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Linh B. Truong, David Medina-Cruz, Ebrahim Mostafavi
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Genetic medicine, including ribonucleic acid (RNA) therapy, has delivered numerous progresses to the treatment of diseases thanks to the development of lipid nanoparticles (LNPs) as a delivery vehicle. However, RNA therapeutics are still limited by the lack of safe, precise, and efficient delivery outside of the liver. Thus, to fully realize the potential of genetic medicine, strategies to arm LNPs with extrahepatic targeting capabilities are urgently needed. This review explores the current state of next-generation LNPs that can bring RNA biomolecules to their targeted organ. The main approaches commonly used are described, including the modulation of internal lipid chemistries, the use of conjugated targeting moieties, and the designs of clinical administration. This work will demonstrate the advances in each approach and the remaining challenges in the field, focusing on clinical translation.
基因药物，包括核糖核酸（RNA）疗法，因脂质纳米颗粒（LNPs）作为递送载体的发展，已经在疾病治疗方面取得了许多进展。然而，RNA 治疗仍然受到在肝脏以外安全、精确和高效递送的限制。因此，为了充分发挥基因医学的潜力，迫切需要装备 LNPs 具备肝脏以外靶向能力的策略。本综述探讨了下一代 LNPs 的当前状态，这些 LNPs 可以将 RNA 生物分子输送到其靶向器官。描述了常用的主要方法，包括调节内部脂质化学成分、使用偶联靶向基团以及临床管理设计。本工作将展示每种方法的进展以及该领域尚存的挑战，重点关注临床转化。
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Nucleic acid therapeutics have gained significant momentum and acknowledgement over the past few years due to their ability to manipulate cellular machinery at the source – targeting the earlier steps in the central dogma. With the rise in biological applications, the need for safe, effective delivery of these therapeutics presents a hurdle in translational efforts. Prior delivery methods using viral vectors had their limitations, especially surrounding their safety profiles. However, the development of lipid nanoparticles (LNPs) presents a potential solution to some of these drawbacks, allowing for safe, effective, and transient delivery of RNA macromolecules. Decades of research in both RNA biology and lipid chemistry have cumulated in Onpattro, the first siRNA therapy that was delivered by LNP. The first messenger RNA (mRNA)-LNP drug product was approved shortly after for SARS-CoV-19 vaccine, developed by both Pfizer and Moderna These approvals have validated LNPs as effective carrier to deliver RNA macromolecules, and thus paved the way for the clinical development of countless therapeutic RNA modalities, including antisense oligonucleotides, mRNA and gene editing constructs. The breakthrough of LNP technology has opened the doors for genetic medicine to truly fulfill its potential.
在过去几年中，由于核酸疗法能够在源头处操纵细胞机制——针对中心法则的早期步骤，因此获得了显著的发展动力和认可。随着生物应用的增加，对这些治疗方法的安全、有效递送的需求成为转化工作中的一道障碍。先前使用病毒载体的递送方法存在其局限性，尤其是在安全性方面。然而，脂质纳米颗粒（LNPs）的发展提供了对其中一些缺点的潜在解决方案，允许安全、有效和短暂地递送 RNA 大分子。几十年来，在 RNA 生物学和脂质化学领域的研究已经累积成果，导致了 Onpattro 的诞生，这是第一种通过 LNP 递送的 siRNA 疗法。随后不久，第一款信使 RNA（mRNA）-LNP 药物产品也获得了批准，用于 SARS-CoV-19 疫苗，由辉瑞和莫德纳联合开发。这些批准验证了 LNPs 作为有效的载体来传递 RNA 大分子的有效性，从而为众多治疗 RNA 模式的临床开发铺平了道路，包括反义寡核苷酸。LNP 技术的突破为基因医学真正发挥其潜力打开了大门。
A diverse library of lipid composition has been used to formulate RNA-LNPs, but these LNPs are generally composed of four types of components: cationic or ionizable lipids, helper lipids, sterols and PEG-anchored lipids, such as 1,2-Dioleoyl-3-trimethylammonium (DOTAP), and ionizable lipids, such as Dlin-MC3-DMA, form electrostatic interactions with the negatively charged nucleic acid backbone, thus enhancing the encapsulation efficiency. Helper lipids are usually inspired by membrane bilayers and used to fill the nanoparticles (NPs) and stabilize the aqueous phase that the nucleic acids are in with the hydrophobic lipid tails. Sterols, most commonly naturally occurring cholesterol, further maintains the stability of the LNPs, while induces cellular uptake via ApoE-binding and low-density lipoprotein (LDL) receptor-mediated endocytosis. Lastly, PEGylated lipids control the size of LNPs by limiting the fusion rate of NPs during formation and enhance biocompatibility as well as pharmacology in vivo. Several review articles have detailed the advances in research to uncover more optimal LNPs formulation to enhance RNA delivery.
脂质纳米颗粒（RNA-LNPs）的制备已使用了多样化的脂质组成，但这些 LNPs 通常由四种类型的组分组成：阳离子或可电离脂质、辅助脂质、固醇和 PEG 锚定脂质。阳离子脂质，如 1,2-二油酰-3-三甲基铵（DOTAP），以及可电离脂质，如 Dlin-MC3-DMA，与带负电的核酸骨架形成静电相互作用，从而提高了包封效率。辅助脂质通常受到膜双层的启发，用于填充纳米颗粒（NPs）并稳定核酸所在的水相，具有疏水脂质尾巴。固醇类，通常是天然存在的胆固醇，进一步维持了 LNPs 的稳定性，同时通过 ApoE 结合和低密度脂蛋白（LDL）受体介导的内吞作用诱导细胞摄取。最后，PEG 化脂质通过限制 NPs 在形成过程中的融合速率来控制 LNPs 的大小，增强体内的生物相容性和药理学效应。一些综述文章详细介绍了研究的进展，以发现更优化的 LNPs 制剂，以增强 RNA 递送。
Additionally, more research has shed light on the systematic understanding of RNA delivery using LNPs. Three important aspects of LNP-mediated delivery stood out: the formation and structural determination of LNP containing RNA cargo; the adsorption of serum protein and biodistribution of LNPs; the cellular uptake and the intracellular fate decisions of LNPs. These novel discoveries have enabled more rational design of LNP formulations and more promising clinical translation. However, most LNPs can only deliver their cargoes into the hepatocytes due to the preferential liver accumulation. To truly enable genetic medicine to fulfill its potential, the LNP delivery toolbox needs to expand to other organs. In this review, strategies for extrahepatic delivery and their advances are detailed. Specifically, several pathways to target other organs are explored, demonstrated in Fig. 1, including changes in the inherent formulation of LNP, additions of conjugated modalities, and variations in the route of administrations. In each of these pathways, this work aims to provide some perspectives on the rationalities, mechanisms and current directions that researchers might consider. Furthermore, this review serves as a overarching view of the current state of RNA-encapsulated LNP-mediated extrahepatic delivery and offers strategic guidelines for future work.
此外，更多的研究已经阐明了对使用 LNPs 进行 RNA 递送的系统性理解。LNP 介导递送的三个重要方面突出表现为：含有 RNA 载荷的 LNP 的组成和结构确定；血清蛋白吸附和 LNPs 的生物分布；LNPs 的细胞摄取和细胞内命运决策。这些新的发现使得 LNP 制剂的更加合理化设计以及更有前景的临床转化成为可能。然而，由于其偏好于在肝脏中积聚，大多数 LNPs 只能将其载荷递送到肝细胞中。为了真正实现基因医学的潜力，LNP 递送工具箱需要扩展到其他器官。在本综述中，详细介绍了用于非肝脏递送的策略及其进展。具体而言，探讨了几种用于靶向其他器官的途径，如图 1 所示，包括改变 LNP 的固有配方、添加偶联模式以及改变给药途径的变化。在每个途径中，本综述旨在为研究人员可能考虑的合理性、机制和当前方向提供一些观点。此外，本综述还提供了关于 RNA 包裹的 LNP 介导非肝脏递送的当前状态的总体视图，并为未来的工作提供了战略指导。
Fig. 1. Overview of mechanism of RNA delivery via lipid nanoparticles and strategies for extrahepatic targeting, including (1) optimization of lipid chemistries, (2) addition of targeting modalities and (3) choices for routes of administration.
图 1. 通过脂质纳米颗粒传递 RNA 的机制和肝外靶向策略概述，包括 (1) 脂质化学的优化，(2) 增加靶向方式和 (3) 给药途径的选择。
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The search for extrahepatic LNP formulation was challenging, especially since the mechanistic understanding of cellular fate decision for LNPs is still in its infant stage. Widely accepted proposal for LNP delivery includes the shedding of LNPs during circulation prior to penetrating tissues, followed by protein adsorption. Specifically, apolipoprotein E (ApoE) becomes a particular protein of interest that was found to be influential in cellular delivery, activating LDLR-mediated endocytosis into cellular cytoplasm. While ApoE is highly expressed in the liver, other cell types also express it, thus theoretically can receive LNPs. Yet, historically, traditional LNP formulation shows limited to no delivery into extrahepatic tissues. Tissue penetration, biological barriers and circulation time play a factor, as most LNP's characteristic favors the accumulation in the liver. However, these factors only present incremental opportunities to target extrahepatic tissues. Therefore, research focus on screening for lipid formulations that can enable extrahepatic uptake of LNPs and subsequent functional nucleic acid delivery.
实现非肝脏 LNP 制剂的搜索具有挑战性，特别是因为对 LNPs 细胞命运决策的机制理解仍处于初级阶段。广泛接受的 LNP 递送建议包括在穿透组织之前在循环过程中脱落 LNPs，随后发生蛋白质吸附。具体来说，载脂蛋白 E（ApoE）成为一个特别引人关注的蛋白质，发现它在细胞递送中具有影响力，激活 LDLR 介导的内吞作用进入细胞质内。虽然 ApoE 在肝脏中高度表达，但其他细胞类型也表达它，因此从理论上讲可以接受 LNPs。然而，历史上，传统的 LNP 制剂显示出有限或没有递送到非肝脏组织的能力。组织穿透、生物屏障和循环时间都起着作用，因为大多数 LNP 的特性都有助于在肝脏中积聚。然而，这些因素只能提供渗透非肝脏组织的增量机会。因此，研究重点是筛选能够实现 LNPs 非肝脏摄取和随后功能性核酸递送的脂质配方。
Thanks to the adoption of sequencing technology, high-throughput screening of formulation via DNA barcoding replaced iterative screening and present higher hit rate for novel discoveries. By creating different cargo with unique barcoding sequence, LNP formulations can be pooled together, and lead candidates can be identified via sequencing of the interested tissue. Such concepts, whose schematics are shown in Fig. 2A, were demonstrated by Dahlman et al., whose study screened 30 different formulation and observed relatively preferential delivery into tissues outside of the liver. The technique became widely adapted, with novel lipid candidates, varying in lipid chemistries and lipid compositions, that incrementally increase delivery efficiency to extrahepatic tissues. However, despite the ability to evaluate numerous formulations simultaneously, a systematic understanding for extrahepatic delivery remains limited.
通过采用测序技术，通过 DNA 条形码的高通量制剂筛选取代了迭代筛选，并为新的发现提供了更高的命中率。通过创建具有独特条形码序列的不同载体，LNP 制剂可以混合在一起，通过对感兴趣的组织进行测序来识别主导候选者。达尔曼等人的研究证明了这种概念，其示意图如图 2A 所示，该研究筛选了 30 种不同的制剂，并观察到相对优先递送到肝脏以外的组织。这种技术得到了广泛的应用，采用了新的脂质候选物，这些脂质候选物在脂质化学和脂质组成上有所变化，递送效率逐渐增加到非肝脏组织。然而，尽管能够同时评估众多制剂，但对于非肝脏递送的系统理解仍然有限。
Fig. 2. Strategies for uncovering different lipidchemistries to facilitate extrahepatic delivery. (A) Schematics of LNP barcoding for high-throughput in vivo screening to identify tissue-specific LNP formulations; (B) Proteomic analysis of serum proteins binding to SORT LNPs surface indicates different corona composition in tissue-targeting LNPs, suggesting mechanistic understanding of different organ's trafficking of RNA-LNPs.
图 2. 揭示不同脂质化学以促进肝外递送的策略。(A) 用于高通量体内筛选以鉴定组织特异性 LNP 制剂的 LNP 条形码示意图；(B) 与 SORT LNP 表面结合的血清蛋白的蛋白质组学分析表明，组织靶向 LNP 中的冠成分不同，这表明了对不同器官 RNA-LNP 运输的机制理解。
Fortunately, promising candidates generated from screening efforts allowed for clearer understanding of desirable properties to enable tissue-specific targeting. These candidates include novel ionizable structures, such as those produced by the polymerization of traditional lipids and the inclusion of amide bonds into the lipid head groups. Clearly, the chemistry of ionizable component of LNP influence the subsequent biodistribution of such LNPs. However, the exact mechanisms of the observed preferential uptake remained a big scientific question in early screening experiments. Similar experiments identified optimal helper lipid structures and sterol additives that can de-target the liver to favor other organs, most commonly spleen or lung tissues. However, as more structures were identified, more mechanistic studies using these structures were carried out, elucidating how tissue-specific delivery can be achieved. This trial-to-mechanism journey was demonstrated best by the Selective Organ Targeting (SORT) LNPs development. SORT LNPs incorporate a fifth lipid component with various ionic charges, with permanently cationic component favoring lung delivery, and anionic component favoring spleen delivery. Further experiments indicate that SORT LNPs exhibit deviations in pKa, which recruit different serum protein via electrostatic interactions compared to traditional liver targeting LNPs (Fig. 2B). Moreover, SORT LNPs possess low ApoE binding on their surfaces, and maintain their delivery efficiency in low ApoE models, which suggested a pathway independent of ApoE binding. In fact, LNPs favoring extrahepatic tissues were observed to have lower interaction with ApoE, differential ionic potential and unique protein corona layers. Additionally, more evidence has shown that LNP uptake can occur in ApoE-independent manner, opening more possibilities to target tissues previously thought to be unreachable . However, a systematic understanding of the main drivers of uptake in extrahepatic cells has not been elucidated consistently, leaving some gaps in the rational design of LNP's chemistries. Additionally, despite the increase in throughput during screenings, the hit rate for these screens has been low, and new, innovative breakthroughs have become scarce. Even so, changes in lipid chemistries have enabled LNPs to reach lung and spleen tissues, opening up new avenues to treat diseases in the fields of pulmonary, oncology and immunology.
幸运的是，从筛选工作中产生的有前途的候选物使人们对实现特定组织靶向提供了更清晰的了解。这些候选物包括新型的离子化结构，例如通过传统脂质的聚合和将酰胺键包含到脂质头基中产生的结构。显然，LNP 的离子化成分的化学性质影响了这些 LNP 的生物分布。然而，早期筛选实验中观察到的优先摄取的确切机制仍然是一个重大科学问题。类似的实验识别出了最佳的辅助脂质结构和固醇添加剂，可以减少肝脏的靶向作用，有利于其他器官，最常见的是脾脏或肺组织。然而，随着更多结构的被识别出来，使用这些结构进行的更多机制研究阐明了如何实现特定组织的递送。这种从试验到机制的过程最好由选择性器官靶向（SORT）LNP 的开发来展示。SORT LNP 包括具有不同离子电荷的第五种脂质成分，其中永久阳离子成分有利于肺递送，而阴离子成分有利于脾脏递送。进一步的实验表明，与传统的以肝脏为靶标的 LNP 相比，SORT LNP 在 pKa 方面存在偏差，通过电荷静电相互作用招募不同的血清蛋白质（如图 2B 所示）。此外，SORT LNP 在其表面具有较低的 ApoE 结合，并在低 ApoE 模型中保持其递送效率，这表明了一种与 ApoE 结合无关的途径。事实上，观察到有利于非肝脏组织的 LNP 与 ApoE 的相互作用较低，具有不同的离子电位和独特的蛋白质包被层。此外，越来越多的证据表明，LNP 的摄取可以以独立于 ApoE 的方式发生，为以前认为无法到达的组织提供了更多可能性。然而，对于在非肝脏细胞中摄取的主要驱动因素的系统理解尚未得到一致阐明，这在 LNP 的化学有理设计中留下了一些空白。此外，尽管筛选过程中的吞吐量增加了，但这些筛选的命中率却很低，新的创新性突破变得稀缺。尽管如此，脂质化学的变化使 LNP 能够抵达肺和脾组织，为肺部、肿瘤学和免疫学领域的疾病治疗开辟了新的途径。
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The extrahepatic delivery of RNA can be challenging due to various biophysical barriers, such as the blood-brain barrier (BBB) and the blood-tumor barrier (BTB), both of which prevent the transport of RNA molecules from the vasculature into tissues. A pivotal strategy for improving the delivery of RNA to extrahepatic tissues is to use conjugation chemistries to modify the LNPs and add targeting modalities that facilitate its uptake by specific organs and cells.
由于血脑屏障（BBB）和血肿瘤屏障（BTB）等各种生物物理屏障，RNA 的非肝脏递送可能会很具挑战性，这些屏障都阻止了 RNA 分子从血管进入组织。改善 RNA 递送到非肝脏组织的关键策略之一是使用偶联化化学方法修改 LNPs，并添加有助于其被特定器官和细胞摄取的靶向模块。
One of the most widely used strategies involving conjugation chemistry has come in the form of ligand-mediated delivery – which involves conjugating LNPs to ligands that bind to specific receptors on the surface of cells in the target tissue. This strategy is incredibly advantageous for precise delivery into tumor cells, whose membrane protein expressions are differentiated from healthy targets. Beyond precision oncology, this approach can take advantage of the unique proteomic atlas on each cell types, and thus not only enhance uptake of a certain tissue of interest, but also delivery the right cargo to specific cell types. To date, ligands conjugation has been the main targeting modality for several class of biologics, most notably in CAR-T applications. It is of note that these ligand-conjugated LNPs would still accumulate mainly in the liver, but would enhance tissue-specific uptake. In order to further encourage these ligand-protein interactions, LNPs should stay in circulation for a longer period, and thus changes in the lipid composition should be further optimized. Some examples of this approach include the conjugation of anisamide, BLT1 inhibitor, urea, and Epi-1 among others, to target overexpressed receptors in cancer cells. For instance, LNPs conjugated with the Epi-1 peptide and integrating a monoacyl derivative of PEG can prevent aggregation and allow for higher molar ratios of the peptide, although transport efficiency decreases. Still, Epi-1-modified LNP have showed a substantial silencing effect in an ovarian cancer peritoneal dissemination model, showing that the modified LNP has better features for targeted transport of siRNA to cancer cells. An additional improvement upon systems like this was developed using a nonstandard macrocyclic peptide, which was discovered by a random nonstandard peptide integrated discovery (RaPID) system. The authors developed a liposomal siRNA delivery system with an Epi-1 lipid-derivative to boost cellular absorption in EpCAM-positive cell types by more than 27-fold.
其中一种广泛使用的涉及偶联化学的策略是通过配体介导的递送，即将 LNPs 与配体偶联，这些配体结合到目标组织中的细胞表面的特定受体。这种策略对于精确递送到肿瘤细胞非常有优势，因为肿瘤细胞的膜蛋白表达与健康细胞有所不同。除了精准肿瘤学之外，这种方法可以利用每种细胞类型上的独特蛋白质组图谱，因此不仅增强了对感兴趣的特定组织的摄取，还将正确的载荷传递到特定的细胞类型。迄今为止，配体偶联已成为几类生物制剂的主要靶向模式，尤其是在 CAR-T 应用中。值得注意的是，这些配体偶联的 LNPs 仍然主要积聚在肝脏，但会增强特定组织的摄取。为了进一步促进这些配体-蛋白质相互作用，LNPs 应在循环中停留更长的时间，因此应进一步优化脂质组成的变化。这种方法的一些示例包括将茴香酰胺、BLT1 抑制剂、尿素和 Epi-1 等配体偶联，以靶向癌细胞中过表达的受体。例如，将 Epi-1 肽与 PEG 的单酰衍生物偶联的 LNPs 可以防止聚集并允许更高摩尔比的肽，但递送效率降低。尽管如此，Epi-1 修饰的 LNP 在卵巢癌腹膜播散模型中显示出了显著的沉默效应，表明修改后的 LNP 对 siRNA 以靶向方式运送到癌细胞具有更好的特性。对于这样的系统，进一步的改进是使用非标准的大环肽，这是通过随机非标准肽整合发现（RaPID）系统发现的。作者开发了一种脂质体 siRNA 递送系统，其中包含 Epi-1 脂质衍生物，可以将 EpCAM 阳性细胞中的细胞吸收增加了 27 倍以上。
Furthermore, LNPs encapsulating mRNA have been covalently conjugated with antibodies, specifically with those binding to plasmalemma vesicle-associated protein (PV1), showing a significant increase in delivery of mRNA to the lungs and improved protein expression by 40 times compared to control LNPs. The particular study examined the effect of LNP size on tissue distribution and transfection, finding that larger-size PV1-targeted LNPs are more effective in targeting PV1 expressed in the caveolae and in robust mRNA expression in the lungs. Alternatively, the LNPs-assisted delivery of mRNA-based drugs to T cells has been demonstrated in the past, but the use of nucleoside-modified mRNA encapsulated in LNPs conjugated antibodies appears as a novel platform that enables binding to the CD4 protein found on T cells. This method allows for specific targeting and delivery of the mRNA to T cells, an approach that was tested in mice, resulting in 30-fold higher signal of reporter mRNA in T cells isolated from the spleen, while 40–60 % of CD4+ T cells in spleen and lymph nodes showed specific, dose-dependent genetic recombination. The encapsulation of siRNA inside LNPs conjugated with an antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) has also been reported, with the LNPs modified with Fab’ portions of the antibody to improve their binding affinity to cells that express HB-EGF. This protein is extensively expressed in certain cancers, hence the αHB-EGF LNP-siRNA was highly selective in binding to cells expressing HB-EGF in laboratory testing and efficiently delivered siRNA to these cells, silencing genes. What's more, siRNA has been delivered to lymphatic endothelial cells as a strategy for cancer therapy using LNPs that were rapidly and conveniently modified with antibodies. This method delivered siRNA utilizing antibody-modified LNPs in vitro and in vivo faster and more versatilely than amide coupling.
此外，已经将封装 mRNA 的 LNPs 与抗体共价结合，特别是那些与质膜囊泡相关蛋白（PV1）结合的抗体，显示将 mRNA 递送到肺部的数量显著增加，与对照组的 LNPs 相比，蛋白表达增加了 40 倍。该研究考察了 LNP 大小对组织分布和转染的影响，发现较大尺寸的 PV1 靶向 LNPs 在靶向洞室内 PV1 和肺部的 mRNA 表达方面更为有效。另外，过去已经证明了 LNPs 辅助递送基于 mRNA 的药物到 T 细胞，但是使用封装在 LNPs 中的核苷酸修饰 mRNA 与偶联抗体似乎是一种新颖的平台，它可以结合到 T 细胞上的 CD4 蛋白。这种方法允许将 mRNA 具体靶向和递送到 T 细胞，这种方法在小鼠中进行了测试，结果显示来自脾脏的 T 细胞中的报告 mRNA 信号增加了 30 倍，而脾脏和淋巴结中 40-60%的 CD4+ T 细胞显示出特异性、剂量依赖性的遗传重组。还报告了将 siRNA 封装在与肝素结合表皮生长因子样生长因子（HB-EGF）抗体结合的 LNPs 中，该 LNPs 以提高与表达 HB-EGF 的细胞的结合亲和力的抗体的 Fab'部分进行改性。由于在某些癌症中广泛表达，因此αHB-EGF LNP-siRNA 在实验室测试中高度选择性地与表达 HB-EGF 的细胞结合，并有效地将 siRNA 递送到这些细胞中，沉默基因。此外，siRNA 已经作为一种癌症治疗策略递送到淋巴内皮细胞，使用了快速便捷地修饰了抗体的 LNPs。与酰胺偶联相比，这种方法在体外和体内利用修饰抗体的 LNPs 更快、更多样化地递送 siRNA。
Cell penetrating peptides (CPPs) have also been conjugated to LNPs to improve the delivery of RNA into cells. Novel protamine-derived CPP compounds were employed to make CPP-decorated LNP (CPP-LNP). Encapsulating siRNA in CPP-LNP enhanced its stability in serum, and unlike LNP without CPP, it was readily internalized into B16F10 murine melanoma cells in a time-dependent manner. The study demonstrated that macropinocytosis and heparan sulfate-mediated endocytosis predominantly absorbed siRNA. The CPP-LNP-encapsulated siRNA also silenced reporter genes in B16F10 cells expressing luciferase and HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. Other LNPs-based systems for siRNA delivery, for instance, the one named sTOLP and based on the CPP oleoyl-octaarginine (OA-R8) modified multifunctional LNPs has been tested. sTOLP NPs had excellent gene silencing activity due to their protamine-complexed siRNA core, OA-R8, cationic and PEGylated lipids, and transferrin as a targeting ligand. In vivo experiments showed that the siRNA encapsulated in sTOLP inhibited tumor growth and was preferentially taken up by tumor cells and hepatocytes in a mouse model without causing immunogenicity or toxicity. sTOLP-loaded siRNA circulated more stablely than free siRNA.
细胞穿透肽（CPPs）也已经与 LNPs 结合，以改善 RNA 递送到细胞的效果。采用新型胰蛋白衍生的 CPP 化合物来制备修饰有 CPP 的 LNP（CPP-LNP）。在 CPP-LNP 中封装 siRNA 增强了其在血清中的稳定性，并且与没有 CPP 的 LNP 不同，它以时间依赖的方式迅速内化到 B16F10 小鼠黑色素瘤细胞中。研究表明，巨细胞吞噬作用和肝素硫酸盐介导的内吞作用主要吸收了 siRNA。CPP-LNP 封装的 siRNA 还在表达荧光素酶的 B16F10 细胞和表达增强型绿色荧光蛋白的 HT1080 人类纤维肉瘤细胞中沉默了报告基因。还测试了基于 LNPs 的其他 siRNA 递送系统，例如名为 sTOLP 的系统，该系统基于 CPP 油酸-八精氨酸（OA-R8）修饰的多功能 LNPs。sTOLP NPs 由于其胰蛋白-复合 siRNA 核心、OA-R8、阳离子和 PEG 化脂质以及靶向配体转铁蛋白，因此具有出色的基因沉默活性。体内实验表明，封装在 sTOLP 中的 siRNA 抑制了肿瘤生长，并且在小鼠模型中更倾向于被肿瘤细胞和肝细胞摄取，而不会引起免疫原性或毒性。与游离 siRNA 相比，sTOLP 载荷的 siRNA 循环更稳定。
LNPs can be customized with functional molecules to influence their pharmacokinetics and intracellular fate. pH-responsive linkers allow endosome membrane fusion to release siRNA into the cytoplasm. LNPs can also be functionalized with peptides and saccharides to target certain cells. For instance, recently, a phosphatidylserine (PS)-based LNP that mimics the endocytic activity of phagocytes and cellular entry of enveloped viruses has been created, showing efficient protein expression in both lymph nodes and the spleen after IV administration with a delivery mechanism mediated by monocyte/macrophage cells. Within the same tissue target, an efficient, endogenously lymph node-targeting LNP was developed for the targeted delivery of mRNA vaccines. The authors used a specific LNP formulation called 113-O12B and found that it increased and specifically expressed mRNA in the lymph nodes compared to a synthetic lipid commonly used in mRNA vaccines. This targeted delivery resulted in an increased immune response and improved the protective and therapeutic effects of the mRNA vaccine in a melanoma model. The LNP was also effective in delivering mRNA encoding a tumor-specific peptide and, when combined with anti-PD-1 therapy, resulted in complete tumor response.
LNPs 可以通过功能分子进行定制，以影响它们的药代动力学和细胞内命运。pH 响应性连接子允许内体膜融合，将 siRNA 释放到细胞质中。LNPs 还可以与肽和糖类功能化，以靶向特定的细胞。例如，最近发明了一种基于磷脂酰丝氨酸（PS）的 LNP，模拟噬菌细胞的内吞活性和包膜病毒的细胞入侵，显示经静脉给药后在淋巴结和脾脏中均有高效的蛋白质表达，其递送机制是由单核/巨噬细胞介导的。在相同的组织靶点内，还开发了一种高效的内源性淋巴结靶向 LNP，用于靶向递送 mRNA 疫苗。作者使用了一种特定的 LNP 配方称为 113-O12B，并发现与 mRNA 疫苗中常用的合成脂质相比，它在淋巴结中增加了 mRNA 的特异性表达。这种靶向递送导致了免疫应答的增强，并在黑色素瘤模型中提高了 mRNA 疫苗的保护和治疗效果。该 LNP 还有效地递送了编码肿瘤特异性肽的 mRNA，当与抗 PD-1 治疗联合使用时，导致了完全的肿瘤反应。
The examples mentioned above, as well as others in the past, have allowed researchers to explore the potential of RNA molecules as therapeutic agents for cancer, autoimmune diseases and other disorders. However, as we have discussed, delivering siRNAs beyond the liver remains a significant challenge, as they are rapidly cleared from the circulation by the kidneys and have limited tissue penetration and bioavailability. Although promising, conjugation of targeting moiety presents a novel and poorly characterized pathway, with challenging translational power. The complexity during the manufacturing of conjugated LNPs can often require rigorous quality control and present a regulatory hurdle. However, by understanding the relationship between the structure of RNA conjugates and their pharmacokinetics, researchers hope to develop more effective and selective delivery systems for the treatment of a wide range of diseases. Overall, the use of conjugation chemistries to modify RNA-LNPs should warrant further consideration due to their improved specificity and effectiveness in getting large nucleic acids to their desired locations (Fig. 3).
如上所述的例子以及过去的其他例子，使研究人员能够探索将 RNA 分子作为癌症、自身免疫性疾病和其他疾病的治疗药物的潜力。然而，正如我们所讨论的，将 siRNA 递送到肝脏以外的地方仍然是一个重大挑战，因为它们会被肾脏迅速清除，且组织渗透和生物利用度有限。尽管有希望，但靶向部分的偶联表现出一种新颖且鲜为人知的途径，其具有挑战性的转化潜力。偶联 LNPs 制造过程的复杂性通常需要严格的质量控制并存在法规障碍。然而，通过了解 RNA 偶联物结构与其药代动力学之间的关系，研究人员希望开发更有效和选择性的递送系统，以治疗各种疾病。总的来说，使用偶联化学修饰 RNA-LNPs 应该值得进一步考虑，因为它们在将大型核酸送达到所需位置方面具有改进的特异性和有效性（图 3）。
Fig. 3. (A) Schematic of innovative five-element nanoparticles (FNPs) using helper-polymer poly(β-amino esters) (PBAEs) and DOTAP to achieve high stability and lung targeting. After lyophilization, PBAEs and DOTAP increase hydrophobic force and charge repulsion, resulting in good stability at 4 °C. Systemically given FNPs bind to the αvβ3 receptor on pulmonary endothelial cells via a protein corona adsorbed on FNPs and endogenous vitronectin-enriched; (B) A method to increase small interfering RNA delivery by adding monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into lipid nanoparticles (LNPs) (siRNA). A multipurpose envelope type nanodevice (MEND) customized with an Epi-1 peptide, a ligand with high affinity for the epithelial cell adhesion molecule, was previously produced by the scientists (EpCAM). SiRNA delivery was improved using this peptide-modified MEND. When the ratio of modification was increased to improve cellular absorption, LNP aggregation occurred, especially in large-scale preparations; (C) A schematic is provided showing the components used to generate LNPs and the expected structure of the resulting LNPs; (D) The size distribution of a representative sample of LNPs is presented using dynamic light scattering, showing a diameter of approximately 70 nm. (C) A schematic is also provided showing how these LNPs, when loaded with CAR mRNA, can be used to induce CAR expression in T cells, which then target and kill tumor cells; and (E) sTOLP, a new siRNA delivery technology, uses cell-penetrating peptide oleoyl-octaarginine (OA-R8) modified multifunctional lipid nanoparticles with a protamine-complexed siRNA core, cationic and PEGylated lipids, and transferrin as a targeting ligand.
图 3. （A）创新的五元纳米颗粒（FNPs）示意图，使用辅助聚合物聚（β-氨基酯）（PBAEs）和 DOTAP 实现高稳定性和靶向肺部。在冻干后，PBAEs 和 DOTAP 增加了疏水力和电荷排斥力，导致在 4°C 下保持良好的稳定性。系统给予的 FNPs 通过在 FNPs 上吸附的蛋白质包被和内源性富含维脱诺结合到肺内皮细胞上的αvβ3 受体；（B）通过将单酰基和二酰基基团共轭的聚乙二醇（PEG）添加到脂质纳米颗粒（LNPs）（siRNA）中来增加小干扰 RNA 递送的方法。科学家以前曾制造过定制的多功能信封型纳米器（MEND），其中包括 Epi-1 肽，一种与上皮细胞粘附分子具有高亲和力的配体（EpCAM）。使用这种肽修饰的 MEND 可以改善 siRNA 递送。当增加修饰比例以改善细胞吸收时，在大规模制备中会发生 LNP 聚集；（C）示意图显示了生成 LNPs 所使用的组分以及所产生的 LNPs 的预期结构；（D）使用动态光散射法显示了代表性 LNPs 样品的大小分布，显示直径约为 70 纳米；（E）sTOLP，一种新的 siRNA 递送技术，使用了经油酸八精氨酸（OA-R8）修饰的多功能脂质纳米颗粒，其中包括蛋白质结合的 siRNA 核心，阳离子和 PEG 化脂质，以及转铁蛋白作为靶向配体。
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The current standard for clinical applications for RNA-LNP products still is limited with intravenous and intramuscular delivery. As mentioned, traditional formulation of LNP preferentially target liver tissues after intravenous injection. In contrast, intramuscular delivery resulted in expression at the injection site, with any particles entering the bloodstream accumulating in the liver thereafter. Optimal formulations are not conserved across different route of administration, pointing to different biophysical barriers and cellular uptake efficiency between different method of delivery. In fact, it has been evident that route of administration governs the pharmacological properties of nanoparticles, including circulation time and biodistribution. Such translational hurdles necessitate a separate development of formulation that can optimally perform in the administrative route of interest. However, these findings also suggest the possibility of RNA delivery across several different routes if suitable lipid chemistries were identified.RNA-LNP 产品的临床应用目前仍然受到静脉和肌肉注射的限制。正如前面提到的，传统的 LNP 制剂在静脉注射后首选靶向肝脏组织。相比之下，肌肉注射会导致在注射部位表达，然后进入血液循环的任何颗粒都会在随后积聚在肝脏中。最佳制剂在不同的给药途径之间并不保持一致，这表明不同的给药方法之间存在不同的生物物理障碍和细胞摄取效率。事实上，很明显，给药途径决定了纳米颗粒的药理学特性，包括循环时间和生物分布。这种转化障碍需要单独开发能够在感兴趣的管理途径中最佳执行的制剂。然而，这些发现也暗示了如果找到合适的脂质化学成分，RNA 可以通过几种不同的途径进行递送的可能性。
Local delivery via intramuscular (IM) or subcutaneous (SC) injection is gaining more attention in vaccine applications. IM and SC injection release the RNA-LNPs directly to local tissues at a high concentration, which diffuse and enter cellular cytoplasm. Significantly, the site of injection is in proximity to various lymph nodes, which house immune cells such as dendritic cells and macrophages. These cells either activate innate immune response, or present specific antigens that can recruit other immune cells to enable adaptive immune activation. Continuous effort in optimizing LNP formulations, specifically to mitigate uptake of non-lymphatic cells and prevent systemic circulation, have yielded therapeutic candidates for both infectious diseases and cancers.
通过肌肉注射（IM）或皮下注射（SC）进行局部递送在疫苗应用中越来越受到关注。IM 和 SC 注射会将 RNA-LNPs 直接释放到局部组织中，浓度较高，然后扩散并进入细胞质。值得注意的是，注射部位靠近各种淋巴结，这些淋巴结内有免疫细胞，如树突状细胞和巨噬细胞。这些细胞要么激活先天免疫反应，要么呈递特定抗原，可以招募其他免疫细胞以实现适应性免疫激活。持续努力优化 LNP 制剂，特别是为了减轻非淋巴细胞的摄取并防止全身循环，已经产生了用于传染病和癌症的治疗候选药物。
Delivery of RNA to the lung can be improved by the switch to intranasal route, where nebulized LNPs can enter into the airway directly to reach the lung tissues of interest. However, aside from the distinct biodistribution and cellular heterogeneity compared to systemic delivery, the challenge in producing inhalable, stable and functional LNPs has significantly impacted the progress in this field. LNPs candidate need to be optimized differently than systemically or locally administered formulations, while able to undergo the physical stress that nebulization processes can exert with minimal structural damage. In fact, traditional LNP formulations often becomes unstable and aggregated after nebulization. Fortunately, iterative screening has yielded several candidates that can both survive the nebulization step and overcome physical barriers associated with pulmonary pathway. From these findings, composition and structure of PEG lipid and sterol additives present an important optimization parameter that can enable therapeutic delivery of nebulized LNPs.
通过切换到鼻内途径可以改善 RNA 到达肺部的递送，在这种途径下，雾化的 LNPs 可以直接进入气道，达到感兴趣的肺部组织。然而，除了与全身递送相比的明显生物分布和细胞异质性之外，生产可吸入、稳定和功能性 LNPs 的挑战显著影响了该领域的进展。LNPs 候选药物需要以不同于全身或局部给药制剂的方式进行优化，能够承受雾化过程可能施加的物理压力，同时将结构损伤降至最低。事实上，传统的 LNP 制剂在雾化后往往变得不稳定并聚集。幸运的是，反复筛选已经产生了几个候选药物，既可以在雾化步骤后存活，又能克服与肺途径相关的物理障碍。从这些发现中，PEG 脂质和固醇添加剂的组成和结构成为一个重要的优化参数，可以实现雾化 LNPs 的治疗递送。
As more and more routes of administration are explored for RNA delivery using LNPs, a common theme emerge: parallel development of formulations are necessary for each platform. The interactions and fates of LNPs in different delivery pathways remained limited, and within each pathway, how cellular internalization and cargo release occur is still poorly understood. Thus, iterative screening is still the dominant approach in uncovering novel constructs that efficiently perform for any certain routes of administration. Current advances in utilizing various routes of administration for LNP delivery have enabled RNA to reach several different organs, including the heart, the eyes, the peripheral nervous system and the central nervous system.
随着越来越多的递送途径利用 LNPs 进行 RNA 递送，一个共性问题浮现出来：必须为每个平台并行开发制剂。在不同的递送途径中，LNPs 的相互作用和命运仍然有限，在每个途径内，细胞内摄取和载体释放发生的方式仍然知之甚少。因此，迭代筛选仍然是揭示对任何一种给药途径有效执行的新构建的主要方法。利用各种给药途径进行 LNP 递送的当前进展已经使 RNA 能够抵达多个不同的器官，包括心脏、眼睛、外周神经系统和中枢神经系统。
Targeted delivery can also be achieved through other clinical regimens, including co-injections or sequential injections with other modalities. These modalities either lower the physical barriers that the nanoparticles have to cross, or favorably target the organ of interest. Research in these approaches are at their infancy, but it's worth mentioning due to the burgeoning understanding of different mechanism of actions of uptake and barriers across different tissues. For instance, the blood-brain-barrier presents a significant challenge for any meaningful delivery to the central nervous system from intravenous injections. However, by utilizing microbubbles, in combination with focus ultrasound, the blood-brain-barrier was opened in a targeted manner, allowing LNPs carrying cargo to infiltrate the space. However, these openings are still small, which only allows nanoparticles of certain sizes to cross. This limitation on the nanoparticles' size can heavily influence the PEG composition, and the RNA loading of LNPs, leading to potential reduction in efficacy. On the other hand, detargeting the cargo's expression in the liver can lead to preferential targeting elsewhere, as detailed by the work of Sago et al. Additionally, by attacking components involved in LNP trafficking, further enhancement can be made to selectively deliver cargo to specific tissues. Unfortunately, the clinical translations for these approaches remain complex, hindering progress in the field.
通过其他临床方案，包括与其他疗法的联合注射或顺序注射，也可以实现有针对性的递送。这些疗法要么降低了纳米颗粒必须穿越的物理屏障，要么有利地靶向了感兴趣的器官。这些方法的研究正处于初级阶段，但值得一提，因为对不同组织中的各种摄取机制和屏障的机制有了更加深入的了解。例如，血脑屏障对于从静脉注射到中枢神经系统的任何有意义的递送都构成了重大挑战。然而，通过利用微泡，结合聚焦超声，可以有针对性地打开血脑屏障，允许携带载荷的 LNPs 渗透进入其中。然而，这些开口仍然很小，只允许特定大小的纳米颗粒穿越。这种对纳米颗粒大小的限制可能会严重影响 PEG 组成和 LNPs 的 RNA 装载，从而可能降低疗效。另一方面，通过抵消与 LNP 运输有关的组分，可以进一步增强对特定组织的选择性递送。不幸的是，这些方法的临床应用仍然复杂，阻碍了该领域的进展。
Overall, by using various clinical administration of RNA-LNP drug product, extrahepatic delivery has been demonstrated. However, some platforms of delivery either require harsh processing of LNPs or invasive procedures on the patients. Therefore, while promising, further understanding of tissue-specific uptake mechanisms, particles' stability and targeting strategies are necessary to enable precise delivery of RNA.
总的来说，通过使用 RNA-LNP 药物产品的各种临床途径，已经实现了肝外递送。然而，一些递送平台要么需要对 LNPs 进行严格的处理，要么需要对患者进行侵入性程序。因此，尽管有很多前景，但为了实现 RNA 的精确递送，仍然需要进一步了解特定组织摄取机制、颗粒稳定性和靶向策略。
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This review summarizes the current strategies for enabling RNA-carrying LNPs to reach extrahepatic environments. By tuning the internal lipid chemistry, LNPs can possess a wide range of physicochemical properties, including size and pKa values, and thus drive plasma protein adsorption and regulate intracellular fate. By conjugating known targeting modality, LNPs can preferentially bind to specified cell types that activate ligand-protein interactions and enable selective uptake. Lastly, by changing the point of entry, LNPs can obtain differentiated biodistribution and pharmacological profiles, thus enabling them to reach hard-to-target organs. The future of targeted RNA therapy will most likely incorporate one, if not multiple, of these strategies. However, while it is important to celebrate the progress made, it is equally as important to acknowledge the hurdles that still need to be overcome. First, the discoveries of new formulations are challenging due to the lack of clinically relevant models in vitro. Therefore, screening of LNPs either occurs slowly and iteratively in animals or risks translational barriers in cells. The lack of in vitro model has hindered the development of new formulations, and the development of more relevant models will be crucial to study the mechanistic pathways as well as to enhance LNP discovery processes. Additionally, the biology of LNP trafficking is still poorly understood, including the biodistribution, internalization and functional delivery of LNPs in clinical settings. The lack of such understanding limits rational designs of LNP formulations, and thus result in inefficient trial-and-error approach. Lastly, complicated delivery methods present huge risks to the quality of the final products, jeopardizing manufacturability and regulatory path to market.
本综述总结了使携带 RNA 的 LNPs 能够到达肝外环境的当前策略。通过调整内部脂质化学成分，LNPs 可以具有各种物理化学特性，包括大小和 pKa 值，从而驱动血浆蛋白吸附并调控细胞内命运。通过连接已知的靶向模块，LNPs 可以优先结合到指定的细胞类型，激活配体-蛋白相互作用并实现选择性摄取。最后，通过改变进入点，LNPs 可以获得不同的生物分布和药理特性，从而使其能够到达难以靶向的器官。有针对性的 RNA 疗法的未来很可能会采用这些策略中的一个，如果不是多个策略。然而，尽管庆祝取得的进展很重要，但同样重要的是要承认仍然需要克服的障碍。首先，由于体外缺乏临床相关模型，新配方的发现具有挑战性。因此，LNPs 的筛选在动物体内要么进行缓慢而迭代，要么在细胞中面临翻译障碍的风险。缺乏体外模型已经阻碍了新配方的开发，因此开发更相关的模型将对研究机制途径以及增强 LNP 发现过程至关重要。此外，LNPs 运输的生物学机制仍然知之甚少，包括在临床环境中的生物分布、内吞和功能递送。这种缺乏理解限制了 LNP 配方的合理设计，从而导致低效的试错方法。最后，复杂的递送方法对最终产品的质量构成巨大风险，从而危及了可制造性和市场监管途径。
Therefore, to fully enable the extrahepatic targeting of LNPs, and the subsequent potential of RNA therapy, new advances must be made in both cellular biology, material chemistries, manufacturing technologies, and regulatory advocacy. While genetic modification has reached the point of precise engineering, the delivery has not been able to be programmable quite yet. Therefore, unlocking this bottleneck is crucial to advancing precision medicine in general, and genetic medicine in particular. This review, therefore, hopes to serve as a useful tool for the next generation of research in this field by discussing and addressing the above-mentioned issues.
因此，要完全实现 LNPs 的肝外靶向，以及 RNA 疗法的潜力，必须在细胞生物学、材料化学、制造技术和监管倡导方面取得新的进展。尽管基因修饰已经达到了精确工程的程度，但递送尚不能被精确控制。因此，突破这一瓶颈对于推动精准医学总体，特别是遗传医学，至关重要。因此，本综述希望通过讨论和解决上述问题，为这一领域的下一代研究提供一个有用的工具。
