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Thermal stabilization of biologics with photoresponsive hydrogels. Balaji V. Sridhar, John R. Janczy, Øyvind Hatlevik, Gabriel Wolfson, Kristi S. Anseth, and Mark W. Tibbitt Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01507 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biomacromolecules

Thermal stabilization of biologics with photoresponsive hydrogels Balaji V. Sridhar,†,⊥ John R. Janczy,†,⊥ Øyvind Hatlevik,† Gabriel Wolfson,† Kristi S. Anseth,‡,¶,§ and Mark W. Tibbitt∗,k †Nanoly Bioscience, Inc., Denver, CO, USA ‡Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA ¶BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, USA §Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, CO, USA kMacromolecular Engineering Laboratory, Department of Mechanical and Process Engineering, ETH Zürich, Zürich, Switzerland ⊥these authors contributed equally to this work E-mail: [email protected] Phone: +41 44 632 25 16 Abstract Modern medicine, biological research, and clinical diagnostics depend on the reliable supply and storage of complex biomolecules. However, biomolecules are inherently susceptible to thermal stress and the global distribution of value-added biologics, including vaccines, biotherapeutics, and Research Use Only (RUO) proteins, requires an integrated cold chain from point of manufacture to point of use. To mitigate reliance on the cold chain, formulations have been engineered to protect biologics from thermal stress, including materials-based strategies that impart thermal stability via direct encapsulation of the molecule. While direct encapsulation has demonstrated pronounced

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stabilization of proteins and complex biological fluids, no solution offers thermal stability while enabling facile and on-demand release from the encapsulating material, a critical feature for broad use. Here we show that direct encapsulation within synthetic, photoresponsive hydrogels protected biologics from thermal stress and afforded userdefined release at the point of use. The poly(ethylene glycol) (PEG) based hydrogel was formed via a bioorthogonal, click reaction in the presence of biologics without impact on biologic activity. Cleavage of the installed photolabile moiety enabled subsequent dissolution of the network with light and release of the encapsulated biologic. Hydrogel encapsulation improved stability for encapsulated enzymes commonly used in molecular biology (β-galactosidase, alkaline phosphatase, and T4 DNA ligase) following thermal stress. β-galactosidase and alkaline phosphatase were stabilized for 4 weeks at temperatures up to 60 ◦ C, and for 60 min at 85 ◦ C for alkaline phosphatase. T4 DNA ligase, which loses activity rapidly at moderately elevated temperatures, was protected during thermal stress of 40 ◦ C for 24 h and 60 ◦ C for 30 min. These data demonstrate a general method to employ reversible polymer networks as robust excipients for thermal stability of complex biologics during storage and shipment that additionally enable on-demand release of active molecules at the point of use.

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Introduction Biomacromolecules are used widely as therapeutics (e.g., vaccines and engineered antibodies) in the clinic and as reagents (e.g., enzymes) in industry and research laboratories. 1 However, biomacromolecules are inherently unstable on account of their chemical and structural complexity. Their activity or efficacy often declines during storage and transport as thermal fluctuations can lead to aggregation, denaturation, or oxidative degradation and render them ineffective. 2,3 Even ambient temperature storage can lead to a significant reduction in biological activity. Therefore, in order to retain activity of biomacromolecules during storage and shipping, most biologics are maintained within a continuous cold chain (2–8 ◦ C) from production to end use. 4 The global cold chain results in exorbitant cost (estimated at $8 billion USD in 2015) and risk associated with potential cold chain disruption, and it remains one of the major challenges faced by global vaccination programs according to the World Health Organization. 4,5 Further, cold chain requirements restrict efficient distribution to many critical areas of the developing world. To mitigate reliance on the cold chain, many biomacromolecules are engineered during development to increase thermal stability. 6 However, efficient protein engineering is difficult and requires new approval by the regulatory authorities; therefore, formulation or excipient strategies are often preferred. Perhaps the most common approach to improve the thermal stability of formulated biomacromolecules has been to lyophilize or freeze-dry formulations of the active molecule with or without excipients prior to storage and transport. 7 However, during lyophilization, the sample is subjected to extreme changes in temperature, concentration, pressure, hydration level, and/or pH, each of which can cause the biomacromolecule to denature and/or aggregate. 8,9 Further, if the cargo survives the initial freeze-drying process, it can still be damaged by thermal stress when formulated as a lyophilized powder. 7,10 A range of formulation additives or excipients have been employed to prevent or slow the degradation of biomacromolecule activity during lyophilization, storage, and shipping. 11 Common excipients include amino acids, sugars and carbohydrates, as well as salts and other osmolytes. 10,11 3



The most common and effective excipient to date is trehalose, a disaccharide of glucose that is produced by many thermostable organisms and in response to desiccation. 12,13 Excipients often function by maintaining a preferred hydration state, through direct interactions with the molecule of interest, or by limiting aggregation. Yet, even when the excipients are tailored to the molecule of interest, formulated and lyophilized biomacromolecules can exhibit significant thermal instability. Therefore, polymeric or nanoparticle excipients have been designed to confer broad temperature stability to biomacromolecules that obviate the need for lyophilization. The thermal stability of adenoviruses was enhanced by formulation with low concentrations of PEG or anionic gold nanoparticles. 14 Engineered trehalose glycopolymers protected proteins during thermal stress up to 30 min at 70 ◦ C. 8,15 Further, direct encapsulation in synthetic or natural materials has conferred long-term thermal stability for a wide-range of biologics. Pioneering work by Kaplan and coworkers have shown thermal stability for proteins and complex biological fluids via encapsulation within silk fibroin biomaterials. 16–18 A hydrogel with covalent incorporation of trehalose exhibited improved stability at elevated temperatures for controlled drug delivery of active biotherapeutics. 19 While encapsulation approaches confer thermal stability without the need for lyophilization or direct conjugation, a major challenge remains to release the biologic on-demand at the point of use. Here we describe the use of a photoresponsive, PEG-based hydrogel to protect biologics from thermal stress during storage and transport via direct encapsulation. The crosslinked hydrogel was engineered with a photolabile moiety in the network backbone to afford ondemand release of the entrapped cargo at the point of use via network dissolution with controlled light exposure. The hydrogel was used to encapsulate three different proteins, and the samples were subjected to a range of thermal challenges. After storage, UV light dissolved the protecting gel, releasing the encapsulated biomacromolecules. The activity of the encapsulated proteins were quantified after release. Model enzymes (β-galactosidase and alkaline phosphatase) were stabilized for 4 weeks at 60 ◦ C; alkaline phosphatase was

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further stabilized for 60 min at 85 ◦ C. A common and highly temperature sensitive ligase (T4 DNA ligase) was stabilized for 24 hours at 40 ◦ C and for 30 min at 60 ◦ C. Thermal stabilization required network formation as each constituent alone did not confer full stability and thermal stability was improved with the inclusion of trehalose in the gel formulation. This work presents a synthetic materials-based strategy that enables direct encapsulatation of proteins of interest without conjugation, stabilization from thermal damage during storage and transport, and on-demand release at the point of use as an approach to mitigate reliance on the cold chain.

Experimental Section Materials. All chemical reagents were purchased from Sigma-Aldrich and used without purification except as noted.

Analytical Techniques. NMR spectroscopy.

1

H-NMR and

13

C-NMR spectra were obtained using a 400 MHz

Bruker Avance III 400. All chemical shifts were reported in ppm and relative to the peaks of the deuterated solvents used: CDCl3 and DMSO-d 6 at δ 7.26 and 2.50 ppm, respectively, for the 1 H spectra and DMSO-d 6 at δ 39.51 ppm for the 13 C spectra.. Multiplicities of 1 H-NMR spin couplings are reported as s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, p = pentet.

Matrix-assisted laser desorption–ionization time-of-flight spectrometry. Matrixassisted laser desorption–ionization time-of-flight (MALDI-TOF) spectra were obtained usR R ing a Waters Micromass MALDI micro MXTM . Samples were loaded on and run using

α-cyano-4-hydroxycinnamic acid matrix. 5



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Rheometry. Hydrogel mechanical properties were assessed with shear rheometery on a Discovery HR-3 (TA Instruments) photorheometer and analyzed with TRIOS Software (TA Instruments). Formation and degradation tests were performed with an 8 mm quartz parallel plate geometry and a steel Peltier plate (T = 25 ◦ C) at an angular frequency of 10 rad/s with a strain of 1.0%, confirmed to be in the linear viscoelastic regime for this material. The gap height was 300-500 µm. Gel precursor solutions were mixed and placed on the rheometer within 20 to 60 seconds. Samples were exposed to irradiation (λ = 365 nm, I0 ≈ 10 mW/cm2 ) after complete gelation (∼240 seconds after the start of the experiment). The storage (G 0 ) and loss (G 00 ) moduli were recorded for 1200 seconds.

High-performance liquid chromatography. As indicated, reaction products were purified using a Waters Delta 600 semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) system. A 10 mL/min flow using a 70 min linear gradient of 5-95% acetonitrile with 0.1% trifluoroacetic acid was used. Product separation was achieved with a Waters Xbridge Prep C18 5um OBD 19 x 50 mm column.

Methods. Synthesis of octyne-functionalized PEG. The synthesis of dibenzocyclooctynefunctionalized

multiarm

PEG

was

based

on

similar

published

protocols. 20,21

Dibenzocyclooctyne-carboxylic acid (DBCO-COOH, 100 mg, 0.3 mmol, Jena Biosciences) and

2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl

uronium

hexafluorophosphate

methanaminium (HATU, 167 mg, 0.44 mmol, Anaspec) were dissolved in minimal dimethylformamide (DMF, 5 mL). 4-methylmorpholine (100 mg, 0.99 mmol) was added to the DBCO-COOH/HATU solution and reacted for 15 min at room temperature. In parallel, four-arm, amine-terminated PEG (PEG-4-NH2; Mn ≈ 20 kDa, 1.0 g, 0.2 mmol of NH2 , JenKem Technology USA) was dissolved in DMF (5 mL). The activated DBCO-COOH solutions was added to the PEG-4-NH2 solution and stirred overnight at room temperature.

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The reaction was concentrated, dissolved in dH2 O, dialyzed (SpectraPor 7 MWCO ≈ 8 kDa, Spectrum Laboratories), filtered, and lyophilized. A white powder (0.81 g, 81%) was recovered and the degree of functionalization was confirmed to be ∼90% by 1 H-NMR and the functionalization was confirmed by 13 C-NMR (Supporting Information; Fig. S1 and S2).

Synthesis of 4-azidobutanoic acid The synthesis of 4-azidobutanoic acid was based on a previous protocol. 21 Ethyl-4-bromobutyrate (50.0 g, 256 mmol) was dissolved in dimethyl sulfoxide (DMSO; 250 mL) in a 500 mL round-bottom flask and stirred under an argon atmosphere at 55 ◦ C overnight with sodium azide (25.0 g, 1.5 eq., 385 mmol). The reaction mixture was diluted with dH2 O (250 mL) and the product was extracted 3x into diethyl ether (250 mL). The recovered organic phase was washed 1x with dH2 O (250 mL), 1x with brine (250 mL), dried over Na2 SO4 , filtered, and concentrated to yield 39.0 g (248 mmol) of ethyl-4-azidobutanoate. Ethyl-4-azidobutanoate was dissolved in a solution of 1N NaOH (250 mL) and methanol (150 mL) and stirred for 3 h at room temperature. The methanol was removed and the pH of the aqueous phase was brought to 1 with dropwise addition of HCl. The product was extracted 3x into diethyl ether (250 mL) and the recovered organic phase was dried over Na2 SO4 , filtered, and concentrated to yield 31.0 g (240 mmol) of 4azidobutanoic acid. 1 H-NMR (400 MHz, CDCl3 , δ): 9.64 (br s, 1H), 3.34 (t, 2H), 2.44 (t, 2H), 1.88 (p, 2H).

Synthesis

of

4-[4-(1-(4-azidobutanoyloxy)ethyl)-2-methoxy-5-nitrophenoxy]-

butanoic acid. The synthesis of the photolabile azide was based on a previous protocol. 21 In brief, 4-azidobutanoic anhydride was prepared by reacting 4-azidobutanoic acid (31.0 g, 240 mmol) and N,N0 -dicyclohexylcarbodiimide (DCC; 15.9 g, 77 mmol) in anhydrous dichloromethane (DCM; 200 mL) under an argon atmosphere and stirring for 45 min at room temperature. The reaction was concentrated and the dicyclohexylurea byproduct precipitated, which was removed by filtration over celite. The reaction was redissovled in DCM (40 mL), concentrated, and filtered over celite. 7


This was repeated until no


dicyclohexylurea was observed upon concentration.

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4-[4-(1-Hydroxyethyl)-2-methoxy-5-

nitrophenoxy]butanoic acid (NBE; 5.0 g, 16.7 mmol) and 4-dimethylaminopyridine (DMAP; 100.0 mg, 0.82 mmol) was added to the anhydride mixture and dissolved in DCM (125 mL). Pyridine (1.35 mL, 17.1 mmol) was added to the reaction mixture, which was stirred under an argon atmosphere overnight at room temperature. The crude reaction mixture was washed 1x with aq. NaHCO3 (125 mL), 1x with 1N HCl (125 mL), and 1x with brine (125 mL) and the recovered organic phase was dried over Na2 SO4 , filtered, and concentrated. The intermediate mixture was dissolved in a 50:50 solution of acetone:dH2 O (1500 mL) and stirred overnight. Acetone was removed by rotary evaporation and the product was extracted into DCM (750 mL). The recovered organic phase was washed 1x with 1N HCl (500 mL) and 1x with brine (500 mL). The recovered organic phase was dried over Na2 SO4 , filtered, and concentrated.

The product was purified by flash chromatography (5:1 to

1:1 hexanes:ethyl acetate with 1% acetic acid) to yield a yellow solid (4.9 g, 11.9 mmol). 1

H-NMR (400 MHz, DMSO, δ): 12.18 (s, 1H), 7.57 (s, 1H), 7.10 (s, 1H), 6.21 (q, 1H), 4.07

(t, 2H), 3.93 (s, 3H), 3.32 (t, 2H), 2.4 (p, 4H), 1.95 (p, 2H), 1.76 (p, 2H), 1.58 (d. 3H).

Synthesis of azide-functionalized photolabile peptide. The synthesis of bis(azide)functionalized photolabile peptide was based on a similar published protocol. 21 In brief, the base peptide H-RGGRK(dde)-NH2 was synthesized via Fmoc solid-phase peptide synthesis with HATU activation (0.5 mmol, Tribute peptide synthesizer, Protein Technologies). 4-Azidobutanoic acid 21 was coupled to the N-terminal amine with HATU activation, the 1-(4,4-dimethyl-2-,6-dioxacyclohexylidene)ethyl (Dde) group was removed with 2% hydrazine monohydrate in DMF (3 x 10 min), 4-(4-(1-(4-azidobutanoxy)ethyl)-2-methoxy-5nitrophenoxy)butanoic acid (PLazide) 21 was coupled to the ε-amino group of the C-terminal lysine. The resin was treated with trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5) for 2 hrs. The product was precipitated in and washed (2x) with ice-cold diethyl ether. The crude peptide was purified via semi-preperative reversed-phase HPLC, and lyophilized

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to give the product (Azide-RGGRK(PLazide)-NH2 ) as a fluffy, yellow solid. Peptide purity was confirmed with 1 H-NMR,

13

C-NMR, and MALDI-TOF (Supporting Information; Fig.

S3, S4, and S5): calculated ([M + H]+ 1075.54); observed ([M + H]+ 1075.52). NMR peak assignment was assisted by homonuclear COSY spectra.

Formation of photoresponsive networks. Hydrogel networks were prepared by mixing solutions of PEG-4-DBCO (20 wt% in dH2 O) and PLP-2-N3 (4 wt% in dH2 O) at equal stoichiometry of DBCO:N3 . That is, 40 µL 6 wt% hydrogel precursor solutions were prepared by combining 10.84 µL PEG-4-DBCO (20 wt%), 5.82 µL PLP-2-N3 , and 23.34 µL of dH2 O. For encapsulation of enzymes, stock solutions of alkaline phosphatase (ALP; 20 mg/mL in dH2 O), β-galactosidase (β-gal, 40 mg/mL in DPBS), T4 DNA ligase (2000 U/µL, New England Biolabs), and trehalose (56.8 mg/mL in dH2 O) were prepared. The balance volume of dH2 O was replaced with corresponding volumes of ALP, β-gal, or T4 DNA ligase and trehalose (2.5x protein by mass) to prepare hydrogels of the respective formulations. The hydrogels were prepared at final protein concentrations in the hydrogel of 5 µg/µL for β-gal, 2.5 µg/µL for ALP, and 1000 U/µL for T4 DNA ligase. Hydrogels were prepared between R two round glass coverslips (22 mm diameter, 1 oz., VWR; one treated with Sigmacote and

one untreated) to prepare thin hydrogel films. 5 µL of precursor solution were placed on the untreated glass cover slip and covered with the treated glass cover slip. The solution was allowed to gel for 4–5 min after which hydrogel films ∼25–40 µm in thickness were formed. After gelation, the treated cover slip was removed and the gels were dehydrated under house vacuum for 2-16 h to remove residual moisture. Samples were stored at test temperature (4 ◦

C to 85 ◦ C) for the indicated period of time. All gel formulations were reported as network

wt% as well as concentrations of protein and trehalose in the as prepared state.

Hydrogel photodegradation and release of encapsulated protein. Encapsulated enzymes were recovered from the hydrogels by photodegradation of the thin film gels in the presence of a recovery solution. All protein-loaded gels were stored at temperature in 9



the dehydrated state during thermal treatment as described above. Trehalose (2.5x protein by mass) was included in all gel preparations during thermal treatment. Treated protein controls were stored at temperature in the as delivered powdered form. Following thermal treatment, recovery solution (245 µL DPBS for β-gal; 245 µL dH2 O for ALP; 95 µL dH2 O for T4 DNA ligase) was added to each 5 µL gel. To release its contents into solution, the gel was then exposed to light to dissolve the network. Gels were photodegraded with one of two release conditions: i. a handheld LED (λ = 365 nm, I0 ≈ 10 mW/cm2 , t = 20 min; ThorLabs) or ii. a UV transilluminator (λ = 365 nm, I0 ≈ 5 mW/cm2 , t = 40 min; Fotodyne, Inc.). All protein analyses were conducted without purification after gel dissolution. That is, the degraded hydrogel products remained in the assay solution and control experiments demonstrated protein activity remains intact (> 90%) in the presence of degraded hydrogel products. In all experiments, enzyme activity was normalized to a freshly prepared enzyme solution at the same concentration (storage: -20 ◦ C) and then compared amongst the control and treatment groups for that experiment.

β-galactosidase activity assay. β-galactosidase (β-gal) activity was measured using onitrophenyl-D-galactopyranoside (ONPG) (Thermo Scientific) according to the manufacturer’s protocol.

Briefly, room temperature ONPG (16 mM in DPBS) was mixed 2:1

(ONPG:β-gal test solution, 100 µL:50 µL) with the test β-gal solution (100 µg/mL) in a 96well plate. β-gal catalyzes the hydrolysis of ONPG to release o-nitrophenol. o-Nitrophenol is a chromogenic substrate with maximal absorbance at 405 nm. Absorbance at 405 nm was measured every 60 s for 10 min, and the mean slope of the resultant curve was recorded. The β-gal test solution was compared to a freshly prepared and non-treated standard of the same β-gal concentration.

Alkaline phosphatase activity assay. Alkaline phosphatase (ALP) activity was measured using para-nitrophenyl phosphate disodium salt (pNPP) (Thermo Scientific) per manufacturer’s protocol. Briefly, room temperature pNPP was mixed 1:1 (pNPP:ALP test so10


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Biomacromolecules

lution, 50 µL:50 µL) with the test ALP solution in a 96-well plate. ALP catalyzes the hydrolysis of pNPP into free phosphate and para-nitrophenol (pNP). pNP is a chromogenic substrate with maximal absorbance at 405 nm. Absorbance at 405 nm was measured every 60 s for 10 min, and the mean slope of the resultant curve was recorded. The ALP test solution was compared to a freshly prepared and non-treated standard of the same ALP concentration (50 µg/mL).

T4 DNA ligase activity assay. T4 DNA ligase (New England Biolabs) activity was measured by ligation of λ DNA/HinDIII digest fragments (New England Biolabs) according to the manufacturer’s protocol. Briefly, 1 µL T4 DNA ligase (50 U/µL), 1 µL of λ DNA/HinDIII digest fragments (500 ng/µL), 2 µL of 10x Ligase Buffer (New England Biolabs), and 16 µL of nuclease free water were combined. The ligation reaction was allowed to proceed for 16 h at 16 ◦ C. After incubation, 5 µL of 6x DNA loading dye without SDS (New England Biolabs) was added to the reaction mixture. The reaction mixture was resolved on a 1% TAE agarose gel, and visualized with ethidium bromide. Gel images were analyzed using ImageJ. The largest, i.e., slowest migrating, band in each lane was selected for quantification. The signal of this band was normalized to the total signal in each lane. The normalized band signal was then compared to the corresponding normalized band signal in the freshly prepared and non-treated ligase lane. Quantification was performed and averaged for three independent experiments.

Statistical analysis. Statistical analysis was performed using Prism 5.0a (GraphPad Software). Unless otherwise noted, statistical significance for single comparisons was determined by Student’s t-test; ANOVA with a Bonferroni post-test was used for multiple comparisons.

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Results Formation of photoresponsive hydrogel. In order to investigate thermal stabilization of biologics via direct encapsulation in reversible hydrogels, we designed a step-growth, photoresponsive hydrogel amenable to encapsulation of biologics and on-demand release (Fig. 1). 22,23 An alkyne-functionalized multi-arm poly(ethylene glycol) (PEG) was synthesized by reacting dibenzocyclooctyne-carboxylic acid (DBCO-COOH) to 4-arm PEG-amine (Mn ≈ 20 kDa) via HATU coupling to generate PEG4-DBCO (Fig. 1). A photolabile azide was synthesized by reacting 4-azidobutanoic anhydride with 4-[4-(1-hydroxyethyl-2-2methoxy-5-nitrophenoxy]butanoic acid (PL) to generate PLN3 . A photocleavable, bis(azide) peptide (PLP-2-N3 ; N3 -RGGRK(PLN3 )-NH2 ) was synthesized via Fmoc solid-phase peptide synthesis with HATU activation. PEG-4-DBCO was reacted with PLP-2-N3 via a strain-promoted azide-alkyne cycloaddition (SPAAC) between terminal DBCO and –N3 moieties with 1:1 stoichiometry at 6 wt% total macromer concentration to form the step-growth, photoresponsive hydrogel. The bioorthogonal, SPAAC reaction proceeded rapidly in aqueous media and in the presence of biologics without crossreaction. 20,24 Network formation was complete ∼4–5 min after mixing, as measured by the plateau in the shear storage modulus (G0 ; Fig. 2A). The hydrogels formed with shear storage moduli of G0 = 630 ± 90 Pa (Fig. 2B; mean ± s.e.m.; n = 5).

Hydrogel photodegradation and release of encapsulated proteins. To provide a method for on-demand release of encapsulated biologics, the photolabile peptide was engineered such that PLN3 moieties were installed into the backbone of the formed network. This enabled subsequent photodegradation and dissolution of the gel following irreversible cleavage of PLN3 with controlled light exposure. 21,25,26 Network dissolution occurred as each active chain in the network contains a PLN3 moiety, which can cleave upon exposure to 365 nm light. Cleavage of a sufficient fraction of the PLN3 moieties (Prg = 0.42 from the 12


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A. N

N

PEG-4-DBCO O

O

HN

PLP-2-N3

O

O

O H 2N

NH O

O n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

O

O

O n

O

O

O

HN

HN

n

NO2

O H N

HN O n

HN

NH

O N H

O

O

H N

O

O N H

O

O

NH2 O

N3

NH HN

O

O

O

O

N

N3

HN

NH2

N

B. Hydrogel formation: +

N

N

-

+

Hydrogel dissolution:

N

N

N

O

Aqueous O

37 °C N

O

λ

O

O

N

NO2

O

O

N O

NO

+ HO

UV light

PEG-4-DBCO +

PLP-2-N3 +

Biologic

Figure 1: Hydrogel components, formation, and dissolution. (A.) Chemical structures of the hydrogel forming components: PEG-4-DBCO (n ≈ 114) and PLP-2-N3 . (B.) Hydrogel formation proceeded via a strain-promoted azide-alkyne cycloaddition between PEG4-DBCO and PLP-2-N3 in the presence of a biologic. The biologic was encapsulated in the network without direct conjugation, providing thermal protection during storage and shipment. At the point of use, the hydrogel was dissolved with light via cleavage of nitrobenzyl ether moieties in the network backbone to release the active biologic. The squiggles indicate that the chemical moieties are attached to the network backbone. Flory-Stockmayer Theory for this network) 27,28 induced network dissolution into a soluble form, releasing the encapsulated proteins into the recovery solution. 29,30 To characterize the kinetics of photodegradation, formed gels were exposed to UV light and the evolution of G0 was monitored via in situ photorheometry as a measure of the rate of PLN3 cleavage. From rubber elasticity, the normalized storage modulus is directly 0

related to the number density of elastically active chains in the network ( GG0 = 0

ν , ν0

where

ν is the number density of elastically active chains). As the photocleavage reaction of the

13



0

PLN3 moiety follows first-order kinetics, log GG0 decayed linearly with time and a characteristic 0

timescale of network degradation was defined (τP D ) from a linear fit of these data (Supporting Information; Fig. S6). 30–32 For this gel formulation and irradiation conditions (λ = 365 nm; I0 ≈ 10 mW cm−2 ), τP D = 50 ± 10 s (Fig. 2B; mean ± S.D.; n = 5), indicating complete dissolution of the network should occur within several minutes . β-galactosidase (β-gal) was then encapsulated within the photoresponsive gel at 10 mg mL−1 to determine whether encapsulation affects protein activity and to quantify release as a function of light exposure. Thin films of gel with encapsulated β-gal were exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ) immediately after drying and the activity of β-gal was measured at each time point (Fig. 2C). The released enzyme retained full activity, indicating that the network formation and dissolution did not afffect protein stability. Complete release was observed after 5 min of irradiation, consistent with the characteristic timescale of network degradation. Control experiments confirmed that UV light exposure is required to liberate encapsulated protein (β-gal) from the hydrogel as no release (enzyme activity) was observed after incubation of the gel in recovery solution for 20 min without UV light (Fig. 2D). Further, enzyme activity remained above 90% of control levels when exposed to UV light or network degradation products alone (Supporting Information; Fig. S7, S8, and S9), indicating that enzyme activity was minimally impacted by the UV light exposure or degraded network products.

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A.

B. 10 G' G''

102

Modulus (Pa)

G',G'' (Pa)

103

101 100

10-1

0

500

1000

100 80

102

60 40

101

20 100

1500

Time (s)

C.

0

τ (s )

G' (Pa)

D.

100

100

Normalized -gal Activity


3

Time (s)

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80 60 40 20 0 0

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20

80 60 40 20 0 UV:

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Figure 2: Characterization of photoresponsive hydrogels and payload release. (A.) In situ dynamic photorheometry of a photoresponsive hydrogel sample quantifying shear storage modulus (G0 ) and shear loss modulus (G00 ) during network formation and degradation. Upon complete network formation, the sample was exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; indicated by purple shading) triggering network degradation, causing a monotonic decrease in shear storage modulus (G0 ). Data shown are representative of five independent experiments. Samples were measured in a parallel plate geometry between a Peltier plate (25 ◦ C) and an 8 mm quartz plate; gels were 20 µL in volume; γ = 1.0 %; ω = 10 rad s−1 . (B.) Repeat measurements of the plateau shear storage modulus (G0 ) and characteristic time scale for photodegration (τP D ) of the photoresponsive hydrogel. Data shown are pooled from five independent experiments and plotted as mean ± s.e.m. (C.) Protein release was characterized from β-gal loaded photoresponsive hydrogels as a function of exposure time. The supernatant was collected after UV exposure (λ = 365 nm; I0 ≈ 10 mW cm−2 ) for the indicated period of time and β-gal activity was measured and normalized to freshly prepared β-gal. Data shown are pooled from three independent experiments and plotted as mean ± SD. (D.) Protein release was characterized from β-gal loaded photoresponive hydrogels with and without UV light exposure. In each case, gels were prepared and immersed in recovery buffer for 20 min prior to measuring enzyme activity. Samples exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ) demonstrated activity while samples that were not exposed to UV light demonstrated no activity. Data pooled from three independent experiments and plotted as mean ± SD.

Thermal stabilization of β-galactosidase. After establishing the photoresponsive hydrogel as a platform for protein encapsulation and on-demand release, we measured the ability of our gel to stabilize β-gal during thermal stress.

15



β-gal is a commonly used research enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides and is used industrially for the removal of lactose from milk. 33 However, β-gal is thermally unstable and broad use requires engineered derivatives or stable formulations. 33,34 In this work, β-gal activity was measured by incubating with ONPG, which, in the presence of active β-gal, releases o-nitrophenol and the concentration of o-nitrophenol was measured by quantifying 405 nm absorbance. After storage with or without gel encapsulation at 60 ◦ C for 2 or 4 weeks, all samples were exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; t = 20 min) to release the encapsulated β-gal and the enzyme activity in each sample was quantified. Protein release was not observed without UV light exposure for gels stored at 60 ◦ C for 2 weeks (Supporting Information; Fig. S10). Enzyme activity was reduced to less than 10% activity for samples stored as a conventionally dried powder, while enzyme stored within our gel retained ≥ 70% activity after 2 or 4 weeks (Fig. 3A). To assess if the observed thermal stabilization required intact network formation around the payload, β-gal was formulated via gel encapsulation and with each of the constituents of the photoresponsive gel (PEG-4-DBCO, PLP-2-N3 , or trehalose) alone and stored at 60 ◦ C for 4 weeks. While the gel constituents provided some thermal stabilization, full protection was observed only when the enzyme was encapsulated within the covalently crosslinked gel (Fig. 3B).

Thermal stabilization of alkaline phosphatase. The ability of our gel to stabilize alkaline phosphatase (ALP) was assessed for a range of thermal stresses. ALP is a commonly used research enzyme in molecular biology that catalyzes the removal of 50 -phosphate groups from nucleic acids. 35 In this work, ALP activity was measured by incubating with pNPP, which, in the presence of active ALP, releases pnitrophenol and the kinetic generation of o-nitrophenol was quantified by monitoring 405 nm absorbance. After storage with or without gel at room temperature (25 ◦ C) for 2 weeks, samples were exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; t = 20 min) to release the 16


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-gal -gal + Hydrogel

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60 40 20

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0 2 4 Time (weeks)

PEG-4-DBCO:

+

+

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Trehalose:

+

-

+

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PLP-2-N3:

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Figure 3: Hydrogel protects β-gal from thermal stress. (A.) β-gal stored with or without hydrogel protection at 60 ◦ C for 2 or 4 weeks. Following storage, each sample was exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; t = 40 min) and β-gal activity in the release supernatant was measured and normalized to freshly prepared and untreated β-gal, which was stored at 4 ◦ C from reconstitution to analysis. Data shown are representative of at least three independent experiments and plotted as mean ± SD. *** indicates P ≤ 0.001 by Student’s t-test. (B.) β-gal was stored at 60 ◦ C for 4 weeks under the specified conditions. Following storage, each sample was exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; t = 40 min) and β-gal activity in the release supernatant was measured and normalized to freshly prepared and untreated β-gal, which was stored at 4 ◦ C from reconstitution to analysis. Data shown are pooled from three independent experiments and plotted as mean ± SD. ** P ≤ 0.01, *** P ≤ 0.001 by one-way ANOVA with Dunnett’s multiple comparison test. encapsulated ALP and the enzyme activity in each sample was quantified. In this experiment, ALP activity was reduced to less than 25% activity when stored as delivered whereas ALP stored within our gel retained ≥ 75% activity (Fig. 4A). Encapsulation of ALP within our gel also provided thermal protection for up to 4 weeks when stored at 60 ◦ C, demonstrating long-term thermal protection at elevated temperature (Fig. 4B). Additionally, gel-based thermal stabilization of ALP was observed for 60 min at 85 ◦ C (Fig. 4C).

Thermal stabilization of DNA ligase. After demonstrating that our photoresponsive hydrogel was capable of stabilizing model enzymes during thermal stress, the ability of the gel to protect T4 DNA ligase was tested. T4 DNA ligase, isolated from bacteriophage T4, is by far the most popular DNA ligase, and is used in virtually all applications that require DNA ligase. Important uses of T4 ligase

17


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80 60 40 20 0 2 4 Time (weeks)

85 ºC

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**

**

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30 45 Time (minutes)

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Figure 4: Hydrogel protects ALP from thermal stress. ALP stored with or without hydrogel protection at (A.) room temperature (25 ◦ C) for 2 weeks, (B.) 60 ◦ C for 2 or 4 weeks, and (C.) 85 ◦ C for up to 60 min. Following storage, each sample was exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; t = 20 min) and ALP activity in the release supernatant was measured and normalized to freshly prepared ALP, which was stored at 4 ◦ C from reconstitution to analysis. Each time point was compared to control non-encapsulated ALP. Data shown are representative of at least three independent experiments and plotted as mean ± SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by Student’s t-test. include: nick repair in dsDNA, circularization of DNA for cloning, and blunt-end dsDNA ligation. 36 However, T4 DNA ligase loses activity rapidly at room temperature and requires precise storage conditions often resulting in large rates of spoilage. The enzyme must be stored at -20 ◦ C with proper buffer support to ensure enzyme function and T4 DNA ligase will lose approximately 50% of its activity when stored at 0 ◦ C for 6 months. 36,37 We observed full ligase activity following direct encapsulation in our gel for 24 h at 40 ◦ C (Fig. 5A,B). Further, the gel protected T4 DNA ligase at 60 ◦ C for 35 min (Fig. 5C,D)

Discussion In total, these data indicate that our photoresponsive hydrogel is capable of conferring thermal stabilization to biologics via direct encapsulation. For each enzyme studied here, gel

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D. Relative Band Intensity

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-

+ + + + + + Fresh 60°C -20°C 60°C

100 80 60

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Figure 5: Hydrogel protects T4 DNA ligase from thermal stress. T4 DNA ligase, with or without hydrogel protection, was stored at (A., B.) 40 ◦ C for 24 h, or at (C., D.) 60 ◦ C for 35 min. For each experiment, a hydrogel encapsulated control was stored at -20 ◦ C, the storage temperature recommended by the manufacturer, for the duration of the experiment. Following storage all samples were exposed to UV light (λ = 365 nm; I0 ≈ 10 mW cm−2 ; t = 10 min) to induce payload release. Ligase activity was assayed by ligating DNA restriction fragments from phage λ. Additionally, a freshly prepared ligase control was assayed. (A., C.) The ligation products were separated on 1% TAE agarose gels and visualized by ethidium bromide staining. Data shown are representative of three independent experiments. (B., D.) To quantify enzyme activity, the relative intensity of the highest molecular weight band was determined by densitometry. Each lane was compared to the freshly prepared ligase control. Data shown are pooled from three independent experiments (line represents the mean, *** P ≤ 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test). encapsulation provided significantly improved stability as compared to untreated conventionally dried controls. Enzyme activity remained intact following on-demand release as well as with direct exposure to UV light or network degradation products. We have demonstrated the use of this system to protect enzymes that are of broad interest to the research and industrial communities and the results suggest that the gel should find broad application, including for biotherapeutic and vaccine stabilization. Of particular note, the system is fully synthetic and may demonstrate less batch-to-batch variation than other encapsulation systems based on natural polymers as well as offer easier quality control during manufacture.

19



The growing supply of biotherapeutics and vaccines places increasing pressure on the integrated cold chain and advanced solutions that mitigate the need for continual refrigeration from point of manufacture to point of use will significantly improve global distribution of value-added biologics. The precise mechanism of how direct encapsulation in the photoresponsive gel stabilizes biologics is not known. Molecular crowding has been proposed as a mechanism by which dense macromolecular excipients may stabilize biomolecules. 38 In this manner, the presence of a crosslinked polymer network may slow or prevent protein-protein interactions and limit aggregation even at elevated temperatures. Alternatively, the network is composed of a hydrophilic PEG backbone and this may provide a favorable hydration state during storage, which shifts the thermodynamic landscape away from denaturation. In total, the results suggest a more general mechanism to employ reversible polymer networks for the long-term thermal stabilization of complex biomolecules. Macromolecular engineering of synthetic materials for direct encapsulation, thermal protection, and on-demand release may provide a simple and effective method to avoid cold chain demands. Further studies are required to provide mechanistic insight as to how synthetic polymer networks stabilize encapsulated proteins. A more complete understanding would have signficant impact not only on the design of next-generation thermal stabilization platforms but also on the design of in vivo drug delivery platforms whose efficacy depends on sustained biotherapeutic activity at 37 ◦ C. A clear criterion for use of this system as a stabilization platform for vaccines and therapeutic biologics use is minimal impact on protein function and in vivo tolerability of the degraded hydrogel products. In this work, the stabilized network was dissolved at the point of use and remained in solution with the protected product during testing. For the enzymes tested, activity remained high in the presence of the degraded hydrogel products (> 90% activity; Supporting Information; Fig. S8 and S9). However, this remains a question for each new biologic and investigation of the in vivo toxicity of the degraded products is needed to

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confirm the clinical utility of this system for vaccines and therapeutic biologics.

Conclusion In this work, we described the synthesis and formation of a synthetic, photoresponsive hydrogel as a thermal stabilization plaform for biologics via direct encapsulation. The gel stabilized thermally-labile enzymes β-galactosidase, alkaline phosphatase, and T4 DNA ligase during exposure to thermal stress. Critically, the network allowed for user-defined release of the stabilized biologics with light. The results demonstrate that our gel platform can serve as a broadly applicable excipient in the biotechnology industry to protect biotherapeutics and research use enzymes from thermal stress. Further, these findings suggest a generalized mechanism for thermal stabilization of biomacromolecules via direct encapsulation in hydrogels and that additional reversible polymer networks should be explored as stabilizers in biologic formulations.

Associated Content The Supporting Information is available free of charge on the ACS Publications website. The Supporting Information includes 1 H-NMR and 1

H-NMR,

13

13

C-NMR spectra of PEG-4-DBCO;

C-NMR, and MALDI-TOF spectra of PLP-2-N3 ; rheology for the timescale

of photodegradation; additional controls for protein activity in response to UV light and photodegraded hydrogel products; and release without UV light following thermal treatment.

Acknowledgement This work was funded by Nanoly Bioscience, Inc., The State of Colorado through the Advanced Industries Accelerator Grant program, and ETH Zürich. The authors thank J. Heaps, S. Liu (Alta Biotech), D.L. Alge, M.A. Azagarsamy, and B.L. Barthel for their assistance 21



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with this work.

References (1) Leader, B.; Baca, Q. J.; Golan, D. E. Nat. Rev. Drug Discovery 2008, 7, 21–39. (2) Wang, W. Int. J. Pharm. 1999, 185, 129–188. (3) Wang, W. Int. J. Pharm. 2005, 289, 1–30. (4) The

2015

Biopharma

Cold

Chain

Landscape.

2015;

http://pharmaceuticalcommerce.com/special-report/ the-2015-biopharma-cold-chain-landscape/. (5) Wolfson, L. J.; Gasse, F.; Lee-Martin, S.-P.; Lydon, P.; Magan, A.; Tibouti, A.; Johns, B.; Hutubessy, R.; Salama, P.; Okwo-Bele, J.-M. Bulletin of the World Health Organization 2008, 86, 27–39. (6) Yang, H.; Liu, L.; Li, J.; Chen, J.; Du, G. ChemBioEng Rev. 2015, 2, 87–94. (7) Wang, W. Int. J. Pharm. 2000, 203, 1–60. (8) Lee, J.; Lin, E.-W.; Lau, U. Y.; Hedrick, J. L.; Bat, E.; Maynard, H. D. Biomacromolecules 2013, 14, 2561–2569. (9) Kasper, J. C.; Friess, W. Eur. J. Pharm. Biopharm. 2011, 78, 248–263. (10) Carpenter, J. F.; Pikal, M. J.; Chang, B. S.; Randolph, T. W. Pharm. Res. 1997, 14, 969–975. (11) Kamerzell, T. J.; Esfandiary, R.; Joshi, S. B.; Middaugh, C. R.; Volkin, D. B. Adv. Drug Delivery Rev. 2011, 63, 1118–1159. (12) Sola-Penna, M.; Meyer-Fernandes, J. Arch. Biochem. Biophys. 1998, 360, 10–14. 22


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(13) Guo, N.; Puhlev, I.; Brown, D. R.; Mansbridge, J.; Levine, F. Nat. Biotechnol. 2000, 18, 168–171. (14) Pelliccia, M.; Andreozzi, P.; Paulose, J.; D’Alicarnasso, M.; Cagno, V.; Donalisio, M.; Civra, A.; Broeckel, R. M.; Haese, N.; Jacob Silva, P.; Carney, R. P.; Marjomäki, V.; Streblow, D. N.; Lembo, D.; Stellacci, F.; Vitelli, V.; Krol, S. Nat. Commun. 2016, 7, 13520. (15) Messina, M. S.; Ko, J. H.; Yang, Z.; Strouse, M. J.; Houk, K. N.; Maynard, H. D. Polym. Chem. 2017, 8, 4781–4788. (16) Lu, S.; Wang, X.; Lu, Q.; Hu, X.; Uppal, N.; Omenetto, F. G.; Kaplan, D. L. Biomacromolecules 2009, 10, 1032–1042. (17) Guziewicz, N. A.; Massetti, A. J.; Perez-Ramirez, B. J.; Kaplan, D. L. Biomaterials 2013, 34, 7766–7775. (18) Kluge, J. A.; Li, A. B.; Kahn, B. T.; Michaud, D. S.; Omenetto, F. G.; Kaplan, D. L. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5892–5897. (19) O’Shea, T. M.; Webber, M. J.; Aimetti, A. A.; Langer, R. Adv. Healthcare Mater. 2015, 4, 1802–1812. (20) DeForest, C. A.; Polizzotti, B. D.; Anseth, K. S. Nat. Mater. 2009, 8, 659–664. (21) DeForest, C. A.; Anseth, K. S. Nat. Chem. 2011, 3, 925–931. (22) Tibbitt, M. W.; Han, B. W.; Kloxin, A. M.; Anseth, K. S. J. Biomed. Mater. Res., Part A 2012, 100A, 1647–1654. (23) Azagarsamy, M. A.; Alge, D. L.; Radhakrishnan, S. J.; Tibbitt, M. W.; Anseth, K. S. Biomacromolecules 2012, 13, 2219–2224. (24) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974–6998. 23



(25) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Science 2009, 324, 59. (26) Kloxin, A. M.; Tibbitt, M. W.; Anseth, K. S. Nat. Protoc. 2010, 5, 1867–1887. (27) Flory, P. J. J. Phys. Chem. 1942, 46, 132–140. (28) Stockmayer, W. H. J. Chem. Phys. 1944, 12, 125–131. (29) Tibbitt, M. W.; Kloxin, A. M.; Sawicki, L. A.; Anseth, K. S. Macromolecules 2013, 46, 2785–2792. (30) Tibbitt, M. W.; Kloxin, A. M.; Anseth, K. S. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 1899–1911. (31) Kloxin, A. M.; Tibbitt, M. W.; Kasko, A. M.; Fairbairn, J. A.; Anseth, K. S. Adv. Mater. 2010, 22, 61–66. (32) Norris, S. C. P.; Chou, T.; Kasko, A. M. Macromol. Theory Simul. 2017, 26, 1700007. (33) Husain, Q. Crit. Rev. Biotechnol. 2010, 30, 41–62. (34) Edwards, R. A.; Jacobson, A. L.; Huber, R. E. Biochemistry 1990, 29, 11001–11008. (35) Rittié, L.; Perbal, B. Cell Commun. Signaling 2008, 2, 25–45. (36) Lohman, G. J. S.; Tabor, S.; Nichols, N. M. Current Protocols in Molecular Biology; John Wiley & Sons, Inc., 2011; Vol. 94; Chapter 3.14, pp 1–7. (37) Weiss, B.; Jacquemain-Sablon, A.; Live, T. R.; Fareed, G. C.; Richardson, C. C. J. Biol. Chem. 1968, 243, 4543–4555. (38) Zhou, H.-X.; Rivas, G.; Minton, A. P. Annu. Rev. Biophys. 2008, 37, 375–397.

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