Hypericin

Phase 3 clinical study of HyBryte ™ in CTCL initiating in 2024 PRINCETON, N.J., Nov. 19, 2024 /PRNewswire/ -- Soligenix, Inc. (Nasdaq: SNGX) (Soligenix or the Company), a late-stage biopharmaceutical company focused on developing and commercializing products to treat rare diseases where there is an unmet medical need, announced today the formation of a European Medical Advisory Board (MAB) to provide additional medical/clinical strategic guidance to the Company as it advances its confirmatory Phase 3 multicenter, double-blind, placebo-controlled study evaluating the safety and efficacy of HyBryte™ (synthetic hypericin) in the treatment of cutaneous T-cell lymphoma (CTCL) patients with early-stage disease. This confirmatory, 18-week study is expected to enroll approximately 80 patients in the United States (U.S.) and Europe, and is targeted to begin patient enrollment by the end of 2024 with top-line results anticipated in the second half of 2026. "Stemming from the positive feedback from the European Medicines Agency (EMA) on the key design components of our confirmatory Phase 3 placebo-controlled study evaluating the safety and efficacy of HyBryte™, we consider expert European involvement a necessary component for the development of this program. As such, it is an honor to have such a prestigious and dedicated group of clinicians and leaders in the field committed to working with us as we develop HyBryte™," stated Christopher J. Schaber, PhD, President and Chief Executive Officer of Soligenix. "We have assembled some of the leading CTCL experts, who collectively have the experience of treating thousands of patients with this rare condition, and complement the strong CTCL MAB we've established in the U.S. The support of these European experts is a further testament to the opportunity HyBryte™ could provide for patients suffering from this disease worldwide." Comprised of internationally renowned physicians with extensive experience in treating and running clinical research trials in CTCL, this esteemed MAB will play an important advisory role in the conduct and interpretation of the upcoming Phase 3 clinical study and the associated regulatory interactions with health authorities. The MAB will provide feedback, input, and guidance on clinical strategies and their implementation, as well as on other critical items, such as health economics and reimbursement, to assist Soligenix in meeting the needs of patients suffering from CTCL. European MAB Members Martine Bagot, MD, PhD – France Martine Bagot is Professor and Head of the Department of Dermatology at the Hôpital Saint Louis in Paris, France and co-directs the INSERM Unit 976 Human Immunology, Pathophysiology and Immunotherapy. Dr. Bagot also chairs the French Group for the Study of Cutaneous Lymphomas. Dr. Bagot has co-authored more than 750 peer-reviewed publications, most of which are in the complementary fields of dermatology, immunology, and oncology. Main publications include numerous clinical trials in dermato-oncology. Dr. Bagot is involved with major European international societies such as the International Society for Cutaneous Lymphoma (ISCL), The European Society for Dermatological Research (ESDR), and the European Organisation for Research and Treatment of Cancer (EORTC) Cutaneous Lymphoma Task Force, where she previously served as its President and is currently a steering committee member. Pietro Quaglino, MD – Italy Pietro Quaglino is Associate Professor of Dermatology at the Department of Medical Sciences, University of Turin Medical School, Italy. His clinical and research activities focus on melanoma, cutaneous lymphoma, and immune dermatology. He is the principal investigator of several clinical trials in melanoma and cutaneous lymphoma. He is board member of the GIPMe (Gruppo Italiano Polidisciplinare sul Melanoma), board member and treasurer of the IMI (Italian Melanoma Intergroup), past chairman and current steering committee member of the EORTC Cutaneous Lymphoma Task Force, and member of the Board Directors of the ISCL. He has published more than 160 peer-reviewed scientific papers. Dr. Quaglino is Assistant Editor of the Giornale Italiano di Dermatologia e Venereologia, reviewer of international journals on dermatology and referee for ANVUR (National Agency for the Evaluation of Universities and Research Institutes). Pablo Luis Ortiz-Romero, MD, PhD – Spain Pablo Luis Ortiz-Romero is Professor of Dermatology and Head of the Dermatology Department at Hospital Universitario 12 de Octubre, Spain. He is a distinguished dermatologist and researcher specializing in cutaneous lymphomas, particularly mycosis fungoides and Sézary syndrome forms of CTCL. His experience is extensive, having contributed to over 200 publications and numerous clinical studies in the field. Dr. Ortiz-Romero is affiliated with the ISCL and has served on its Board of Directors. His research includes innovative treatments for CTCL, and he is one of the leading physicians focused on novel treatments for CTCL in Spain. He served as the Secretary General of the EORTC Cutaneous Lymphoma Tumor Group and is currently a member of its steering committee. About HyBryte™ HyBryte™ (research name SGX301) is a novel, first-in-class, photodynamic therapy utilizing safe, visible light for activation. The active ingredient in HyBryte™ is synthetic hypericin, a potent photosensitizer that is topically applied to skin lesions that is taken up by the malignant T-cells, and then activated by safe, visible light approximately 24 hours later. The use of visible light in the red-yellow spectrum has the advantage of penetrating more deeply into the skin (much more so than ultraviolet light) and therefore potentially treating deeper skin disease and thicker plaques and lesions. This treatment approach avoids the risk of secondary malignancies (including melanoma) inherent with the frequently employed DNA-damaging drugs and other phototherapy that are dependent on ultraviolet exposure. Combined with photoactivation, hypericin has demonstrated significant anti-proliferative effects on activated normal human lymphoid cells and inhibited growth of malignant T-cells isolated from CTCL patients. In a published Phase 2 clinical study in CTCL, patients experienced a statistically significant (p=0.04) improvement with topical hypericin treatment whereas the placebo was ineffective. HyBryte™ has received orphan drug and fast track designations from the U.S. Food and Drug Administration (FDA), as well as orphan designation from the European Medicines Agency (EMA). The published Phase 3 FLASH trial enrolled a total of 169 patients (166 evaluable) with Stage IA, IB or IIA CTCL. The trial consisted of three treatment cycles. Treatments were administered twice weekly for the first 6 weeks and treatment response was determined at the end of the 8th week of each cycle. In the first double-blind treatment cycle (Cycle 1), 116 patients received HyBryte™ treatment (0.25% synthetic hypericin) and 50 received placebo treatment of their index lesions. A total of 16% of the patients receiving HyBryte™ achieved at least a 50% reduction in their lesions (graded using a standard measurement of dermatologic lesions, the CAILS score) compared to only 4% of patients in the placebo group at 8 weeks (p=0.04) during the first treatment cycle (primary endpoint). HyBryte™ treatment in this cycle was safe and well tolerated. In the second open-label treatment cycle (Cycle 2), all patients received HyBryte™ treatment of their index lesions. Evaluation of 155 patients in this cycle (110 receiving 12 weeks of HyBryte™ treatment and 45 receiving 6 weeks of placebo treatment followed by 6 weeks of HyBryte™ treatment), demonstrated that the response rate among the 12-week treatment group was 40% (p<0.0001 vs the placebo treatment rate in Cycle 1). Comparison of the 12-week and 6-week treatment responses also revealed a statistically significant improvement (p<0.0001) between the two timepoints, indicating that continued treatment results in better outcomes. HyBryte™ continued to be safe and well tolerated. Additional analyses also indicated that HyBryte™ is equally effective in treating both plaque (response 42%, p<0.0001 relative to placebo treatment in Cycle 1) and patch (response 37%, p=0.0009 relative to placebo treatment in Cycle 1) lesions of CTCL, a particularly relevant finding given the historical difficulty in treating plaque lesions in particular. The third (optional) treatment cycle (Cycle 3) was focused on safety and all patients could elect to receive HyBryte™ treatment of all their lesions. Of note, 66% of patients elected to continue with this optional compassionate use / safety cycle of the study. Of the subset of patients that received HyBryte™ throughout all 3 cycles of treatment, 49% of them demonstrated a positive treatment response (p<0.0001 vs patients receiving placebo in Cycle 1). Moreover, in a subset of patients evaluated in this cycle, it was demonstrated that HyBryte™ is not systemically available, consistent with the general safety of this topical product observed to date. At the end of Cycle 3, HyBryte™ continued to be well tolerated despite extended and increased use of the product to treat multiple lesions. Overall safety of HyBryte™ is a critical attribute of this treatment and was monitored throughout the three treatment cycles (Cycles 1, 2 and 3) and the 6-month follow-up period. HyBryte's™ mechanism of action is not associated with DNA damage, making it a safer alternative than currently available therapies, all of which are associated with significant, and sometimes fatal, side effects. Predominantly these include the risk of melanoma and other malignancies, as well as the risk of significant skin damage and premature skin aging. Currently available treatments are only approved in the context of previous treatment failure with other treatment modalities and there is no approved front-line therapy available. Within this landscape, treatment of CTCL is strongly motivated by the safety risk of each product. HyBryte™ potentially represents the safest available efficacious treatment for CTCL. With very limited systemic absorption, a compound that is not mutagenic and a light source that is not carcinogenic, there is no evidence to date of any potential significant safety issues. Following the first Phase 3 study of HyBryte™ for the treatment of CTCL, the FDA and the EMA indicated that they would require a second successful Phase 3 trial to support marketing approval. With agreement from the EMA on the key design components, the second, confirmatory study, called FLASH2, is expected to be initiated before the end of 2024. This study is a randomized, double-blind, placebo-controlled, multicenter study that will enroll approximately 80 subjects with early-stage CTCL. The FLASH2 study replicates the double-blind, placebo-controlled design used in the first successful Phase 3 FLASH study that consisted of three 6-week treatment cycles (18 weeks total), with the primary efficacy assessment occurring at the end of the initial 6-week double-blind, placebo-controlled treatment cycle (Cycle 1). However, this second study extends the double-blind, placebo-controlled assessment to 18 weeks of continuous treatment (no "between-Cycle" treatment breaks) and a more aggressive titration of the light doses with the primary endpoint assessment occurring at the end of the 18-week timepoint. In the first Phase 3 study, a treatment response of 49% (p<0.0001 vs patients receiving placebo in Cycle 1) was observed in patients completing 18 weeks (3 cycles) of therapy. In this second study, all important clinical study design components remain the same as in the first FLASH study, including the primary endpoint and key inclusion-exclusion criteria. The extended treatment for a continuous 18 weeks in a single cycle is expected to statistically demonstrate HyBryte's™ increased effect over a more prolonged, "real world" treatment course. Given the extensive engagement with the CTCL community, the esteemed Medical Advisory Board and the previous trial experience with this disease, accelerated enrollment in support of this study is anticipated, including the potential to enroll previously identified and treated HyBryte™ patients from the FLASH study. Discussions with the FDA on an appropriate study design remain ongoing. While collaborative, the agency has expressed a preference for a longer duration comparative study over a placebo-controlled trial. Given the shorter time to potential commercial revenue and the similar trial design to the first FLASH study afforded by the EMA accepted protocol, this study is being initiated. At the same time, discussions with the FDA will continue on potential modifications to the development path to adequately address their feedback. In addition, the FDA awarded an Orphan Products Development grant to support the evaluation of HyBryte™ for expanded treatment in patients with early-stage CTCL, including in the home use setting. The grant, totaling $2.6 million over 4 years, was awarded to the University of Pennsylvania that was a leading enroller in the Phase 3 FLASH study. Additional supportive studies have demonstrated the utility of longer treatment times (Study RW-HPN-MF-01), the lack of significant systemic exposure to hypericin after topical application (Study HPN-CTCL-02) and its relative efficacy and tolerability compared to Valchlor® (Study HPN-CTCL-04). About Cutaneous T-Cell Lymphoma (CTCL) CTCL is a class of non-Hodgkin's lymphoma (NHL), a type of cancer of the white blood cells that are an integral part of the immune system. Unlike most NHLs which generally involve B-cell lymphocytes (involved in producing antibodies), CTCL is caused by an expansion of malignant T-cell lymphocytes (involved in cell-mediated immunity) normally programmed to migrate to the skin. These malignant cells migrate to the skin where they form various lesions, typically beginning as patches and may progress to raised plaques and tumors. Mortality is related to the stage of CTCL, with median survival generally ranging from about 12 years in the early stages to only 2.5 years when the disease has advanced. There is currently no cure for CTCL. Typically, CTCL lesions are treated and regress but usually return either in the same part of the body or in new areas. CTCL constitutes a rare group of NHLs, occurring in about 4% of the more than 1.7 million individuals living with the disease in the U.S. and Europe (European Union and United Kingdom). It is estimated, based upon review of historic published studies and reports and an interpolation of data on the incidence of CTCL that it affects approximately 31,000 individuals in the U.S. (based on SEER data, with approximately 3,200 new cases seen annually) and approximately 38,000 individuals in Europe (based on ECIS prevalence estimates, with approximately 3,800 new cases annually). About Soligenix, Inc. Soligenix is a late-stage biopharmaceutical company focused on developing and commercializing products to treat rare diseases where there is an unmet medical need. Our Specialized BioTherapeutics business segment is developing and moving toward potential commercialization of HyBryte™ (SGX301 or synthetic hypericin sodium) as a novel photodynamic therapy utilizing safe visible light for the treatment of cutaneous T-cell lymphoma (CTCL). With successful completion of the second Phase 3 study, regulatory approvals will be sought to support potential commercialization worldwide. Development programs in this business segment also include expansion of synthetic hypericin (SGX302) into psoriasis, our first-in-class innate defense regulator (IDR) technology, dusquetide (SGX942) for the treatment of inflammatory diseases, including oral mucositis in head and neck cancer, and (SGX945) in Behçet's Disease. Our Public Health Solutions business segment includes development programs for RiVax®, our ricin toxin vaccine candidate, as well as our vaccine programs targeting filoviruses (such as Marburg and Ebola) and CiVax™, our vaccine candidate for the prevention of COVID-19 (caused by SARS-CoV-2). The development of our vaccine programs incorporates the use of our proprietary heat stabilization platform technology, known as ThermoVax®. To date, this business segment has been supported with government grant and contract funding from the National Institute of Allergy and Infectious Diseases (NIAID), the Defense Threat Reduction Agency (DTRA) and the Biomedical Advanced Research and Development Authority (BARDA). For further information regarding Soligenix, Inc., please visit the Company's website at and follow us on LinkedIn and Twitter at @Soligenix_Inc. This press release may contain forward-looking statements that reflect Soligenix's current expectations about its future results, performance, prospects and opportunities, including but not limited to, potential market sizes, patient populations, clinical trial enrollment, the expected timing for closing the offering described herein and the intended use of proceeds therefrom. Statements that are not historical facts, such as "anticipates," "estimates," "believes," "hopes," "intends," "plans," "expects," "goal," "may," "suggest," "will," "potential," or similar expressions, are forward-looking statements. These statements are subject to a number of risks, uncertainties and other factors that could cause actual events or results in future periods to differ materially from what is expressed in, or implied by, these statements, and include the expected amount and use of proceeds from the offering and the expected closing date of the offering. Soligenix cannot assure you that it will be able to successfully develop, achieve regulatory approval for or commercialize products based on its technologies, particularly in light of the significant uncertainty inherent in developing therapeutics and vaccines against bioterror threats, conducting preclinical and clinical trials of therapeutics and vaccines, obtaining regulatory approvals and manufacturing therapeutics and vaccines, that product development and commercialization efforts will not be reduced or discontinued due to difficulties or delays in clinical trials or due to lack of progress or positive results from research and development efforts, that it will be able to successfully obtain any further funding to support product development and commercialization efforts, including grants and awards, maintain its existing grants which are subject to performance requirements, enter into any biodefense procurement contracts with the U.S. Government or other countries, that it will be able to compete with larger and better financed competitors in the biotechnology industry, that changes in health care practice, third party reimbursement limitations and Federal and/or state health care reform initiatives will not negatively affect its business, or that the U.S. Congress may not pass any legislation that would provide additional funding for the Project BioShield program. In addition, there can be no assurance as to the timing or success of any of its clinical/preclinical trials. Despite the statistically significant result achieved in the first HyBryte™ (SGX301) Phase 3 clinical trial for the treatment of cutaneous T-cell lymphoma, there can be no assurance that the second HyBryte™ (SGX301) Phase 3 clinical trial will be successful or that a marketing authorization from the FDA or EMA will be granted. Additionally, although the EMA has agreed to the key design components of the second HyBryte™ (SGX301) Phase 3 clinical trial, no assurance can be given that the Company will be able to modify the development path to adequately address the FDA's concerns or that the FDA will not require a longer duration comparative study. Notwithstanding the result in the first HyBryte™ (SGX301) Phase 3 clinical trial for the treatment of cutaneous T-cell lymphoma and the Phase 2a clinical trial of SGX302 for the treatment of psoriasis, there can be no assurance as to the timing or success of the clinical trials of SGX302 for the treatment of psoriasis. Additionally, despite the biologic activity observed in aphthous ulcers induced by chemotherapy and radiation, there can be no assurance as to the timing or success of the clinical trials of SGX945 for the treatment of Behçet's Disease. Further, there can be no assurance that RiVax® will qualify for a biodefense Priority Review Voucher (PRV) or that the prior sales of PRVs will be indicative of any potential sales price for a PRV for RiVax®. Also, no assurance can be provided that the Company will receive or continue to receive non-dilutive government funding from grants and contracts that have been or may be awarded or for which the Company will apply in the future. These and other risk factors are described from time to time in filings with the Securities and Exchange Commission (the "SEC"), including, but not limited to, Soligenix's reports on Forms 10-Q and 10-K. Unless required by law, Soligenix assumes no obligation to update or revise any forward-looking statements as a result of new information or future events. SOURCE SOLIGENIX, INC. WANT YOUR COMPANY'S NEWS FEATURED ON PRNEWSWIRE.COM? 440k+ Newsrooms & Influencers 9k+ Digital Media Outlets 270k+ Journalists Opted In GET STARTED

“ 点击蓝字 关注我们 顾景凯 吉林大学唐敖庆卓越教授、药物代谢研究中心主任，超分子结构与材料国家重点实验室教授，国家药品注册审评专家咨询委员会专家。中国药理学会药物代谢专业委员会副主委、分析药理专业委员会副主委；中国医药生物技术协会药物分析技术分会副主委；《药学学报》副主编；Drug Metab Dispos，Acta Pharm Sin B，Asian J Pharm Sci，J Pharm Anal等期刊编委。长期从事纳米药物、高分子药物、多糖药物、蛋白肽类药物体内时空命运分析和基于药代动力学的前体药物设计与开发。先后主持11项国家自然科学基金面上项目、重点项目和国家重大新药创制专项子课题，在 Adv Drug Deliv Rev，Anal Chem，Acta Pharm Sin B，Adv Mater，Biomacromolecules，Clin Pharmacokinet等期刊发表SCI论文200余篇，获授权发明专利15项，作为主要发明人之一的1类新药于2022年上市。 纳米药物递送系统体内命运分析新方法与新技术研究进展 PPS  王心怡1, 2#，付淑军3#，孟祥骏1，吴伟4*，顾景凯2** （1. 天津大学医学部药物科学与技术学院，天津300072；2. 吉林大学超分子结构与材料国家重点实验室超分子化学生物学研究中心 生命科学学院药物代谢研究中心，吉林 长春130012；3. 国家药品监督管理局药品审评中心，北京100022；4. 复旦大学药学院， 上海201203） [摘要] 纳米药物递送系统（NDDSs）能够改善药物的药代动力学行为，将活性药物成分靶向递送至病灶，并控制药物释放。近年来， 随着纳米技术的快速发展，NDDSs在医药领域展现出巨大潜力，已成为该领域的研究热点。对NDDSs的体内命运进行精准分析，是揭示其复杂体内过程，实现纳米药物安全、有效递送的关键之一。NDDSs在体内包含负载型药物、游离型药物、纳米载体和载体材料等多种形态成分，在复杂的生命体系中全面精准分析这些成分的动态变化具有极高的技术挑战。受分析方法与分析技术的制约，目前对于NDDSs的体内命运研究仍存在许多挑战，极大阻碍了NDDSs的快速发展与临床转化。综述对NDDSs中游离型药物、负载型药物、纳米载体和纳米材料的分析新方法、新技术及其在纳米药代动力学中的应用进行介绍，并对该领域未来研究方向提出展望，以期为NDDSs的体内命运相关研究提供参考。 1995 年，美国食品和药品管理局（food and drug administration，FDA）批准了首个抗癌纳米药物Doxil [1]。在过去的30年里，纳米药物递送系统 （nanocarrier drug delivery systems，NDDSs）发展迅速，迄今已有几十万篇相关论文发表[2]。然而， 尽管论文数量庞大， NDDSs的实际临床转化却十分有限。截至2023年末，仅有90多个纳米药物上市[2]。与普通制剂相比，这些纳米药物在临床依从性方面有了极大提高，但大多数在临床疗效方面并未取得显著的改善[1, 3]。 与普通药物制剂不同，NDDSs在体内始终存在 “载药粒子-游离型药物-解聚载体材料”多种形态的动态变化过程，该过程与其体内药代动力学行为、 药效作用和毒性反应密切相关。其中，载药粒子是药物的运输工具和储库，靶部位/靶点（如肿瘤组织） 中的游离药物是药效的物质基础，而其他组织中的游离药物、载药粒子、解聚载体材料等则可能导致 毒性/不良反应。因此，充分了解NDDSs各形态组分的体内药代动力学信息，对其安全性和有效性评价以及制剂处方的设计与优化具有重要意义[4-5]。以往由于缺少前瞻性的理论指导，尤其是缺少有效的分析策略与技术手段，在复杂的生命系统中对 NDDSs 游离药物、载药粒子、解聚载体材料的药代动力学过程进行精准分析，无疑存在巨大的挑战， 已成为制约 NDDSs 临床转化成功率的最主要瓶颈之一[6]。 针对上述问题，本文聚焦NDDSs在体内存在的不同形态成分，对近年来用于追踪NDDSs体内时空命运的先进分析策略与分析技术进行综述，以期为NDDSs的药代/毒代动力学、安全性与有效性评价以及NDDSs的处方设计与工艺优化研究提供比较全面的技术支持，进而提高NDDSs的临床转化成功率。 1 游离型、负载型药物和纳米载体分离分析技术 传统的药物分析技术只关注NDDSs中药物的总浓度，无法阐明药物是负载于载药粒子还是已经从载体中释放出来[7]。由于NDDSs改变了药物的分布，其所负载药物的吸收、分布、代谢、排泄行为相比普通制剂往往会发生显著变化[7]。因此，为了能够更准确地评估纳米药物的有效性与安全性， 区分游离型药物和负载型药物是极为重要的。在复杂的生命系统中，纳米药物的递送过程面临多种挑战，为确保所设计的纳米载体的优良性能，监测纳米载体的体内转运过程也是十分必要的[8]。下文将对NDDSs游离型药物、负载型药物和纳米载体的分离与分析技术进行介绍。 1.1 固相萃取技术 固相萃取（solid phase extraction，SPE）技术基于液-固相色谱理论，通过选择性吸附和选择性洗脱的方式对样品中不同组分进行分离。脂质体是由脂质双层构成的球形囊泡。作为首个成功应用于临床的纳米载体[9-12]，脂质体以独特的两亲性结构，即亲水核心和被脂质层包围的疏水空间，为封装不同极性的药物提供了可能[9, 13]。Wang等[14] 开发了一种SPE结合液相色谱-串联质谱（liquid chromatography tandem mass spectrometry，LC-MS/ MS）检测聚乙二醇（polyethylene glycol，PEG） 化长循环阿霉素（doxorubicin，DOX）脂质体的负载型DOX和游离型DOX的分析方法。该方法利用PEG化长循环DOX脂质体在反相固相萃取小柱上保留能力差，而游离型DOX在反相固相萃取小柱上保留能力强的原理，能够从组织样本中分离出 PEG 化DOX脂质体（负载型DOX）；然后利用 LC-MS/MS 分别检测负载型DOX浓度和DOX总浓度，并通过二者差值计算出游离型DOX的浓度。该研究比较了荷瘤小鼠正常组织和肿瘤组织中PEG 化脂质体内DOX的释放与摄取曲线，结果表明脂质体能有效地将DOX释放到肿瘤中，与直接给药 DOX溶液相比，脂质体使肿瘤对DOX的吸收量增加了1.8倍；脂质体还降低了DOX在心脏的分布量， 从而减少了DOX的心脏毒性。 1.2 荧光成像技术  近年来，荧光成像技术凭借其高灵敏度、操作简便等特性使其在药物递送系统中得到广泛应用[15]， 常被用于标记NDDSs并对其进行可视化示踪[16]。下文将对常用于NDDSs游离型药物、负载型药物和纳米载体的荧光成像技术进行介绍。 1.2.1 聚集诱导发光技术 聚集诱导发光（aggregation- induced emission，AIE）是指某些分子在分散状态下几乎不发光，但当它们聚集在一起时，发光效率显著提高[17]。聚合物胶束是两亲性聚合物分子在水相介质中达到或超过临界胶束浓度（critical micelle concentration，CMC）后自发形成的一种有序结构。两亲性聚合物分子亲脂性尾部因其疏水作用而聚集于胶束内部，亲水性的头部则因具有极性而向外部延伸[18-19]。聚合物胶束的疏水核心能够通过疏水相互作用有效地负载疏水性药物，其亲水外壳与周围的水分子相互作用，保护胶束不被网状内皮系统识别[20]，进而对胶束内部的疏水基团起到保护作用。Sun等[21] 通过将氧化还原敏感的二硫键和 AIE 荧光团Tripp连接于甲氧基聚乙二醇（methoxy polyethylene glycol，mPEG），制备了mPEG-ss-Tripp 聚合物胶束，并以DOX作为药物模型，成功构建出能够同时实现癌症治疗与细胞内成像的智能给药系统。当细胞内谷胱甘肽（glutathione，GSH）还原 mPEG-ss-Tripp 中的二硫键时，聚合物胶束迅速释放药物来抑制肿瘤转移，同时通过监测Tripp的AIE 信号变化来研究胶束的细胞内释药行为。 聚合物纳米粒利用其独特的结构通过吸附或共轭作用使其于聚合物基质或载体表面携带药物[22]， 从而实现对药物的递送。Wang等[23]制备了一种负载姜黄素（curcumin，Cur）的聚乳酸-羟基乙酸 [poly(lactic-co-glycolic) acid，PLGA] 纳米粒（Cur@ PLGA-NPs）。Cur 是一种具有AIE性质的药物， 在与PLGA纳米粒结合后，利用其荧光特性监测载药纳米粒在细胞内的分布。该纳米粒与CT26细胞孵育后，由于Cur的AIE特性，载药Cur纳米粒在细胞中表现出红色荧光，且其荧光强度呈现出浓度依赖性，这表明Cur纳米粒被成功内化到CT26 细胞中。 Wang 等[24] 报道了蛋白冠对阳离子脂质体与细胞相互作用的影响。该研究将具有AIE效应的TR4 作为细胞膜探针，引入阳离子脂质体（TR4脂质体）。当TR4以单体形式存在时不发出荧光，而一旦其插入脂质体中就会发射出较强的荧光。通过成像定位技术，该研究分别对含蛋白冠和不含蛋白冠的脂质体组的观察结果显示，蛋白冠的形成对阳离子脂质 体的转运行为产生了显著的影响，即当脂质体表面形成蛋白冠后，其与细胞的相互作用方式从能量非依赖性的膜融合转变为能量依赖性的内吞作用。 Xu 等[20] 设计并成功合成了一种具有AIE特性的壳聚糖，该壳聚糖通过引入疏水性片段、基于二苯甲酮结构的AIE荧光团、肿瘤靶向配体和酸敏键， 实现了对肿瘤的靶向作用及在酸性条件下的药物释放。该壳聚糖的两亲性使其能够自组装形成包封含紫杉醇的胶束。这种“可视化”胶束系统不仅能够在肿瘤中有效聚集并特异性释放药物，而且能实时监测药物行为。在MCF-7细胞实验中，孵育2 h 即可在细胞质中观察到来自该胶束系统的黄色荧 光，这一现象表明胶束已成功被肿瘤细胞内化。随着孵育时间的延长，荧光强度逐渐增强，进一步说明了肿瘤细胞对胶束的内吞量在不断增加。类似地， Kulkarni 等[25] 将基于四苯乙烯（tetraphenylethylene， TPE）的AIE 荧光团掺入聚乙二醇-聚己内酯 [poly(ethylene glycol)-block-poly(ε-caprolactone)， PEG-b-PCL] 中，该嵌段共聚物可通过自组装形成包 封有DOX和Cur的胶束，可以同时实现肿瘤给药与纳米载体的生物成像示踪。 AIE 效应最显著的优势是在聚集状态下荧光信号的增强，这大大提高了检测的灵敏度。由于AIE 探针在溶液中几乎不发光，其可有效减少背景信号， 提高信噪比。虽然AIE探针在聚集时发光强，但这也可能导致空间分辨率的不足。在AIE探针高度聚集的区域，过强的荧光信号可能掩盖细微的结构信息，特别是在亚细胞结构的研究中。在某些生物环境下，非特异性吸附可能使AIE探针重新聚集，导致产生错误的荧光信号，影响实验结果的准确性。另外，负载的AIE探针在体内从纳米载体上释放行为未必与药物完全一致，这也会影响负载型药物分析结果的准确性。 1.2.2 聚集发光淬灭技术 聚集发光淬灭（aggregation- caused quenching，ACQ）荧光探针具有强疏水性[26]， 在有机溶剂中具有良好的荧光发射性质。随着环境含水量的增加，ACQ荧光探针的荧光发射强度会经历一个显著的变化过程。当含水量达到某一特定值时，ACQ荧光探针的荧光发射强度会迅速下降，而当其处于绝对水环境时，由于分子间的聚集作用， 其荧光发射会完全淬灭[27]。ACQ荧光探针在检测纳米载体完整性方面具有重要的应用价值，当纳米载体保持完整时，其内部的ACQ探针由于处于有机环境而发出强烈的荧光；当纳米载体发生解聚或分解时，ACQ探针由于暴露于水环境中而发生荧光强度降低，甚至完全淬灭。 复旦大学吴伟教授课题组开发了一种利用近红外荧光染料的ACQ特性来监测完整纳米载体体内命运的新策略。该技术已被成功应用于研究多种纳米载体的体内命运[28-33]。He等[34]采用该策略标记和追踪口服聚合物胶束的体内命运发现，大鼠口服聚合物胶束后，其在胃肠道中至少保留4 h，血液和肝脏中出现的荧光直接证明完整聚合物胶束的口服吸收，且约1% ~ 2%的完整胶束通过淋巴途径被吸收。Wu等[35] 也将ACQ探针封装到聚合物胶束中来跟踪胶束的体内命运。 纳米乳是一种由水、油和适当的表面活性剂自组装形成的纳米体系[10]。Jiang等[36]利用ACQ 荧光探针的特性，对石杉碱甲纳米乳（huperzine A-nanoemulsion，HupA-NE）及乳铁蛋白修饰的石杉碱甲纳米乳（lactoferrin-huperzine A-nanoemulsion， Lf-HupA-NE）在大鼠体内的转运进行了示踪。活体成像结果显示，HupA-NE和Lf-HupA-NE经鼻腔给药后，均可通过神经和血液循环转运到脑中。 可溶性微针是近年来研究较多的一类微针，其主要由水溶性高分子材料制备而成，药物分布在针尖的基质中，当微针刺入皮肤后，针尖通过吸收组织液逐渐溶解，并释放出封装的药物[35]。Fu等[26] 将ACQ探针负载于固体脂质纳米粒，进一步将二者一并封装到可溶性微针中，随后追踪固体脂质纳米粒在大鼠不同皮肤部位的透皮扩散情况。活体成像显示，随着时间推移，大鼠不同皮肤部位的荧光强度均逐渐降低，表明负载于纳米载体的ACQ探针因暴露于生物基质而发生荧光猝灭。 Naghibi 等 [37] 将四苯乙烯溴代异丁酸酯 （tetraphenylethene-bromoisobutyrate，TPE-BIB）与 PEG 和肼（hydrazine，Hyd）结合，合成出一种新型刷状聚合物TPE-PEGA-Hyd，其中肼为DOX的结合位点。该聚合物不仅具有PEG的优良特性，还因TPE-BIB 的引入而具备了AIE性能，为药物递送和体内示踪提供了有力的工具。TPE-BIB是能够发出蓝色荧光的AIE荧光团，其荧光强度在聚集状态下显著增强，而DOX在作为药物模型的同时，还具有ACQ特性，其红色荧光在聚集状态下较弱（见 图1）。TPE-PEGA-Hyd-DOX 在肿瘤细胞的低pH 环境中由于肼的裂解而释放DOX。因此，通过该体系410 nm（蓝色）和600 nm（红色）的荧光比率， 可以实现对药物在体内的实时监测。 ACQ是示踪负载型药物和载体粒子的重要技术手段。在许多生物检测中，ACQ效应可减少背景信号，有助于提高检测信噪比。然而，基于ACQ的生物成像不适用于亲水性纳米载体，因为ACQ探针很难被包裹到亲水性基质中，且水分子会迅速吸收到亲水性基质中，导致探针聚集和荧光猝灭。由于ACQ效应依赖于荧光分子的聚集程度，这使得精确的定量分析变得较为复杂。此外，同AIE探针 一样，负载的ACQ探针在体内从纳米载体上释放 行为未必与药物完全一致，这可能会导致对负载型药物的分析不准确。 1.2.3 荧光共振能量转移技术 荧光共振能量转移 （fluorescence resonance energy transfer，FRET）是 2 个荧光团（一个作为供体，另一个作为受体）之间的能量转移过程，当供体的发射光谱与受体的激发光谱在很大程度上重叠，且2种荧光探针之间的距离非常接近时，二者之间就会发生能量转移，处于激发态的供体以非激发方式将能量传递给旁边的受体，使得供体发射的荧光强度衰减，受体荧光分子的荧光强度增加[38]。FRET技术为监测NDDSs的药物释放和载体完整性提供了一种强大的工具 [39]。在实验设计中，将供体和受体分别标记在纳米材料和药物分子上，当药物从纳米材料中释放时，受体与供体之间的距离发生变化，进而引起FRET信号的改变。因此，通过分析FRET信号的变化，不仅 可以用来监测药物的释放，还能够用于指征有机纳米载体的完整性[17, 40]。在选择FRET染料时，通常倾向选择具有近红外范围发射光谱的染料，这是因为近红外光能够更好地穿透细胞和组织，从而避免来自这些生物结构自发荧光的干扰[40]。 Zhong 等[41] 合成了含有TPE的pH响应型纳米复合体，且以DOX作为药物模型。通过监测DOX 与TPE之间的FRET信号变化，在肿瘤细胞内追踪由pH变化触发的DOX动态释放过程。Laskar 等[42] 将 2 种疏水性荧光抗癌药物Cur和喜树碱 （camptothecin，Cpt）分别通过酯键和氧化还原响应型二硫键与PEG连接，成功合成出两亲性的PEG 化前药Cpt-S-S-PEG-Cur。由于前药聚合物能够在水中形成稳定的纳米组装以及Cpt（FRET供体）的发射光谱与Cur（FRET受体）吸收光谱之间的重叠， 供受体之间能够产生FRET信号。在与肿瘤相关的氧化还原环境（存在一定量的GSH）中，这些稳定的纳米组装显示出Cpt的优先释放，导致FRET消失。因此，这种基于GSH响应型FRET的递药系统能够实现以非侵入方式来监测载体中的药物释放。Zhou 等[43] 报道了一种含供体荧光团DAN-聚丙硫醚（polypropylene sulfide，PPS）-mPEG 与受体荧光团DAB-PPS-mPEG的新型氧化响应型聚合物胶束。当活性氧存在时，该胶束中的PPS嵌段发生氧化并分解，导致供体荧光团淬灭，从而触发FRET 过程的改变，这种设计使得药物释放与荧光信号变化直接相关。该研究以尼罗红为模型药物，探究了胶束荧光变化与胶束降解及药物释放之间的关系。 纳米晶在改善水溶性差的药物口服生物利用度方面展现出巨大潜力，然而，关于纳米晶如何提高口服生物利用度的具体机制，目前仍存在一定争议，这主要是由于在研究中无法同时追踪溶解的药物和纳米晶颗粒所导致。目前对此存在2种不同的观点：一是通过纳米晶减小粒径从而改善溶解度；二是通过纳米晶的内吞作用改善口服吸收[44]。Zhang 等[17] 以香豆素6为药物模型制备纳米晶，通过ACQ信号来检测游离的香豆素6，同时将2-((5 (4-( 二苯基氨基)苯基)噻吩-2-基)亚甲基)丙二腈 [2-((5-(4-(dip-tolylamino)phenyl)thiophen-2-yl) methylene)malononitrile，MeTTMN] 掺入香豆素6 构建FRET对，并通过监测FRET信号跟踪纳米晶体颗粒。研究结果表明，该纳米晶体主要通过快速溶解改善药物的口服吸收，成功阐明了纳米晶如何提高药物的口服吸收。 Tengjisi等[38]通过合成2种类型的FRET纳米粒， 成功构建了能够同时监测药物释放与二氧化硅纳米粒降解的系统。该研究中疏水性FRET对（DiO和 DiI）被共同封装在纳米粒核心（FRET@Si NP）， 用作药物替代物以模拟药物包封和药物释放。当 DiO 和DiI 足够接近时，可以观察到来自DiI的 FRET信号。然而，一旦2种染料因药物释放而分离， DiI 信号消失，DiO信号则会出现（见图 2A）。同时，另一组FRET对（Cy3和Cy5）被用于二氧化硅纳米粒的外表面标记（Si@FRET NP），用于监测二氧化硅的降解。当二氧化硅壳完整时，可观察到来自Cy5的FRET信号；随着二氧化硅的降解， Cy3 信号开始增加，而Cy5信号降低（见图 2B）。通过使用这2个FRET纳米系统，药物释放和二氧化硅纳米粒的降解可以被分别监测。 FRET 信号的产生依赖于供体发射光谱与受体吸收光谱的重叠及两者适当的空间配置，这保证了检测的特异性。FRET技术提供了强大的定性分析能力，但其在定量分析方面仍面临着诸多挑战。例如， FRET 效率的计算需要考虑多种因素，包括供体和受体的光谱重叠、荧光寿命、荧光量子产率等，这些均需要复杂的数学模型和校正方法。因此，FRET 技术尚难以实现全面的药代动力学分析。此外， FRET 荧光染料的生物活性也可能会影响NDDSs的药代动力学特性。 1.2.4 碳量子点 碳量子点（carbon quantum dots， CQDs）是一种零维碳纳米材料，具有众多优于有机荧光团的特性[45]。首先，CQDs高度的光稳定性确保了其在各种环境下均能保持稳定的发光性能。其次，CQDs具有可调节发射特性，使其发射波长可根据需要进行调整，从而满足不同的应用场景。再者，CQDs较大的双光子激发截面使其在较低能量激发下也能实现高效发光，有利于降低高能激光对生物组织的损害。此外，CQDs还具有优异的细胞渗透性和生物相容性。Mazandarani等[46]合成了一种壳聚糖-适体-碳量子点（chitosan-aptamer-carbon quantum dot，CS-Apt-CQD）纳米水凝胶，通过乳化法将5-氟尿嘧啶-灵芝酸负载于水凝胶系统中，其中CQD发挥示踪水凝胶的作用。Rahiminezhad等[47] 开发出一种新颖的纳米复合材料，该材料结合了石墨烯量子点（graphene quantum dots，GQDs）和 PLGA，GQD凭借其独特的光致发光特性而被用作荧光探针，以实现对药物递送过程的精确追踪，进而优化索拉非尼的药物递送效果。 CQDs 具有非常稳定的荧光特性，使其在长时间或复杂环境下仍能保持较高的信号强度。目前CQDs的荧光发光波长多为蓝绿色（400 ~ 520 nm），多需要紫外光波激发，存在生物组织穿透深度低、易造成生物组织光损伤的缺点。 1.3 酶联免疫吸附测定技术 酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）是一种经典的生物分析技术，其基于抗原与抗体之间的特异性结合。当一种特定的抗原被加入到样品板的小孔中时，可与相应的抗体结合。这种结合可通过添加酶标记的二抗来检测，该酶可催化一种底物产生颜色、荧光或其他可检测的信号。邓雪等[48]利用ELISA技术，对尿酸酶和尿 酸酶脂质体在大鼠体内的药代动力学行为进行比较， 结果显示，尿酸酶脂质体组的大鼠体内尿酸酶活性更高，且半衰期更长。ELISA具有高灵敏度、高通量的优点，但使用该方法需要针对每种待测物开发特异性的抗体与检测条件，不仅操作繁琐，且成本高昂。此外，ELISA作为间接检测技术，其选择性、动态范围往往不尽如人意[49]。近年来，直接利用 ELISA 技术对NDDSs进行体内分析的研究相对较少，这可能是因为NDDSs的复杂性与多样性使得开发通用的ELISA检测方法变得困难。 1.4 抗聚乙二醇单链可变片段抗体技术 复旦大学占昌友教授课题组开发了一种利用抗 PEG 单链可变片段抗体（anti-PEG single chain variable fragment antibody，PEG-scFv）来分离PEG 化脂质体的技术[50]。scFv 是一类具有小相对分子质量和高抗原结合活性的抗体片段[51]，因其无Fc片段，在纳 米药物表面吸附后，scFv不会引起补体激活[52-53]。PEG-scFv 与 PEG 化脂质体表面的PEG结合后，能够引起PEG化脂质体的沉淀，通过低速离心，易于将沉淀的脂质体与上清液中的游离型药物分离，进而对二者进行后续的定量分析（见图3）。此外， 这项技术也被证明能够用于分离血浆中的PEG化聚合物胶束，从而实现了紫杉醇胶束的稳定性和体内命运的解析[54]。 PEG-scFv 专门针对PEG分子，具有很高的结合特异性，且这种结合不会导致药物载体中的药物泄漏，确保了该分离方法的准确性和效率。因许多纳米药物均使用PEG来增加溶解性、稳定性或减少免疫原性，该技术对于其他种类的PEG化纳米药物也具有很好的应用潜能。 1.5 稳定同位素示踪超滤技术 超滤是一种膜分离技术，主要用于大分子和小分子的分离。超滤法主要依赖于半透膜的微孔结构。当溶液在外界压力作用下流经超滤膜表面时，小于膜孔径的小分子透过膜，而大于膜孔径的物质被截留。对于纳米药物，在超滤过程中存在一个重要的问题，即血浆蛋白结合的药物成分对负载型药物定量准确性的影响[55]。Stern 团队开发了稳定同位素示踪超滤（stable isotope tracer ultrafiltration assay，SITUA）技术，通过将稳定同位素示踪剂加入到含有纳米药物的血浆样本中，对传统的超滤方法进行改进，从而能够更准确地评估血浆蛋白的结合情况[56]。SITUA假设同位素标记的药物与从纳米药物中释放的药物具有相同的蛋白结合行为。该具体过程为：首先将同位素标记的药物（D*）加入到含有纳米药物的血浆中，D*会迅速与血浆蛋白达到结合平衡；然后将血浆样本转移到超滤装置中并通过离心分离，分别检测血浆样本中D*的总浓度 [Total D*]、超滤液中 D* 的浓度[Ultrafiltrate D*]、血浆样本中非同位素标记药物（D）的总浓度[Total D]以及超滤液中D的浓度[Ultrafiltrate D]。通过图4中的公式（1）计算D*的蛋白结合率（%Bound D*）。由于D与D*具有相同的血浆蛋白结合行为，因此D的血浆蛋白结合率等于D*的血浆蛋白结合率。根据图 4中的公式（2）计算游离型药物的浓度[Unencapsulated D]。确定了%Bound D* 和[Unencapsulated D] 后，可以使用图4中的公式（3）计算蛋白结合型D的浓度 [Protein-bound D]，并使用图4 中的公式（4）计算负载型药物的浓度[Encapsulated D][55]。 SITUA 技术已被用来研究白蛋白结合型紫杉醇Abraxane® 及紫杉醇聚合物胶束制剂Genexol®PM 在大鼠中的药代动力学特征，并进行了生物等效性评价。该研究结果发现，尽管Abraxane®和 Genexol®PM 的总药物、游离型药物的浓度-时间曲线几乎相同，但2种制剂游离型药物的药代动力学参数具有显著的统计学差异，而总药物无此差异[57-58]。这也再次证明了只关注总药物浓度的传统分析方法的局限性。 利用SITUA技术可以获得血浆样本中的负载型药物、蛋白结合药物和游离型药物的浓度信息。然而， SITUA 技术是基于稳定同位素标记药物和从纳米药物中释放的药物具有相同血浆蛋白结合行为的假设。实际上，血浆中释放药物的浓度范围较宽，不同浓度下药物的蛋白结合率可能会发生变化，从而导致潜在的准确性问题。因此，在使用此方法之前，需要检验不同浓度下药物的血浆蛋白结合率是否一致。此外，超滤装置的非特异性吸附问题也需要注意。 1.6 磁共振成像技术 磁共振成像（magnetic resonance imaging，MRI） 是一种无创成像技术，具有高分辨率与深组织穿透能力，能够精确捕捉到纳米载体在体内的动态变化[8]。 氧化铁纳米粒（iron oxide nanoparticle，IONP） 由于其独特的磁性和良好的生物相容性被广泛用作MRI造影剂[59]。Stater 等 [60] 将免疫佐剂单磷酰脂质A（monophosphoryl lipid A，MPLA）装载到纳米氧化铁粒菲立莫妥（ferumoxytol，FH）的碳水化合物外壳中，形成FH-MPLA复合物。随后， 将FH-MPLA通过静脉注射到小鼠体内。FH作为纳米载体，能够将药物有效地递送至肿瘤细胞， 同时也作为MRI造影剂，可通过无创成像技术实时监测纳米载体的体内分布。Zhou等[61]设计并合成了一种装载有金丝桃素的锰基树枝状聚合物纳米粒（manganese-based and hypericin-loaded polyester dendrimer nanoparticle，MHD），当 MHD 到达肿瘤部位时，MHD可被肿瘤细胞内化并降解释放出金丝桃素和锰离子。锰离子一方面具有优异的MRI成像性能，另一方面可通过将内源性H2O2转化为毒性 羟基自由基，从而强化金丝桃素的光动力治疗过程。MHD整体显示出良好的成像能力与治疗效果。 MRI 技术能够提供高分辨率的三维成像，这对于纳米载体在体内的行为和分布的研究至关重要。MRI 技术和相关纳米载体的研究涉及复杂的物理学、化学和生物学问题，需要跨学科的知识和技术， 涉及材料科学、生物医学工程、药学等多个领域。MRI 设备及维护成本高昂，对于初期纳米载体的研究和开发造成较高的经济压力。另外，不是所有的纳米材料均适合作为MRI造影剂，需要具有特定的磁学性质，这进一步限制了MRI的应用范围。 2 载体材料分析技术 纳米载体依赖载体材料来维持其结构，从而实现药物递送与控制释放的目的。载体材料不仅能够影响其所负载药物的药代动力学过程，导致药效和毒副作用的改变，也能通过影响机体免疫、代谢等过程，产生新的毒副作用。因此，阐释载体材料的体内命运对于评估NDDSs的安全性具有重要意义。纳米载体包括有机载体和无机载体。尽管无机载体 相关报道很多，但在生物相容性、生物降解性及载药性能等方面有机载体优于无机载体。目前，绝大多数已上市或处于临床阶段的纳米载体均基于有机载体。由于有机载体的组成材料具有生物降解性， 其降解产物较为复杂，且检测过程易受生物基质的干扰。有机载体材料多为高分子聚合物，通常具有多分散的性质，不同相对分子质量的同系物组成和比例千变万化，因此其体内定性、定量分析均具有很大的挑战。下文将对常用的纳米载体材料分析技术进行介绍。 2.1 液相色谱-串联质谱技术 近几十年来LC-MS/MS技术的发展令人瞩目， 不仅在小分子药物的定量分析中展现出高灵敏度， 还在大分子如蛋白质的定量生物分析中逐渐获得更多关注。LC-MS/MS技术具有优异的选择性、良好的准确度与精密度以及更宽的动态范围[62]，使得其在分析复杂生物基质中的待测物时具有更高的可靠性。 小分子纳米载体材料相对分子质量固定，采用基于多反应监测（multiple reaction monitoring， MRM）扫描模式的传统LC-MS/MS技术即可实现其在生物样品中的定量分析，这已是成熟技术体系。然而纳米载体材料多数为高分子聚合物，相对分子质量呈多分散性，在电喷雾质谱中离子化后会形成多电荷离子，进而产生大量的前体离子。上述生物 样品定量分析常用的MRM扫描模式，因其分辨率和扫描速度的局限，无法全面采集到聚合物的质谱信息，导致无法精准分析聚合物纳米材料。Zhou等[63] 开发了一种基于MSALL采集模式的LC-MS/MS技术。该技术采用信息独立采集方式，允许所有PEG前体 离子通过质谱第一个四极杆并进入碰撞池，在碰撞池通过施加适当的碰撞能量从而裂解产生PEG特异性碎片，利用PEG特异性碎片实现对具有多分散相对分子质量和众多前体离子的PEG的绝对定量分析 （见图5）。该项技术的主要优势包括能够全面分析聚合物，具有出色的特异性、高灵敏度和可重复性。基于该技术，Meng等[64]研究了两嵌段共聚物单甲氧基聚乙二醇-聚乳酸[monomethoxy poly(ethylene glycol)-block-poly(D，L-lactic acid)，PEG-PLA] 的大鼠体内命运，包括大鼠静脉注射后的药代动力学、 生物分布、代谢和排泄情况。该研究结果表明， PEG-PLA 原形主要分布在脾脏、肝脏和肾脏，主要通过尿液排泄，其中超过80%以代谢物PEG排出。Ren 等[65] 使用该技术研究了D-α-生育酚聚乙二醇 1000 琥珀酸酯（D-α-tocopheryl polyethylene glycol 1000 succinate，TPGS）及其代谢物PEG1000 在大鼠口服和静脉注射后的药代动力学、组织分布和排泄情况，还考察了TPGS与人肝微粒体中细胞色素 P450 的相互作用。 2.2 放射性同位素标记技术 使用放射性同位素标记技术，能够在体内对纳米材料和载体进行示踪，从而定量研究其生物分布与清除过程[66-67] ，进而评估纳米药物的治疗潜力。 Anane-Adjei 等 [68] 制备了一种经89Zr 标记的超支化聚N-(2-羟丙基)甲基丙烯酰胺[hyperbranched N-(2-hydroxypropyl)methacry-lamide,HPMA]聚合物， 并将其静脉注射到小鼠体内，随后利用正电子发射断层扫描-计算机断层扫描（positron mmission tomography-computedtomography，PET-CT）成像技术来监测聚合物在小鼠体内的实时生物分布。Imlimthan 等[69] 开发了一种177Lu 标记的载有维罗非尼的纤维素纳米晶（cellulose nanocrystal，CNC）颗粒，并通过单光子发射计算机断层成像术（single-photon emission computed tomography，SPECT）对 177Lu-CNC 在肺转移性黑色素瘤小鼠的体内命运进行了示踪。该研究结果显示，随着孵育时间的延长，177Lu-CNC在 肿瘤细胞内的放射性增高，而在48 h后放射性降低。  放射性同位素示踪剂为医学领域提供了强大的监测工具。然而，放射性同位素示踪剂也存在一些不可忽视的缺点，主要集中在成本、安全性以及同位素标记脱落对结果准确性的影响等。 2.3 同步辐射X射线技术 同步辐射X射线技术，凭借其高灵敏度和空间分辨率来研究纳米材料的电子配置、配位几何或氧化状态的能力，已被用于分析纳米材料的体内行为[70]。Yao 等[71] 研究了[Gd@C82(OH)22]n 在巨噬细胞中的分布情况，发现[Gd@C82(OH)22]n主要分布在溶酶体 中。该技术还可以提供高分辨率的三维亚细胞定位， 观察纳米材料在完整细胞中的位置。此外，该技术可根据[Gd@C82(OH)22]n 的密度和分布情况指征纳米材料在3种不同类型的溶酶体中的细胞内转运情况。同样，具有足够样品穿透力的纳米尺度三维成像在研究细胞内纳米材料的分布方面起重要作用，其能够将细胞内纳米材料的积累情况可视化，提供了进 一步研究其生物转化行为的新方向。 3 结语与展望 过去几十年NDDSs的研发投入巨大，但仅有数十个产品上市，这在很大程度上可能归因于对 NDDSs 的体内命运缺乏最基本的认知。NDDSs在复杂生命体系中不仅存在多种形态成分，且多种形态成分之间始终处于动态平衡之中，给其体内命运研究带来极大的技术挑战。本文对近年来用于追踪 NDDSs 体内命运的分析技术进行了综述，希望能够为NDDSs的体内命运研究提供技术参考。 尽管NDDSs体内命运的分析技术已取得一定进展，但仍面临诸多挑战，如检测的分辨率、灵敏度和特异性等方面仍有待提高。此外，目前对NDDSs 体内命运的研究仍主要依赖单一技术或方法，缺乏多种技术综合应用以对其进行系统性研究。质谱具备同时检测药物、载体材料及其代谢物的能力，且具有高灵敏度和高特异性的优势。通过将质谱技术与操作简便、条件温和、分离高效的新型分离技术结合，有可能在同时定量纳米药物的游离型药物、 负载型药物、纳米载体和纳米材料方面进一步取得进展。多种成像技术如同步辐射光源、MRI、荧光成像的结合，有望实现优势互补，从而精准、实时、 动态地观察纳米药物在体内的分布和代谢过程，并从不同角度揭示纳米药物的生物分布和作用机制， 最终获得更全面、更准确的纳米药物体内命运信息。 随着纳米药物体内命运研究的不断深入，研究纳米药物与不同类型细胞（如肿瘤细胞、免疫细胞、 内皮细胞等）之间的相互作用，分析纳米药物在细胞内的分布和运动轨迹，探讨纳米药物与生物大分子之间的相互作用，深化对纳米药物与体内系统相互作用的理解。为实现这些目标，需要克服更多技术挑战， 探索和发展更为高效、灵敏和精准的分析新技术，从而更加全面、深入地揭示NDDSs的体内命运，更高效地推动纳米药物的设计与优化，最终实现纳米药物在临床应用中的疗效最大化与毒副作用最小化。 参考文献： [1] Zingg R, Fischer M. 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