Thermochromism-induced temperature self-regulation and alternating photothermal nanohelix clusters for synergistic tumor chemo/photothermal therapy

Abstract

To improve the inherent defects of chemotherapy and photothermal therapy (PTT), we design a novel thermochromism-induced temperature self-regulation and alternating photothermal system based on iodine (I₂)-loaded acetylated amylose nanohelix clusters (ILAA NHCs) under the guidance of molecular dynamic simulation in which I₂ is loaded into the helical cavity of acetylated amylose (AA) by hydrophobic interaction. ILAA NHCs perform versatile photothermal conversion through their unique reversible thermochromism. Upon irradiation, I₂ is gradually released and the ILAA NHCs turn into colorless. The laser is then penetrated deeply into the tissue for deep-seated heating, and the ILAA NHCs' color can be recovered by reversible thermochromism because of I₂ reloading into the ILAA NHCs. When the process is repeated, the temperature can be controlled in a certain range. This alternating light-to-heat conversion significantly improve the effect of PTT. Meanwhile, I₂ efficiently acts dual functions of chemotherapy and PTT. Results show that the photothermal depth by ILAA NHCs is 2.1-fold than other common photothermal agents (PTAs), and the irradiated region exhibits a lower surface temperature. In vitro and in vivo experiments both provide ILAA NHCs an excellent comprehensive antitumor effect with synergistic chemo/PTT, indicating versatile potential for tumor chemo/PTT.

Introduction

Photothermal therapy (PTT) is an efficient treatment mode that converts light especially near-infrared (NIR) light energy to heat through photothermal agents (PTAs) and increases the tumor temperature above 50 °C to kill cells quickly [1,2]. NIR light is a safe remote external stimulus that allows deep penetration, low water absorption, and high temporal and spatial resolution, thereby making PTT a safe, highly efficient, non-invasive, and fix-pointed treatment with few side effects [[3], [4], [5]]. Hence, PTT has attracted much interest of researchers. However, owing to the inhomogeneous heat distribution during irradiation, PTT alone cannot inhibit tumor growth completely [6]. To enhance the antitumor effect, researchers combined chemotherapy, [7] radiotherapy, [8] photodynamic therapy, [[9], [10], [11]] gene therapy, [12] or immunotherapy [13] with PTT to perform synergistic treatment. Among them, chemo/photothermal therapy (chemo/PTT) is considered to be one of the most efficient methods. Hyperthermia caused by PTT could enhance the cellular uptake and release of chemotherapeutic drug and even inhibit multidrug resistance of tumor which are beneficial for chemotherapy [14]. Complementarily, chemotherapy is a continuous treatment without limitation on space, which could offset the scarcity of PTT. Many researches have verified this synergistic therapy and make great progress using various type of nanosystem. Recently, multifunctional nanosystems which integrated chemo/photothermal agent and another components such as contrast agent in one nanoplatform to achieve multimodal imaging-guided chemo/PTT, [15] targeted nanosystems including cell membrane coated nanosystem [16], and smarted chemo/photothermal nanosystems for programmed treatment [17] have been developed quickly and make effective treatment. The introduce of nanoplatform in tumor chemo/PTT not only allow for long circulating time in bloodstream and large accumulation of drug in tumor by enhanced permeability and retention (EPR) effect, but also enhance the cellular uptake and provide stimuli-responded drug release for programmed treatment [2]. In detail, Lei et al. designed a pH-responsive cluster bomb that used WS₂ as PTA and doxorubicin as therapeutic agent. Such design achieved the smart release of drugs and performed programmed combined therapy that killed different cells with different treatments [18]. Meng et al. applied thermosensitive macromolecule nanogels to wrap doxorubicin and Cu₂S, and the drug molecules were released for synergistic therapy once PTT acted. These ingenious designs intensely inhibited the growth and metastasis of tumors [17].

Although researches about chemo/PTT have made great progress, some issues needed to be overcome: (1) PTAs are designed to have strong light absorption in the NIR region. Hence, PTAs considerably attenuate and hinder the deep penetration of NIR light. Hirsch et al. reported that the maximum temperature during photothermal process appeared at 1 mm deep of the subcutaneous layer [19]. Thus, PTT alone only eliminates shallow tumor cells and triggers a large proportion of tumor recurrence due to short effective photothermal depth. (2) Recent photothermal processes are robust, and real-time temperature adjustment in a proper region cannot be achieved with PTAs themselves due to the loss of thermo-feedback [20]. This drawback causes extra reverse effect and pain to patients, such as skin burn and inflammation. (3) Most PTAs are synthetic materials. Thus, their metabolism and degradation in the body remain a huge challenge. Moreover, most systems for chemo/PTT are complicated and contain numerous components for different applications. However, these components only achieve one component for one action and hence do not satisfy efficient utilization. Complicated components increase the difficulty of preparation with limited antitumor effect, and also enhance the risk in clinical application. It's reported that tellurium nanorods play dual roles as PTA and chemotherapy agent and exert better antitumor effect than traditional chemo/PTT system [21]. However, tellurium nanorods also suffer unconscious toxicity and degradation in the body, which limit their further application. Therefore, an ideal nanoplatform for chemo/PTT must meet simple preparation, highly integrated, natural products, and exhibit smart temperature self-regulation and uniform deep-seated heating properties.

To the best of our knowledge, amylose is a natural biomacromolecule and can form deep-blue complex when mixed with iodine (I₂). The I₂ molecules arrange into various lengths of polyiodine chains under the constraint of a special V-type helical structure of amylose [22]. Different lengths of polyiodine chain induce different fields of absorption redshift, thus causing a broad absorption band from the visible to NIR region. This property makes I₂-loaded amylose a potential PTA. Unfortunately, native amylose suffers unfavorable solubility, and the loaded I₂ cannot be released at a suitable temperature. In the past few years, our group devoted to exploit novel amylose-based drug delivery systems under the guidance of molecular dynamic (MD) simulation and developed rapid blood-brain barrier cross-transport system [23] and sequential molecule-triggered release system [24] based on the acylated amylose helical structure. Most importantly, we found that acylation provided amylose excellent solubility and the helix thermosensitivity for I₂ release, [25] thereby inducing I₂-loaded acylated amylose a special reversible thermochromic property (upon irradiation, temperature increases as the color changes from deep blue to colorless due to the release of I₂ and then the color recovers as the I₂ is reloaded). Meanwhile, I₂ is also a low-toxic antitumor drug that suppresses various tumor cells, which could induce tumor cells apoptosis by changing the members of bcl-2 family proteins and leading the activation and translocation of Bax to mitochondria [26,27]. Inspired by these, we assumed that I₂-loaded acylated amylose can integrate the roles of chemotherapy and PTT in I₂, and achieve deep-seated heating and temperature self-regulation through its unique reversible thermochromism, which could cleverly improve the abovementioned challenges.

Hence we design I₂-loaded acetylated amylose nanohelix clusters (ILAA NHCs) under the guidance of MD simulation. I₂ is loaded in the acetylated amylose (AA) helical cavity by hydrophobic interaction, then these helices self-aggregate to nanoclusters. Optimal ILAA NHCs display reversible thermochromism in which the color fades and recovers repeatedly at proper temperature with respect to the release and reload of I₂. Thus, ILAA NHCs exhibit unique alternating PTT and on-demand drug release (Scheme 1). Once exposed to laser, ILAA NHCs perform excellent photothermal conversion due to their high NIR absorbance. Once the temperature increases above a critical value, the helices tend to unfold, and the I₂ is gradually released from the ILAA NHCs. The ILAA NHCs change from deep-blue to colorless and lose their photothermal capability. Then the color and photothermal capability partially recover when the temperature decreases below the critical value through heat dissipation. Intermittent but repeatable photothermal state effectively mitigates the brutality of continuous heating and controls the in situ temperature in a proper range that avoids unwanted burns. Simultaneously, the colorless ILAA NHCs allow the NIR light to penetrate to deep tissues. The light energy is absorbed by deep-seated ILAA NHCs and converted to heat killing deep-seated tumor cells. Thus, alternating photothermal effect from shallow to deep is achieved for homogeneous PTT. In addition, the released I₂ efficiently functions as a chemotherapeutic drug and continuously hunt residual cells to ensure complete clearance of tumor cells. Therefore, ILAA NHCs are highly efficient thermo-sensitive nanoclusters in which I₂ integrates the functions of PTT and chemotherapy. Whole in vitro and in vivo experiments clearly verified our initial design. The ILAA NHCs exerted a comprehensive antitumor effect, including high inhibition, low risk of recurrence, and excellent survival status. This kind of system with alternating photothermal strategy can be used as a novel program for synergistic tumor chemo/PTT and has great potential in clinical applications.