ホーム組織工学2次元および3次元細胞培養用生分解性ポリエチレングリコール

2次元および3次元細胞培養用生分解性ポリエチレングリコール

Andrea M. Kasko

Department of Bioengineering, University of California, Los Angeles

はじめに

組織工学や薬物送達などをはじめとするバイオテクノロジー分野の進歩に伴い、さまざまな機能性生体材料の需要が増大しています。過去数十年にわたり、高分子生体材料の研究では、他の用途のために開発されたポリマーの生体適合性の確認やその加工技術の開発（エレクトロスピニング、溶媒キャスト/孔物質溶出（porogen leaching）法、3次元印刷など）に重点が置かれてきました。最近では生体医学用に特化した材料の合成、すなわち合成タンパク質や糖類似体、水媒体との相溶性の高いポリマーの合成に加え、自然界に存在するポリマーの化学修飾（ゲル化やin vivoでの安定性の強化など）へと研究の流れが変化しています。この十年の間では、細胞足場としての利用や治療薬の送達を目的として設計された生体材料の開発などが行われています。

研究者から強い関心が寄せられている生体材料の1つに、ヒドロゲルがあります¹。ヒドロゲルは2次元および3次元の細胞足場として広く研究されていますが、それは化学的にも物理的にも本来の細胞の環境に非常に近い状態を模倣しているためです²。ヒドロゲルは、合成ポリマー（ポリエチレングリコール、ポリヒドロキシエチルメタクリラートなど）や天然に存在するポリマー（コラーゲン、ヒアルロン酸、ヘパリンなど）から合成され^1b、その高い水分含有量と、細胞、タンパク質、DNAの存在下で作製可能であるために細胞培養の3次元モデルとして有用な材料です。構成する材料の反応性にもよりますが、pH³、温度⁴、クーロン相互作用、共有結合/非共有結合性相互作用⁵、または重合反応を用いることで、ゲル化を行うことが可能です。

PEG

ポリエチレングリコール（PEG）は親水性ポリマーのひとつで、架橋してネットワークを構成させることで大量の水を保持することができます。一般的にPEGは免疫反応を引き起こさないため、生物学的応用に適した材料です⁶。1970年代以降、治療用タンパク質やペプチドを修飾することによる溶解度の向上や毒性の低下、循環滞留性の向上のためにPEGは使われてきました⁷。そして、1970年代後半になると、PEGヒドロゲルを用いた細胞培養の実験が始まりました。PEGヒドロゲルは化学組成が明確であり、その合成や化学修飾に多様な化学を利用することができます。

PEGマクロモノマー

PEGはエチレンオキシドのリビングアニオン開環重合によって合成されます。そのため、多様な末端基（例えば、アルコール、メチルエーテル、アミン、N-ヒドロキシスクシンイミジル（NHS）エステル）を持ち、広い範囲にわたってさまざまな分子量を持つ、よく定義された（多分散性が低い）PEGを容易に得ることができます。

ヒドロゲルの形成にはPEGの架橋が必要です。初期の頃は、電離放射線を用いて非特異的に架橋されていました⁸。現在では、PEGヒドロゲルの合成には、PEGマクロモノマーの反応性鎖末端を利用して共有結合的に架橋させる方法が一般的に用いられています。

アクリラート、メタクリラート、アリルエーテル、マレイミド、ビニルスルホン、NHSエステル、ビニルエーテル基などの反応性鎖末端を持つPEGマクロモノマー（表1）は、入手の容易な出発物質から簡便に合成されます。PEGのアルコール鎖末端は、塩基存在下で、塩化アクリロイル、塩化メタクリロイルなどの酸性塩化物を用いてエステル化することができます。PEG鎖末端は、塩基性条件下で、2-chloroethyl vinyl etherやallyl bromideなどのハロゲン化アルキルと反応させることでエステル化が可能です。PEGジビニルスルホンは、PEGを大過剰のジビニルスルホンとカップリングさせるか、あるいは、クロロエチルスルホン鎖末端を調製した後に、塩基を除くことでジビニルスルホン基を導入する多段階プロセスによって合成します⁹。

Poly(ethylene glycol) is a hydrophilic polymer that, when cross-linked into networks, can have a high water content.PEG is a suitable material for biological applications because it does not generally elicit an immune response.⁶ Since the 1970s, PEG has been used to modify therapeutic proteins and peptides to increase their solubility, lower their toxicity and to prolong their circulation half-life.⁷ In the late 1970s, researchers began to experiment with PEG hydrogels for cell culture.PEG hydrogels are chemically well-defined, and multiple chemistries can be used both for their formation and chemical modification.

PEG Macromers

PEG is easily synthesized by the living anionic ring-opening polymerization of ethylene oxide; well-defined (low polydispersity) PEGs with a range of molecular weights and a variety of end groups (e.g., alcohol, methyl ether, amine, N-hydroxysuccinimidyl (NHS) ester) are widely available.

In order to form a hydrogel, PEG must be cross-linked.Initially, PEG was cross-linked non-specifically using ionizing radiation.⁸ PEG hydrogels are now typically synthesized via covalent cross-linking of PEG macromers with reactive chain ends.

PEG macromers with reactive chain ends such as acrylate, methacrylate, allyl ether, maleimide, vinyl sulfone, NHS ester and vinyl ether groups (Chart 1) are easily synthesized from readily available starting materials.The alcohol chain ends of PEG can be esterified using acid chlorides (e.g., acryloyl chloride, methacryloyl chloride) in the presence of base.PEG chain ends can be etherified under basic conditions by reaction with alkyl halides such as 2-chloroethyl vinyl ether or allyl bromide.PEG divinyl sulfone is prepared by coupling PEG to a large excess of divinyl sulfone or by a multistep process to prepare chloroethyl sulfone chain ends that undergo basic elimination to form vinyl sulfone groups⁹.

表1PEGマクロモノマーのさまざまな末端基

マクロモノマーはホモ二官能性、あるいはヘテロ二官能性です。ホモ二官能性のマクロモノマーは主にネットワーク形成に使われ、ヘテロ二官能性マクロモノマーはヒドロゲルネットワーク内への治療薬分子の結合に使われます。

ヒドロゲル形成のメカニズム

ヒドロゲルを形成する架橋メカニズムは、PEGマクロモノマーの鎖末端の特性に依存します。多くの場合、一般的にはラジカル開始剤の存在下で反応性のビニル鎖末端が重合化されたときに架橋が起こります。例えば、レドックス生成ラジカル（過硫酸アンモニウムやTEMEDなど）、あるいは光によって生成するラジカル（例えばIrgacure^® 651, λ ＝ 365 nm）を用いて、マクロモノマーの重合を開始させることができます（スキーム1）。アクリラートやメタクリラートの鎖末端は連鎖重合します。一方、遂次重合によるネットワーク形成では、多官能性架橋剤（f > 2）はPEG鎖末端と化学量論的に反応しますが、多官能性PEG（f > 2）を二官能性架橋剤と架橋させることもできます（スキーム1）。アクリラート、メタクリラート、ビニルスルホン、マレイミド、ビニルエーテル、アリルエーテルはすべて、反応条件を調整してチオールへ変換させることで、遂次重合によるネットワーク形成が可能です。代表的な架橋剤には、チオールまたはアミン系の構造が含まれています。混合モードでの重合は、同一の反応槽中で両方のメカニズムが生じることによるもので、アクリラート基とメタクリラート基は混合モードでネットワーク形成することができます。どちらのヒドロゲル生成メカニズムも生細胞のカプセル化に利用可能であり、ペプチドやタンパク質、その他治療薬の活性を保ちながら導入することができます。

スキーム1.連鎖重合と遂次重合反応

異なるメカニズムの結果生じたメッシュ構造を図1に示します。連鎖重合で形成されたネットワークでは、成長鎖は架橋サイトに形成されますが、一方の遂次成長によるネットワークでは、欠陥を除く架橋サイトは多官能性架橋剤と同じ官能性を持ちます。連鎖重合と遂次重合のどちらの場合でも、ループ状や完全に絡み合った状態の鎖、ダングリング鎖末端などのネットワーク欠陥が存在することがあります。マクロモノマーの化学的特性とヒドロゲル形成のメカニズムは、ヒドロゲルネットワークの架橋密度に影響を与える点で、どちらも重要です。2次元、3次元の培養に重要な材料特性は、ヒドロゲル形成の際の化学によって容易に制御できます。架橋密度を高めることで、メッシュサイズは小さくなり、膨潤比が減少し、貯蔵弾性率は増加します。また、PEGマクロモノマーの分子量を変化させると、ヒドロゲルの性質を大まかに制御することができます（架橋密度に大きな違いが生じます）。さらに、ヒドロゲル生成に用いる反応メカニズムを変えることで、ヒドロゲルの性質を細かく制御することが可能です（系の架橋密度の調整に利用されます）。

図1生成メカニズムが与える、ヒドロゲルのネットワーク構造やネットワーク欠陥への影響

分解性ヒドロゲル

細胞分化や組織の形態形成の研究に3次元ヒドロゲル足場を利用するには、ゲルの物理的および化学的性質を空間的、時間的に制御された方法でコントロールすることが極めて重要となります¹⁰。一般的には、高分子材料の性質は、重合/架橋（結合の形成現象）、あるいは制御された分解や放出（結合の切断現象）によって変化します。結合生成には、低分子量の化合物（開始剤、触媒、モノマー、材料に結合させる配位子）が使われることがありますが、結合の切断には外部から化合物を追加する必要はまずありません。低分子化合物は高分子化合物よりもin vitroやin vivoで大きな副作用を示すことが多いため、多くの研究グループが高分子生体材料のin situの手段として分解反応を用いています。

加水分解

ヒドロゲルの分解で最も一般的に利用される反応は加水分解であり、ポリマー骨格に水分子を添加することで鎖の切断を引き起こします。無水物、エステル類、アミド類は、いずれも加水分解に対し敏感です。通常、無水物の加水分解は早く、アミド類の無触媒加水分解は非常に遅い反応であるため、加水分解によって分解するヒドロゲルの多くはエステル結合を利用しています。生理学的に妥当な分解時間で加水分解するヒドロゲルを得るためには、一般的に、ラクチドやグリコリドを用いた分解性エステル結合によってPEGを官能基化します。

PEGのアルコール鎖末端は3,6-dimethyl-1,4-dioxane-2,5-dioneと1,4-Dioxane-2,5-dioneの開環反応を引き起こし、それぞれPEGラクチドおよびPEGグリコリドを生成します（スキーム2）¹¹。開環反応には一般的にtin(II)-2-ethylhexanoateを触媒に用いますが¹²、4-(dimethylamino)pyridine（DMAP）を触媒として用いても容易に反応が進みます¹³。この場合、おそらく残存スズよりも容易に触媒を除去できます。PEGラクチドあるいはPEGグリコリドのアルコール鎖末端は、アクリラートやメタクリラートなどの反応性二重結合によって容易に官能基化されます。ヒドロゲルの分解に加えて、エステル結合の加水分解はヒドロゲルにカプセル化された薬物の細胞への送達に利用されています。例えば、デキサメタゾン¹⁴やスタチン¹⁵などの治療薬は分解性ラクチド結合によってヒドロゲル中に結合され、持続的に放出されます。そのため、間葉幹細胞（MSCs）を骨芽細胞へ分化させるために使用されています。

スキーム2PEGラクチドとPEGグリコリドの合成

酵素分解

エステル結合は酵素によって分解が可能ですが、多くの場合、エステルやアミドの非配列特異的な酵素分解よりも、ヒドロゲル中に導入したペプチドの配列特異的な酵素分解が利用されています。Hubbell のグループはこの手法の草分け的存在で¹⁶、アクリラート、マレイミド、ビニルスルホンを介してシステイン官能基化ペプチドをマイケル付加させることによって、マトリックスメタロプロテイナーゼ（MMP）感受性結合をヒドロゲルに導入しました（スキーム3）¹⁷。

MMP分解性結合はまた、治療薬剤をヒドロゲル中に結合するためにも用いられています。例えば、血管内皮増殖因子（VEG-F）のような増殖因子はMMP感受性結合の酵素分解によって放出され、血管形成を誘発します¹⁸。

加水分解、酵素分解のいずれにおいても、マクロモノマーの化学的性質により分解速度をあらかじめ決めることができます。加水分解の場合、その材料の分解速度は材料特性（例えば疎水性や親水性）や加水分解性官能基の数によって決定され、材料を一度合成してしまえば変更することはできません。酵素分解の場合、反応は一般的に酵素を産生する細胞の近傍で起こります。加水分解や酵素分解はどちらも持続的なヒドロゲルの分解や治療薬の放出に有効な手法ですが、ヒドロゲルを合成した後では放出速度の調整や停止を行うことができず、また、空間的に放出を制御することもできません。

スキーム3ビニルスルホン基へのシステイン含有ペプチドのマイケル付加によって合成される酵素分解性ヒドロゲル

光分解性ヒドロゲル

加水分解や酵素分解が可能な結合に比べ、光分解性結合の場合には、分解と放出を空間的、時間的に正確に制御できます。多くの研究者が光重合性ヒドロゲルや光機能性ヒドロゲルについて報告していますが、生体適合性光分解性ヒドロゲルの報告はごくわずかです。KloxinとKaskoは2-methoxy-5-nitro-4-(1-hydroxyethyl)phenoxybutanoateを含むPEGマクロモノマー（スキーム4）から合成した光分解性ヒドロゲルネットワークについて報告しており¹⁹、オルト-ニトロベンジル（o-NB）結合基の光分解性挙動についての詳細を明らかにしています。光分解性マクロモノマーから合成したヒドロゲルは、露光すると、照射時間、波長、光の強さに応じてバルク分解を起こします。遮光すると分解は停止し、照射を再開すると試料は光分解を続けます。光放出性の細胞接着性配位子RGDS（Arg-Gly-Asp-Ser）を含むヒドロゲルにカプセル化されたhMSCs（ヒト間葉幹細胞）は、10日目にRGDSが放出されると（軟骨形成におけるフィブロネクチンの抑制に対応）、軟骨形成の過程を経て分化します。一方で、この分解性ヒドロゲルの表面エロージョン（侵食）やゲルを用いたリソグラフィは、10^-7から10^-2 m以上の大きさの形状の作製に用いることができます²⁰。局所的な部分分解による架橋密度の低下と膨潤の増加によって、ヒドロゲルの上に、ゲルから盛り上がった状態のより軟らかい形状をエッチングすることが可能です。

スキーム4治療薬の放出を目的として、ヒドロゲル骨格中に導入された光分解性o-NB結合基部分

o-NBを含むヒドロゲルは、一光子光分解に加えて二光子光分解に対しても感受性があり、3次元エッチングに利用できます^19,20。一光子反応では、光が照射されたあらゆる領域で反応が起きます。これに対して、多光子リソグラフィでは、複数の光子が同時に吸収された場所のみ、つまり光源の焦点領域で起きます（図2）。生体材料の一光子リソグラフィで使われる一般的な波長は長波長の紫外線（≧365 nm）から可視領域ですが、二光子リソグラフィでは赤外線（一般的におよそ740～800 nm）が使用されます。赤外線は生体適合性がより高いうえに、生細胞に対する破壊力が小さく、大きな侵入深度が得られます。二光子吸収が起こり得るのも、光路全体に沿った部分というよりは収束光の焦点付近に限られており、三次元的な励起の制御が可能となります。一光子、多光子のどちらの反応も、500 nmよりも微細な形状をパターニングできる可能性を持っており、これは哺乳類の細胞よりも小さな大きさです²¹。このことは、ヒドロゲルの足場構造や化学的性質をこれまでにない精度で空間的制御することが可能であることを意味しています。

図2一光子光分解（左）は紫外可視の光で露光されたヒドロゲル全域で起こり、二光子光分解（右）は赤外光の2つの光子が同時に吸収された部分でのみ起こります。

o-NBリンカーはまた、治療薬をヒドロゲル中に結合して生細胞へと送達するのにも使われます。Griffinらは、o-NB-PEGマクロモノマーを用いてヒドロゲル中に結合したフルオレセインを制御放出できることを示しました²²。このモデル治療薬の放出は、さまざまな波長（365～436 nm）や強度（5～20 mW/cm²）、照射時間（0～20分）の光に対する関数として定量的に示されます。最も速い放出が起こるのは波長が365 nm（これはこの波長でo-NBリンカーのモル吸光係数が高くなることと一致しています）のときですが、波長405 nmのときにも顕著な放出が見られます。この放出は化合物の物理定数（モル吸収光係数など）によって容易にモデル化できます。これらの系では、光の減衰によって化学的、機械的な勾配を容易に作製することができます。

結論

ポリエチレングリコールは入手が容易で、簡便に修飾が可能なポリマーであり、組織培養用2次元、3次元の足場をはじめとするヒドロゲルの作製に広く使われています。また、PEGヒドロゲルへの分解性結合の導入は容易です。加水分解性ゲルでは持続的分解や治療薬の放出が可能であり、酵素分解性ゲルではその分解や放出は細胞に大きく影響されます。光分解を用いることで、ヒドロゲルの化学的、物理的性質を、ユーザーの要望に合わせてリアルタイムで外部から操作することが可能となります。

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West, J. L.; Hubbell, J. A. Macromolecules 1999, 32, 241-244.
(a) Lutolf, M. P.; Hubbell, J. A. Biomacromolecules 2003, 4, 713-722.
(b) Lutolf, M. P.; Lauer-Fields, J. L.; Schmoekel, H. G.; Metters, A. T.; Weber, F. E.; Fields, G. B.; Hubbell, J. A. P. Natl.Acad.Sci. USA 2003, 100, 5413-5418.
Zisch, A. H.; Lutolf, M. P.; Ehrbar, M.; Raeber, G. P.; Rizzi, S. C.; Davies, N.; Schmokel, H.; Bezuidenhout, D.; Djonov, V.; Zilla, P.; Hubbell, J. A. Faseb.J. 2003, 17, 2260.
(a) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Science 2009, 324, 59-63.
(b) Kloxin, A. M.; Tibbitt, M. W.; Kasko, A. M.; Fairbairn, J. A.; Anseth, K. S. Adv.Mater.2010, 22, 61-66.
Wong, D. Y.; Griffin, D. R.; Reed, J.; Kasko, A. M. Macromolecules 2010, 43, 2824-2831.
Kasko, A. M.; Wong, D. Y. Future Med. Chem. 2010, 2, 1669-1680.
Griffin, D. R.; Patterson, J. T.; Kasko, A. M. Biotechnol.Bioeng.2010, 107, 1012-1019.

PEG Macromers

Chart 1.End groups of different PEG macromers.

Macromers can be homobifunctional or heterobifunctional.Homobifunctional macromers are typically used to form networks, while heterobifunctional macromers may be used to tether a therapeutic molecule into a hydrogel network.

Mechanisms of Hydrogel Formation

The cross-linking mechanism to form hydrogels depends on the identity of the chain ends of PEG macromers.In most cases, cross-linking occurs when the reactive vinyl chain ends are polymerized, usually with a free radical initiator.For example, polymerization of macromers can be initiated using redox-generated radicals (e.g., ammonium persulfate and TEMED), or radicals generated with light (e.g., Irgacure^® 651, λ=365 nm Scheme 1).Acrylate and methacrylate chain ends undergo chain polymerization.In step growth network formation, a multifunctional (f>2) cross-linker reacts with the PEG chain ends in a stoichiometric manner; alternatively, multifunctional PEGs (f>2) can be crosslinked with difunctional crosslinkers (Scheme 1).Acrylate, methacrylate, vinyl sulfone, maleimide, vinyl ether and allyl ether are all capable of step growth network formation, through conversion to thiols depending on reaction conditions.Typical cross-linkers may include thiol or amine moieties.Mixed-mode polymerizations are the result of both mechanisms occurring in the same reaction vessel; acrylate and methacrylate groups can undergo mixed mode network formation.Both mechanisms of hydrogel formation can be used to encapsulate live cells, and both mechanisms allow for the reactive incorporation of peptides, proteins and other therapeutics.

Scheme 1.Chain growth and step growth reactions.

The mesh structure that results from different mechanisms is depicted in Figure 1.In chain growth networks, a kinetic chain is formed at the crosslink site, while in step growth networks, the crosslink sites bear the same functionality as the multifunctional cross-linker, neglecting defects.In both chain and step growth, network defects such as loops, permanent entanglements and dangling chain ends may exist.The chemical identity of the macromer and the mechanism of hydrogel formation are both important as each influences the cross-link density of the hydrogel network.Material properties that are important to 2D and 3D culture are easily controlled through the chemistry of hydrogel formation.As cross-linking density increases, mesh size decreases, swelling ratio decreases, and storage modulus increases.Varying the molecular weight of the PEG macromer results in coarse control over hydrogel properties (large differences in cross-linking density).Varying the reaction mechanism used to produce the hydrogels results in fine control over hydrogel properties (can be used to tune cross-linking density of a system).

Figure 1.Formation mechanism affects hydrogel network structure and network defects.

Degradeable Hydrogels

In order to use 3D hydrogel scaffolds to study cell differentiation and tissue evolution, it is critical to be able to control the physical and chemical properties of the gel in a spatially and temporally controlled manner.¹⁰ Polymeric material properties are typically changed through polymerization/cross-linking (bond forming events) or through controlled degradation and/or release (bond breaking events).Bond forming events typically often use small molecule reagents (initiators, catalysts, monomers, ligands to be conjugated to the material) while bond breaking typically does not rely on exogenous reagents.Small molecules often have more adverse effects in vitro and in vivo than polymeric reagents so many research groups use degradation as a tool for in situ manipulation of polymeric biomaterials.

Hydrolytic Degradation

The mechanism of degradation most commonly utilized in hydrogels is hydrolysis, in which a molecule of water adds to the polymer backbone, causing chain scission.Anhydrides, esters and amides are all susceptible to hydrolysis.Anyhydrides typically hydrolyze too quickly, and the uncatalyzed hydrolysis of amides is too slow, so most hydrogels that degrade hydrolytically utilize ester linkages.In order to obtain hydrolytically degradable hydrogels with physiologically relevant time scales of degradation, researchers typically functionalize PEG with degradable ester linkages using lactide or glycolide segments.

Alcohol chain ends on PEG can initiate ring-opening reactions of 3,6-dimethyl-1,4-dioxane-2,5-dione and 1,4-Dioxane-2,5-dione to generate PEG-lactide and PEG-glycolide, respectively (Scheme 2).¹¹ The ring-opening reaction is typically catalyzed by tin(II)-2-ethylhexanoate,¹² although the reaction is also easily accomplished using dimethylaminopyridine as a catalyst,¹³ which may be easier to remove than the residual tin.The alcohol chain ends of PEG-lactide or PEG-glycolide are easily functionalized with reactive double bonds such as acrylate and methacrylate.

Scheme 2.Synthesis of PEG-lactide and PEG-glycolide.

Enzymatic Degradation

Although ester linkages are enzymatically degradable, most researchers utilize sequence-specific enzymatic degradation of peptides incorporated into hydrogels rather than non-specific enzymatic degradation of esters and amides.Hubbell′s group pioneered this approach¹⁶ by incorporating matrix metalloproteinase (MMP) sensitive linkages into hydrogels via Michael addition of cysteine-functionalized peptides across acrylates, maleimides and vinyl sulfones (Scheme 3).¹⁷

MMP-degradable linkages have also been used to tether therapeutic agents into hydrogels.For example, growth factors such as vascular endothelial growth factor (VEG-F) can be released via enzymatic degradation of an MMP-sensitive tether to induce angiogenesis.¹⁸

In both hydrolysis and enzymolysis, the rate of degradation is predetermined by the chemistry of the macromer.In hydrolysis, the degradation rate of the material is pre-engineered through the identity (e.g., hydrophobicity or hydrophilicity) and number of the hydrolysable groups, and cannot be changed once the material is fabricated.In enzymolysis, the degradation typically occurs in an area local to the cells producing the enzyme.While hydrolysis and enzymolysis are both effective methods for sustained hydrogel degradation and sustained release of therapeutic agents, the rate of release cannot be adjusted or arrested after the hydrogel is fabricated, and release is not spatially controlled.

Scheme 3.Enzymatically degradable hydrogels via Michael addition of cysteinecontaining peptides to vinyl sulfone groups.

Photodegradable Hydrogels

In contrast to hydrolytically and enzymatically degradable linkages, photodegradable linkages allow precise spatial and temporal control over degradation and release.While many researchers have reported photopolymerizable hydrogels, and photofunctionalizable hydrogels, very few reports exist of biocompatible photodegradable hydrogels.Kloxin and Kasko reported photodegradable hydrogel networks formed from 2-methoxy-5-nitro-4-(1-hydroxyethyl) phenoxybutanoate-containing PEG macromers (Scheme 4)¹⁹; the photodegradation behavior of the ortho-nitrobenzyl (o-NB) linker group is well-characterized.Hydrogels formed from the photodegradable macromer show bulk degradation upon exposure to light that is dependent on exposure time, wavelength, and light intensity.When the light is shuttered, degradation is arrested; the sample continues photolyzing once light exposure resumes. hMSCs (human mesenchymal stem cells) encapsulated in a hydrogel containing the photo-releasable cell-adhesive ligand RGDS (Arg-Gly-Asp-Ser) differentiate down the chondrogenic pathway when the RGD is released at day ten (corresponding to the downregulation of fibronectin during chondrogenesis).Surface erosion and through-gel lithography of this degradable hydrogel can be used to form features over a range of lengths scales, from 10-⁷ m to 10-² m or larger.²⁰ Partial degradation in a local area results in decreased cross-link density and increased swelling, providing a means to etch softer features onto a hydrogel that protrude out from the gel.

Scheme 4.Photodegradable o-NB moieties incorporated into hydrogel backbone and for therapeutic agent release.

In addition to single photon photolysis, the o-NB containing hydrogels are also susceptible to two-photon photolysis, allowing for 3D etching.^19-20 In single photon reactions, any area exposed to the light will react.In contrast, multi-photon lithography should occur only where multiple photons are simultaneously absorbed, which occurs at the focal volume of the light source (inset).Typical wavelengths in single photon lithography of biomaterials range from long wave UV (≥365 nm) into the visible region, while two-photon lithography uses IR light (typically ~740–800 nm).IR light is more biocompatible and less destructive to live tissues and offers greater penetration depth.The probability of twophoton absorption occurring is also tightly limited to the focal point of the focused light, rather than along the entire path of the light, providing 3D control over excitation.Both single- and multi-photon reactions have the potential to pattern materials with features smaller than 500 nm, much smaller than the size of a mammalian cell.²¹ This represents an unprecedented level of spatial control over hydrogel scaffold structure and chemistry.

Figure 2.Single photon photolysis (left) occurs in the entire area of the hydrogel exposed to UV-visible light, and two photon photolysis (right) results only in the area where simultaneous absorption of two photons of IR light occurs.

The o-NB linker can also be used to tether therapeutic agents into hydrogels for delivery to live cells.Griffin et al. demonstrated the controlled release of fluorescein tethered into a hydrogel through an o-NB-PEG macromer.²² The release of this model therapeutic as a function of light exposure at multiple wavelengths (365–436 nm), intensities (5–20 mW/cm2) and durations (0–20 minutes) was quantified.While the fastest release occurs at 365 nm (which corresponds to a higher molar absorptivity of the o-NB linker at that wavelength), significant release is also seen at 405 nm; the release is easily modeled from physical constants of the molecules (such as molar absorptivity).Light attenuation allows the facile formation of chemical and mechanical gradients in these systems.

Conclusion

Poly(ethylene glycol) is a readily available, easily modifiable polymer.It has found widespread use in hydrogel fabrication, including as 2D and 3D scaffolds for tissue culture.Degradable linkages are easily introduced into PEG hydrogels.Hydrolytically degradable gels allow for sustained material degradation and/or therapeutic agent release.Degradation and release is cell-dictated in enzymatically degradable gels.Photodegradation allows for real-time user tailored external manipulation of the chemical and physical properties of hydrogels.

Materials

Product #	Description	Add to Cart
689882	O-(2-Aminoethyl)-O′-[2-(biotinylamino)ethyl]octaethylene glycol ≥95% (oligomer purity)	pricing

671487	O-(2-Aminoethyl)-O′-(2-carboxyethyl)polyethylene glycol hydrochloride M_p 3,000	pricing

671592	O-(2-Aminoethyl)-O′-(2-carboxyethyl)polyethylene glycol 5,000 hydrochloride M_p 5,000	pricing

672130	O-(2-Aminoethyl)polyethylene glycol M_p 5,000	pricing

671924	O-(2-Aminoethyl)polyethylene glycol M_p 10,000	pricing

07969	O-(2-Aminoethyl)polyethylene glycol 3,000 M_p 3,000	pricing

672165	O-(2-Aminoethyl)-O′-(2-(succinylamino)ethyl)polyethylene glycol hydrochloride M_p 10,000	pricing

689440	O-(2-Azidoethyl)heptaethylene glycol ≥95% (oligomer purity)	pricing

669946	O-[2-(Biotinyl-amino)ethyl]-O′-(2-carboxyethyl)polyethylene glycol M_p 3,000	pricing

689998	O-[2-(Biotinylamino)ethyl]-O′-(2-carboxyethyl)undecaethylene glycol ≥95% (oligomer purity)	pricing

670049	O-[2-(Biotinyl-amino)ethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]polyethylene glycol M_p 3,000	pricing

689653	O-(2-Carboxyethyl)-O′-[2-(Fmoc-amino)-ethyl]heptacosaethylene glycol ≥90% (oligomer purity)	pricing

670812	O-(2-Carboxyethyl)polyethylene glycol 3,000	pricing

671037	O-(2-Carboxyethyl)polyethylene glycol 10,000	pricing

712515	O-(3-Carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]-polyethylene glycol M_w 3000	pricing

712523	O-(3-Carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]-polyethylene glycol M_w 5000	pricing

259268	Hexaethylene glycol 97%	pricing

670162	O-[N-(6-Maleimidohexanoyl)aminoethyl]-O′-(2-carboxyethyl)polyethylene glycol M_p 3,000	pricing

670278	O-[N-(6-Maleimidohexanoyl)aminoethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]polyethylene glycol 3,000 M_p 3,000	pricing

689777	O-[N-(3-Maleimidopropionyl)aminoethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]heptacosaethylene glycol ≥90% (oligomer purity)	pricing

719080	PEG-ditosylate average M_n 1,300	pricing

705047	PEG-ditosylate average M_n 10,000	pricing

701750	PEG-di-p-tosylate average M_n 3,500	pricing

335754	Pentaethylene glycol 98%	pricing

81300	Poly(ethylene glycol) average M_n 20,000	pricing

202444	Poly(ethylene glycol) average M_n 3,350, powder	pricing

81210	Poly(ethylene glycol) average M_w 1,500	pricing

295906	Poly(ethylene glycol) average M_n 2,050, chips	pricing

309028	Poly(ethylene glycol) average M_n 10,000, flakes	pricing

14509	Poly(ethylene glycol) bis(amine) M_w 20,000	pricing

14501	Poly(ethylene glycol) bis(amine) M_w 2,000	pricing

14502	Poly(ethylene glycol) bis(amine) M_w 3,000	pricing

407038	Poly(ethylene glycol) bis(carboxymethyl) ether average M_n 600	pricing

406996	Poly(ethylene glycol) bis(carboxymethyl) ether average M_n 250	pricing

699810	Poly(ethylene glycol) diacetylene average M_n 2,000	pricing

475629	Poly(ethylene glycol) diacrylate average M_n 250	pricing

701963	Poly(ethylene glycol) diacrylate average M_n 6,000, contains ≤1500 ppm MEHQ as inhibitor	pricing

729094	Poly(ethylene glycol) diacrylate average M_n 10,000, contains MEHQ as inhibitor	pricing

729086	Poly(ethylene glycol) diacrylate average M_n 1,000, contains MEHQ as inhibitor	pricing

475696	Poly(ethylene glycol) diglycidyl ether average M_n 500	pricing

409510	Poly(ethylene glycol) dimethacrylate average M_n 550, contains 80-120 ppm MEHQ as inhibitor, 270-330 ppm BHT as inhibitor	pricing

687537	Poly(ethylene glycol) dimethacrylate average M_n 6,000, contains 1000 ppm 4-methoxyphenol as inhibitor	pricing

725692	Poly(ethylene glycol) dimethacrylate average M_n 20,000, contains MEHQ as inhibitor	pricing

725684	Poly(ethylene glycol) dimethacrylate average M_n 10,000, contains MEHQ as inhibitor	pricing

717142	Poly(ethylene glycol) dithiol average M_n 1,000	pricing

704539	Poly(ethylene glycol) dithiol average M_n 3,400	pricing

705004	Poly(ethylene glycol) dithiol average M_n 8,000	pricing

410195	Poly(ethylene glycol) divinyl ether average M_n 250	pricing

752398	Poly(ethylene glycol), 4 hydroxyl dendron, generation 1 average M_n 20,300	pricing

579319	Poly(ethylene glycol), α-maleimidopropionamide-ω-formyl terminated average M_n 3,000	pricing

409537	Poly(ethylene glycol) methacrylate average M_n 360, contains 500-800 ppm MEHQ as inhibitor	pricing

409529	Poly(ethylene glycol) methacrylate average M_n 500, contains 900 ppm monomethyl ether hydroquinone as inhibitor	pricing

81323	Poly(ethylene glycol) methyl ether average M_n 5,000	pricing

202487	Poly(ethylene glycol) methyl ether average M_n 550	pricing

202495	Poly(ethylene glycol) methyl ether average M_n 750	pricing

732621	Poly(ethylene glycol) methyl ether average M_n 10,000	pricing

732613	Poly(ethylene glycol) methyl ether average M_n 20,000	pricing

699802	Poly(ethylene glycol)methyl ether acetylene average M_n 2,000	pricing

730289	Poly(ethylene glycol) methyl ether acrylate average M_n 5,000, contains MEHQ as inhibitor	pricing

730270	Poly(ethylene glycol) methyl ether acrylate average M_n 2,000, contains MEHQ as inhibitor	pricing

740705	Poly(ethylene glycol) methyl ether 2-(dodecylthiocarbonothioylthio)-2-methylpropionate average M_n 1,100	pricing

731765	Poly(ethylene glycol) methyl ether maleimide average M_n 2,000	pricing

447951	Poly(ethylene glycol) methyl ether methacrylate average M_n 950, contains 300 ppm BHT as inhibitor, 100 ppm MEHQ as inhibitor	pricing

730327	Poly(ethylene glycol) methyl ether methacrylate average M_n 4,000, contains ≤300 ppm monomethyl ether hydroquinone as inhibitor	pricing

730319	Poly(ethylene glycol) methyl ether methacrylate average M_n 1500, contains ≤300 ppm monomethyl ether hydroquinone as stabilizer	pricing

729108	Poly(ethylene glycol) methyl ether thiol average M_n 800	pricing

729140	Poly(ethylene glycol) methyl ether thiol average M_n 2,000	pricing

729159	Poly(ethylene glycol) methyl ether thiol average M_n 6,000	pricing

729116	Poly(ethylene glycol) methyl ether tosylate average M_n 900	pricing

729124	Poly(ethylene glycol) methyl ether tosylate average M_n 2,000	pricing

729132	Poly(ethylene glycol) methyl ether tosylate average M_n 5,000	pricing

309524	Poly(ethylene glycol) tetrahydrofurfuryl ether average M_n 200	pricing

181986	Poly(ethylene oxide) average M_v 100,000, powder	pricing

182028	Poly(ethylene oxide) average M_v 600,000, powder	pricing

181994	Poly(ethylene oxide) average M_v 200,000, powder	pricing

110175	Tetraethylene glycol 99%	pricing

712507	O-[2-(3-Tritylthiopropionylamino)ethyl]polyethylene glycol M_p 3,000	pricing

References

(a) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337-4351.
(b) Lee, K. Y.; Mooney, D. J. Chem. Rev. 2001, 101, 1869-1879.
(c) Hoffman, A. S. Bioartificial Organs III: Tissue Sourcing, Immunoisolation, and Clinical Trials;Hunkeler, D., Ed.; New York Academy of Sciences: New York, 2001; pp 62-73.
(d) Hoffman, A. S. Adv.Drug Delivery Rev. 2002, 54, 3-12.
(a) Tibbitt, M. W.; Anseth, K. S. Biotechnol.Bioeng.2009, 103, 655-663.
(b) Lin, C. C.; Anseth, K. S. Pharm. Res. 2009, 26, 631-643.
(a) Richter, A.; Paschew, G.; Klatt, S.; Lienig, J.; Arndt, K. F.; Adler, H. J. P. Sensors 2008, 8, 561-581.
(b) Gupta, P.; Vermani, K.; Garg, S. Drug Discovery Today 2002, 7, 569-579.
Ruel-Gariepy, E.; Leroux, J. C. Eur. J. Pharm. Biopharm.2004, 58, 409-426.
Yang, Z.; Xu, B. J. Mater.Chem. 2007, 17, 2385-2393.
Zalipsky, S.; Harris, J. M. Poly(Ethylene Glycol) 1997, 680, 1-13.
Davis, F. F. Adv.Drug Deliver Rev. 2002, 54, 457-458.
Merrill, E. W.; Salzman, E. W.; Wan, S.; Mahmud, N.; Kushner, L.; Lindon, J. N.; Curme, J. T. Am. Soc.Art. Int. Org.1982, 28, 482-487.
Morpurgo, M.; Veronese, F. M.; Kachensky, D.; Harris, J. M. Bioconjugate Chem. 1996, 7, 363-368.
Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol.2005, 23, 47-55.
Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A. Macromolecules 1993, 26, 581-587.
Du, Y. J.; Lemstra, P. J.; Nijenhuis, A. J.; Vanaert, H. A. M.; Bastiaansen, C. Macromolecules 1995, 28, 2124-2132.
Nederberg, F.; Connor, E. F.; Moller, M.; Glauser, T.; Hedrick, J. L. Angew.Chem. Int. Edit.2001, 40, 2712-2715.
Kim, H.; Kim, H. W.; Suh, H. Biomaterials 2003, 24, 4671-4679.
Benoit, D. S. W.; Nuttelman, C. R.; Collins, S. D.; Anseth, K. S. Biomaterials 2006, 27, 6102-6110.
West, J. L.; Hubbell, J. A. Macromolecules 1999, 32, 241-244.
(a) Lutolf, M. P.; Hubbell, J. A. Biomacromolecules 2003, 4, 713-722.
(b) Lutolf, M. P.; Lauer-Fields, J. L.; Schmoekel, H. G.; Metters, A. T.; Weber, F. E.; Fields, G. B.; Hubbell, J. A. P. Natl.Acad.Sci. USA 2003, 100, 5413-5418.
Zisch, A. H.; Lutolf, M. P.; Ehrbar, M.; Raeber, G. P.; Rizzi, S. C.; Davies, N.; Schmokel, H.; Bezuidenhout, D.; Djonov, V.; Zilla, P.; Hubbell, J. A. Faseb.J. 2003, 17, 2260.
(a) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Science 2009, 324, 59-63.
(b) Kloxin, A. M.; Tibbitt, M. W.; Kasko, A. M.; Fairbairn, J. A.; Anseth, K. S. Adv.Mater.2010, 22, 61-66.
Wong, D. Y.; Griffin, D. R.; Reed, J.; Kasko, A. M. Macromolecules 2010, 43, 2824-2831.
Kasko, A. M.; Wong, D. Y. Future Med. Chem. 2010, 2, 1669-1680.
Griffin, D. R.; Patterson, J. T.; Kasko, A. M. Biotechnol.Bioeng.2010, 107, 1012-1019.

Mechanisms of Hydrogel Formation

The cross-linking mechanism to form hydrogels depends on the identity of the chain ends of PEG macromers.In most cases, cross-linking occurs when the reactive vinyl chain ends are polymerized, usually with a free radical initiator.For example, polymerization of macromers can be initiated using redox-generated radicals (e.g., ammonium persulfate and TEMED), or radicals generated with light (e.g., Irgacure^® 651, λ=365 nM Scheme 1).Acrylate and methacrylate chain ends undergo chain polymerization.In step growth network formation, a multifunctional (f>2) cross-linker reacts with the PEG chain ends in a stoichiometric manner; alternatively, multifunctional PEGs (f>2) can be crosslinked with difunctional crosslinkers (Scheme 1).Acrylate, methacrylate, vinyl sulfone, maleimide, vinyl ether and allyl ether are all capable of step growth network formation, through conversion to thiols depending on reaction conditions.Typical cross-linkers may include thiol or amine moieties.Mixed-mode polymerizations are the result of both mechanisms occurring in the same reaction vessel; acrylate and methacrylate groups can undergo mixed mode network formation.Both mechanisms of hydrogel formation can be used to encapsulate live cells, and both mechanisms allow for the reactive incorporation of peptides, proteins and other therapeutics.

Scheme 1.Chain growth and step growth reactions.

The mesh structure that results from different mechanisms is depicted in Figure 1.In chain growth networks, a kinetic chain is formed at the crosslink site, while in step growth networks, the crosslink sites bear the same functionality as the multifunctional cross-linker, neglecting defects.In both chain and step growth, network defects such as loops, permanent entanglements and dangling chain ends may exist.The chemical identity of the macromer and the mechanism of hydrogel formation are both important as each influences the cross-link density of the hydrogel network.Material properties that are important to 2D and 3D culture are easily controlled through the chemistry of hydrogel formation.As cross-linking density increases, mesh size decreases, swelling ratio decreases, and storage modulus increases.Varying the molecular weight of the PEG macromer results in coarse control over hydrogel properties (large differences in cross-linking density).Varying the reaction mechanism used to produce the hydrogels results in fine control over hydrogel properties (can be used to tune cross-linking density of a system).

Figure 1.Formation mechanism affects hydrogel network structure and network defects.

Degradeable Hydrogels

Hydrolytic Degradation

Alcohol chain ends on PEG can initiate ring-opening reactions of 3,6-dimethyl-1,4-dioxane-2,5-dione and 1,4-Dioxane-2,5-dione to generate PEG-lactide and PEG-glycolide, respectively (Scheme 2).¹¹ The ring-opening reaction is typically catalyzed by tin(II)-2-ethylhexanoate,¹² although the reaction is also easily accomplished using dimethylaminopyridine as a catalyst,¹³ which may be easier to remove than the residual tin.The alcohol chain ends of PEG-lactide or PEG-glycolide are easily functionalized with reactive double bonds such as acrylate and methacrylate.

Scheme 2.Synthesis of PEG-lactide and PEG-glycolide.

Enzymatic Degradation

Scheme 3.Enzymatically degradable hydrogels via Michael addition of cysteinecontaining peptides to vinyl sulfone groups.

Photodegradable Hydrogels

In contrast to hydrolytically and enzymatically degradable linkages, photodegradable linkages allow precise spatial and temporal control over degradation and release.While many researchers have reported photopolymerizable hydrogels, and photofunctionalizable hydrogels, very few reports exist of biocompatible photodegradable hydrogels.Kloxin and Kasko reported photodegradable hydrogel networks formed from 2-methoxy-5-nitro-4-(1-hydroxyethyl) phenoxybutanoate-containing PEG macromers (Scheme 4)¹⁹; the photodegradation behavior of the ortho-nitrobenzyl (o-NB) linker group is well-characterized.Hydrogels formed from the photodegradable macromer show bulk degradation upon exposure to light that is dependent on exposure time, wavelength, and light intensity.When the light is shuttered, degradation is arrested; the sample continues photolyzing once light exposure resumes. hMSCs (human mesenchymal stem cells) encapsulated in a hydrogel containing the photo-releasable cell-adhesive ligand RGDS (Arg-Gly-Asp-Ser) differentiate down the chondrogenic pathway when the RGD is released at day ten (corresponding to the downregulation of fibronectin during chondrogenesis).Surface erosion and through-gel lithography of this degradable hydrogel can be used to form features over a range of lengths scales, from 10-⁷ m to 10-² m or larger.²⁰ Partial degradation in a local area results in decreased cross-link density and increased swelling, providing a means to etch softer features onto a hydrogel that protrude out from the gel.

Scheme 4.Photodegradable o-NB moieties incorporated into hydrogel backbone and for therapeutic agent release.

In addition to single photon photolysis, the o-NB containing hydrogels are also susceptible to two-photon photolysis, allowing for 3D etching.^19-20 In single photon reactions, any area exposed to the light will react.In contrast, multi-photon lithography should occur only where multiple photons are simultaneously absorbed, which occurs at the focal volume of the light source (inset).Typical wavelengths in single photon lithography of biomaterials range from long wave UV (≥365 nM) into the visible region, while two-photon lithography uses IR light (typically ~740–800 nM).IR light is more biocompatible and less destructive to live tissues and offers greater penetration depth.The probability of twophoton absorption occurring is also tightly limited to the focal point of the focused light, rather than along the entire path of the light, providing 3D control over excitation.Both single- and multi-photon reactions have the potential to pattern materials with features smaller than 500 nM, much smaller than the size of a mammalian cell.²¹ This represents an unprecedented level of spatial control over hydrogel scaffold structure and chemistry.

Figure 2.Single photon photolysis (left) occurs in the entire area of the hydrogel exposed to UV-visible light, and two photon photolysis (right) results only in the area where simultaneous absorption of two photons of IR light occurs.

The o-NB linker can also be used to tether therapeutic agents into hydrogels for delivery to live cells.Griffin et al. demonstrated the controlled release of fluorescein tethered into a hydrogel through an o-NB-PEG macromer.²² The release of this model therapeutic as a function of light exposure at multiple wavelengths (365–436 nM), intensities (5–20 mW/cm²) and durations (0–20 minutes) was quantified.While the fastest release occurs at 365 nM (which corresponds to a higher molar absorptivity of the o-NB linker at that wavelength), significant release is also seen at 405 nM; the release is easily modeled from physical constants of the molecules (such as molar absorptivity).Light attenuation allows the facile formation of chemical and mechanical gradients in these systems.

Conclusion

参考文献

Drury JL, Mooney DJ. 2003. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials. 24(24):4337-4351. https://doi.org/10.1016/s0142-9612(03)00340-5

Lee KY, Mooney DJ. 2001. Hydrogels for Tissue Engineering. Chem. Rev.. 101(7):1869-1880. https://doi.org/10.1021/cr000108x

HOFFMAN AS. Hydrogels for Biomedical Applications. 944(1):62-73. https://doi.org/10.1111/j.1749-6632.2001.tb03823.x

Hoffman AS. 2002. Hydrogels for biomedical applications. Advanced Drug Delivery Reviews. 54(1):3-12. https://doi.org/10.1016/s0169-409x(01)00239-3

Tibbitt MW, Anseth KS. 2009. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol.Bioeng.. 103(4):655-663. https://doi.org/10.1002/bit.22361

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2次元および3次元細胞培養用生分解性ポリエチレングリコール

はじめに

PEG

PEGマクロモノマー

PEG Macromers

ヒドロゲル形成のメカニズム

分解性ヒドロゲル

加水分解

酵素分解

光分解性ヒドロゲル

結論

関連製品

参考文献

Related Links

PEG Macromers

Mechanisms of Hydrogel Formation

Degradeable Hydrogels

Hydrolytic Degradation

Enzymatic Degradation

Photodegradable Hydrogels

Conclusion

Materials

References

Related Links

Mechanisms of Hydrogel Formation

Degradeable Hydrogels

Hydrolytic Degradation

Enzymatic Degradation

Photodegradable Hydrogels

Conclusion

参考文献