無(wú)線移動(dòng)微型機(jī)器人預(yù)期將在通過(guò)微創(chuàng)療法治療某些難以觸及、受限和脆弱的體內(nèi)部位方面，精確而有效地發(fā)揮作用。然而，設(shè)計(jì)這樣一款無(wú)線移動(dòng)微型機(jī)器人，需要綜合考慮多種因素，包含動(dòng)力、控制、環(huán)境感知、醫(yī)療功能和生物降解性等。德國(guó)馬克斯·普朗克智能系統(tǒng)研究所的Metin Sitti團(tuán)隊(duì)研發(fā)了一種基于水凝膠、磁動(dòng)力和控制的，可酶降解的微型游動(dòng)裝置（Figure 1）。該裝置可對(duì)體內(nèi)微環(huán)境中的病理標(biāo)志物產(chǎn)生響應(yīng)，并遞送藥物，以完成治療和診斷的任務(wù)。研究者擬通過(guò)一種雙螺旋結(jié)構(gòu)來(lái)負(fù)載藥物和實(shí)現(xiàn)磁場(chǎng)作用下的游動(dòng)。最終，研究者基于3D打印和雙光子聚合技術(shù)，制備了由甲基丙烯酸明膠和生物功能化超順磁性氧化鐵納米粒子組成的3D微型游動(dòng)裝置（長(zhǎng)度：20 μm，直徑：6 μm）。他們發(fā)現(xiàn)，在正常生理濃度下，該微型游動(dòng)裝置在可被基質(zhì)金屬蛋白酶-2（MMP-2）在118小時(shí)內(nèi)完全降解為可溶性的無(wú)毒物。該微型游動(dòng)裝置通過(guò)溶脹來(lái)實(shí)現(xiàn)對(duì)病理濃度的MMP-2的快速響應(yīng)，從而實(shí)現(xiàn)負(fù)載藥物的釋放。該微型游動(dòng)裝置完全降解后，除了可釋放負(fù)載的治療診斷學(xué)的藥物，還可作為釋放其它載體，如功能化的納米顆粒等。

Figure 1. Design and 3D fabrication of biodegradable hydrogel microrobotic swimmers.

      為了實(shí)現(xiàn)3D微型游動(dòng)裝置的移動(dòng)，研究者用自制的六線圈電磁裝置來(lái)構(gòu)建旋轉(zhuǎn)磁場(chǎng)，并在微型游動(dòng)裝置的長(zhǎng)軸上施加計(jì)算機(jī)控制的磁矩。我們測(cè)定了3D微型游動(dòng)裝置的失步頻率。該3D微型游動(dòng)裝置中氧化鐵納米顆粒的載入量為6 mg mL-1，在20 mT條件下和1-6 Hz的范圍內(nèi)，每10秒增加一次輸入頻率，步長(zhǎng)為1Hz。結(jié)果表明，3D微型游動(dòng)裝置的游動(dòng)速度隨激發(fā)頻率的增加呈線性增加(Figure 2a)。在1-3 Hz的頻率范圍內(nèi)，微型游動(dòng)裝置表現(xiàn)出擺動(dòng)行為。在3-5 Hz范圍內(nèi)觀察到螺旋狀運(yùn)動(dòng)。微型游動(dòng)裝置的步進(jìn)頻率為6 Hz。通過(guò)在5 Hz時(shí)驅(qū)動(dòng)微型游動(dòng)裝置的長(zhǎng)距離運(yùn)動(dòng)軌跡，測(cè)定的微型游動(dòng)裝置的平均游動(dòng)速度(3.36-0.71 μms-1) (Figure 2b)。工作區(qū)內(nèi)的時(shí)間恒定的旋轉(zhuǎn)場(chǎng)可實(shí)現(xiàn)微型游動(dòng)裝置的導(dǎo)航。通過(guò)磁轉(zhuǎn)向控制，可以控制微型游動(dòng)裝置從給定的從A點(diǎn)到達(dá)D點(diǎn)(Figure 2c)。

Figure 2. Swimming trajectory of a double-helixmicroswimmer under a rotating magnetic field. (A) The step-out frequency of the microswimmerscontaining 6 mg mL-1 iron oxidenanoparticles was found to be around 6 Hz. (B)Image sequence of the hydrogel microswimmer actuated under a rotating magneticfield with the magnetic field strength and excitation frequency being 20 mT and5 Hz, respectively. The average velocity of the microswimmers was found to be3.36 ± 0.71 μm s-1. (C) Magnetic steering control of themicroswimmer. A microswimmer is given the task of reaching each checkpoint fromA to D.

      據(jù)報(bào)道，在健康個(gè)體中的不同組織中，MMP-2以不同濃度存在，但通常范圍在140-200 ngmL-1范圍內(nèi)。在高濃度MMP-2條件下（100 μgmL-1），在37℃條件下，微型游動(dòng)裝置可在1小時(shí)內(nèi)完全降解，并且其完全降解時(shí)間是初始MMP-2濃度的函數(shù)(Figure 3a, b)。與體內(nèi)更接近濃度條件下（如4 μgmL-1），該微型游動(dòng)裝置完全降解約需要5小時(shí)，在另外濃度條件（0.500 μg mL-1），該微型游動(dòng)裝置完全降解約需要67小時(shí)，在生理水平下（0.125 μgmL-1），降解時(shí)間為118小時(shí)。由于不同組織的特異性蛋白酶和其他局部條件存在差異，應(yīng)根據(jù)微型游動(dòng)裝置應(yīng)用的目標(biāo)組織，研究該微型游動(dòng)裝置的降解動(dòng)力學(xué)。近年來(lái)，基于鹽-侵蝕的無(wú)機(jī)微型游動(dòng)裝置在體內(nèi)環(huán)境中的降解是一種常規(guī)途徑。本研究提出了一種基于生理環(huán)境中酶條件下的降解新途徑，并且該過(guò)程的降解產(chǎn)物完全無(wú)毒（Figure 3c）。

Figure 3. Biodegradation of the hydrogel microswimmers by the MMP-2enzyme. (A) DIC images of adegrading microswimmer array in the presence of 4 μg mL-1 enzyme.Degradation starts with the rapid swell of the microswimmers followed by thecollapse of the entire network. (B) Enzymatic degradation of the microswimmers.At the physiological level, MMP-2 degrades the microswimmers within 118 h.Enzymatic susceptibility introduces a concept of operational lifetime, definedas the time period that microswimmer preserves its original morphology forproper navigation. The operational lifetime is a function of the enzymeconcentration in the microenvironment. Data are presented as mean ± standarddeviation. (C) Live (green) and dead (red) SKBR3 breast cancer cells treatedwith the degradation products of the microswimmers. (D) Quantitative analysisof the acute toxicity induced by the degradation products of the microswimmersin comparison with 5 μg mL-1 iron oxide nanoparticles and untreatedcells. Data are presented as mean ± standard deviation.

      MMP-2酶可以作為一種有價(jià)值的生物標(biāo)志物，用于微型游動(dòng)裝置所感知組織的病理狀態(tài)和發(fā)揮作用。初期，高濃度的MMP-2可作為微型游動(dòng)裝置釋放藥物的開(kāi)關(guān)。當(dāng)組織的局部MMP-2濃度升高時(shí)，初期微型游動(dòng)裝置的會(huì)快速溶脹，并釋放藥物。由于藥物釋放速度與溶脹程度有關(guān)，所以初始MMP-2酶濃度可以調(diào)節(jié)微型游動(dòng)裝置的釋放動(dòng)力學(xué)。溶脹動(dòng)力學(xué)與初始酶濃度的關(guān)系(Figure 4a)。在酶濃度為0.250 μgmL-1時(shí)，微型游動(dòng)裝置在加入酶20分鐘后開(kāi)始腫脹。在0.5和1 μgmL-1時(shí)，微型游動(dòng)裝置在接觸酶時(shí)就會(huì)開(kāi)始溶脹。此外，微型游動(dòng)裝置對(duì)病理性MMP-2濃度表現(xiàn)出高度的敏感性，而在生理?xiàng)l件下，微型游動(dòng)裝置可保持原來(lái)的大小，表明微型游動(dòng)裝置具有MMP-2酶響應(yīng)行為。水凝膠的降解對(duì)于充分利用水凝膠中的藥物，具有重要意義。如果材料不降解，將有近一半的藥物留在水凝膠網(wǎng)絡(luò)內(nèi)，造成利用率低。該研究制備的微型游動(dòng)裝置可完全降解，從而具有較高的藥物利用率（Figure 4b, c）。

Figure 4. Enzymatically-controlled drug release from the hydrogelmicroswimmers. (A)Elevated concentrations of MMP-2 cause rapid swelling of the microswimmer body,thereby acting as a switch for accelerated drug release. Data are presented asmean ± standard deviation. (B) At 1 μg mL-1 MMP-2 concentration,accelerated drug release in the first few hours (the pale pink region) isattributed to swelling-mediated mesh size increase. At the end of two days,almost all the payload is released from the degraded microswimmers whereas halfof the content is retained in the non-degraded one, which severely reduces thebioavailability of a significant portion of the drug to be delivered (palegreen region). Data as presented with mean ± standard deviation. (C)Epifluorescence images of microswimmers with loaded dextran-FITC cargo used asa model macromolecular drug equivalent. Enhanced drug bioavailability isevidenced by the enzymatic degradation of the network, which releases itsentire content.

       微型游動(dòng)裝置中水凝膠的降解可進(jìn)一步釋放磁性納米顆粒，這些納米顆粒原用來(lái)為運(yùn)動(dòng)提供磁性力矩。我們?cè)O(shè)想，用這些局部釋放的磁性造影劑對(duì)腫瘤細(xì)胞進(jìn)行靶向標(biāo)記，可以后續(xù)評(píng)估前期治療性藥物釋放的效果。ERBB2受體在乳腺癌細(xì)胞株SKBR3中過(guò)表達(dá)，我們對(duì)納米顆粒表面修飾anti-ErbB 2 抗體，可靶向結(jié)合ERBB2受體，從而標(biāo)記腫瘤細(xì)胞。為了便于體外分析，納米顆粒也同時(shí)被熒光基團(tuán)修飾（Figure 5a）。與未修飾的納米顆粒類似，抗體標(biāo)記的納米顆粒仍可嵌入到微型游動(dòng)裝置內(nèi)（Figure 5b）。改性的納米粒子的加入不會(huì)影響微型游動(dòng)裝置的整體游動(dòng)性能。我們研究了通過(guò)MMP-2酶介導(dǎo)的微型游動(dòng)裝置體內(nèi)降解后釋放的磁性納米顆粒的細(xì)胞標(biāo)記性能。微型游動(dòng)裝置降解后，釋放到環(huán)境中的功能性納米顆?？蓸?biāo)記39.3%的SKBR3細(xì)胞，用熒光活化細(xì)胞分選法檢測(cè)（Figure 5c和S 14）。另一方面，未經(jīng)表面修飾的納米顆粒也可標(biāo)記約9.5%的細(xì)胞，這表明細(xì)胞與納米顆粒之間也存在著一定非特異性相互作用（Figure 5d）。

 Figure 5. Targeted cell labeling with the magnetic nanoparticles releasedfrom collapsed microswimmers towards diagnostic in vivo imaging. (A) The design of superparamagnetic iron oxidenanoparticles of 50 nm size functionalized with a fluorophore and anti-ErbB 2antibody for targeted labeling of ERBB2-overexpressing breast cancer SKBR3cells. (B) Epifluorescence image of microswimmers embedded with the nanoparticles.(C) Targeted labeling of SKBR3 with the anti-ErbB 2 modified magneticnanoparticles released upon the MMP-2-mediated degradation of themicroswimmers. In the absence of anti-ErbB 2, the nanoparticles fail to targetSKBR3 cells.

本研究是由德國(guó)馬克斯·普朗克智能系統(tǒng)研究所的MetinSitti團(tuán)隊(duì)完成，并于2019年3月在線發(fā)表于ACS Nano。
   論文信息：Hakan Ceylan, Immihan Ceren Yasa, Oncay Yasa, Ahmet Fatih Tabak, Joshua Giltinan, andMetin Sitti*. 3D-Printed Biodegradable Microswimmer for Theranostic CargoDelivery and Release. ACS Nano. 13(3):3353-3362.