为了提高对肺癌的治疗效果，吸入纳米药物在近二十年来受到广泛关注。与全身给药相比，吸入给药纳米递送系统在提高肺部生物利用度、降低转移率和减少全身不良反应等方面具有极大的优势。纳米药物在肿瘤中的蓄积必须克服肺部的清除作用，并加强向肿瘤组织的转运。然而，单一尺寸的纳米粒无法同时满足肿瘤高渗透性和肺组织驻留。基于此矛盾，本论文构建了一种具有黏液穿透能力的粒径可变的纳米给药系统，用于克服肺部递送过程中的各种生物屏障从而发挥抗肿瘤疗效。此外，目前人们对吸入性纳米药物转运至肿瘤部位的具体过程知之甚少，有鉴于此，本文对肺组织和肿瘤组织中的纳米粒进行了量化，通过测定游离药物和纳米粒的含量，研究了纳米粒在肺组织和肿瘤组织的过程，进而阐明了其与肺组织驻留、向肿瘤中转运以及肿瘤累积和渗透相关的生物学特性。

论文首先合成了近红外荧光染料吲哚菁绿衍生物（Indocyanine green derivative, ICGD）标记的线性-树枝状嵌段共聚物K4单体，此两亲性多肽材料能够在水中自组装并包裹化疗药物7-乙基-10-羟基喜树碱（7-Ethyl-10-hydroxycamptothecin, SN38）形成粒径为28 nm的K4纳米粒，并通过其表面的苯硼酸被修饰有多巴胺和叶酸的透明质酸（Hyaluronic acid, HA）包裹，形成粒径在120 nm左右的HA-K4纳米粒。在体外模拟肿瘤微环境的条件下，HA-K4纳米粒可以响应性地转化为K4纳米粒。并且雾化性能表征显示，K4纳米粒和HA-K4纳米粒均具有较好的吸入给药潜力。

然后对K4纳米粒和HA-K4纳米粒的呼吸道黏液和瘤组织穿透能力以及体外抗肿瘤活性进行了评价。相比于小粒径的K4纳米粒，HA-K4纳米粒表现出更强的黏液渗透和肺组织驻留能力。体内外肿瘤穿透能力结果表明，K4纳米粒具有更快速的肿瘤穿透性，而HA-K4纳米粒同样具有良好的穿透能力，但存在6-12小时的迟滞现象。两种纳米粒在体外均具有较强的声动力学转化能力，能够在超声条件下产生活性氧从而引起细胞损伤。体外细胞抗肿瘤实验表明，K4纳米粒和HA-K4纳米粒的化疗和声动力学治疗似乎具有协同抗肿瘤作用。

接着对纳米粒的体内过程进行评价，研究了纳米粒经肺部给药和静脉给药后在体内的过程。研究表明，气管滴注HA-K4纳米粒的肺组织SN38含量和荧光强度AUC分别为静脉注射的43.7倍和99.7倍，表明气管内滴注纳米粒能够极大地提高肺组织暴露量。并且相比于容易快速清除的K4纳米粒，大粒径的HA-K4纳米粒由于较强的黏液渗透和肺组织驻留能力，使得SN38的肺部生物利用度提高了58.5%。肺部给药的纳米粒能够通过与肿瘤细胞直接接触、纳米粒的转胞吞作用及释放药物的扩散到达肿瘤组织。到达瘤组织的大粒径纳米粒可显著增加其驻留，ICGD标记的大粒径纳米粒的瘤组织生物利用度是小粒径纳米粒的5.99倍，并提高SN38的瘤内生物利用度1.99倍。体内药效结果显示，HA-K4纳米粒具有更好的肺部安全性，经肺部给药后在肿瘤内提供的持续高浓度的ICGD和SN38可实现化疗-声动力学联合治疗，将荷原位Lewis肺癌肿瘤小鼠的生存期从15天延长至22天，发挥了显著的抗肿瘤药效。

最后为了探究巨噬细胞的吞噬作用对肺部递送纳米药物抗肺癌疗效的影响，采用主动靶向修饰的策略，设计合成和构建了三种对LLC肿瘤细胞和巨噬细胞具有不同识别能力的纳米载体，分别为苯硼酸靶向LLC肿瘤细胞的ICP纳米粒、在ICP材料上进一步修饰叶酸基团的FCP纳米粒和在ICP材料上进一步修饰甘露糖基团的MCP纳米粒。三种纳米粒的粒径分布、电位、形态及临界胶束浓度等理化性质接近，仅有靶向基团会影响纳米粒进入肺组织后的过程。为模拟体内环境下不同细胞对纳米粒的竞争性摄取行为，论文构建了3D海藻酸钠/聚乙烯醇水凝胶肿瘤细胞和巨噬细胞共培养模型，在此竞争性摄取模型下，FCP纳米粒能被肿瘤细胞选择性摄取，MCP纳米粒能被巨噬细胞选择性摄取。将抗肿瘤药物淫羊藿素（Icaritin, ICT）包载进入三种纳米粒中，在原位Lewis肺癌小鼠模型上进行体内药效评价。结果显示，MCP纳米粒与对照组的肿瘤重量无显著性差异，ICP纳米粒则在一定程度上抑制了肿瘤生长，其抑瘤率为29.46%；而FCP纳米粒的肿瘤抑制作用显著优于其它两种纳米粒，其抑瘤率为48.51%。MCP纳米粒的抗肿瘤作用显著弱于FCP纳米粒和ICP纳米粒，总体上显示出巨噬细胞拦截减弱肺部给药纳米粒的抗肿瘤疗效的趋势。

综上所述，本论文研究了吸入纳米粒的粒径和靶向修饰对抗肺癌疗效的影响，研究发现巨噬细胞对吸入纳米粒的拦截可降低药物的抗肿瘤疗效，大小可变的吸入纳米粒能够在保证黏液穿透能力的同时，通过延迟药物释放和纳米粒向血液和肺外器官的易位从而延缓吸收清除，并通过粒径转变改善其瘤组织蓄积与渗透能力，为肺癌的无创化治疗提供了一种有前景的联合化疗和声动力学治疗的方法。

In order to improve therapeutic efficacy for the treatment of lung cancer, inhaled nanomedicines have been increasingly drawing attention in the last two decades. Extensive work has demonstrated that there are several advantages associated with inhalation delivery of nanoparticles, including improved lung bioavailability, reduced potential of metastasis and minimized systemic adverse effects when compared to systemic administration. The tumor accumulation of drug and/or nanoparticles has to overcome lung clearances and to enhance the transport to tumors. However, nanoparticles with single size could not simultaneously achieve prolonged lung retention and high tumor accumulation and penetration. To deal with this dilemma, the present thesis developed a size-transformable nano drug delivery system with mucus penetration ability to overcome various biological barriers of pulmonary delivery and thereby enhancing antitumor efficacy. In addition, very little is currently known about how inhaled nanomedicine for lung cancer treatment overcomes biological barriers hampering the tumor availability of drug and nanoparticles. To fill this gap, we set out to quantify the pulmonary and tumor fate of nanoparticles with a view to elucidating their biological features relevant to lung retention, transport to tumor, and tumor accumulation and penetration.

Firstly, the thesis synthesized the linear-dendritic block copolymer modified with near-infrared fluorescent dye indocyanine green derivative (ICGD), called K4 monomer, and its self-assembly could form K4 NPs to load antineoplastic agent 7-Ethyl-10-hydroxycamptothecin (SN38) with particle size of 28 nm. K4 NPs could be coated with hyaluronic acid (HA) modified with dopamine and folic acid via phenylboric acid on its surface to form HA-K4 NPs with particle size of about 120 nm. Under conditions mimicking tumor microenvironment in vitro, HA-K4 NPs could be transformed to small-sized K4 NPs in a responsive manner. In addition, the nebulization performance of K4 NPs and HA-K4 NPs showed good inhalability.

Then the thesis evaluated the mucus and tumor penetration abilities of K4 and HA-K4 NPs in vitro and in vivo and their cytotoxicity against Lewis lung carcinoma cells (LLCs) in vitro. Compared with small-size K4 NPs, HA-K4 NPs showed stronger mucus penetration and lung retention. In vitro and in vivo tumor penetration results showed that K4 NPs showed better tumor penetration at the initial stage, while HA-K4 NPs penetrate the tumor tissues in a delayed manner. In addition, both nanoparticles have strong sonodynamic transformation ability and could produce reactive oxygen species (ROS) under ultrasonic conditions. The cytotoxicity of K4 and HA-K4 NPs was further enhanced after ultrasound treatment, demonstrating a synergistic or additive effect of chemo-sonodynamic therapy on the cytotoxicity in vitro.

Next, the thesis investigated the pulmonary and tumor pharmacokinetics of K4 and HA-K4 NPs after intratracheal instillation to mice bearing orthotopic Lewis lung carcinoma tumors. It was found that the AUC values of SN38 and fluorescence intensity in lung tissues from instilled HA-K4 NPs were 43.7-99.7 folds of that by injected HA-K4 NPs, indicating that pulmonary delivery could significantly increase lung tissue exposure. Relative to small-size nanoparticles, the large-size counterparts exhibit superior ability of mucus penetration and lung retention, leading to an increase in lung bioavailability of SN38 by 58.5%. In addition, HA-K4 NPs could enhance the tumor accumulation via direct access or transcytosis of nanoparticles and diffusion of the released drug, and the large nanoparticles penetrate within tumor tissues after size-transformation, leading to increases in tumor bioavailability of sonosensitizer by 5.99-fold and SN38 by 1.99-fold compared with the small-size nanoparticles. HA-K4 NPs exhibits good safety profile, and the sustained and high levels of sonosensitizer and SN38 conferred in the tumor after pulmonary delivery triggered chemo-sonodynamic combination therapy, extending the survival time of mice bearing orthotopic Lewis lung carcinoma tumors from 15 days to 22 days, which achieved significant anti-tumor efficacy.

Finally, to explore the effect of macrophage phagocytosis on pulmonary delivery of nanoparticles, the thesis synthesized three polypeptide derivatives modified with different targeting groups, which self-assembled to form three nanocarriers, namely phenylboric acid modified ICP nanoparticles for tumor cell targeting, phenylboric acid and folic acid dual-modified FCP nanoparticles for enhanced tumor cell targeting and phenylboric acid and mannose dual-modified MCP nanoparticles for macrophage targeting. The particle size distribution, zeta potential, morphology and critical micellar concentration of the three nanoparticles were similar, and only the target groups affected the fate of the nanoparticles after pulmonary administration. Upon establishing a sodium alginate/PVA hydrogel 3D LLC and macrophage co-culture system, the competitive uptake of nanoparticles by cells was evaluated in vitro and the results showed that FCP NPs could be selectively uptake by tumor cells and MCP NPs could be selectively uptake by macrophages. Subsequently, in vivo efficacy evaluation in mice bearing orthotopic Lewis lung carcinoma tumors showed that there was no significant difference in tumor weight between MCP NPs and the control group, while ICP NPs inhibited tumor growth to a certain extent with an inhibition rate of 29.46%. The tumor inhibition rate of FCP NPs was 48.51%, markedly higher than the other two nanoparticles. The antitumor effect of MCP NPs was significantly weaker than that of FCP NPs and ICP NPs, indicating that targeting macrophages decreased the anti-tumor efficacy of pulmonary delivery of nanoparticles.

In summary, this thesis has investigated the effect of particle size and ligand modification of inhaled nanoparticles on the pulmonary and tumor fates. The present results demonstrated that macrophage phagocytosis may restrict the drug availability to tumor cells, reducing the anti-tumor efficacy of inhaled nanomedicine. In addition, an inhaled size-transformable nanosystem could not only restrict mucociliary clearance by immediate penetration through the mucus, but also retard absorption clearances by delaying the drug release and translocation of nanoparticles into the bloodstream and extrapulmonary organs, and improved the tumor accumulation and penetration through size transformation. Pulmonary delivery of such a size-transformable nanocarrier represented a promising means to co-deliver chemotherapeutics and sonosensitizers for noninvasive lung cancer management.