基於金奈米液滴汽化之聲穿孔效應研究

Abstract

金奈米液滴為近年光熱治療研究中常使用到的載體，相對於早期研究所使用的微氣泡載體，不僅擁有較高的穩定度，更因為其相當微小的粒徑，將能通過腫瘤組織之血管新生區域，並透過增強滲透滯留效應增進釋放效率。金奈米液滴受到聲壓或熱能激發後將汽化成微氣泡，當聲壓足夠時更可引起穴蝕效應。如此短時間內所發生的體積變化將會在鄰近區域內產生衝擊波，因此可在液滴周遭細胞的細胞膜上造成聲穿孔作用，藉此達到運送藥物或奈米金桿至細胞內的效果。然而誘發液滴產生穴蝕效應所需的能量通常較微氣泡來得高，很可能會有對周邊正常組織造成傷害的疑慮。此外，穴蝕效應發生的同時也將迅速消耗載體的數目，導致治療成效與超音波對比度的下降。為了減少激發能量的同時兼顧物質釋放效率及載體的耗損量，本研究提出以液滴汽化過程作為誘發聲穿孔作用的機制，係基於汽化與穴蝕效應皆屬於液滴所產生之快速體積變化的觀察，因此提出汽化過程應能造成聲穿孔作用的假說。此外，利用汽化後微氣泡可再凝結回液態的特性，將提供液滴再次汽化來增進聲穿孔作用的可能性，預期將可透過反覆較低能量的激發過程提高物質釋放的效果。本研究首先將液滴固定於仿體結構中，並透過聲學方法誘發液滴汽化，確認了以人類血清白蛋白液滴包覆全氟戊烷製備之液滴具有可重複性汽化特性。另一方面，相較於傳統研究中普遍使用純光學或純聲學方法激發液滴，本研究則結合光學與聲學方法同步激發，大幅降低了誘發汽化與穴蝕效應所需的能量。於此架構下，將激發條件控制在能夠使液滴汽化但並不足以引發穴蝕效應的狀態時，觀察到汽化事件確實能夠引起聲穿孔效應，且其效率隨激發次數增加而有加強的趨勢，甚至可達到與穴蝕效應產生之聲穿孔效率相當的程度。本研究結果證實液滴汽化事件即可造成聲穿孔效應，於液滴作為藥物載體的臨床用途上，將可用較安全的激發能量促進藥物的局部釋放，並由於汽化後的微氣泡較不容易受到破壞，除了在促進釋放的過程中可降低液滴用量，更在多次激發過程中仍能維持超音波影像中的對比度。

Gold nano-droplets (AuNDs) are commonly used vehicles in photothermal therapies. In comparison to gold microbubbles (AuMBs) that have been deployed in early research, AuNDs are not only stable but also capable of passing through angiogenesis regions in a tumor because of their relatively small size. Thus, AuNDs possess a high releasing efficiency with the aid of enhanced permeability effect (EPR). AuNDs can be vaporized into microbubbles either acoustically or optically. When sufficient energy is given, cavitation takes place and results in shock waves around the microbubbles. It is the mechanical force generated from the volumetric shift that gives rise to sonoporation effect on nearby cells so that drugs or therapeutic agents can be transferred from the vehicles to the cells. However, it requires a high level of energy to drive cavitation leaving potential to damage normal tissue during the treatment. In addition, since cavitation resulting from micro bubble destruction cannot be repeated, its delivery performance and ultrasound echogenicity can drastically change. With the aim of reducing the driving energy and allowing repeatable effects, in this research, we propose a mechanism that induces sonoporation based on droplets vaporization. It is based on the fact that vaporization and cavitation both present rapid volumetric changes. We hypothesize that the subsequent mechanical force generated from the vaporization can also induce sonoporation. Under this proposed mechanism, we can leverage recondensation of the vaporized nano-droplets (i.e. from gas phase to liquid phase), and further enhance the sonoporation efficiency through the repeated recondensation-vaporization process. In our research, we immobilized AuNDs into an agarose phantom and activated the droplets through acoustic vaporization. We confirmed the ability to perform repeatable vaporization on human serum albumin (HSA)-coated perfluoropentane (PFP) droplets. On the other hand, we developed a more efficient vaporization method by synchronizing the acoustic pulse with the laser pulse. Under the same framework, we have found several activation conditions that can vaporize the droplets without significant cavitation effects. Besides, it is evidenced that the vaporization events can induce sonoporation. Although vaporization-based sonoporation was not as effective as that of cavitation-based sonoporation, we found a significant increment when multiple activations were given. Eventually, the sonoporation effects can reach to a similar level as that of the cavitation-based approach. In summary, we demonstrated that vaporization leads to sonoporation. It has the potential to be a safer strategy for enhancing the delivery efficiency in clinical applications. In other words, the vaporization-based approach requires a relatively low driving energy, reduces droplet consumption and preserves the ultrasound contrast at the same time.