Doxorubicin Hydrochloride liposomal(Imunon)

“ 点击蓝字 关注我们 孙会敏 二级研究员，中国食品药品检定研究院化学药品检定所所长、首席专家。兼任国家市场监督管理局标准物质技术评审专家；中国药品监督管理研究会药品辅料与包材监督管理研究专委会主任委员；国家药典委员会第十一、十二届药用辅料与药包材专委会委员；农业部第七、八届兽药评价委员会委员；农业农村部兽药评审中心专家；中国合格评定国家认可委员会实验室技术委员会药品专业委员会委员；美国药典委员会药用辅料专委会委员；中国食品药品检定研究院第八、九届学术委员会委员；中国食品药品检定研究院第八、九届国家药品标准物质委员会委员；国家药品监督管理局保健食品技术规范专门委员会委员；国家药品监督管理局药品审评中心专家；国家药品监督管理局药包材审评专家；全国工商联医药业商会专家指导委员会委员；中国颗粒学会生物颗粒专业委员会委员；全国包装标准技术委员会第三届玻璃容器分技术委员会副主任委员；中国医药质量管理协会理事会常务理事；Asian J Pharm Sci 编委；《药物分析杂志》《分析测试学报》《中国医药工业杂志》等编委。长期从事药品检定、进口药品的标准复核、药用辅料和药包材检验、药用辅料和药包材质量控制和安全性评价、药包材注册审评指导原则制定、标准物质质量研究等工作。承担国家药典委员会课题和国家重大科技专项课题几十项，发表学术论文 130 余篇，编写专著 9 部，获国家发明专利 10 项。 赵霞 主任药师，药物分析专业博士，硕士生导师，现任中国食品药品检定研究院药用辅料和包装材料检定所副所长（主持工作）兼包装材料室主任。兼任第十二届国家药典委员会药包材专委会委员，国家药品监督管理局药用辅料质量研究与评价重点实验室主任，中国药品监督管理研究会药品辅料与包材监督管理研究专委会副主任委员，药物制剂及辅料研究与评价重点实验室学术委员会委员，CNAS 药品专业委员会委员，国家药品监督管理局药品审评中心外聘专家，北京市科学技术奖评审专家。专注于药用辅料检验、药包材的质量研究。主要从事药包材的检验、标准制定、质量研究工作。多年开展玻璃类、塑料类、橡胶类及金属类药包材与药物的相容性研究以及药品包装系统的密封完整性的评价工作。承担国家级课题多项。组织完成 130 个国家药包材标准的首次制修订工作、国家药监局玻璃专项调研与验证工作。完成国家药典委员会标准提高课题 20 余项，为《中华人民共和国药典》2025 年药包材新标准体系构建与起草的核心专家。获中国药学发展奖食品药品质量检测技术杰出青年学者奖、中国石油和化学工业联合会科技进步二等奖。已累计发表学术论文 100 余篇，参与编写专著 7 部，获国家专利 7 项。 刺激响应性药物载体及其材料的研究进展 PPS  张雪妍 #，王珏 #，王晓锋，贺刘莹，王会娟，杨锐，赵霞 *，孙会敏 ** （国家药品监督管理局药用辅料质量研究与评价重点实验室，中国食品药品检定研究院，北京 100050） [ 摘要 ] 刺激响应性药物载体因具有载药稳定性好、生物相容性好等特点而成为研究热点，通过各种内在或外在条件给予适当刺激，刺激响应性药物载体能达到药物靶点精准控制释放的目的。简介天然和人工合成的刺激响应性药物载体材料，内源性和外源性刺激响应性载体的作用机制及设计策略，刺激响应性药物载体及其材料的评价指标与技术，旨在为刺激响应性药物的研发、临床转化、评价监管提供参考。 随着医学的进步，传统给药系统在治疗疾病时的局限性日益突出。为了实现更好的靶向性及可控性，研究人员设计了刺激响应性药物递送系统（stimuli-responsive drug delivery systems）。刺激响应性药物递送系统是一种能够对体内外环境刺激如pH、温度、光、磁场等化学或物理信号以及酶等生化物质发生特定响应的药物递送系统。该类系统的研究始于 20 世纪后期，随着化学、材料学、生物学、医学等相关学科的发展研究不断深入，刺激信号种类日益增多，响应性材料的种类和功能不断增加，灵敏度也不断提高。目前刺激响应性药物递送系统可用于肿瘤治疗、炎症治疗、抗菌治疗 [1]、糖尿病管理 [2]、手术辅助等。 针对不同部位微环境的不同，已开发多种刺激响应性递送系统，与传统的递送系统相比，刺激响应性药物递送系统具有以下优势：1）靶向给药，响应特定的体内外刺激，实现药物在病灶部位的定向聚集和释放。2）定点定时定量释放，根据治疗需要，精确控制药物的释放速率和模式，提高药物的生物利用度和疗效。3）减少药物在非靶部位的释放，降低药物的全身毒性和副作用。4）改变药物的释放模式和靶向部位，减少耐药性的发生。2000 年美国食品药品监督管理局（Food and Drug Administration，FDA）批准了首款刺激响应性药物 Visudyne，用于光动力治疗的眼科疾病。2024 年 4 月，Lumisight也获得 FDA 批准，作为一种酶响应性光学成像剂，在乳房肿瘤切除术中作为辅助手段。 目前常见的刺激响应性药物载体主要包括：聚肽纳米材料、纳米纤维、纳米管、脂质体、聚合物胶束、嵌段共聚物 [3]、树枝状大分子 [4]、纳米水凝胶 [5]、金属有机框架 [6] 等。目前对刺激响应性载体材料的研究集中在人工合成材料和天然材料 [7]。人工合成材料包括聚丙烯酸树脂类、非离子表面活剂、聚酯类的高分子材料；天然材料包括壳聚糖、透明质酸、纤维素、明胶等。本文综述了天然和人工合成的刺激响应性药物载体材料响应性原理及应用实例，内源性和外源性刺激响应性载体作用机制、设计策略及部分进入临床和上市阶段的刺激响应性药物，刺激响应性药物载体及材料常用的评价指标与技术，旨在为未来刺激响应性药物的研发、临床转化、评价监管提供参考。 1 刺激响应性载体材料 刺激响应性药物递送载体的刺激响应性来源于材料的响应性。刺激响应性材料可分为人工合成材料和天然材料。除了满足传统辅料改善溶解度、提高生物利用度等作用，刺激响应性材料还能够主动与生物功能相互作用，感知身体内部或外部的生理状态，从而调节其构型及性能，达到靶向用药、降低副作用、个性化医疗等作用。 1.1 人工合成材料 人工合成的刺激响应性材料通常是通过化学合成方法制备的高分子材料。该种材料可通过化学合成精确调控其分子结构与性质，如相对分子质量、结构、官能团等，其性能可广泛调节，以满足多样化的应用需求。人工合成的刺激响应性材料常展现出卓越的化学稳定性和机械性能，长时间不降解，保持长期稳定性。部分用于合成刺激响应性载体的人工合成材料如表 1 所示。 1.1.1 聚丙烯酸衍生物 聚丙烯酸衍生物如聚丙烯 酸 [poly(acrylic acid)，PAA]、聚甲基丙烯酸 [poly(methacrylic acid)，PMAA]、聚丙烯酰胺[poly(acrylamide)，PAAM]、聚 N-异丙基丙烯酰胺[poly(N-isopropylacrylamide)，PNIPAAM] 等具有良好的生物相容性、稳定性、pH 和温度的敏感性。PAA 是由丙烯酸聚合而成，丙烯酸是由一个乙烯基直接连接到羧酸末端组成。丙烯酸中的羧基可通过氢键的形成和解离对 pH 变化做出响应，并通过静电相互作用携带正电荷的粒子。PAA 可进行羧基反应与其他功能团共轭，获得多功能的聚丙烯酸衍生物。弱酸性环境可质子化 PAA 或其衍生物的羧基和胺基，导致亲水部分收缩，溶酶体膜不稳定破裂，药物被释放到细胞质基质中，这被称为质子海绵效应 [25]。 杨龙花 [26] 通过原子转移自由基聚合技术，合 成 了 聚（ 甲 基 丙 烯 酸-N，N-二甲氨基乙酯）（PDMAEMA）， 再 与 苯 丙 氨 酰-赖 氨 酸（Phe-Lys）二肽结合，合成了两代线形-树枝状嵌段共聚物 PDMAEMA-b-dendr(Phe-Lys)n（n = 1, 2），PDMAEMA 中的叔胺基团 pKa 值约为 7。当 pH 值低于 pKa，PDMAEMA 的胺基被质子化，聚合物表现为亲水性，聚合物链之间的静电斥力增加，胶束结构变得疏松，从而加速药物的释放；而在 pH 值高于 pKa 时，胺基去质子化，聚合物表现为疏水性。这种性质使得 PDMAEMA 能够在不同的 pH 值下实现对药物的控制释放。 PNIPAAM 具有较低的低临界溶液温度（lower critical solution temperature，LCST），其在低温下高度水合并在水溶液中溶解良好，加热会导致其疏水性增加。聚 N-丙烯酰基甘氨酰胺具有上临界溶液温度（upper critical solution temperature，UCST），其在一定温度以下不溶，而在该温度以上则可溶。目前研究最多的热敏性 PAA 是具有 LCST 的聚合物即 PNIPAAM 的纳米颗粒。PNIPAAM 由疏水性的异丙基侧链 [-CH(CH3)2-] 和亲水性的酰胺基团（-CONH-）组成。当温度低于 LCST 时，PNIPAAM 中具有亲水性的酰胺基团与水分子之间形成氢键，从而展现出良好的水溶性；随着温度逐渐升高，PNIPAAM 由亲水性变为疏水性，其侧链会先发生坍塌，随后进一步聚集成球形结构。PNIPAAM 的 LCST 约 为 32 ℃， 低于人体正常体温 37℃，通过将其与亲水性聚合组分如 N，N-二甲基丙烯酰胺和丙烯酰胺 [27] 杂化，可将 PNIPAAM的 LCST 调节至更高的温度，以达到在体内运用的目的。 1.1.2 泊洛沙姆 泊洛沙姆又称普朗尼克，是一种聚（环氧乙烷）-聚（环氧丙烷）-聚（环氧乙烷）（PEO-PPO-PEO）三嵌段共聚物，由中心疏水聚（环氧丙烷）核心组成 , 两侧有亲水性聚（环氧乙烷）链。升温后，泊洛沙姆胶束内的疏水性聚环氧丙烷基团脱水发生溶胀，达到一定温度时，胶束之间开始互相接触并形成网络结构，最终形成凝胶，这一变化过程中最直接的表现是体系的黏度显著上升。泊洛沙姆家族中作为温敏凝胶材料最常用的是泊洛沙姆407（F127）和泊洛沙姆 188（F68），F127 相对分子质量为 9 840 ~ 14 600，其具有独特的反向热凝胶作用，低温时为液体，高温时为固体，在 17.5% ~ 30% 浓度范围内具有成胶性 , 且随浓度增大胶凝温度逐渐下降。F68 作为一种温度调节剂，2% ~ 10% 的 F68 加入到 F127 中可升高胶凝温度，F68 浓度越大，胶凝温度越高 [28]。Dang 等 [15] 设计了一种基于海藻酸钠和 F127 的温度响应性递送载体，用于甲氨蝶呤的按需释放。通过动态光散射测试，低温时F127 段膨胀，纳米粒子结构松弛；37℃下，F127段收缩，尺寸缩小了 36.71%，结构紧凑。体外释放研究发现，37℃下，甲氨蝶呤的释放速率会因 F127段的收缩而增加。泊罗沙姆的温敏性使其在温度刺激响应性载体中应用广泛。 1.1.3 聚酯类材料 可生物降解的聚乳酸（polylactic acid，PLA）、聚己内酯（polycaprolactone，PCL）、乳酸-羟基乙酸共聚物 [poly(lactic-co-glycolic acid)，PLGA] 等聚酯类材料，被广泛应用于刺激响应性载体的制备，可在聚酯类材料表面键合敏感基团，从而产生相应的响应性[21]。其中聚乙二醇（polyethylene glycol，PEG）可与聚丙交酯乙交酯形成 B-A-B 三嵌段共聚物 PLGA-PEG-PLG，溶于水后会形成一种具有核壳结构的球状胶束，除了键合其他敏感基团产生相应响应性，该共聚物自身有良好的温敏性。温度低于相转变温度时 , 疏水的 PLGA 内核和亲水性的 PEG 外壳自组装形成胶束呈现溶液状态；当温度上升至相转变温度时，PLGA 链之间的相互作用力增强并伴有 PEG 外壳脱水，胶束聚集增加，使聚合物溶液形成凝胶。聚合物发生溶胶-凝胶转变的温度可通过 PEG、PLGA 的比例来调控，当 PLGA-PEG-PLGA 共聚物中 PEG 与 PLGA 比值为 0.56 或更低以及该共聚物在溶质中浓度超过 15% 时，在37℃的环境下，该共聚物可实现温度响应型的溶胶-凝胶转变 [18]。 Ran 等 [19] 以 PLGA-PEG-PLGA 作为原始材料，负载黑磷纳米片自组装成水凝胶。通过动态光散射和流变学测试评估该水凝胶的温度响应性。该实验结果显示，在 25℃时，该水凝胶呈现溶液状态；当温度升至 37℃转变为凝胶状态，该水凝胶的储能模量、损耗模量及黏度均增加，这种转变归因于PLGA 嵌段的温度敏感性。基于上述特征，该水凝胶可设计应用于递送药物，在体内循环时其保持溶液状态，到达病变部位时，转变为凝胶状态，从而实现药物的可控释放。 1.2 天然材料 天然的刺激响应性材料主要来源于生物体内的天然高分子，如蛋白质、多糖和核酸等。与合成材料相比，天然材料及其衍生物因其具有生物降解性、生物相容性、天然丰度高等优势 [29] 而受到越来越多的关注，同时其有望解决传统给药过程中难以实现的水溶性靶向，实现更精确的靶向性、特异性分散和低毒性 [30-31]，为有效治疗各种疾病提供了新的选择。部分用于合成刺激响应性载体的天然材料如表2 所示。 1.2.1 多糖 壳聚糖（chitosan，CS）是天然甲壳素（从甲壳类动物中提取）脱乙酰化后的产物，是由 β-(1-4)糖苷键连接的葡萄糖胺和葡萄糖的 N-乙基酰胺基团组成的碱性多糖。在酸性条件下，壳聚糖的氨基被质子化，可通过静电相互作用、氢键和偶极相互作用与溶液中其他带负电的阴离子或聚阴离子发生溶胶-凝胶转化过程，具有优异的 pH 响应性 [45]。此外，壳聚糖可被溶菌酶和壳聚糖酶降解并转化为寡糖和单糖，促进药物在特定的目标区域释放，从而更好地发挥药效 [46]。Zhao 等 [47] 利用静电自组装将氧化石墨烯纳米颗粒与壳聚糖结合，并基于静电相互作用涂覆二甲基顺丁烯酸酐改性的壳聚糖，设计了一种电荷逆转的药物递送系统。该药物递送系统通过引入 pH 响应性、可切割的 2，3-二甲基马来酸酐，带负电荷的纳米复合材料处于肿瘤细胞外基质中可逆转为正电荷，从而提高了多柔比星被肿瘤细胞摄取内化的效率，可用于潜在的抗癌治疗。 海藻酸盐是从褐藻中提取的一种天然阴离子多糖，具有优异的生物降解性和生物相容性，其主链分子由 α-L- 乌糖酸和 β-D- 甘露糖醛酸通过 β-1，4-葡萄糖苷键连接而成。海藻酸盐含有大量的-COO-基团，在水溶液中可表现出聚阴离子行为，且具有黏附性，可作为治疗黏膜组织的药物载体 [48]。在酸性条件下 -COO- 可转化为 -COOH，电离度和亲水性降低，分子链收缩；当 pH 值升高时 -COOH基团持续解离，亲水性增加，分子链伸展，显示出明显的 pH 响应性。由于海藻酸盐中 -COO- 的分子间相互作用，可通过与阳离子的离子交联形成水凝胶，用于药物输送。Sarika 等 [49] 合成了一种阳离子明胶和海藻酸钠的 pH 响应性聚电解质复合纳米颗粒。酸性环境中，阳离子明胶的正电荷增加，增强了与海藻酸钠的静电相互作用，该复合物稳定性增加；中性或碱性环境中，羧基的质子化减少，阳离子明胶的正电荷减少，与海藻酸钠的静电相互作用减弱，该复合物的稳定性降低。该复合物用于姜黄素递送时，在 pH 7.4 时约 65% 的姜黄素释放，在pH 5.0 时 87% 的姜黄素释放，显示出较好的 pH 响应性。 透明质酸（hyaluronic acid，HA）是一种由 D-葡萄糖醛酸和 D-N-乙酰氨基葡萄糖交替单元组成的天然黏多糖，并通过 β-1，3 和 β-1，4 糖苷键连接。HA 广泛存在于脊椎动物的表皮和结缔组织中，是细胞外基质的主要成分，具有良好的生物相容性、生物降解性和非免疫原性。HA 可通过与细胞表面受体 CD44 的特异性结合来调节细胞运动、黏附、分化和聚集等生理功能，此外，与 CD44 的结合使HA 具有靶向肿瘤细胞的能力 [50]。在疾病部位（如肿瘤组织）高表达的透明质酸酶（HAase）可以降解HA以控制药物释放。Hu等 [51]以HAase为引发剂，将近红外激光敏感的一氧化氮供体引入 HA 纳米颗粒中，以多柔比星为化疗药，吲哚菁绿为光热剂制备纳米颗粒（IDDHN-Dox-ICG）。在近红外光照射下，该纳米颗粒借助 HAase 介导的酶降解一氧化氮供体壳和激光增强的一氧化氮释放触发的强热疗效应，实现了体内外协同的深穿透作用。 纤维素是地球上最丰富、最广泛且成本最低的天然多糖聚合物，由通过 β-1，4-葡萄糖苷键连接的葡萄糖单元组成，其生物相容性好、高模量、低密度、改性潜力大。羧甲基纤维素是一种水溶性的阴离子纤维素衍生物，其分子链中含有大量的羧基，可通过多价金属阳离子的交联形成凝胶 [52]。由于羧基的存在，该凝胶适合作为 pH 触发的药物载体。羟丙基纤维素是一种热响应性衍生物，在水溶液中表现出41℃的LCST，当处于接近LCST的水溶液中，随着相应的反应变化，其表现出明显的水合 - 脱水转变 [39]。 1.2.2 蛋白质 明胶是从动物胶原中提取的一种 常 用 的 生 物 聚 合 物， 具 有 由 18 种氨基酸交联而 成 直 链。明 胶 可 被 基 质 金 属 蛋 白 酶（matrix metalloproteinase，MMP）降解为低分子多肽，从而引起药物释放，可用于制备酶响应性药物载体 [43]。此外，氨基的质子化和去质子化引起的电荷变化以及羟基和羧基共同作用引起的亲水性变化，使明胶在肿瘤酸性环境中药物释放增强。Ruan 等 [42] 设计了一种 pH 和酶响应型纳米复合材料（G-AuNPs-DOX-PEG），由明胶纳米粒子涂层、结合多柔比星的金纳米粒子和长链聚乙二醇组成，肿瘤中高度表达的 MMP-2 可降解该明胶纳米粒子，其尺寸从186.5 nm减至59.3 nm，进而增强了多柔比星的释放。丝素蛋白（silk fibroin，SF）主要来源于蚕茧和蜘蛛丝，具有不溶性 β- 折叠晶体结构，含有通过二硫键共价结合的重链和轻链。SF 富含易于修饰的官能团，对官能团的修饰可调节自组装过程和实现药物有效负载与释放。作为一种天然蛋白质，SF 具有良好的生物相容性和生物降解性，引起的免疫反应和毒性反应极小 [53]。通过调整制备条件和加工方法对 SF 的结构和性质进行控制，以满足特定的药物输送要求。 2 刺激响应性载体 刺激响应性载体是指由刺激响应性材料构成的能够根据环境变化（如温度、pH、光照、磁场等）而发生特定物理或化学变化的载体。根据刺激类型，刺激响应性载体可分为内源刺激响应性载体和外源刺激响应性载体。 2.1 内源刺激响应性载体 内源刺激响应性载体会对体内的刺激如 pH、氧化还原、蛋白酶等产生响应，从而调节药物的释放。病变部位环境的改变会引起递送系统的化学或物理变化，从而实现有效载荷的释放。pH 响应性载体利用人体内不同区域间的显著pH 差异，实现药物的局部精准递送。人体正常生理环境 pH 为 7.4。感染、炎症和肿瘤的病理部位的pH 与正常组织不同，病变区域的微环境显示出低pH。口腔内 pH 为 6.6 ~ 7.1，胃内 pH 为 1.0 ~ 3.0，呈强酸性；小肠 pH 为 4.8~8.2，结肠 pH 为 7.0~7.5，接近中性；肿瘤细胞外环境的 pH 为 6.5 ~ 7.0，略低于正常组织；早期内体的 pH 为 6.0 ~ 6.5，次级内体的 pH 更低（4.5 ~ 5.0），随着内体的成熟，其酸性逐渐增强。当 pH 发生变化时，刺激响应性载体可通过膨胀、收缩、裂解、降解、质子化和去质子化官能团响应并释放药物。开发该类载体主要采用 2种策略，第 1 种是使用能够进行 pH 依赖性质子化的聚阳离子或聚阴离子。常用的阴离子 pH 响应单元有丙烯酸、甲基丙烯酸，阳离子 pH 响应单元有甲基丙烯酸二甲氨基乙酯、甲基丙烯酸二乙氨基乙酯、丙烯酰胺 [54]，已应用于各种类型的纳米材料，如脂质纳米颗粒、脂质体、两亲性聚合物纳米颗粒、纳米疫苗以及纳米凝胶。第2种策略是将pH响应性、可切割的酸敏感键结合到治疗载体材料中 [34]，主要应用于酸敏感聚合物、金属有机框架、杂化纳米颗粒和交联聚合物。 Du 等 [55] 开发了一种基于不对称的聚（乙二醇）- 聚（2，4，6-三甲氧基苯亚甲基 -1，1，1-三（羟甲基）乙烷甲基丙烯酸酯）-聚（丙烯酸）（PEG-PTTMA- PAA）三嵌段共聚物。该聚合物在 pH 7.4 时很稳定，但在酸性环境（pH 4.0~5.0）下，聚合物中的缩醛基团易发生快速水解，导致聚合物体膨胀并最终解体；同时丙烯酸部分在酸性环境下离子化状态发生变化，使得 PAA 从亲水性转变为疏水性，并从随机卷曲结构转变为 α- 螺旋结构，从而导致聚合物体的破坏，实现药物的释放。体外释放研究发现，盐酸多柔比星从该聚合物体中的释放过程高度依赖pH的变化，在 pH 7.4 时释放率较低，而在 pH 5.0 时释放率显著增加。 酶在许多生理和生物过程中均扮演至关重要的角色，在部分病变组织中酶的表达也会出现异常。肿瘤细胞中有多种酶的过表达，如基质金属蛋白酶、透明质酸酶、组织蛋白酶 B、吲哚胺 2，3-双加氧酶 1、成纤维细胞活化蛋白-α 和 γ-谷氨酰转肽酶 [56]。酶响应性载体通过酶的作用，可转变为解体结构，导致尺寸缩小，从而促进其渗透至病变和受损组织，有助于实现药物的精准释放，可应用于癌症诊断、预后评估及免疫治疗。设计酶响应性载体，最常见的策略是利用与免疫治疗药物相连的肽序列，在肿瘤部位由特定酶特异性切割，从而实现免疫治疗剂的精准释放。 骆飞翔 [57] 制备了一种 MMP-9 响应性纳米颗粒，其疏水性核用于递送他克莫司，亲水性外壳含有甲氧基聚乙二醇和 MMP-9 的底物多肽片段，当其暴露于移植肝脏内过表达的 MMP-9 酶时，MMP-9 酶会特异性切割 MMP-9 底物多肽片段，导致甲氧基聚乙二醇从纳米颗粒上脱落。该纳米颗粒从纳米级胶束转变为微米级聚集体，这种结构变化促进了他克莫司的释放，从而在移植肝脏内实现了药物的局部高浓度，减少了其他器官的药物分布，降低了系统性副作用。 谷胱甘肽（glutathione，GSH）是一种广泛存在于生物细胞内的重要物质，其在细胞内区室（细胞质、线粒体和细胞核）含量比细胞外液高 2~3 个数量级。肿瘤细胞通过异常的线粒体呼吸链和烟酰胺腺嘌呤二核苷酸磷酸氧化酶途径，产生比健康细胞高出 100倍的活性氧（reactive oxygen species，ROS），有助于肿瘤的发生和发展。ROS 在体内一般通过 GSH 进行调节，减少过量的 ROS 并维持组织的氧化还原状态。因此，肿瘤组织中的 GSH 浓度比健康组织高4 ~ 7 倍，有利于 GSH 在肿瘤细胞中特异性触发药物释放。设计氧化还原性载体常采用键合可还原键的策略，特别是二硒键（-Se-Se-）[58]、二硫键（-S-S-）[59-60]、硫代酰基键（-C=S-）[61]、半胱氨酸 - 丝氨酸 - 丝氨酸（CSS）键和硫酮键 [61]，用于连接和递送各种免疫治疗负载物，包括抗原、抗体、抑制剂、激动剂和 mRNA 疫苗。 Trastuzumab-DM1（T-DM1）[62] 是氧化还原响应性药物，已于 2013 年在美国、欧洲获得批准，用于治疗人表皮生长因子受体 2（human epidermal growth factor receptor 2，HER2）阳性转移性乳腺癌。2020 年 1 月 T-DM1 在中国获批，用于 HER2 阳性早期乳腺癌的辅助治疗。T-DM1 由抗 HER2 单克隆抗体曲妥珠单抗与微管抑制剂 DM1 通过稳定的硫醚键连接而成。在肿瘤细胞内，T-DM1 的抗体部分被溶酶体蛋白酶降解，释放出 DM1。T-DM1 在血液循环中保持稳定，其不可裂解的硫醚连接体结构有助于保持 DM1 的惰性状态，避免过早地释放 DM1，保证 DM1 在靶细胞内精准释放，从而减少了系统性毒性。 2.2 外源刺激响应性载体 外源刺激响应性载体是一类可在体外特定刺激下进行调控的载体。通过对外部刺激的持续时间和强度进行精确调控，可实现从时空维度对该类载体药物释放曲线的精确控制。目前针对该类载体的研究涉及热、光、超声、磁和电等多种刺激形式。 温度响应性载体是控制药物释放最方便、最有效的递送载体之一。炎症和肿瘤等病灶处的温度常高于正常组织 [63]，纳米颗粒可利用肿瘤组织与正常组织之间的温差，从而被激活以强化其在肿瘤中的药物释放能力。据此原理可开发温敏凝胶，低于其LCST 时，温敏凝胶呈现为溶胶状态 [64]；当温度升高至 LCST 或接近体温时，高分子链之间的相互作用增强，导致凝胶形成。此外，当肿瘤细胞所处的环境温度达到 42.5℃以上时，肿瘤细胞的生存率快速下降；正常细胞和组织可耐受较高的温度，因此高温可选择性地杀死肿瘤细胞而不会对正常组织造成过大损害。因此，采用温敏性递送载体，并施以外部温度刺激，高温可部分杀死肿瘤细胞，同时载体包裹药物可在肿瘤部位精准释放，以达到更好的疗效。对于温度的调控有多种加热策略，如超声波、近红外光和磁场。设计温度响应载体常用的温敏材料包括泊洛沙姆类、聚 N-异丙基丙烯酰胺 [65-66]、壳聚糖。温度响应性载体可用于鼻腔、眼部、阴部给药。 ThermoDox [67] 是一种新型抗癌药物，其温敏特性主要源于脂质体的组成成分产生。正常体温下，ThermoDox 的脂质体外壳稳定，药物不会释放；当通过射频消融技术、红外加热等方式将肝脏温度升高至 42℃并维持一段时间后，脂质体外壳的物理结构发生改变，形成多个微小开口，促使抗癌药物多柔比星快速释放，实现将高剂量药物直接输送至肝癌组织的目的并降低对正常组织的毒性影响。尽管ThermoDox在肝癌治疗方面已进行了Ⅲ期临床试验，但该药物并未成功上市。2021 年 2 月，Celsion 公司鉴于 ThermoDox 针对肝癌的Ⅲ期临床研究未取得预期效果，决定终止其临床研究进程。磁响应性载体在外部磁场作用下被引导并集中于身体特定区域，从而实现药物的靶点释放 [68]。磁性材料（包括超顺磁性纳米颗粒、磁铁矿、铁铂合金和钴铁合金）已被广泛研究，作为磁驱动靶向、磁激活远程药物输送和磁共振成像 [69] 的平台。靶向免疫治疗输送中最常见的磁响应材料是超顺磁性氧化铁纳米粒子，通常与聚合物、脂质或蛋白质递送系统相结合赋予其磁性。 NanoTherm [70] 是一种基于铁氧体纳米材料的磁响应性药物，于 2011 年在欧盟获得批准，临床上用于脑胶质瘤的局部颅内磁热治疗。NanoTherm 由悬浮于铁磁流体中的磁性纳米粒子组成，可直接注射至肿瘤组织内部。该磁性纳米粒子在交变磁场作用下被激活，持续产生热量，从而实现对肿瘤细胞的热损伤；也可与其他治疗方式相结合，以增强对脑胶质瘤的治疗效果。 光响应性载体由含有光敏感基团的材料制备而成。在特定波长光照下，光敏感基团吸收光子能量引发光化学反应，改变药物递送系统组成或构象，从而达到控制药物释放的目的。目前 , 根据光响应机制的不同 , 光敏感基团有 4 种：1）光致断裂型，如邻硝基苄基氧和香豆素基团，此类基团能够吸收光子的能量，促使特定共价键的断裂。2）光致异构型，如偶氮苯和螺吡喃，光照刺激使此类基团内部的双键进行顺反异构化反应或环闭合的反应。3）光致重排型，如 2-重氮-1，2-萘醌基团，其吸收特定光子能量能够进行 Wolff 重排反应。4）光致交联光型，如香豆素、肉桂酰和蒽，光照刺激下 , 香豆素或肉桂酰进行 [2 + 2] 环加成反应 , 蒽进行 [4 + 4] 的环加成反应形成二聚体。常用的近红外光可通过加热光热剂触发热敏感载体外的药物释放。近红外响应性的载体材料主要有纳米金 [71]、吲哚菁绿等。紫外 - 可见光响应性一般通过引入光活性基团来实现，如偶氮苯 [72]、肉桂酰、螺旋苯并吡喃或三苯基甲烷。此外，光敏生物材料主要包括抗体-siRNA 偶联物 [73] 和聚酰胺树状大分子 [74]（对紫外线光作出反应）以及聚合物纳米粒子和上转换纳米粒子 [75]（对近红外光作出反应），这些材料具备物理化学性质可调节的特点，表现出高度模块化的光敏化学性质。 Visudyne [76] 是用于治疗湿性年龄相关性黄斑变性的光响应性药物，通过静脉注射进入血液循环，并与脂蛋白结合，进而聚集于黄斑中央处的新生血管。在（689±3）nm 的非致热红光源照射下，Visudyne的卟啉基团在有氧环境下发生光化学反应，产生活性氧自由基，损伤局部新生血管内皮细胞，帮助血管闭合。Visudyne 疗效明确，对正常视网膜组织损伤小，且可重复治疗，目前已获得美国 FDA的批准，也已在日本上市。 Nanobiotix 公司开发的 NBTXR3 [77] 由氧化铪纳米粒子构成，是一种光响应性药物。在头颈部鳞状细胞癌治疗中 NBTXR3 具有显著疗效，客观缓解率高达 81.8%。目前该药在中国和美国均已进入Ⅲ期临床试验阶段。NBTXR3 是一种无机结晶的氧化铪纳米粒子水悬液，其原子序数高（Z = 72）、电子密度大，在无X射线时该纳米粒子呈惰性“关闭”状态，在其被 X 射线激活时则呈“开启”状态，并显著增强放射治疗的疗效。 AuroShell [78] 纳米粒子是用于前列腺癌的金-硅纳米壳疗法，目前已应用于临床试验。该纳米粒子通过静脉注射递送，并在肿瘤部位积累。利用近红外激光照射肿瘤，该纳米粒子吸收光能并转化为热能，从而破坏肿瘤组织及其供应血管。AuroShell 的光响应性来源于其金属壳在近红外区域具有强烈的消光系数，能够有效吸收光能并转化为热能。此外，还有超声波响应性载体和电响应性载体。超声波响应性载体能够接收并响应超声波信号，实现特定的功能，因而在医学成像和无损检测等领域得到广泛应用。电响应性载体能够感应电场变化，借助导电聚合物对于电脉冲的响应特性，实现药物在靶部位的按需释放 [79]。 2.3 刺激响应性载体小结 内源刺激响应性载体的优势体现在其能够借助体内固有的刺激因素调控药物的释放过程，从而实现对病变区域的精准靶向治疗。该方法减少了外部干预，降低了对机体的创伤性，更适用于个性化医疗策略的实施。然而，内源刺激响应性载体在体外精准控制其于体内的释药行为较为困难，且由于体内刺激因素并非全是疾病所特有，可能导致药物在非病灶部位的意外释放。此外，不同肿瘤类型、生理阶段及个体差异均会限制内源刺激响应性载体的临床应用。目前针对 pH 响应性和酶响应性药物递送载体研究较多，但难以精确控制病灶处的药物释放，可重复性差，进入到临床阶段的相关药物较少。 外源刺激响应性载体的显著优势是具有远程可控性。目前，上市的大多数刺激响应性药物均为外源性响应型。药物载体可通过外部信号如温度、光、磁、超声等触发药物释放，为疾病的精准治疗提供可能。外源刺激响应性载体的缺点在于实施外源性刺激的时空位置难以精确定位，需要引入指示定位功能的部件，使得载体设计更为复杂；同时，外源性刺激源的组织穿透深度也限制了其应用。 ThermoDox 曾作为一款极具潜力的温度响应性药物用于肿瘤治疗，在Ⅱ期临床试验中期其治疗效果表现良好。然而，Ⅲ期临床试验 ThermoDox 未能取得预期成功，主要原因在于射频消融技术对肿瘤的杀伤范围有限。在 ThermoDox 的Ⅲ期临床试验中，选取的肿瘤大小为 3 ~ 7 cm，需要采用多点叠加射频消融技术。然而，ThermoDox 在相变温度时释放速度最快，超过相变温度释放速度变慢；同时在轻微高温下长时间加热或在更高温度下短时间加热均会引发血管分流，进而导致转运至此区域的 ThermoDox 减少，阻碍药物输送。基于此，Regenold 等 [80] 提出关于 ThermoDox 在体内分布和释放的研究理论，用以解释其临床试验失败的原因。该研究将肿瘤分成了 3 个区域，最中间的区域是消融区，直接通过射频消融术杀死肿瘤细胞；最外层是轻度加热区域，在此温度环境下，ThermoDox 能够大量输送至该区域，药物释放快且完全，进而有效杀死外围的肿瘤细胞；中间区域的温度介于外层和消融区域之间，这一温度不足以杀死肿瘤细胞，且存在血管分流，致使此区域的 ThermoDox 输送减少，过高的温度也使药物释放量降低。该中间区域的存在导致了ThermoDox 的临床疗效不理想。因此，在外源刺激响应性载体设计时，需要综合考虑更全面的因素，例如肿瘤的大小、形状以及肿瘤内部的温度分布。同时，ThermoDox 的配方和释放机制也需改进，以便在更广的温度范围内能保持高效的药物释放，从而提高治疗效果。 3 刺激响应性载体及其材料的评价技术及指标 3.1 刺激响应性载体材料的评价 刺激响应性载体材料的评价，主要是针对其理化性质与表征的评价。只有确保所使用的材料的质量达到标准，才能制造出符合规定要求的药物递送载体。具体评价指标主要包括高分子链结构、聚集态结构、相对分子质量、溶解性与溶胀性、黏度、吸湿性、降解与老化机制等。 高分子链结构研究材料的基本组成并预测其可能的反应性，核磁共振 [81]、红外光谱 [82] 等技术可用于分析高分子链的结构和化学组成。聚集态结构是研究材料内部的微观排列方式及分子间的相互作用，对于了解材料的物理化学性质和优化材料性能具有重要意义。X 射线衍射和差示扫描量热法（differential scanning calorimetry，DSC）[83] 可用来研究高分子材料的晶体结构。 相对分子质量及其分布的测定对于评估材料的加工性能和最终产品的稳定性至关重要，即使相同种类不同相对分子质量的聚合物也展现出不同的物理化学特性，所以对于材料的相对分子质量及其分布须做出明确规定。凝胶渗透色谱是测定材料相对分子质量及其分布的主要技术 [84]。 溶解性与溶胀性的研究是指通过将材料置于不同溶剂中，观察其溶解或溶胀行为来评估溶解性和溶胀性，有助于确定材料在不同溶剂中的行为。黏度的测量可以反映材料在加工过程中的流动特性，流变学测试可用来测量材料的黏度和流动特性，包括剪切应力与剪切速度的关系 [85]。吸湿性分析对于预测材料在储存和使用过程中的稳定性具有重要意义，通过热重分析可测定材料中水分的含量，从而评估吸湿性 [86]。 降解与老化机制的研究有助于了解材料在体内或环境中的长期稳定性 [87]，热重分析和 DSC 法可用来研究材料的热稳定性和降解机制。 3.2 刺激响应性载体的评价 作为纳米递送系统的一种，刺激响应性载体的评估方法和指标与大多数纳米载体基本一致，主要围绕物理化学特性、药物包封率和载药量及药物体外释放 3 个维度的评价指标及评价方法（见表 3）。 分子大小及其分布的控制对于刺激响应性载体至关重要。NanoTherm [70] 纳米颗粒由氧化铁核和氨基硅烷涂层构成。这些纳米颗粒悬浮于水中，形成胶态分散体。纳米颗粒的结构与稳定性受其尺寸与形态的影响。不同的合成技术可能导致尺寸相近而磁性各异的纳米颗粒。最小化尺寸分布范围对于精确控制关键特性至关重要，这直接决定了治疗效果的一致性。 针对特定刺激响应性载体，须对其特殊性能指标进行评估。大多数温度响应性载体是基于 LCST设计的，故评价其 LCST 对于温度响应性载体尤为重要。光谱法是最常用的一种 LCST 的测定方法，通过测量溶液在不同温度下的吸光度或透光率变化来确定 LCST，具体将为溶液透光率降至一半时的温度定义为 LCST。此外，透射电子显微镜可通过观察在不同温度下聚合物溶液的形态变化确定LCST；DSC 法通过在 LCST 附近检测 DSC 曲线上的吸热峰或放热峰确定 LCST；温度扫描测试法则通过测定载体黏度变化确定 LCST。Zong 等 [94]分别采用光谱法和 DSC 法测定 LCST。采用光谱法时，将水凝胶样品置于不同温度（24 ~ 42℃）下浸泡 5 min，以模拟体温变化对其透明度的影响。该研究结果显示，随着温度的升高，水凝胶的透光率下降，表明水凝胶从亲水状态转变为疏水状态，发生了相变；在 15 ~ 60℃的温度范围内进行 DSC 分析，最终确定了纤维素纳米纤维-聚 N-异丙基丙烯酰胺（CNF-PNIPAM）水凝胶的 LCST 为 39.78℃，高于纯 PNIPAM 水凝胶的 LCST（约 32℃）。 基 于 光 热 效 应 设 计 的 光 响 应 性 载 体， 如AuroShell 纳米粒，其功能和性能的验证需通过光热效应检测来实现。光热效应是指载体在吸收光能后，通过非辐射跃迁将光能转化为热能的过程。通过测量载体在特定波长下，温度随着光照强度及光照时间的变化，采用热成像相机监测样品表面温度，以记录样品温度的动态变化，评估其热转换效率 [97]。药物体外释放度指药物在特定条件下从制剂中释放的速度和程度，是纳米药物质量控制的关键指标之一。通常情况下，刺激响应性药物载体在装载药物后，会通过体外释放实验以评估其对特定刺激的响应性。体外释放的主要测定方法包括取样分离法、透析法、流通池法等 [98]。若刺激响应性药物载体在药物释放前后的粒径、形态或其他性质有明显变化，也可通过对药物释放前后的载体进行粒径、形态或其他性质进行分析，辅助证明载体对特定刺激的响应性释放。 4 结语与展望 虽然刺激响应性药物载体和材料在医药研究中具有重要的应用前景，国内对这一领域的研究投入和关注也较高，但目前的研究多处于理论研究阶段，导致其在药物开发和应用方面存在一定的滞后性。此外，一些刺激响应性药物载体在动物模型实验中表现出较好的靶向效果，能够精准地将药物递送至病变部位，但在进入临床试验阶段时，却常常出现脱靶效应，从而影响治疗效果。从基础研究到临床应用的转化过程中，载体的设计和功能还需进一步优化。同时，大分子物质的代谢物在研究中往往未得到足够重视，可能导致对药物安全性评估的不全面，进而影响药物的临床应用。因此，加强对大分子物质代谢物的研究，确保药物的安全性和有效性，是当前该类药物研究中亟待解决的问题。 国内外关于刺激响应性药物载体及其材料的评价指标研究相对较少，尚未形成完善的评价体系，这在一定程度上限制了相关药物的市场准入速度。目前关于刺激响应性药物载体的结构性能表征及体外释放性能的研究逐渐趋于成熟，但在细胞摄取和毒性评估、体内分布和稳定性测试等方面的评价指标研究相对较少。为确保上述药物载体在临床应用中的有效性和安全性，进一步深入研究其在细胞水平和整体生物体中的行为和稳定性至关重要。 参考文献： [1] Zhang J, Tang W, Zhang X, et al. 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[98]  杨子毅 , 盛利娟 , 朱霖 , 等 . 纳米药物体外溶出测定方法研究进展 [J]. 医药导报, 2022, 41(11): 1615-1621. 美编排版：史鑫宇 感谢您阅读《药学进展》微信平台原创好文，也欢迎各位读者转载、引用。本文选自《药学进展》2025年第 2 期。 《药学进展》杂志由国家教育部主管、中国药科大学和中国药学会共同主办，中国科技核心期刊（中国科技论文统计源期刊）。刊物以反映药学科研领域的新方法、新成果、新进展、新趋势为宗旨，以综述、评述、行业发展报告为特色，以药学学科进展、技术进展、新药研发各环节技术信息为重点，是一本专注于医药科技前沿与产业动态的专业媒体。 《药学进展》注重内容策划、加强组稿约稿、深度挖掘、分析药学信息资源、在药学学科进展、科研思路方法、靶点机制探讨、新药研发报告、临床用药分析、国际医药前沿等方面初具特色；特别是医药信息内容以科学前沿与国家战略需求相合，更加突出前瞻性、权威性、时效性、新颖性、系统性、实战性。根据最新统计数据，刊物篇均下载率连续三年蝉联我国医药期刊榜首，复合影响因子1.216，具有较高的影响力。 《药学进展》编委会由国家重大专项化学药总师陈凯先院士担任主编，编委由新药研发技术链政府监管部门、高校科研院所、制药企业、临床医院、CRO、金融资本及知识产权相关机构近两百位极具影响力的专家组成。 联系《药学进展》↓↓↓ 编辑部官网：pps.cpu.edu.cn； 邮箱：yxjz@163.com； 电话：025-83271227。 欢迎投稿、订阅！ 往期推荐 聚焦“兴药为民·2023生物医药创新融合发展大会”“兴药为民·2023生物医药创新融合发展大会”盛大启幕！院士专家齐聚杭城，绘就生物医药前沿赛道新蓝图“兴药强刊”青年学者论坛暨《药学进展》第二届青年编委会议成功召开“兴药为民·2023生物医药创新融合发展大会”路演专场圆满收官！校企合作新旅程已启航 我知道你在看哟

Advances Non-Viral DNA-Mediated Immunotherapy and Next-Generation Vaccine Programs with Multiple Near-Term Milestones Supported by a Strong Balance Sheet Conference Call Begins Today at 11:00 a.m. EDT LAWRENCEVILLE, N.J., Aug. 10, 2023 (GLOBE NEWSWIRE) -- IMUNON, Inc. (NASDAQ: IMNN), a clinical-stage drug-development company focused on developing non-viral DNA-mediated immuno-oncology therapies and next-generation vaccines, today announced financial results for the three and six months ended June 30, 2023. The Company also provided an update on its clinical development programs with IMNN-001 (formerly GEN-1), a DNA-based interleukin-12 (IL-12) immunotherapy in Phase 2 clinical development for the treatment of advanced-stage ovarian cancer, and on its PlaCCine modality, a proprietary mono- or multi-cistronic non-viral and synthetic DNA technology for the expression of pathogen antigens, being evaluated in preclinical studies for the development of next-generation vaccines. Highlights of the second quarter of 2023 and recent weeks include: On track to submit an Investigational New Drug (IND) application in the first quarter of 2024 for a proposed Phase 1/2 program with a seasonal COVID-19 booster vaccine, following positive pre-IND feedback from the U.S. Food and Drug Administration (FDA) Reported data suggesting PlaCCine vaccines elicit robust and more durable T-cell responses than commercial mRNA vaccines, signaling that these vaccines may provide greater protection against reinfection, hospitalization or death Unveiled new current Good Manufacturing Practices (cGMP) clinical materials production facility to support R&D efficiencies and lower costs for infectious disease and cancer vaccines, and non-viral DNA-based immune-oncology therapies Reported cash and cash equivalents of $24.1 million as of June 30, 2023, which along with planned sales of State of New Jersey net operating losses (NOLs) is expected to fund operations through 2024 “We have now successfully de-risked our PlaCCine modality preclinically across many pathogens of interest by demonstrating in animal models the immunogenicity and safety of our vaccines. We have generated compelling data in SARS-CoV-2 and with IMNN-101, a next-generation COVID-19 seasonal booster, and expect to be in the clinic in first quarter of 2024. In addition, we have generated excellent immunological responses for vaccines against other pathogens of concern including monkeypox, flu and arenaviruses. Our DNA vaccines are well positioned to become the next generation of vaccines and I am excited about their potential with the demonstration preclinically of durability of IgG antibody response, without a booster dose, and higher T-cell activation than mRNA vaccines. Another significant advantage over commercial mRNA vaccines is the demonstration of more than 12 months stability at standard refrigerated temperature of 4°C,” said Dr. Corinne Le Goff, IMUNON’s President and Chief Executive Officer. “Based on positive FDA feedback in July from a pre-IND meeting for our seasonal COVID-19 booster vaccine, we are on track to submit an IND application in the first quarter of 2024. The FDA confirmed in a written response that our plug-and-play strategy for our platform approach was acceptable. This confirms the flexibility and versatility of our platform, which allows for the rapid production and development of any vaccine simply by changing the antigen coding cassette. In addition, we expect to announce our next pathogen target for our PlaCCine modality in the coming weeks.” Dr. Le Goff continued, “In June we unveiled our new pilot manufacturing capability for DNA plasmids and nanoparticle delivery systems. Our scientists are now able to select any protein from the human or pathogen proteomes to be engineered. Our existing labs also have the ability to conduct testing and to run experiments in a variety of animal disease models. These internal capabilities will allow us to control both the costs and the process. The objective of our vaccine program is to establish the platform’s safety and efficacy in various Phase 1 studies, and then seek to license this powerful technology and/or establish non-dilutive partnerships to develop vaccines for pathogens of interest,” she added. “We expect to reach several value-creating milestones over the next six to 18 months. Among them is reporting additional interim data on IMNN-001 from our OVATION 2 Study and the combination study with bevacizumab in advanced ovarian cancer, reporting topline data from the OVATION 2 Study, filing the IND for our SARS-CoV-2 vaccine and announcing proof-of-concept vaccine data for our next pathogen target. We intend to discuss all of this and more during a virtual R&D Day event we are planning for this fall,” Dr. Le Goff concluded. RECENT DEVELOPMENTS PlaCCine: Developing the Prophylactic Vaccines of the Future Publication of Preclinical Data with PlaCCine DNA-based Vaccines Modality Available Online on bioRxiv. In August 2023 the Company announced that a manuscript titled “Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer” is available on the preprint server bioRxiv [here]. The study used IMUNON’s proprietary formulation against the spike proteins from two SARS-CoV-2 variants, both alone and in combination. These results add to the growing body of preclinical data confirming the efficacy and superior desirable features of IMUNON’s PlaCCine vaccine modality. Data from the study show: IMUNON’s proprietary formulation of functionalized polymer protected DNA from degradation, while the combination with an adjuvant led to an increase in protein expression DNA formulated with PlaCCine resulted in a DNA vaccine product that was stable for up to one year at 4°C DNA formulated in PlaCCine resulted in the induction of spike-specific neutralizing antibodies and cytotoxic T cells In the in vivo challenge model, the vaccine-induced immune response was capable of suppressing viral replication Multiple inserts can be cloned into the PlaCCine backbone (a plug-and-play strategy), therefore allowing for an immune response with broader protection Presentation at the 2023 Viruses and Cells – Gordon Research Conference Describes Compelling Preclinical Data ShowingContinued Durability of Response Over14 Months with Humoral Immune Response Increasing Over Time. In May 2023 Khursheed Anwer, Ph.D., IMUNON’s Executive Vice President and Chief Science Officer, presented new PlaCCine preclinical data at the 2023 Viruses and Cells – Gordon Research Conference in Barcelona. Dr. Khursheed’s presentation, titled “A Novel DNA Vaccine Approach to Prophylactic and Therapeutic Vaccines,” described IMUNON’S PlaCCine technology platform for the development of next-generation vaccines, and is available here. Results from preclinical studies in a PlaCCine COVID-19 vaccine demonstrated characteristics that address the limitations of current commercial vaccines by offering enhanced breadth of protection to emerging variants, persistence and robust cellular immunity, as well as stability at workable temperatures. Importantly, humoral immune responses specific to the SARS-CoV-2 spike antigen were persistent over a 14-month post-vaccination period in mice, while the T-cell responses from PlaCCine COVID-19 vaccines after 14 months were higher than a commercial mRNA vaccine. In another mouse study, the humoral response to a single dose of a commercial mRNA vaccine plateaued within 14 days after vaccination while the response continued to increase over time with a PlaCCine vaccine, demonstrating improved durability. In addition, PlaCCine was stable for at least nine months at refrigerated temperatures and for at least one month at room temperature. Presentation at the Vaccine Technology Summit 2023Describes Compelling Preclinical Data Supporting Continued Development of PlaCCine as a Differentiated, Next-Generation Vaccine. In March 2023 Dr. Anwer presented data on the Company’s PlaCCine platform at the Vaccine Technology Summit 2023 in Boston. Dr. Anwer’s presentation is titled “A Novel DNA Vaccine Platform with Potential to Create Next Generation Vaccines” and is available here. Dr. Anwer reviewed the Company’s work in advancing its PlaCCine modality and the promising preclinical data generated to date. Among topics presented was the ability of this multi-valent technology to achieve broad-spectrum immunity from a single DNA plasmid with a synthetic delivery system that is independent of virus, device or liquid nanoparticle formulations. The data presented showed: Robust immunogenicity and protection in SARS-CoV-2 models Durable cellular or humoral responses detectable for more than 12 months Comparable protection activity to a commercial mRNA vaccine in a booster-dose comparison Superior immune quality versus the mRNA vaccine in a single-dose comparison In addition, the PlaCCine modality had important advantages for a commercial vaccine, including a shelf-life at 4⁰C for greater than nine months, and the ability for simple, rapid and scalable manufacturing. IMNN-001 Immunotherapy Presentation at the American Association for Cancer Research (AACR)Describes Findings from Mouse Model of Peritoneally Disseminated Ovarian Cancer that Suggest Biweekly Dosing Regimen for Further Evaluation in Human Clinical Studies. In April 2023 Jean Boyer, Ph.D., IMUNON’s Vice President of Preclinical Research, presented a poster titled “Efficacy of IMNN-001, an Interleukin-12 Immune Gene Therapy, at Different Dose Frequencies” at the AACR in Orlando, which is available here. Researchers concluded that IMNN-001 demonstrated stimulation of the immune response in the ID8 ovarian tumor model. Of the three dosing regimens tested, the once every 2-week regimen demonstrated comparability to the weekly regimen while showing superiority over the once every 3-week regimen, particularly with respect to mortality and tumor burden. These findings suggest exploring once every 2-week dosing of IMNN-001 in human studies, which is already incorporated into the protocol of a new study evaluating IMNN-001 in combination with bevacizumab in the treatment of advanced ovarian cancer. This study is sponsored by the Break Through Cancer Foundation. Corporate Developments IMUNON Unveils New Manufacturing Facilityat Huntsville’s HudsonAlpha Biotech Campus. In June 2023, the Company unveiled its new cGMP clinical materials production facility on the Huntsville, Alabama campus of the HudsonAlpha Institute for Biotechnology. The facility is intended to support R&D efficiencies and lower development costs for infectious disease and cancer vaccines, and non-viral DNA-based immune-oncology therapies. This new capability complements the Company’s existing cGMP quality control facility for testing clinical products at the Huntsville site. IMUNON has designed and built its own manufacturing capabilities to produce GMP-grade plasmid DNA (pDNA) and DNA-facilitating agents to support Phase 1 clinical studies with its PlaCCine infectious disease modality and its IndiPlas and FixPlas cancer vaccine modalities. The new facility’s specifications follow the 2008 FDA guidance cGMP for Phase 1 investigational drugs. The pDNA and DNA facilitating agents are key components of the final vaccine formulation, with GMP fill and finish carried out at a CDMO partner site. IMUNON’s CEO Presents Business Overview at BIO 2023 International Convention and Mass General Brigham World Medical Innovation Forum 2023. Dr. Le Goff provided an overview of IMUNON’s business progress to an audience of investors and biopharmaceutical professionals at the BIO 2023 International Convention and at the Mass General Brigham World Medical Innovation Forum 2023, both in Boston. She highlighted the strength of IMUNON’s leadership team, and the status of the Company’s TheraPlas nucleic acid therapeutics platform and its PlaCCine nucleic acid vaccine platform while providing context on the promise of DNA as a therapeutic and a vaccine. For the PlaCCine and TheraPlas technologies, Dr. Le Goff described mechanisms of action and provided a closer look at the promising clinical results generated to-date. She provided background on the use of DNA in these medicines, characterizing its performance in terms of durability, development speed and ease of manufacturing, shipping and storage. Dr. Le Goff also highlighted the potential of IMNN-001 for the treatment of ovarian cancer during a panel discussion. Dr. Le Goff’s presentation is available here. SECOND QUARTER FINANCIAL RESULTS IMUNON reported a net loss for the second quarter of 2023 of $5.6 million, or $0.61 per share, compared with a net loss of $6.0 million, or $0.87 per share, for the second quarter of 2022. Operating expenses were $5.5 million for the second quarter of 2023, a decrease of $0.6 million, or 10%, from $6.1 million for the second quarter of 2022. Net cash used for operating activities was $6.8 million for the second quarter of 2023 compared with $5.4 million for the comparable prior-year period. The increase was primarily due to the cash settlement in April 2023 along with related legal fees for arbitration with a former contract manufacturer for ThermoDox. Cash used by financing activities of $6.2 million during the second quarter of 2023 resulted from the early repayment of the Company’s loan facility with Silicon Valley Bank, offset by equity sales under the Company’s At-the-Market Equity Facility. The Company had $24.1 million in cash, investments and accrued interest receivable as of June 30, 2023. Combined with $1.8 million in planned future sales of IMUNON’s State of New Jersey NOLs, the Company believes it has sufficient capital resources to fund its operations through 2024. Research and development (R&D) expenses were $3.1 million for the second quarter of 2023 compared with $3.2 million for the comparable period in 2022. R&D costs to support the OVATION 2 Study as well as the Phase 3 OPTIMA Study decreased to $0.3 million for the second quarter of 2023 compared with $0.8 million for the same period of 2022. Other clinical and regulatory costs were $0.4 million for the second quarter of 2023 compared with $0.7 million for the second quarter of 2022. R&D costs associated with the preclinical development of the PlaCCine DNA vaccine modality increased to $1.3 million for the second quarter of 2023 compared with $0.6 million for the same period of 2022. R&D costs associated with the preclinical development of IMNN-001 decreased to $0.4 million for the second quarter of 2023 compared with $0.8 million for the same period of 2022. Chemistry, manufacturing and controls (CMC) costs increased to $0.7 million for the second quarter of 2023 compared with $0.3 million for the second quarter of 2022 due to higher costs related to the development of in-house pilot manufacturing capabilities for DNA plasmids and nanoparticle delivery systems. General and administrative expenses were $2.3 million for the second quarter of 2023 compared with $2.9 million for the comparable prior-year period. The decrease was primarily due to lower non-cash stock-compensation expense and lower professional fees, including legal fees to defend various lawsuits filed after the announcement in July 2020 of the Phase 3 OPTIMA Study results, offset by higher compensation expenses related to the CEO succession plan announced in July 2022 and higher staffing costs. Other non-operating expenses were $85 thousand for the second quarter of 2023 compared with $65 thousand for the prior-year period. The Company incurred early debt extinguishment expense on its loan facility with Silicon Valley Bank totalling $0.3 million, offset by higher investment income from the Company’s short-term investments due to higher returns on these investments. FIRST HALFFINANCIAL RESULTS For the six months ended June 30, 2023, the Company reported a net loss of $11.2 million, or $1.28 per share, compared with a net loss of $16.5 million, or $2.59 per share, for the same period of 2022. Operating expenses were $11.2 million for the first six months of 2023, a decrease of $0.9 million, or 8%, from $12.1 million for the same period of 2022. Net cash used for operating activities was $10.8 million for the first six months of 2023 compared with $13.4 million for the same period in 2022. The decrease was primarily due to the one-time payment of $4.5 million in interest expense resulting from the sale and subsequent redemption of $30 million of Series A & B convertible redeemable preferred stock in the first quarter of 2022. The Company’s projected cash utilization for the balance of 2023 is approximately $4.5 million per quarter. Cash used by financing activities of $3.7 million during the first six months of 2023 resulted from the early repayment of the Company’s loan facility with Silicon Valley Bank ($6.4 million), offset by sales of equity under the Company’s At-the-Market Equity Facility ($2.7 million). The Company also received net proceeds of $1.6 million from the sale of its unused New Jersey NOLs in the first quarter of 2023. R&D expenses were $5.8 million for the first six months of 2023 compared with $6.3 million for the comparable period in 2022. R&D costs to support the OVATION 2 Study as well as the Phase 3 OPTIMA Study decreased to $0.6 million for the first six months of 2023 compared with $1.3 million for the comparable 2022 period. Other clinical and regulatory costs were $0.7 million for the first six months of 2023 compared with $1.5 million for the same period of 2022. R&D costs associated with the preclinical development of the PlaCCine DNA vaccine modality increased to $2.3 million for the first six months of 2023 compared with $1.2 million for the same period of 2022. R&D costs associated with the preclinical development of IMNN-001 decreased to $0.8 million for the first half of 2023 compared with $1.7 million for the same period of 2022. CMC costs increased to $1.4 million for the first six months of 2023 compared with $0.6 million for the comparable 2022 period due to higher costs related to the development of in-house pilot manufacturing capabilities for DNA plasmids and nanoparticle delivery systems. General and administrative expenses were $5.4 million for the first six months of 2023 compared with $5.7 million for the same period of 2022. The $0.3 million decrease was primarily attributable to lower non-cash stock-compensation expense, offset by higher compensation expenses related to the CEO succession plan announced in July 2022 and higher staffing costs. Other non-operating income was $9 thousand for the first six months of 2023 compared with $4.7 million for the comparable prior-year period. The decrease was primarily attributable to the one-time payment of $4.5 million in interest expense resulting from the sale and subsequent redemption of $30 million of Series A & B convertible redeemable preferred stock in the first quarter of 2022. VIRTUAL R&D DAY EVENT IMUNON management along with several guest key opinion leaders plan to host a virtual R&D Day event this fall to discuss the Company’s progress in developing its PlaCCine platform, IMNN-001, and other achievements. Presenters will also review strategic plans and opportunities for IMUNON. Additional information including date, time of day and instructions to participate will be announced in a separate news release. CONFERENCE CALL AND WEBCAST The Company is hosting a conference call to provide a business update, discuss second quarter 2023 financial results and answer questions at 11:00 a.m. EDT today. To participate in the call, please dial 866-777-2509 (Toll-Free/North America) or 412-317-5413 (International/Toll) and ask for the IMUNON Second Quarter 2023 Earnings Call. A live webcast of the call will be available here. The call will be archived for replay until August 24, 2023. The replay can be accessed at 877-344-7529 (U.S. Toll-Free), 855-669-9658 (Canada Toll-Free) or 412-317-0088 (International Toll), using the replay access code 1202020. A webcast of the call will be available here for 90 days. About IMUNON IMUNON is a fully integrated, clinical stage biotechnology company focused on advancing a portfolio of innovative treatments that harness the body’s natural mechanisms to generate safe, effective, and durable responses across a broad array of human diseases, constituting a differentiating approach from conventional therapies. IMUNON is developing its non-viral DNA technology across 4 modalities. The first modality, Theraplas®, is developed for the coding of proteins and cytokines in the treatment of solid tumors where an immunological approach is deemed promising. The second modality, PlaCCine® is developed for the coding of viral antigens that can elicit a strong immunological response. This technology may represent a promising platform for the development of vaccines in infectious diseases. The third modality, Fixplas®, concerns the application of our DNA technology to produce universal cancer vaccines also called tumor associated antigen cancer vaccines. Finally, the fourth modality, which is still in the discovery phase, Indiplas®, will focus on the development of personalized cancer vaccines, or neoepitope cancer vaccines. The Company’s lead clinical program, IMNN-001, is a DNA-based immunotherapy for the localized treatment of advanced ovarian cancer currently in Phase 2 development. IMNN-001 works by instructing the body to produce safe and durable levels of powerful cancer fighting molecules, such as interleukin-12 and interferon gamma, at the tumor site. Additionally, the Company is conducting IND enabling preclinical studies for the development of a COVID-19 booster vaccine: IMNN-101. The Company has also initiated preclinical studies to develop a Trp2 tumor associated antigen cancer vaccine in melanoma: IMNN-201. We will continue to leverage these modalities and to advance the technological frontier of plasmid DNA to better serve patients with difficult-to-treat conditions. For more information on IMUNON, visit . Forward-Looking Statements IMUNON wishes to inform readers that forward-looking statements in this news release are made pursuant to the “safe harbor” provisions of the Private Securities Litigation Reform Act of 1995. Readers are cautioned that such forward-looking statements involve risks and uncertainties including, without limitation, unforeseen changes in the course of research and development activities and in clinical trials; the uncertainties of and difficulties in analyzing interim clinical data; the significant expense, time and risk of failure of conducting clinical trials; the need for IMUNON to evaluate its future development plans; possible acquisitions or licenses of other technologies, assets or businesses; possible actions by customers, suppliers, competitors or regulatory authorities; and other risks detailed from time to time in IMUNON’s periodic reports and prospectuses filed with the Securities and Exchange Commission. IMUNON assumes no obligation to update or supplement forward-looking statements that become untrue because of subsequent events, new information or otherwise. Contacts: IMUNON LHA Investor Relations Jeffrey W. Church Kim Sutton Golodetz Executive Vice President, CFO 212-838-3777 609-482-2455 Kgolodetz@lhai.com jchurch@imunon.com (Tables to Follow) IMUNON, Inc. Condensed Statements of Operations (in thousands except per share amounts) Three Months Ended June 30, Six Months Ended June 30, 2023 2022 2023 2022 Licensing revenue $ - $ 125 $ - $ 250 Operating expenses: Research and development 3,134 3,226 5,754 6,322 General and administrative 2,340 2,877 5,405 5,748 Total operating expenses 5,474 6,103 11,159 12,070 Loss from operations (5,474 ) (5,978 ) ($ 11,159 ) (11,820 ) Other income (expense): Investment income 281 40 535 52 Interest expense (37 ) (105 ) (197 ) (4,751 ) Loss on debt extinguishment (329 ) - (329 ) - Other income - - - 2 Total other (expense) income, net (85 ) (65 ) 9 (4,697 ) Net loss $ (5,559 ) $ (6,043 ) $ (11,150 ) $ (16,517 ) Net loss per common share Basic and diluted $ (0.61 ) $ (0.87 ) $ (1.28 ) $ (2.59 ) Weighted average shares outstanding Basic and diluted 9,137 6,982 8,711 6,373 IMUNON, Inc. Selected Balance Sheet Information (in thousands) June 30, 2023 December 31, 2022 ASSETS Current assets Cash and cash equivalents $ 6,611 $ 11,493 Investment securities and interest receivable 17,480 21,384 Money market investments, restricted cash - 1,500 Advances, deposits and other current assets 2,640 2,778 Total current assets 26,731 37,155 Property and equipment 651 548 Other assets Restricted cash invested in money market account - 4,500 Deferred tax asset - 1,567 Operating lease right-of-use assets, deposits, and other assets 1,339 206 Total other assets 1,339 6,273 Total assets $ 28,721 $ 43,976 LIABILITIES AND STOCKHOLDERS’ EQUITY Current liabilities Accounts payable and accrued liabilities $ 5,846 $ 8,381 Note payable – current portion - 1,425 Operating lease liabilities – current portion 278 231 Total current liabilities 6,124 10,037 Notes payable – noncurrent portion - 4,611 Operating lease liabilities – noncurrent portion 1,082 - Total liabilities 7,206 14,648 Stockholders’ equity Common stock 92 74 Additional paid-in capital 401,164 397,980 Accumulated other comprehensive gain (loss) 162 27 Accumulated deficit (379,818 ) (368,668 ) 21,600 29,413 Less: Treasury stock (85 ) (85 ) Total stockholders’ equity 21,515 29,328 Total liabilities and stockholders’ equity $ 28,721 $ 43,976 # # #