development for PEG-based materials have both illustrated benefits and revealed shortcomings. Side effects include hypersensitivity and immunogenicity against PEG, accumulation of PEG in organs, and non-biodegradability.[1–3] In addition, protein-PEG conjugates are known to be unstable to increased temperatures.[4] These disadvantages provide a strong motivation for the development of PEG alternatives.

In recent years, synthesis of polymers with additional benefits other than improved pharmacokinetic properties is of great interest.[5–7] For example, polymers that stabilize proteins against environmental stressors can be used to eliminate the loss of protein activity associated with storage and handling.[8] Charged materials such as poly(sulfonates), poly(carboxybetaines), and cationic dendronized polymers have protected conjugates against environmental and thermal stressors.[9–12] Additionally, synthetic carboxylated polyamidosaccharides have been shown to retain lysozyme activity after multiple lyophilization cycles.[13] And our group has previously reported on glycopolymers with trehalose side chains for enhancing stability of a wide range of proteins against lyophilization, mechanical, and heat stress.[14–17] Yet, most of the reported stabilizers are not degradable.

Hydrolytically or enzymatically degradable alternatives have been used, but may present problems such as undesired biological side effects or difficulty in attaching protein-reactive units. For instance, hydroxyethyl starch has been extensively used for the preparation of protein conjugates with increased half-lives, but may accumulate in some organs increasing risk of death for critically ill patients.[18, 19] To address this need, we recently described stabilizing polymers with polyester backbones.[17] Specifically, polycaprolactone was prepared with zwitterionic and trehalose side chains. These polymers were highly effective protein stabilizers and also degraded through ester hydrolysis. We were thus interested to investigate other degradable protein stabilizers, specifically, those that could be made by controlled radical polymerizations (CRP). We had previously prepared degradable comb PEG polymers by CRP and conjugated them to proteins.[20] However, these polymers were not protein stabilizers.

An effective method of introducing degradable units by CRP is by copolymerization of cyclic ketene acetals (CKAs).[21–23] This class of monomers undergoes radical ring-opening polymerization to insert degradable esters into the growing polymer chain and is compatible with various CRP techniques. In particular, 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) has been extensively employed,[24–26] including in degradable glycopolymers. For example, Lu and coworkers described the synthesis of a styrenyl galactopyranose-based copolymer,[27] and recently Stenzel and coworkers copolymerized BMDO with an acryloyl fructopyranose monomer.[28] Herein, we describe the synthesis and initial biological evaluation of trehalose side chain polymers with BMDO units incorporated in the backbone by reversible addition-fragmentation chain transfer (RAFT) polymerization.

2. Results and Discussion

2.1. Polymer Synthesis and Characterization

Initial attempts to copolymerize BMDO with a trimethylsilyl (TMS)-protected trehalose monomer were unsuccessful, presumably due to steric hindrance between bulky protected trehalose units (data not shown). Therefore, a system of post-polymerization modification was employed. First, RAFT polymerization was performed with a trithiocarbonate chain transfer agent (CTA)[16], but-3-enyl methacrylate (bMA), BMDO, and azobisisobutyronitrile (AIBN) to yield p(BMDO-co-bMA) (see Scheme 1 for synthetic steps and Figure S2 for 1H NMR characterization). The final polymer contained 29 bMA units and 5 BMDO units, with a number-average molecular weight (Mn) of 5200 by 1H-NMR and 2900 by gel permeation chromatography (GPC) and a molecular weight dispersity (Ð) of 1.76. Consistent with the literature,[29] no crosslinking of alkene units was observed and there was no consumption of alkene protons (5.81ppm) compared to methylene protons (4.06 ppm) in the 1H-NMR spectrum (Figure S2). The incomplete control over the polymerization is hypothesized to be due to the mismatch between BMDO and the activated bMA monomer. Thiolated trehalose[17] was then coupled via thiol-ene chemistry to prepare p(BMDO-co-BMA-trehalose-OAc). GPC analysis revealed a clean increase in molecular weight (Mn = 20,900) and decrease in Ð to 1.22, consistent with attachment of trehalose side chains (Figure S5). The acetyl groups were then removed with K2CO3 to yield p(BMDO-co-BMA-trehalose) (see Figure S3 and Figure S4 for 1H NMR characterization). By GPC the Mn shifted to a value of 3300 Da and Ð of 1.29. (Mn = 4600 and Ð = 1.30 by aq. SEC), which is smaller than would be anticipated. The Mn by 1H-NMR could not be determined accurately because a complete loss of the aldehyde end group was observed. However, we believe the low molecular weight was not due to cleavage of the backbone during these mild deprotection conditions because treatment with KOH further reduces the molecular weight (Figure S6) and we have previously shown that BMDO copolymers are stable for greater than 7 days in carbonate solution.[20] Instead, the low Mn is likely a result of interactions of the polymer with the GPC column, as we have previously observed with other trehalose polymers.[30]

Scheme 1.

Scheme 1

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Synthesis of p(BMDO-co-bMA) and modification with thiolated trehalose to yield p(BMDO-co-BMA-trehalose).

Degradation of p(BMDO-co-BMA-trehalose) was evaluated by exposure to a 5% KOH solution. KOH is commonly used to accelerate conditions for polymer degradation.[20, 24, 25, 31] The trehalose copolymer was found to be hydrolytically degradable, and degradation was observed within 24 hours as demonstrated by a decrease in GPC molecular weight to 1900 Da (Figure S6). No further degradation was observed upon further incubation in KOH. The degraded products were neutralized and dialyzed in water to remove salts for cytotoxicity evaluation. Hydrolytic degradation in cell-conditioned media and upon esterase exposure was also explored (Table S1), but no decrease in molecular weight was observed on the time scale tested (3 weeks). Slight increases in observed Mn were hypothesized to be due to overlap between biomolecule peaks and p(BMDO-co-BMA-trehalose) in the SEC trace.

2.2. Expression and Purification of G-CSF

To explore the ability of the trehalose copolymer to stabilize proteins, G-CSF was chosen as the target protein because of its clinical relevance. The soluble expression and purification of G-CSF was achieved by fusing G-CSF to maltose binding protein (MBP). To help with purification, a polyhistidine-tag was appended to the N-terminus of MBP, and an enterokinase (EK) recognition site (DDDDK) was inserted between MBP and G-CSF (His6-MBP-GCSF). The designed fusion protein sequence was synthesized and inserted into Saci/BamHI sites of a pMAL-p2E plasmid. The resulting plasmid was then transformed into BL21(DE3) competent E. coli cells and His6-MBP-GCSF was overexpressed (details in SI). Purification of the His6-MBP-GCSF fusion protein was performed on a nickel(II)-nitrilotriacetic acid (Ni-NTA) column. This process removed most of the non-specific proteins (Figure S7) except for His-tagged MBP without the attached G-CSF. Removal of endotoxins was also performed following this step.

The His6-MBP-GCSF fusion protein was cleaved with EK, releasing the desired G-CSF protein. The expected cleavage products were observed by SDS-PAGE and Western blot analysis showing increased amounts of His6-MBP and the desired G-CSF proteins released with increasing EK concentrations (Figure S8). A second Ni-NTA column was then used to selectively bind the His-tagged proteins, allowing the cleaved G-CSF protein to freely elute from the column. Fast protein liquid chromatography (FPLC) purification was then performed to remove any other contaminating proteins. SDS-PAGE analysis showed the protein content following the second Ni-NTA column (Figure 1a, lane 1), and subsequent FPLC purification of G-CSF (Figure 1a, lanes 3 and 4).

Figure 1.

Figure 1

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Characterization of purified G-CSF by a) SDS-PAGE visualized with silver staining and b) matrix-assisted laser desorption/ionization (MALDI).

The purified G-CSF protein was characterized by MALDI, enzyme-linked immunosorbent assay (ELISA), and bioactivity in NFS-60 cells. The MALDI spectra showed a sharp peak at m/z = 18777.4, confirming the expected G-CSF size of 18.8 kDa (Figure 1b). The peak at m/z = 37575.7 likely indicates the presence of G-CSF dimer. The concentration of purified G-CSF was determined by ELISA. Bioactivity of the purified G-CSF protein was assayed by proliferation of mouse NFS-60 myelogenous leukemia lymphoblast cells (Figure S9).[32] The dose-response curve was sigmodal and the EC50 of the purified G-CSF protein was 0.23 ng/mL. The results suggest that the purified protein is slightly less active compared to commercially available G-CSF, which has a reported EC50 of 0.1 ng/mL.[33]

2.3. Cytocompatibility of degradable trehalose polymers

Cytotoxicity assessment of the degraded and non-degraded products of p(BMDO-co-BMA-trehalose) from 0.01 to 1 mg/mL concentrations was carried out in NFS-60 cells and human dermal fibroblasts (HDFs) using a LIVE/DEAD assay (Figure 2). Benzyl trehalose and p(styrenyl acetal trehalose) (Figure S1) both previously shown to be non-cytotoxic,[15] were included in the assays as controls; benzyl trehalose was selected to be analogous with the degraded trehalose fragments. In HDFs, degraded p(BMDO-co-BMA-trehalose) products were shown to reduce cell viability to 74% at 1 mg/mL, the highest concentration tested, while the non-degraded polymer and benzyl trehalose was shown to be non-cytotoxic at that concentration (Figure 2a). These results indicate that the degraded BMDO unit, and not the trehalose monomer unit, is most likely associated with the observed cytotoxicity. Similar effects were observed in NFS-60 cells. p(Styrenyl acetal trehalose) exhibited no cytotoxicity (100% cell viability relative to no excipient), p(BMDO-co-BMA-trehalose) was 95%, and the degraded polymer fragments dropped to 90% cell viability at a concentration of 1 mg/mL (Figure 2b). Again, degraded p(BMDO-co-BMA-trehalose) had a significantly greater adverse effect on cells compared to the non-degraded polymer, which suggests that the degraded products are cytotoxic at high concentrations. To our knowledge, no previous investigation of BMDO degradation product cytocompatibility at concentrations above 0.2 mg/mL has been carried out. At lower concentrations of degraded fragments, p(BMDO-co-BMA-trehalose) is equivalent to those previously described.[25, 28]

Figure 2.

Figure 2

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Cytotoxicity assay of p(BMDO-co-BMA-trehalose) before and after degradation and benzyl trehalose in a) HDFs by LIVE/DEAD assay (n = 4), and of the non-degraded, degraded products and p(styrenyl acetal trehalose) in NFS-60 cells based on b) cell viability by LIVE/DEAD assay (n = 4), and c) cell proliferation (n = 6). Results are shown as the average with standard deviation and normalized to 100% to the condition of no excipients. ** = p < 0.01, *** = p < 0.005 relative to no excipients.

The effects of p(BMDO-co-BMA-trehalose) and p(styrenyl acetal trehalose) on cell proliferation were evaluated at concentrations up to 1 mg/mL in NFS-60 cells (Figure 2c). At the highest concentration, the percent cell proliferation was reduced to 84% for the control trehalose polymer, 60% for p(BMDO-co-BMA-trehalose), and 38% for degraded polymer relative to no additive control. Because these values are significantly higher than the decreases in cell viability previously observed, this indicates that the nondegraded polymers are inhibiting NFS-60 cell growth rather than killing the cells. This combination of inhibition and slight cytotoxicity observed for both p(BMDO-co-BMA-trehalose) and the degraded products resulted in a significant decrease in NFS-60 cell proliferation at higher polymer concentrations.

2.4. Stabilization of G-CSF

Next, the trehalose glycopolymers were evaluated for their ability to stabilize the purified G-CSF protein to heat stress. G-CSF was heated at 40 °C for 30 minutes with and without excipients and the bioactivity was assayed by NFS-60 cell proliferation. The excipients included PEG, trehalose, p(BMDO-co-BMA-trehalose), and p(styrenyl acetal trehalose), and were screened with weight equivalents from 1 to 500 relative to G-CSF. The resulting G-CSF bioactivities with and without excipients are shown in Figure 3. The activity of G-CSF was reduced to about 30% following heat treatment. The addition of PEG did not stabilize G-CSF as the activity levels (37% ± 3.8) were similar to the negative control (Figure 3a). In the presence of trehalose, 77% ± 7.6 activity was retained at 500 wt. eq. relative to G-CSF, which showed moderate stabilization (Figure 3b). Moderate stabilization of G-CSF was also observed with the addition of p(BMDO-co-BMA-trehalose) showing 66% ± 4.6 and 51% ± 5.9 of the original activity with 10 and 500 wt. eq., respectively (Figure 3c). P(styrenyl acetal trehalose), especially at 500 wt. eq., showed full retention of activity (Figure 3d). In all of the trehalose glycopolymers, even at the lowest concentration of 1 wt. eq., stabilization with statistical significance was observed compared to the negative control.

Figure 3.

Figure 3

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Bioactivity of G-CSF without any additive or with 1, 10, 100, or 500 weight equivalents of excipient to protein without heating (untreated) and with heating (treated) to 40 °C for 30 minutes. Excipients shown are a) 20 kDa PEG, b) trehalose, c) p(BMDO-co-BMA-trehalose), and d) p(styrenyl acetal trehalose). Data shown as the average (n = 6) and standard deviation. *** = p < 0.005 relative to heated G-CSF control.

Additionally, the excipients were added without heating the protein to determine any inherent effects the additives had on GCSF activity. With PEG, trehalose, and p(BMDO-co-BMA-trehalose), no significant change in activity was observed. However, the addition of the control p(styrenyl acetal trehalose) increased G-CSF activity to over 100% relative to the positive control. A similar increase in activity has been observed with other proteins,[15] and suggests that the polymer may help with enhancing protein/substrate binding or stabilization of the active site as does trehalose.[34] We then calculated the percent decrease of the degradable and control polymers and found the values to be similar until 500 wt. eq. (Figure S10). This suggests that the ability of the trehalose polymers to stabilize GCSF are similar at 1–100 weight equivalents, but GCSF with p(styrenyl acetal trehalose) starts at a higher activity leading to higher overall retention of activity upon heat stress.

This work demonstrates evaluation of a hydrolytically degradable trehalose glycopolymer containing BMDO units. p(BMDO-co-BMA-trehalose) stabilized G-CSF against heat stress similar to the non-degradable control until the highest concentration tested; although the actual percent bioactivities were lower overall. The polymer itself was non-cytotoxic. However, the degradation products demonstrated slight cytotoxicity at higher concentrations and inhibition of cell growth, likely due to the BMDO units. Further optimization of CKA structure may decrease this observed incompatibility. For instance, 2-methylene-1,3-dioxepane (MDO) ring opens into the well-known and FDA-approved poly(caprolactone).[35] Alternatively, 2-methylene-4-phenyl-1,3-dioxolane (MPDL) could be used.[36, 37] Regardless, these data provide important information about the allowable working concentrations of this polymer within cells.

3. Conclusions

A degradable trehalose copolymer was synthesized by CRP. The polymer was prepared by copolymerizing but-3-enyl methacrylate with BMDO by RAFT polymerization to obtain hydrolysable esters in the backbone followed by thiol-ene modification with trehalose on the side chains. The therapeutic protein G-CSF was expressed and purified. Stability screening with G-CSF and p(BMDO-co-BMA-trehalose) showed 66% retention of activity after heat stress. Characterization of the polymer confirmed that the BMDO-containing polymer was degraded within 1 day. Although the parent polymer was non-cytotoxic at the concentrations tested, the degraded fragments exhibited cytotoxicity at a concentration of 1 mg/mL.

Supplementary Material

ESI

Acknowledgments

The National Science Foundation (CHE-1507735) is thanked for funding. The authors thank Jeong Hoon Ko (UCLA) for the synthesis of the CTA. The authors thank Prof. Yang Liu (Chapman University) and Dr. Mark Arbing (UCLA) for the design of the G-CSF plasmid. UYL thanks the NIH Chemistry Biology Interface Training Fellowship (T32 GM 008496) and UCLA Graduate Division for funding. EMP thanks the NSF Graduate Research Fellowship (DGE-1144087), the Christopher S. Foote Graduate Research Fellowship in Organic Chemistry, and the UCLA Dissertation Year Fellowship.

Footnotes

a

Supporting Information is available online from the Wiley Online Library or from the author.

Supporting Information

Supporting Information is available from the Wiley Online Library

Contributor Information

Dr. Uland Y. Lau, Department of Bioengineering, University of California, Los Angeles, 410 Westwood Plaza, Los Angeles, California 90095, USA

Emma M. Pelegri-O’Day, Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, USA

Prof. Heather D. Maynard, Department of Bioengineering, University of California, Los Angeles, 410 Westwood Plaza, Los Angeles, California 90095, USA. Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, USA

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