1. Introduction

Cardiovascular disease is the most common cause of human mortality in western countries; the single leading cause of human death in the USA is coronary heart disease, which is caused by atherosclerosis of the coronary arteries and produces angina pectoris or heart attack. The use of intravascular stents has revolutionized the treatment of coronary heart disease and other cardiovascular diseases by improving the blood flow in diseased blood vessels, using a less-invasive method. However, the occurrence of early and late stent thrombosis has raised serious safety concerns regarding the use of intravascular stents. Temporary stent materials might reduce the risk of late stent thrombosis because they disappear from the vulnerable tissues after the healing process is completed. Because the surface of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers resembles that of cell membranes, I hypothesized that bioabsorbable stent materials bearing phosphorylcholine groups could enhance tissue compatibility and have the potential to reduce the incidence of both early and late stent thrombosis (Fig. 1).

2. Methods and Results

2.1. Preparation of poly (L-lactic acid) / a water-soluble phospholipid polymer blends

A 6 wt% poly (L-lactic acid) (PLLA) solution in dichloromethane (DCM), 6 wt% Blends of PLLA and a water-soluble amphiphilic poly(MPC-co-n-butyl methacrylate (BMA)) (PMB30W) (weight ratio, 95/5) blend polymer solution in DCM/ methanol (MeOH) (volume ratio, 12/1), and 4 wt% PLLA/PMB30W (weight ratio, 90/10) blend polymer solution in DCM/MeOH (volume ratio, 12/1) were prepared by stirring the solutions sufficiently until they turned transparent. The solutions were sonicated in a cold bath with ice for 30 min for homogenization. In order to prepare cast films, the polymer solutions were cast onto Teflon dishes, and the solvents were dried at room temperature overnight. In order to prepare tubing, the polymer solutions were repeated coated on the rotated Teflon rod. The PLLA/PMB30W cast films demonstrated higher breaking strengths than PLLA cast films, and their Young's modulus was similar to that of PLLA under dry conditions (Fig. 2). A high density of phosphorylcholine head groups on the inner surface of PLLA/PMB30W tubing was developed by repeated coatings with the PLLA/PMB30W blend polymer solutions (Fig. 3). The PLLA/PMB30W tubing showed stable degradation behaviors similar to the PLLA tubing. These materials are characterized by strong mechanical properties and stable degradation behaviors that are either superior or similar to those of high molecular weight PLLA.

2.2. Tissue compatibility of poly (L-lactic acid) / a water-soluble phospholipid polymer blends

PLLA and PLLA/PMB30W were evaluated in vitro and small animals. A high-content automated screening assay (240 random fields per group) confirmed that PLLA induced apoptosis of a mouse macrophage-like cell line (apoptotic population: 73.9% in 0.8mg PLLA/mL) while PLLA/PMB30W remained biologically inert (apoptotic population: 13.8% in 0.8mg PLLA and not quantified PMB30W/mL) (Fig. 4). Human peripheral blood mononuclear cells were cultured on PLLA and PLLA/PMB30W (n=8 cultures per group) to compare inflammatory reactions. Enzyme-linked immunosorbent assay quantified substantial decreased concentrations of proinflammatory cytokines in PLLA/PMB30W, compared to PLLA. PLLA and PLLA/PMB30W (n=3-6 material per group) were implanted into subcutaneous tissues of rats, rat infrarenal aortas, and the internal carotid arteries of rabbits. Subcutaneous degradation profiles showed that both PLLA and PLLA/PMB30W have not been rapidly absorbed. After intravascular implantation of polymer tubing, an average of total cross-sectional areas (CSA) exhibited that PLLA/PMB30W tubing maintained larger cross-sectional areas than PLLA in both rats (0.67mm2 versus 0.24mm2) and rabbits (0.96mm2 versus 0.59mm2) during 30 days of implantation (Fig. 5).

2.3. Design of nanocarrier-mediated delivery of antiproliferatives

To obtain bioabsorbable sirolimus (SRL)-releasing polymer films that could release nanovehicles incorporating SRL, poly (L-lactide-co-caprolactone-co-glycolide) (PLCG), PMB30W, and SRL were blended with different formulations (Table 1.) The SRL -releasing polymer films and the released materials were characterized by differential scanning calorimeter, scanning electron microscopy, transmission electron microscopy, dynamic light scattering, and high-performance liquid chromatography. Nanovehicles were formed with PMB30W chains and could be released from the PLCG/PMB30W/SRL films. The hydrodynamic diameter of the nanovehicles in phosphate-buffered saline was smaller than 20nm. The PLCG/PMB30W/SRL films substantially enhanced the carrier-mediated delivery of SRL and attenuated its degradation product, 34-hydroxy SRL (Fig. 6).

3. Discussion

There are two possible mechanisms to explain why the PLLA/PMB30W (95/5) cast (solvents, DCM/MeOH=12/1, v/v) films demonstrated higher breaking strengths than the PLLA cast (solvent, DCM) films. First, the cracks presented at the air contact surface of PLLA cast films could deteriorate their mechanical properties. Because the PLLA/PMB30W (95/5) cast films had a smooth surface without cracks and limited sub-micron sized pores, the polymer walls of the PLLA/PMB30W (95/5) cast films were more compact and were able to sustain deforming pressures. Second, interlocking networks were formed between the hydrophobic segments (BMA units) of PMB30W and PLLA chains. The mechanical properties of PLLA increase via solution blending with surfactants of optimum concentrations. In the PLLA/PMB30W (95/5) blend system, the homogeneous spreading of PMB30W could bridge the gap among the crystalline regions of PLLA across the amorphous regions of PLLA. Meanwhile, for the efficient surface modification, PMB30W was accumulated with MeOH-rich mixed solvents on the inner surface of PLLA/PMB30W tubing by the repeated coatings with the DCM/MeOH (12/1 by v/v) mixed solvent. Therefore, a high-density phosphorylcholine group coat was formed on the inner surface of the PLLA/PMB30W tubing even without water immersion.

The idea that phosphorylcholine groups might reduce stent thrombosis has not been resolved. Thus, the tissue compatibility of PLLA and PLLA/PMB30W was evaluated. The gravest risk associated with temporary stent materials is that they are heterogeneously degraded and release microparticles that enter circulation and may lead to the formation of thrombotic emboli. The hypothesis that microparticles covered with phosphorylcholine groups could prevent the apoptosis of phagocytes and thus remain biologically inert has been proven by a high-content screening assay. Moreover, an in vitro immunological study has revealed that temporary stent materials bearing phosphorylcholine groups can reduce the inflammatory reaction of human blood cells. Finally, animal studies have revealed that the biodegradation properties of the proposed material are appropriate for its use in stenting and can reduce thrombus formation after intravascular stent implantation in small animals. Because the formation of cell membranes in nature is highly regulated and finely tuned at the molecular level, the surface could improve the vascular tissue compatibility of these stent materials from the time of implantation to the final absorption.

For drug-eluting stent materials, a new drug delivery system was proposed to enhance and stabilize the delivery of SRL incorporated by PMB30W. SRL is an unstable agent; its degradation produces an open-chain isomer, 34-hydroxy SRL; it retains less than 10% of the antiproliferative activity of the parent SRL. An attractive approach to stabilizing bioactive agents is to incorporate them into nanovehicles. The PMB30W chains assemble themselves in an aqueous medium and form colloidal aggregates. These aggregates could be used as nanovehicles to maintain SRL inside of the nanovehicles in a stable manner. The hydrophobic part (BMA units) of PMB30W nanovehicles could inhibit the entrance of water molecules, while the hydrophilic part (phosphorylcholine groups) of PMB30W is endowed with forming the small size of nanovehicles in an aqueous medium. Thus, the shielding effect provided by PMB30W nanovehicles suppressed the hydrolysis of SRL and enhanced the carrier-mediated delivery of SRL. The release of SRL was controlled by tuning the size of PMB30W/SRL-rich domains in the PLCG/PMB30W/SRL films. This approach might reduce the in-stent neointimal hyperplasia and the drug-associated platelet activation.

4. Conclusion

In this thesis, I have proposed a new concept of clinical materials - temporary stent materials bearing phosphorylcholine groups. The preparation of the materials was efficient for the surface modification and comparable with conventional temporary stent materials in terms of bulk properties. The preliminary hypothesis that temporary stent materials bearing phosphorylcholine groups offer enhanced tissue compatibility has been proven in part. Furthermore, temporary stent materials bearing phosphorylcholine groups have been found to have potential as efficient reservoirs for enhancing and modulating an antiproliferative release.

Figure 1. Scheme of temporary stent materials bearing phosphorylcholine groups

Figure 2. Mechanical properties of cast films. Columns and error bars represent means ± standard deviations for n=6. Statistical differences were obtained by ANOVA followed by Tukey's test. p values are given when p value is below 0.05.

Figure 3. Schematic diagram of distribution of phosphorylcholine groups in the PLLA/PMB30W tubing. Dark blue circles indicate phosphorylcholine groups of PMB30W.

Figure 4. The apoptotic population of a mouse macrophage-like cell line after induction of PLLA or PLLA/PMB30W microparticles in the presence of serum for 24.

Figure 5. Cross-sectional areas (CSA) of polymer tubing tubing after the intravascular insertion. Statistical differences (**P < 0.01; ***P < 0.001) were analyzed by the Mann-Whitney U test.

Figure 6. Release profiles of (A) SRL and (B) 34-hydroxy SRL from PLCG/SRL films (blue squares), Film X (red dimonds), Film Y (green triangles), and Film Z (purple crosses) during incubation in PBS with BSA. Data points and error bars represent mean ± SD for n=4-5.

生体内埋入型インプラントデバイスは、循環器系、骨格・運動系などを代表とする器官の機能不全を一部補助する為に重要である。特に、循環器系デバイスは血液と常時接触するために、これを作製するためには血液凝固反応を完全に抑制できるバイオマテリアルが不可欠となる。デバイス表面における血液凝固反応は、界面において誘起され、複数の経路により血栓へと至る。したがって、初期過程に関連したin vitro(生体外)での評価と比較的長期間にわたるin vivo(生体内)での評価を系統的に行うことが求められる。細胞膜表面を模したポリマーバイオマテリアルである2-methacryloyloxyethyl phosphorylcholine (MPC)ポリマーは、効果的に血栓形成反応を阻止できるために医療デバイスの抗血栓性を付与する表面処理として利用されてきている。さらに、他のポリマーと組み合わせて、バルク特性及び表面特性の双方を同時に制御することにより、新しい循環器系デバイスバイオマテリアル創製が期待されている。本研究では、血栓形成を長期間にわたって抑制可能なバイオマテリアルの分子設計としてMPCポリマーを生体内分解性ポリマーと組み合わせた新しいポリマーブレンド系を考案し、マテリアル表面で生じる細胞応答、組織応答を正確に判断できる評価を行っている。これらから新しい循環器系デバイスを創製するために必要なバイオマテリアル設計論を提案している。

本論文は全4章から構成されている。

第1章は、本研究の背景と意義を解説している。すなわち、血管拡張ステントに関連した生体反応の基礎をまとめ、一時的に利用する血管拡張ステントに利用するバイオマテリアルに要求される性能を説明している。さらにin vivoでのバイオマテリアル評価法についても言及し、本研究のための基盤技術を解説している。

第2章では、一時的に利用する血管拡張ステントを作製するためのバイオマテリアルの設計について、代表的な生分解性ポリマーであるポリ(L-乳酸)(PLLA)と水溶性MPCポリマー(PMB30W)とを複合化させたポリマーブレンド(PLLA/PMB30W)系を検討している。PLLAに対してPMB30Wを添加する際の溶媒、添加組成を変化させて、バルク特性と表面組成に対する影響を検討している。まず、PMB30Wが両親媒性を持つ特徴から、PLLAとPMB30Wの良溶媒となる系を確認している。また、PMB30WのPLLAに対する添加組成として5wt%が最適であることを見いだしている。このPLLA/PMB30Wの機械的特性を評価したところ、破断強度が未処理のPLLAよりも高くなることを明らかにした。これは、溶媒組成と添加量の最適化より見いだされた現象であり、ポリマー鎖が効果的に絡み合っていると結論している。これらの基礎的な知見より、ステントの基本形状であるポリマーチューブへの成形加工も実施し、表面近傍に血液適合性のMPCユニットが濃縮できる条件を規定している。さらに、埋入初期の過剰な組織反応を沈静化できる薬物(Sirolimus)をステントから徐放させることを企画している。薬物の溶解性の観点からマトリックスとなる生分解性ポリマーとしてポリ(L-ラクチド/カプロラクトン/グリコチド)(PLCG)を利用し、PMB30Wを薬物リザーバーおよび親水化剤することで、薬物の約30日間にわたる効果的な徐放に成功している。この過程で、PMB30W中に貯蔵した薬物の不安定化が抑制できることを発見し、これまでの薬物徐放ステントで認められていた問題の解決にひとつの知見を与えるに至った。

第3章では、PLLA/PMB30Wの生体親和性について、in vitroおよびin vivoにおける評価を行っている。in vitroでの評価では、細胞応答を評価するために新たにPLLA/PMB30Wより直径450nm程度のポリマー粒子を作製している。この粒子を免疫担当細胞であるマクロファージ由来細胞(J774A.1)に適用した際の、貪食とアポトーシスを観察している。PLLA粒子では、細胞からの貪食を受け、細胞のアポトーシスを誘起することが認められるが、PMB30Wを添加したPLLA/PMB30W粒子では、粒子濃度が0.8mg/mLという比較的高濃度においてもこの反応がPLLA粒子に比較して1/6以下と軽度となっていることが見いだしている。さらに、細胞内反応を追跡する目的で、ヒト末梢血単核細胞(hPBMCs)を利用して、ポリマー粒子と接触した際に生成するマクロファージ遊走阻害因子(MIF)、血管内膜肥厚の指標となるmonocyte chemoattractant protein-1(MCP-1)、インターロイキン1β(IL-1B)および6(IL6)、腫瘍壊死因子(TNF-αを定量している。PLLA粒子では、これらいずれの指標も大きな値を示し、未処理細胞と比較しても明らかな細胞応答を示しているが、PLLA/PMB30W粒子では、これらがいずれも抑制されることを明らかにしている。これらのことから、ポリマー表面にMPCユニットが存在することで、細胞応答を低減できることを結論している。これらの基礎的なin vitroでの結果をふまえ、動物を利用したin vivoでの評価を行っている。PLLA/PMB30Wより作製したチューブを血管内に移植し、開存性を6ヶ月間にわたり経時的に観察している。その結果、移植30日間後に血栓形成により閉塞したチューブ内平均面積割合は、PLLAの87%に対して、67%と有意に低くなっていることを認めている。これらのようにPLLA/PMB30Wにおいては、細胞応答、血栓形成特性ともに反応が抑制されていることを明らかにしている。

第4章では、一時的に利用する血管拡張ステントに利用するバイオマテリアルに関する本研究をまとめている。

以上のように、本研究では、生分解性ポリマーとMPCポリマーとのブレンドを利用して、一時的に利用する血管拡張ステントデバイスのマテリアル設計と生医学的特性解析を実施し、細胞応答性を有意に低減するバイオマテリアル創製とデバイス作製に成功している。また、長期間にわたる生体親和性、血液適合性の評価法を確立してきており、今後の循環器系デバイス開発に有意義な提案できると判断できる。

このように本研究は、バイオマテリアルの研究領域に新しい分子設計概念を導引しており、バイオエンジニアリング分野の新たな発展をもたらす研究と評価できる。

よって本論文は博士(工学)の学位請求論文として合格と認める。