Optimizing the targeting of lipid nanoparticles for gene therapy

Abstract

Gene drugs based on nucleic acid molecules have shown great potential in the treatment of various diseases. Lipid nanoparticles (LNPs) are currently the most advanced carriers for delivering nucleic acids. However, gene therapy fails to meet the clinical needs of organs other than the liver due to accumulation in the liver. Precise delivery of nucleic acids to specific target organs and target cells has become a key challenge in bringing gene therapy to the clinic. In this review, we present the typical composition and targeting properties of LNPs. Then we systematically describe the strategies and research progress to optimize the targeting properties of LNPs from three perspectives: surface modification, formulation optimization, and novel lipid molecule design. This review will further inspire researchers to rationally design targeted LNPs to advance the development of gene therapy.

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Lei Yue

Lei Yue obtained her M.E. degree from Tianjin University, China, in 2025. Her research focuses on the application of peptides and lipid nanoparticles in the field of drug delivery.

Xiulei Gao

Xiulei Gao is currently a master's student in chemical engineering at Tianjin University. She received her B.S. degree from Tianjin University in 2022. Her research interests focus on the modification, screening, and delivery applications of lipid nanoparticles.

Wei Qi

Wei Qi obtained her PhD degree in Chemical Engineering from Tianjin University in 2002 and was a visiting scholar at the University of Illinois at Urbana-Champaign, USA, from 2012 to 2013. She has been a Professor of Chemical and Biochemical Engineering since 2009 and is now the director of the Education Quality Management Office, at Tianjin University. Her present research interest involves the R&D of bio-chemicals, biofuels, and biomaterials via biocatalysis and industrial catalysis.

Lvhong Zhang

Lvhong Zhang obtained her PhD degree from Tianjin University, China, in 1998. She is now a professor at the School of Chemical Engineering and Technology, Tianjin University. She is also a member of the Stirring Technology Expert Committee at the National Chemical Engineering Design Technology Centre Station. Her primary research interests encompass oil production, refining, mass transfer and separation in chemical engineering, and computational fluid dynamics.

Yuefei Wang

Yuefei Wang obtained his PhD degree from Tianjin University, China, in 2015, and worked as a visiting scholar in Prof. Nicholas A. Kotov's lab at the University of Michigan, Ann Arbor, USA, from 2018 to 2019. He is now a professor and assistant dean of the School of Chemical Engineering and Technology, as well as the deputy director of the Chemical Engineering Research Center at Tianjin University. Additionally, he is a principal investigator at the state key laboratory of chemical engineering and low-carbon technology. His research interests mainly focus on the synthesis of biomimetic materials and their applications in medicine, sensing, and catalysis.

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1. Introduction

Gene therapy mainly includes delivery of small interfering RNA (siRNA) to inhibit the expression of disease-causing genes, delivery of messenger RNA (mRNA) to induce the production of therapeutic proteins,¹ and correction of disease-causing mutations through gene editing techniques such as CRISPR-Cas9.² In addition, preclinical studies also use other types of nucleic acids, such as antisense oligonucleotides (ASOs), microRNA, and DNA. In gene therapy, to avoid the degradation of naked nucleic acids by nuclease enzymes during in vivo transport, the development of effective delivery systems is crucial. Currently, delivery vectors are categorized into viral and non-viral vectors. Viral vectors, such as adenoviruses, retroviruses, and lentiviruses, occupied an important position in early gene therapy research due to their excellent gene delivery efficiency. However, viral vectors have limitations such as limited capacity, immunogenicity, and complexity of preparation, which restrict their clinical applications.³ Non-viral vectors, such as lipid nanoparticles (LNPs), polymers, exosomes, and peptides, are gaining attention because of their lower immunogenicity, broad applicability, and low production costs.⁴

Among non-viral vectors, LNPs are the most intensively researched and clinically advanced gene-drug delivery technology with low cytotoxicity, high modifiability, and ease of large-scale production. LNPs have been applied in siRNA drugs^5,6 and COVID-19 (Coronavirus Disease 2019) mRNA vaccines^7,8 and have shown great potential for development in cancer therapeutic vaccines in clinical trials. However, LNPs have inherent liver tropism, which severely limits the targeting and treatment outside the liver.⁹ In order to achieve therapeutic purposes and avoid widespread distribution problems, overcoming liver cell accumulation and delivering nucleic acids to specific target organs or cells has become a key challenge. At present, through library screening and reasonable design, many LNPs that can target different organs or cells have been obtained.

Although several excellent reviews have examined the history of LNPs,¹⁰ there remains a gap in the literature concerning articles that systematically focus on the critical challenge of “targeting optimization”. In this paper, the targeting optimization strategies of LNPs for gene therapy are reviewed. Firstly, we introduced the composition and targeting properties of LNPs, and then discussed the strategy and mechanism of LNPs targeting optimization, as well as the latest research progress. LNPs targeting strategies include (1) surface modification (antibodies, peptides, small molecular sugars, aptamers, etc.) (2) formulation optimization (adding/replacing/simplifying components) (3) design of new lipid molecules (four lipid types) (Scheme 1). Finally, the research and application prospects of LNPs targeting for gene therapy are prospected. We hope this review will inspire the design of targeted LNPs in the future and promote the development of precision gene therapy.

Scheme 1 Optimization of Lipid nanoparticles targeting by three strategies: surface modification, formulation optimization, and design of novel lipid molecules.

2. Lipid nanoparticles

In-depth investigation and continuous optimization of the targeting properties of LNPs are not only crucial to enhance their efficacy and reduce their risks, but also an important direction for the advancement of the entire gene therapy field.¹¹ The unique composition of LNPs offers the possibility of precise delivery of therapeutic nucleic acids to specific cells or tissues. An in-depth understanding of the composition and targeting properties of LNPs is crucial to facilitate the development of targeted LNPs.

2.1. Typical formulations

Typical LNPs consist of four types of lipids: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-derived lipids (PEG-lipids) (Fig. 1). The multiple components of LNPs confer more diversity to LNPs. In addition to key ionizable lipids, other lipid components play important roles in altering the nucleic acid encapsulation, cellular interactions, and biodistribution properties of LNPs.¹²

Fig. 1 Typical components of lipid nanoparticles. Reprinted from ref. 4, with permission from Nature, Copyright 2021.

2.1.1. Ionizable lipids. Permanently charged cationic lipids (e.g., 1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) were initially used to configure LNPs. However, due to the high toxicity resulting from the net positive charge, there has been a shift toward designing ionizable lipids with less toxicity. Ionizable lipids are the most critical components of LNPs and largely determine the stability, delivery efficiency, targeting, and safety of LNPs.¹³ In the marketed formulations of siRNA drugs and mRNA vaccines, all of them have unique ionizable lipids, while the other three components are almost the same.¹⁴ In 2018, DLin-MC3-DMA was used to formulate Onpattro (patisiran), which is the first siRNA drug approved by the U.S. Food and Drug Administration (FDA).^5,6 Two years later, ionizable SM-102 and ALC-0315 configured as COVID-19 (Coronavirus Disease 2019) vaccines mRNA-12737⁷ and BNT162b28⁸ also reached the clinic. In addition, representative ionizable lipid structures are A18-Iso5-2DC18,¹⁵ A6,¹⁶ and 306Oi10.¹⁷

Under acidic manufacturing conditions, ionizable lipids are positively charged by protonation, thereby complexing and encapsulating negatively charged nucleic acids. At physiological pH, ionizable lipids have a near-neutral charge, thus avoiding charge-related toxicity and improving pharmacokinetic properties. Upon cellular uptake, they become charged in the acidic endosomal compartment, which promotes endosomal escape for nucleic acid release. pK_a values (pK_a of the ionizable lipids likely at the LNPs surface) affect the ability of ionizable lipids to deliver gene drugs in vivo.¹⁸ The most effective apparent pK_a values for intrahepatic siRNA delivery¹⁹ and intramuscular mRNA vaccines²⁰ range from 6.2–6.5 and 6.6–6.9.

Ionizable lipids usually consist of an amino head and an alkyl tail connected through a connecting bond.²¹ The structure of the head group, the connecting bond, and the tail of ionizable lipids are all key factors affecting the properties of LNPs. Small structural differences can dramatically improve the performance of LNPs.²² Common ionizable lipids contain single or multiple head groups such as amines (primary, secondary, tertiary, quaternary), guanidines, heterocyclic groups, and combinations thereof. Commonly used linking groups are amide, ester, and ether bonds. Among them, biodegradable ester bonds, as the preferred moiety, have been used in the design of ionizable lipids SM 102, ALC-0315, and Dlin-MC3-DMA, showing better safety.²³ The tail usually consists of 1–4 hydrophobic alkyl chains containing 8–20 carbon atoms, and its structure affects the pK_a value and mobility of ionizable lipids.²⁴ To accelerate the development of ionizable lipids, large lipidoid libraries are often tested using combinatorial chemistry to generate efficient lipid structures. The structural characteristics of ionizable lipids with good delivery ability have been proposed through batch library screening, which provides a reference for rational design.²⁵

2.1.2. Phosphatidylinositol. Phospholipids have hydrophilic heads and hydrophobic tails, can spontaneously form lipid bilayers, and have high phase transition temperatures, so they help stabilize the structure of LNPs.²⁶ In addition, phospholipids also play a role in endosomal escape.²⁷ Structural differences in phospholipids alter the stability,²⁸ delivery efficiency,²⁹ and biodistribution³⁰ of LNPs. Commonly used natural phospholipids include 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC).³¹ It is also possible to chemically synthesize phospholipids to precisely control the physicochemical properties of LNPs. Currently, commercial LNPs formulations contain only 10 mol% DSPC.

For synthetic lipidoids materials, saturated DSPC is suitable for delivery of short RNAs (siRNAs).³² The unsaturated DOPE is more suitable for mRNA delivery. Better mRNA delivery efficiency has been achieved by replacing DSPC with DOPE in many studies of lipidoids.^29,31,33

2.1.3. Polyethylene glycol derived lipids (PEG-lipids). PEG-lipids consist of polyethylene glycol (PEG) conjugated to an anchoring lipid. Although PEG-lipids make up the smallest molar percentage of lipid composition in LNPs (typically about 1.5 mol%), almost all LNPs contain a PEG-lipid component.³⁴ The content of PEG-lipids, the structure and length of PEG-chains and lipid tails determine the properties of PEG-lipids. When PEG-lipids are incorporated into the LNPs, hydrophilic PEG chains stretch over the surface of the LNPs, creating a spatial site-blocking effect that makes it difficult for particles to approach and aggregate with each other, enhancing the dispersion and stability of LNPs.³⁵ After entering the blood circulation, PEG-lipids formed a hydrophilic “protective barrier” on the surface of LNPs, reducing the adsorption of serum proteins. This reduces the rapid clearance of LNPs by the mononuclear phagocyte system (MPS) and prolongs the circulation time. PEG-lipids also affect the properties of nucleic acid encapsulation, transfection efficiency and in vivo distribution.

However, a biocompatible obstacle to the use of PEG-lipids is that repeated administration of PEGylated LNPs triggers phenomena such as hypersensitivity reactions (activation of the complement system) and accelerated blood clearance.³⁶ Researchers are actively exploring the use of PEG-alternative materials (e.g., polysarcosine^37,38), cleavable PEG,³⁹ and branched approaches⁴⁰ to address these issues.

2.1.4. Cholesterol. Cholesterol is a biologically essential molecule that is critical for maintaining cell membrane integrity. Cholesterol accounts for a high percentage of LNPs, and its structure may affect the function of LNPs, such as promoting membrane fusion, affecting intracellular transport,⁴¹ and increasing endosomal escape.⁴² Modification of cholesterol or use of cholesterol analogs has been shown to be important for efficient in vivo delivery.⁴³ LNPs such as β-sitosterol substituted for cholesterol have shown improved mRNA transfection capacity.^44,45 In addition, reports have emphasized the importance of the stereospecificity of cholesterol modifiers in LNPs-mRNA delivery.⁴⁶ Among other things, the sterically pure form reduces the likelihood of mRNA being sorted into the phagocytic pathway, thereby improving functional mRNA delivery.

2.2. Targeting properties of LNPs

Achieving target-specific delivery of LNPs is a complex and challenging process that requires consideration of barriers at the organ, tissue, and cellular levels. For example, the blood–brain barrier (BBB) poses a significant challenge to the delivery of nucleic acids from LNPs to the central nervous system (CNS). The tissue and cellular environments also have a significant impact on the target-specific delivery of LNPs. Local routes of administration such as intravesical administration⁴⁷ and intraperitoneal administration,⁴⁸ although effective in overcoming some of the barriers to systemic administration, are limited in their applicability, and invasive methods of administration may also lead to adverse reactions in local tissues. In contrast, systemic drug delivery offers the possibility of treating more sites of disease, especially delivery to the brain and heart. In order to develop effective systemic delivery gene therapies, the targeting optimization of LNPs is particularly critical.

2.2.1. Liver targeting. The liver is one of the most accessible organs for LNPs. Onpattro, the most successful liver-targeted LNPs, relies on circulating apolipoprotein E (ApoE) adsorbed on the surface of the LNPs to form a protein corona, which binds to low-density lipoprotein receptors (LDLR) on the surface of the hepatocytes, resulting in a highly specific hepatic targeting (Fig. 2A).^5,6 Currently widely used formulations of LNPs were originally developed with the liver as the target organ, such as those based on the ionizable lipids cKK-E12,⁴⁹ C12-200,⁵⁰ and SM-102.⁵¹

Fig. 2 (A) Adsorption of ApoE to the nanoparticle surface results in the binding of the nanoparticle by low-density lipoprotein receptor (LDL-R), highly expressed by hepatocytes and subsequent endocytosis. Reprinted from ref. 57, with permission from Nature, Copyright 2023. (B) The nanoparticle–corona complex interacts with a cell membrane receptor. Reprinted from ref. 56, with permission from Nature, Copyright 2012.

Hepatocytes make up the majority of the liver, which also contains Kupffer cells (KCs), lymphocytes, and liver sinusoidal endothelial cells (LSECs), and these types of cells are also targets of interest for gene therapy. Targeting via the ApoE-LDLR pathway produces high uptake in hepatocytes but limits the delivery of LNPs to other cells within the liver.⁵² With the progress of research, the targeting strategy of LNPs has gradually shifted from the organ level to the cellular level, and there has been a trend toward achieving more precise targeting. In addition to focusing on the overall hepatic uptake efficiency of LNPs, attention needs to be paid to the delivery of LNPs specifically to other cells within the liver.

2.2.2. Extrahepatic targeting. Targeted delivery of nucleic acid drugs to the liver has been clinically effective, however, hepatic accumulation poses a significant challenge to achieve delivery outside the liver. In order to advance the widespread use of LNPs in specific diseases, there is an urgent need to develop new strategies for delivery to organs other than the liver, such as the lungs, spleen, lymph nodes, brain, heart, and bone marrow.⁵³ To date, the lungs and secondary lymphoid organs (SLOs) are the two extrahepatic organs most successfully targeted by LNPs. The SLOs, which include the spleen and lymph nodes, are key sites for immune cell activation and initiation of immune responses. Precise delivery of gene drugs to immune cells in the SLO can effectively activate or modulate immune responses, enabling immunotherapy against cancer or autoimmune diseases as well as in vivo immune cell engineering. Although some progress has been made in the development of LNPs targeting SLOs, these strategies often come at the cost of reduced efficacy.^54,55 The need for LNPs that are both SLO-targeting and efficient has become increasingly urgent. The lung, as an important organ of the respiratory system, is a lesion area for a variety of diseases, such as lung cancer, pulmonary fibrosis, and cystic fibrosis. Inhalation is the most direct way to achieve lung targeting. However, selective delivery to the lungs also faces the unique challenges of alveolar epithelial cells and mucus barriers. Designing LNPs to be able to specifically target the lungs could further improve the precision of gene therapy.

2.2.3. Strategies to improve targeting. Surface modification is a common strategy to improve the targeting of LNPs. It achieves selective binding to target cells by adding molecules that can recognize specific receptors or markers on the surface of LNPs, such as antibodies, peptides, glycans, etc. Although this strategy can improve the targeting of LNPs, it still faces some technical and biosafety challenges. Therefore, in addition to the addition of exogenous targeting ligands, there is an urgent need to develop more efficient, safe, and economical targeting strategies, including formulation optimization of LNPs and design of lipid structure.

During circulation, LNPs interact with specific serum proteins to form a unique protein corona that hinders or promotes delivery to the liver (Fig. 2B). The mechanism of protein corona acting as an endogenous targeting ligand to mediate the specific distribution of LNPs is complex, but the high-abundance proteins contained in protein corona may play an important role. Since the physical and chemical properties of LNPs determine the composition of the protein corona adsorbed to the surface in the cycle,⁵⁶ researchers have achieved endogenous targeting by optimizing the LNPs formulation (adding/replacing/reducing components) and changing the lipid chemical structure.^57,58 The latter strategy is challenging to adjust targeting because the lipid structure-targeting relationship has not been well elucidated.

2.3. The regulatory role of protein corona in LNP targeting

Upon exposure to biological fluids, lipid nanoparticles are rapidly covered by a layer of adsorbed biomolecules, forming a protein corona that dynamically alters their physicochemical identity and ultimately dictates their in vivo fate. The formation of the protein corona is driven by multiple non-covalent interactions, including electrostatic attraction, hydrophobic association, and hydrogen bonding, which collectively reshape the surface characteristics of LNPs and modulate their targeting ability.^56,59

2.3.1. Impact of the protein corona on targeting. The protein corona exerts a dual regulatory effect on LNP targeting behavior. On one hand, it can mask exogenous ligands (e.g., antibodies, peptides, aptamers) on the nanoparticle surface, thereby diminishing receptor recognition and reducing active targeting efficiency.⁶⁰ On the other hand, it can act as an endogenous targeting mediator, conferring new biological identities to the nanoparticles. For example, apolipoprotein E (ApoE) adsorption on LNP surfaces enables their recognition by the LDLR on hepatocytes, thereby facilitating liver-specific accumulation and uptake—an effect underlying the success of Onpattro, the first FDA-approved siRNA LNP drug.⁶¹ Thus, the protein corona functions not merely as a passive barrier but as a dynamic regulator that may either hinder or promote gene delivery depending on its molecular composition and biological context.

2.3.2. Roles of the protein corona in different targeting strategies. The protein corona exerts distinct regulatory effects on LNP targeting depending on the design strategy adopted.

In surface modification, it mainly functions as a passive modulator that influences nanoparticle–cell interactions. Hydrophilic coatings such as PEG or zwitterionic polymers reduce nonspecific adsorption and extend circulation time; however, protein adsorption remains unavoidable. The adsorbed layer, typically enriched with albumin or complement proteins, can alter biodistribution and partially mask targeting ligands, thereby diminishing receptor-specific interactions. Consequently, while surface modification aims to minimize corona formation, the residual corona still defines the biological identity and in vivo fate of LNPs.

In formulation optimization, the protein corona can be deliberately utilized for controlled protein recruitment to enhance organ selectivity. Modulating lipid composition—such as the structure and pK_a of ionizable lipids or the ratio of helper lipids—affects the adsorption of plasma proteins like ApoE, which mediate receptor-dependent uptake in specific tissues. For example, the clinically approved LNP formulation in Onpattro achieves liver targeting through ApoE-mediated LDLR recognition.⁶¹ Thus, formulation optimization represents a strategy that harnesses, rather than suppresses, the protein corona to guide endogenous targeting.

Overall, these two approaches highlight the dual role of the protein corona in gene therapy–oriented LNPs: as both a potential obstacle to exogenous targeting and a natural mediator of selective delivery. Understanding and controlling corona formation is therefore critical for rational LNP design and precise nucleic acid delivery.

3. Surface modification of LNPs

Surface modification strategies are active targeting strategies by which LNPs can be precisely localized to target cells. Targeting molecules (e.g., antibodies, antibody fragments, peptides, glycans, aptamers) are able to interact specifically with target cell surface molecules through ligand-receptor or antibody-antigen mediated interactions.⁶² PEG-lipids containing reactive groups in the PEG tail are currently the most commonly used strategy for anchoring targeting ligands to lipids.⁶³

3.1. Antibody modification

Antibodies have high specificity and affinity to accurately recognize and bind to antigens on the surface of target cells, and there has been an increasing number of studies using antibodies to achieve targeting of LNPs in specific cells and tissues.

3.1.1. Targeting stem cells and immune cells. Antibody modifications can be used to target stem cells with LNPs. CD117 is widely present on the surface of hematopoietic stem cells (HSCs) and their progenitors,⁶⁴ and linking anti-CD117 antibodies to LNPs greatly enhances the efficiency of delivery to hematopoietic stem cells in the bone marrow of mice, enabling in vivo reprogramming of bone marrow stem cells.⁶⁵

Antibodies have also been widely used to mediate the targeting of immune cells by LNPs. CD5 is naturally expressed by T cells and a small subset of B cells, and anti-CD5 antibody-conjugated LNPs can target T cells and transiently generate chimeric antigen-receptor T cells for the treatment of cardiac injury in mice (Fig. 3A).⁶⁶ Similarly, coupling anti-CD4 monoclonal antibodies to LNPs can specifically deliver mRNA⁶⁷ and siRNA⁶⁸ to CD4+ T cells. Anti-CD3-conjugated LNPs can transfect T cells in situ.⁶⁹ β7 integrins are highly expressed in intestinal mononuclear leukocytes.⁷⁰ Covalent attachment of monoclonal antibodies against β7 integrins to hyaluronic acid on the outer surface of LNPs enables specific targeting of intestinal mononuclear leukocytes.⁷¹

Fig. 3 (A) Schematic illustration of the molecular process to create transient FAPCAR T cells using CD5-targeted LNPs. Reprinted from ref. 66, with permission from The American Association for the Advancement of Science, Copyright 2022. (B) Schematic illustration of ASSET incorporation into LNPs, binding to RIg, and targeting. (1) Target cell membrane; (2) targeted cell-surface receptor; (3) encapsulated siRNA; (4) LNPs; (5) ASSET anchored in the LNPs; (6) RIg; (7) interaction between ASSET scFv and RIg Fc. Reprinted from ref. 72, with permission from Nature, Copyright 2018. (C) Schematic illustrations of microfluidic preparation of αPV1 LNPs with lung targeting capability and luciferase mRNA expression in mouse tissues. Reprinted from ref. 80, with permission from American Chemical Society, Copyright 2020.

The complex chemical coupling of antibodies makes rapid screening for effective antibody ligands challenging. To develop antibody-modified LNPs more efficiently, a self-assembled modular cellular targeting platform called ASSET (Anchored Secondary scFv Enabled Targeting) has been proposed (Fig. 3B).⁷² Membrane-anchored lipoproteins (ASSET) were inserted into LNPs, and non-covalent encapsulation of targeting antibodies on the surface of LNPs was achieved through their binding to the Fc structural domain of antibodies. Customizable targeting of different cells can be achieved by simply replacing the antibody, avoiding complex chemical coupling and recalibration. Meanwhile, the high affinity of the antibody in the nanoparticles is preserved. An ASSET strategy has been used to encapsulate anti-Ly6c monoclonal antibodies to LNPs and deliver modified messenger RNA (mmRNA) specifically to Ly6c⁺ inflammatory leukocytes, achieving reduced protein expression in the liver and increased expression at sites of inflammation.⁷³

3.1.2. Targeting cancer cells. Cancer-specific surface markers can be used for targeting LNPs. For example, epidermal growth factor receptor (EGFR) is highly expressed in tumor cells. In an example of the ASSET strategy, gene therapy targeting human ovarian carcinoma cells was achieved with anti-EGFR encapsulated LNPs.⁷⁴ Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is highly expressed in various cancer cell lines and plays a key role in the development of malignant phenotypes such as tumorigenesis, invasion, metastasis, and chemoresistance.⁷⁵ Advanced tumor-targeted delivery of siRNA was achieved by modifying LNPs with Fab′ fragments of anti-HB-EGF monoclonal antibodies.⁷⁶

Antibodies have also been used to modify LNPs to target B-cell malignancy cells, which are resistant to conventional transfection reagents and are dispersed in vivo.⁷⁷ CD 38 is an overexpressed glycoprotein in common B-cell malignancy cells such as multiple myeloma (MM) cells and mantle cell lymphoma (MCL) cells. Targeted LNPs encapsulating siRNA and coated with anti-CD 38 antibodies achieved specific binding to MM cells⁷⁸ and MCL cells⁷⁹ in bone marrow (BM), demonstrating the potential of CD 38 as a target for LNPs to precisely target B-cell malignancies.

3.1.3. Targeting other cells and tissues. Antibody modifications have enabled lung-targeted nucleic acid delivery. Covalent coupling of antibodies that bind plasma membrane vesicle-associated protein, a niche-associated protein, to the surface of LNPs has been used to improve lung-targeted mRNA delivery in vivo by facilitating specific niche-associated protein-mediated endocytosis (Fig. 3C).⁸⁰ Platelet endothelial cell adhesion molecule (PECAM-1) is primarily expressed by endothelial cells, and coupling of PEG-lipids with anti-PECAM-1 enabled ApoE pathway-independent lung endothelial mRNA delivery.⁸¹ There have also been some advances in brain targeting of antibody-modified LNPs. Insulin-like growth factor II (IGF-II) receptor is expressed on brain microvascular endothelial cells, and brain-targeted delivery of plasmids was achieved by conjugating IGF-II monoclonal antibodies to liposomes.⁸²

Antibodies and their fragments as highly specific ligands that mediate targeting have been widely used as a strategy for targeted delivery of nucleic acids by LNPs, showing great potential in targeted delivery of LNPs.

3.2. Peptide modification

Antibodies' immunogenicity, protease sensitivity, large size, complex preparation process, and high production cost hinder their use. Peptide-functionalized targeted LNPs have attracted increasing interest due to the smaller peptides that can easily penetrate tissues to reach target cells and have lower immunogenicity and production costs. Targeted peptides are often acquired in the protein-binding region and can also be identified by phage display technology. Cell-penetrating peptides can promote cell membrane permeation, which may help LNPs to cross the cell membrane directly into the cell interior.

Peptides are able to specifically bind to receptors on the surface of target cells or tissues, thus conferring targeting properties to LNPs. Arg-Gly-Asp (RGD) peptides that bind tumor cell surface overexpression of αvβ3 integrin have been used to construct tumor-targeted LNPs.^83,84 Suga's group identified the targeting macrocyclic peptide Epi-1, which has a high affinity for Epithelial Cell Adhesion Molecule (EpCAM).⁸⁵ Subsequently, Epi-1 was affixed to the head of PEG-DSPE to anchor it to the LNPs surface, dramatically improving siRNA cellular uptake (∼30-fold) in EpCAM-positive cell lines.⁸⁶ GALA (WEAALAEALAEALAEALAEHLAEALAEALEALAAEALA) is a synthetic peptide that specifically binds to plectin receptors on the surface of lung endothelial cells, and also functions to promote endosomal escape.⁸⁷ GALA as a lung-targeting ligand affixed to LNPs has been applied to pulmonary endothelial pDNA delivery in vivo.⁸⁸

In addition, the researchers further applied the targeting strategy to other more challenging organs or cell types, such as the neural retina and the brain. Delivery of LNPs to the neural retina is limited, especially for the photoreceptor (PR). Targeting this key cell type, Herrera-Barrera et al. identified the peptide MH42, which can be targeted to the neural retina, by phage display technology. Attaching it to the surface of LNPs developed the first peptide-directed LNPs capable of efficiently delivering mRNA to the PR (Fig. 4A).⁸⁹ Delivery of nucleic acids to the brain remains a major challenge due to the BBB. Using click chemistry, LNPs functionalized with the peptide RVG 29 (YTIWMPENPRPGTPCDIFTNSRGKRASNGC), which targets the neuronal overexpression of the nicotinic acetylcholine receptor, were able to improve mRNA transfection in neurons and facilitate targeted delivery to the brain after systemic administration (Fig. 4B). Such peptide-functionalized LNPs provide a promising strategy for systemic nucleic acid delivery targeted to the brain.⁹⁰

Fig. 4 (A) Schematic illustration of LNPs formulation and conjugation with peptide via maleimide-thiol chemistry and Cre mouse model depicting both routes of administration trialed. Reprinted from ref. 89, with permission from Science, Copyright 2023. (B) The process of engineering and validating targeted LNPs for brain delivery following systemic administration and ex vivo imaging of luciferase mRNA transfection in the brain in adult C57BL/6 mice. Reprinted from ref. 90, with permission from American Chemical Society, Copyright 2024.

3.3. Other ligands

Other widely used ligands also show potential for targeted delivery by LNPs. Mannose receptors are widely present on a variety of immune cells, including macrophages, dendritic cells (DCs), and LSECs. The modification of mannose ligands on the surface of LNPs enhanced the GFP mRNA targeting efficiency of LSECs (Fig. 5A).^91,92 In addition, studies have also targeted mannose modified on LNPs specifically to hepatic macrophages and dendritic cells. In addition, studies have also shown that mannose-modified LNPs specifically target hepatic macrophages⁹³ and dendritic cells.⁹⁴

Fig. 5 (A) Schematic of mannose-modified LNPs formulation and in vivo and ex vivo IVIS images following IV injection of LNPs that deliver reporter-encoding GFP mRNA. Reprinted from ref. 92, with permission from American Chemical Society, Copyright 2023. (B) N-Acetylgalactosamine (GalNAc) binds asialoglycoprotein receptor 1 (ASGR1) to facilitate nanoparticle uptake by hepatocytes. Reprinted from ref. 57, with permission from Nature, Copyright 2023.

N-Acetylgalactosamine (GalNAc)-targeted ligands can bind to the hepatocyte surface-specific desialylate glycoprotein receptor (ASGPR), which makes the GalNAc-LNPs approach an alternative strategy for ApoE/LDLR-mediated hepatic uptake of LNPs (Fig. 5B).⁹⁵ GalNAc functionalization has enabled liver-targeted delivery of LNPs.⁹⁶ GalNAc-siRNA and GalNAc-ASO affixes have been used in the clinic.^97,98

Aptamers are short sequences of single-stranded (ss) DNA or RNA that bind specific target molecules. Functionalization of LNPs with the osteoblast-targeting aptamer CH6 enhanced osteoblast-specific siRNA delivery in human and rat osteoblasts, elevating the selectivity of osteoblast-targeted delivery from the tissue level to the cellular level.⁹⁹

Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan that has been shown to play a key role in cell growth and migration, embryonic development, inflammatory responses, and neoangiogenesis.¹⁰⁰ As a natural ligand for the CD 44 receptor, which is overexpressed in cancer cells, HA-encapsulated LNPs have been realized in vivo for siRNA-specific delivery to glioblastoma multiforme (GBM) cells¹⁰¹ and ovarian cancer cells.¹⁰²

Transferrin receptor (TfR) expression is upregulated in proliferating cells, including most tumor cells, and transferrin (Tf) can be taken up by cells via TfR-mediated endocytosis. Tf-conjugated LNPs have enabled the selective delivery of antisense oligodeoxynucleotides (ODNs) to leukemia cells.¹⁰³ In addition, TfR is considered to be the most common target for drug delivery to the brain.¹⁰⁴ Sharma et al. designed Tf and cell penetrating peptide (CPP) bi-ligand modified liposomes to enhance pDNA accumulation and transfection in the brain.¹⁰⁵

Although surface modification is a simple approach, surface-modified LNPs are usually not designed to avoid hepatic delivery, but rather to improve delivery to other organs or cell types. Moreover, surface modification strategies require additional purification steps, increasing the complexity of preparing LNPs and leading to product lot-to-lot variation and high costs. Finally, some ligands may also cause immune responses or be potentially toxic, requiring rigorous safety evaluation. Therefore, this approach has not yet progressed in clinical trials.¹⁰⁶ In the future, advanced technologies such as phage display and high-throughput screening should be utilized to discover more ligands with specific binding abilities, broaden the targeting applicability of surface-modified LNPs, and improve their selectivity and stability.

4. Optimization of LNPs formulation

Although exogenous active targeting strategies with surface modifications are effective, the protein corona formed by LNPs in the circulation may override the targeting ligand and reduce targeting function.^107,108 Therefore, there is a need to develop more reliable ligand-independent targeting strategies. While traditional formulations of LNPs contain four essential lipid components, researchers have explored formulations that go beyond the classical four-component formulation by adding additional components/replacement components/reducing components for targeting of LNPs. This approach is much simpler than introducing surface modifications, and the streamlined component strategy in particular achieves targeting while also reducing formulation complexity and safety risks.

4.1. SORT strategy

Optimizing the ratio of lipids to RNA (charge ratio) can affect targeting, suggesting that the nature of the surface charge of LNPs is an important factor influencing their tissue distribution (Fig. 6A).¹⁰⁹ An important strategy for adding additional components called selective organ targeting (SORT) has been reported, establishing a modular and versatile new approach for organ-targeted delivery of LNPs (Fig. 6B).^54,55 The addition of a fifth component (the SORT molecule) to the pre-existing LNPs assemblies without disrupting the core four-component ratio modulates the charge of LNPs and the composition of the circulating protein corona, resulting in attenuation of hepatic accumulation and precise delivery of mRNAs to the spleen or lungs.¹¹⁰

Fig. 6 (A) Bioluminescence imaging of BALB/c mice after i.v. injection of Luc-LPX at various charge ratios. Pie charts show the relative contribution of each organ to the total signal. Reprinted from ref. 109, with permission from Nature, Copyright 2016. (B) The addition of a supplemental component (termed a SORT molecule) to traditional LNPs systematically alters the in vivo delivery profile and mediates tissue-specific delivery as a function of the percentage and biophysical property of the SORT molecule. This methodology successfully redirected multiple classes of nanoparticles. Reprinted from ref. 54, with permission from Nature, Copyright 2020. (C) The top five most abundant plasma proteins that bind different SORT LNPs. Reprinted from ref. 111, with permission from National Academy of Sciences, Copyright 2021. (D) Reprinted from ref. 117 with permission from Nature, Copyright 2024.

4.1.1. Targeting mechanism. SORT LNPs act in vivo through endogenous targeting mechanisms of PEG-lipid desorption, binding of different serum proteins to exposed SORT molecules, and binding of surface-bound proteins to corresponding receptors highly expressed in specific tissues. Different organ-targeted SORT LNPs have a unique protein corona composition (Fig. 6C).¹¹¹ Liver-targeted SORT LNPs have a recognized pK_a in the range of 6 to 7, and the most abundant protein in the protein corona is ApoE, consistent with the known role of ApoE in hepatic delivery. SORT LNPs doped with cationic lipids (e.g., DOTAP) have a high apparent pK_a (>9), and the protein corona composition of such LNPs is enriched in vitronectin (Vtn), which facilitates receptor-mediated endocytosis through the αvβ3 integrin highly expressed in the lung endothelium and contributes to lung-specific targeting.^112–114 Whereas SORT LNPs doped with anionic lipids (e.g., 1,2-dioleoyl-sn-glycero-3-phosphate, 18PA) have smaller apparent pK_a values (2–6), have a β2-glycoprotein I (an anionic phospholipid binding protein) protein corona that facilitates mRNA delivery to the spleen.^110,115

4.1.2. Diversity of SORT molecules. The chemical composition and ratios of SORT molecules can be customized, and initial lung-specific LNPs were implemented using the quaternary ammonium lipid DOTAP as a SORT molecule. The group later tested novel cationic quaternary ammonium lipids as SORT molecules, showing that changes in the chemical structure of the lipids slightly altered the presence and amount of the protein corona component of the LNP, thereby affecting the specificity of organ delivery.¹¹⁶ Recently, it was found that an amidine-incorporated degradable (AID) lipid 12T-O14 could also be used as a complementary cationic lipid to develop SORT LNPs similar to those previously reported. The addition of 12T-O14 to MC3 LNPs formulations can redirect hepatic-enriched LNPs to the lungs or spleen by simply adjusting the ratio of lipids to nucleic acids in the formulation to change the surface charge of the LNPs (Fig. 6D).¹¹⁷

The SORT approach adds complexity to the formulation of LNPs, and a four-component approach that simply replaces phospholipids with permanently charged lipids has been proposed to achieve a SORT-like targeting effect.^118,119 At elevated auxiliary lipid concentrations, the use of amphipathic phospholipids facilitates delivery of mRNA in the liver, negatively charged phospholipids shift delivery efficiency to the spleen, and the use of positively charged phospholipids delivers primarily to the lungs.^120,121

4.1.3. Challenges of the SORT strategy and component design. Unfortunately, the strategy of adding cationic lipids has toxicity issues and has shown potential limitations in future clinical translation.^122,123 Recent studies have found that positively charged SORT LNPs with pulmonary orientation induce massive thrombosis.¹²⁴ This side effect is universal and is independent of the type of ionizable or cationic lipids. Therefore, the design of novel lipids must balance efficacy with safety. Future directions include: (1) biodegradability: designing lipid molecules with readily degradable bonds (such as ester bonds or double bonds) enables rapid metabolism within the body after completing delivery tasks, thereby reducing long-term accumulation and toxicity. (2) Structure-activity relationship optimisation: precisely modulating pK_a values, membrane fusion capabilities, and biocompatibility by adjusting the structure of lipid head groups, linkers, and hydrophobic tails. This maintains neutrality at physiological pH, minimising non-specific interactions with non-target cells.

Secondly, the disconnect between high-throughput screening and in vivo application poses a significant challenge. SORT technology is primarily conducted in vitro (e.g., microfluidic chips, droplet screening),^54,55 where the “optimal” lipids or formulations identified may lose their targeting efficacy in complex in vivo environments due to protein corona formation. Abundant plasma proteins in blood adsorb onto LNP surfaces, forming a protein corona. This outer layer obscures or interferes with surface targeting ligands, impairing binding to target cell receptors.¹¹⁶ Future lipid composition design must account for in vivo interactions with plasma proteins. This challenge may be circumvented by designing surfaces with anti-protein adsorption properties (e.g., optimising PEGylation degree) or by utilising endogenous proteins (such as apolipoprotein E, ApoE) as “endogenous targeting ligands”. For instance, certain lipid components can specifically bind to ApoE, thereby leveraging its affinity for the low-density lipoprotein (LDL) receptor on hepatocytes to achieve liver targeting.

Beyond this, the paramount challenge remains achieving precise targeting to non-hepatic organs. Regardless of the technology employed, LNPs predominantly accumulate in the liver and spleen following intravenous administration. Whilst SORT technology can screen for lipids with potential non-hepatic targeting capabilities, achieving efficient and specific delivery to target organs or cell types such as the lungs, kidneys, heart, or brain remains a formidable challenge. Future lipid design will increasingly emphasise multifunctionality and programmability. This may involve: (1) multicomponent synergy: designing complex LNP formulations comprising multiple lipid components, each performing a specific role (e.g., one lipid responsible for nucleic acid encapsulation and endosomal escape, another for specific cell receptor recognition).¹¹¹ (2) Smart responsive lipids: designing “smart” lipids capable of responding to specific microenvironments (such as the low pH or high enzyme concentrations of tumour tissue) to enable active release and targeting. For example, altering surface charge in acidic tumour microenvironments to enhance cellular uptake.

4.2. Addition/replacement of phospholipids

The surface of LNPs is rich in phospholipids. The strategy of adding/replacing other types of phospholipids can also achieve targeted delivery of LNPs.

4.2.1. Electrically charged phospholipids. The SORT strategy led to a higher accumulation of mRNA in the spleen but resulted in reduced protein expression.^54,109 An anionic phosphatidylserine (PS) molecule, known to promote phagocytic endocytosis activity and enveloped virus entry into cells, was added to a standard four-component formulation of MC3-LNPs. PS acts as an “eat-me” signal for phagocytes by binding to scavenger receptors on the cell surface, facilitating monocyte/macrophage-mediated target delivery of LNPs to the spleen and superficial cervical lymph nodes (SCLN). Notably, this strategy surpassed the currently used charge-driven targeting principle in terms of both target organ accumulation and protein expression.¹²⁵ Subsequently, the strategy of replacing phosphatidylcholine (PC) with PS also enhanced the specific uptake in the spleen of LNPs, supporting this mechanism (Fig. 7A).¹²⁶

Fig. 7 (A) Schematic illustration of the strategy for delivering messenger RNA (mRNA) to secondary lymphoid tissues by phosphatidylserine-loaded lipid nanoparticles (LNPs). Reprinted from ref. 126, with permission from Wiley-VCH, Copyright 2023. (B) Within the liver sinusoids, switching of the helper phospholipid from zwitterionic DSPC to anionic DSPG created anionic srLNPs that are directed to the hepatic RES, via stabilin-receptor-mediated recognition and uptake in LSECs. srLNPs uptake within hepatic RES cells is further enhanced by the inhibition of apoE–LDLr interactions mediated by anionic phospholipids (e.g., DSPG). Reprinted from ref. 127, with permission from Wiley-VCH, Copyright 2022.

The approach of replacing charged phospholipids allows targeting other cell types in addition to the lung and spleen in the intrahepatic delivery of LNPs. Conversion of Onpattro's phospholipids from DSPC to its anionic analog 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) has been engineered to preferentially target anionic LNPs in the hepatic reticuloendothelial system (RES). This strategy is supported by the charge-dependent stabilizing-mediated liver mechanism of LNPs recognition and uptake within LSECs, which redirects LNPs targeted delivery from hepatocytes to the hepatic RES while inhibiting hepatocyte ApoE-LDLR interactions (Fig. 7B).¹²⁷

4.2.2. Neutral phospholipid. In another study, the targeting of LNPs was improved by the addition of a high percentage (40%) of a naturally occurring biocompatible lipid, eggsphingomyelin (ESM). The distribution of these nanoparticles in the spleen and bone marrow was enhanced after intravenous injection.^128,129 This finding provides a new idea for the addition of non-toxic lipids to achieve extrahepatic tissue delivery of RNA.

The selection of suitable neutral auxiliary lipids also provides ideas for the targeted delivery of LNPs.¹³⁰ Several studies have demonstrated that DOPE or DSPC as phospholipids of LNPs promote the accumulation of LNPs in the liver and spleen, respectively.^33,130 This may be due to the fact that the different phospholipids significantly altered the composition of the protein corona on the surface of LNPs. DOPE-LNPs have a stronger affinity for serum ApoE and promote hepatocyte uptake of LNPs through the ApoE-LDLR pathway.

4.3. Streamlined formulations

To achieve targeted delivery of LNPs, their formulations are often increasingly complex. Yet simpler formulations are often safer and easier to translate in the clinic. Many researchers have attempted to streamline the formulation of LNPs to achieve precise targeting of tissues, organs, or cells, but how to maintain the stability and delivery efficiency of LNPs while simplifying the formulations is a major challenge.

4.3.1. Three-component. Su et al. innovatively found that some intrinsic components of LNPs, such as cholesterol and phospholipids, are not essential for delivery and lead to unavoidable hepatic accumulation. For this reason, three-component (3-Comp) LNPs consisting of ionizable cationic lipids/permanent cationic lipids/PEG-lipids were innovatively designed for enhanced lung delivery (Fig. 8A). The simplified 3-Comp LNPs retained the functionality of the LNPs while leading to reduced lipoprotein encapsulation compared with their 4- or 5-Comp counterparts, achieving veritable lung targeting (accumulation and conversion) after systemic administration. Replacing different permanent cationic lipids or different ionizable lipids also preserved the same lung targeting, showing great potential for development.¹³¹ Similarly, Fei et al. removed DSPC and cholesterol from the LNPs system and designed a three-component LNPs platform containing only ionizable cationic lipids, PEG-DMG, and targeting lipids by system optimization. The three-component formulation enabled targeted mRNA delivery to the lung, liver, and spleen, providing a good balance of organ specificity, efficacy, and stability (Fig. 8B). Compared to the four-/five-component, the three-component formulation also circumvents to some extent the problem of mRNA leakage targeting other tissues and organs. This strategy greatly simplifies the optimization process of LNPs and shows great potential in the development of improved targeting strategies for LNPs.¹³²

Fig. 8 (A) Schematic and organ images of 3-Comp nAcx-Cm/permanently cationic lipid/PEG-lipid LNPs (cholesterol- and phospholipid-free) mediating high lung-targeted mRNA delivery. Reprinted from ref. 131, with permission from Nature, Copyright 2024. (B) Simplified targeted LNPs (stLNPs), comprising an ionizable cationic lipid, PEG-DMG, and a targeting lipid. Introduction of DOTAP, 18PA, or cholesterol as the targeting lipids to stLNPs achieved lung-, spleen-, and liver-targeted mRNA delivery, respectively. Reprinted from ref. 132 with permission from Wiley-VCH, Copyright 2024. (C) Cationic lipid/mRNA nanoparticles (CLNs) were formulated, addressing the extrahepatic challenge by conventional LNPs and enabling spleen-specific mRNA delivery post intravenous (IV) administration. Reprinted from ref. 134, with permission from Wiley-VCH, Copyright 2024.

4.3.2. One-component. Simplified nucleic acid delivery systems for LNPs are evolving.¹³³ Recently, Liu et al. reported an ionizable cationic lipid enriched with secondary amines that can be used as a stand-alone carrier to specifically deliver mRNA to spleen and T cells (Fig. 8C). This finding provides a facile alternative for the delivery of LNPs to extrahepatic tissues. Surprisingly, the two-component LNPs formulated with PEG-doped lipids instead promoted effective lung targeting. This shift in organ targeting may be attributed to the formation of the protein corona, reflecting the important synergistic role of helper lipids in targeting regulation.¹³⁴

The selection of appropriate formulations is crucial for the optimization of LNPs targeting. Although significant progress has been made in optimizing the formulation of LNPs, many challenges remain, such as the toxicity of the strategy of adding cationic lipids, and the problem of the unknown specific mechanism of protein corona in the targeting of LNPs. Formulation optimization approaches with lean components have good targeting efficacy and low safety risks. However, this type of research is still in its infancy, and more in-depth studies are needed to fully explore the potential for clinical development. In order to develop more clinically valuable formulations for targeting LNPs, multiple factors such as targeting, stability, delivery efficiency, and safety need to be considered.

5. Design of novel lipid molecules

The structure of lipids has a significant impact on their interaction with cell membranes and subsequent endocytosis processes, and specific chemical structures may allow them to interact with specific components of the cell surface to facilitate targeted delivery. By analyzing the relationship between lipid structure and targeting properties, the traits of ionizable lipids for in vivo targeted delivery can be increased, providing a theoretical basis for the rational design of subsequently targeted LNPs (Table 1).

Table 1 Summary of the structure and targeting properties of novel lipid molecules designed

LNP component	Target organ or cell	Key structural features	Mechanism	Advantages	Disadvantages	Representative molecules
Ionizable Lipid	Secondary lymphoid organs (T cells, lymph nodes, splenic Immune cells)	Cyclic structure^15 (e.g., imidazole, adamantane)	Screening of an imidazole-based lipid library revealed a strong preference for the spleen;^22,135 the unique conformational state of adamantane influences protein interactions and lipophilicity, though the underlying mechanism remains unclear^32	Achieves specific targeting shift from the liver to the spleen	Mechanism remains to be elucidated	93-O17S,^22 A3B7C2^135
		Polyamine core	Provides more headgroup diversity and structural variability, enabling efficient mRNA delivery.^136,137	Enables efficient mRNA delivery to human T cells with lymph node specificity, significantly reducing accumulation in non-target tissues.	—	C14-4,^138 113-O12B,^139 OF-Deg-Lin,^142 OF-C4-Deg-Lin^143
		Introduction of negative charges	Incorporating negatively charged sulfonate groups and amine headgroup–alkyl tail linkages onto a cyclic siloxane structure imparts negative surface charge to LNPs	Enables spleen-specific targeting	Negatively charged phospholipids introduced have poor solubility and are difficult to incorporate	Si12-C10^144
	Lung (endothelial cells, epithelial cells)	Connecting bond variation (ester^145 vs. amide bond)	Alters the composition of the adsorbed protein corona—from apolipoprotein E (ApoE) to fibrinogen—thereby changing targeting specificity^146	Enables targeting redirection simply by modifying the chemical linker structure	The generality of this strategy requires further investigation	306-O12B, 306-N16B,^146 SiLNPs^144
		Novel head groups (e.g., sulfonium,^148 crown ether^155)	Thiol-based lipids possess cationic properties and high positive charge density; crown ether lipids utilize metal ion coordination to modulate targeting behavior	Enables efficient lung targeting, including hard-to-transfect pulmonary epithelial cells	Mechanism requires further clarification	sLNPs,^148 CBILs^155
	Other organs or cells (e.g., aHSC, brain, bone)	—	Novel lipids (e.g., CL15A6) identified through lipid library screening or designed using existing targeting mechanisms—such as BBB transcytosis,^157 calcium coordination in bone, or ROS-responsive degradation in tumors	Allows highly selective delivery to specific pathological cells	Structural–targeting correlations remain unpredictable and require further exploration	CL15A6,^156 NT-lipidoids,^158 BP-LNPs^159,160
Phosphatidylinositol	Liver (short chain) vs. Spleen (long chain)	Short (C9–C12) vs. long (C13–C16) hydrocarbon tails	The hydrophobic tail length of lipids affects mRNA organ tropism	Can be applied in SORT screening to achieve multi-organ targeting in combination with different helper lipids	—	iPhos^164
PEG-Lipids	Extrahepatic (no specific organ or cell)	High molecular weight and long tail length	Controlling the desorption rate of PEG from the LNP surface:^169,170 short-chain PEG detaches rapidly to enhance hepatocyte uptake, while long-chain PEG detaches slowly to extend circulation time and promote accumulation in other tissues	Extends circulation time, increases delivery probability to target tissues, and serves as a scaffold for ligand conjugation	The “PEG dilemma” persists^172—extended circulation often reduces cellular uptake efficiency	PEG-DSPE^171
	Lymph nodes, spleen	Branched PEG or analogous structures	Branched lipid structures enable high lymph node–specific transfection	Provides a feasible approach for lymph node targeting	—	Tween 20, tween 80^173
Cholesterol and its analogs	Liver endothelial cells, kupffer cells, T cells, B cells	Structural oxidation,^176 esterification,^177 bile acids^178	Modifies the composition of the adsorbed protein corona, thus altering targeting specificity; bile acid derivatives may regulate targeting via combined effects of charge, protein corona remodeling, and morphological changes	Enables targeting optimization simply by selecting cholesterol variants	Underlying mechanisms remain to be clarified	20α-OH^176

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5.1. Ionizable lipid

As the most important component of LNPs, the structural modules of ionizable lipids drive targeting specificity. Novel ionizable lipids can be obtained by tuning amine heads, hydrophobic tails, linkers and other functional groups. Researchers have used combinatorial chemistry to rapidly generate large lipid libraries to identify key ionizable lipid structures that can be used for specific delivery.

5.1.1. Targeting secondary lymphoid organs (SLO).
Cyclic structure. The selective liver-to-spleen organ transfer through the structural design of ionizable lipids has achieved some encouraging results. Ionizable lipid structures containing a cyclic moiety exhibit targeted delivery of nucleic acids to secondary lymphoid organs.¹⁵ Ionizable lipids containing imidazole moieties (IMILs) were screened by the lipid library to show a clear preference for spleen in mRNA transfection. Highly efficient transfection of splenic T lymphocytes²² and splenic dendritic cells¹³⁵ (Fig. 9A) has been achieved with IMIL 93-O17S and A3B7C2-based LNPs, respectively. In addition, adamantane is a caged saturated hydrocarbon, and the conformationally bound structure of the adamantyl group has unique properties that affect protein interactions and lipophilicity. Ionizable lipids containing adamantyl tails can deliver siRNA and sgRNA to splenic T cells due to the unique conformational state, but the mechanism of this endogenous transport pathway needs to be further investigated (Fig. 9B).³²

Fig. 9 (A) High-throughput screening identified IMIL A3B7C2 as a candidate with efficient spleen transfection (high IST) coupled with robust spleen specificity (high PST). Reprinted from ref. 135, with permission from American Chemical Society, Copyright 2024. (B) Constrained ionizable lipids can be formulated with cholesterol, lipid-PEG, and DSPC to form stable “constrained LNPs” (cLNPs). Reprinted from ref. 32, with permission from Wiley-VCH, Copyright 2019. (C) OF-Deg-Lin, a bis-lysine-derived ionizable lipid that forms LNPs targeting the spleen. Reprinted from ref. 142, with permission from Wiley-VCH, Copyright 2017. (D) Cyclic siloxane structure Si12 with multiple reaction sites promotes spleen-specific delivery after attaching negatively charged sulfonic group and amine head-alkyl tails. Reprinted from ref. 144, with permission from Nature, Copyright 2024.

Polyamine core. Ionizable lipids containing multiple amines provide more head groups compared to monoamine lipids, offer higher variability, and show good prospects for targeting development.^136,137 By library screening, saturated ionizable lipid C14-4 LNPs containing polyamine nuclei could efficiently deliver mRNA into primary human T cells and induce chimeric antigen receptor (CAR) expression.¹³⁸ Lymph node-specific targeting of polyamine nuclear LNPs 113-O12B can specifically deliver mRNA to lymph nodes to generate robust CD8+ T cell responses and significantly reduce accumulation in off-target tissues.¹³⁹

Piperazine-derived ionizable lipids (Pi-Lipids) were prepared as LNPs that can target hepatic and splenic immune cells in vivo.¹⁴⁰ Fenton et al. developed ionizable lipids with a diketopiperazine core.¹⁴¹ Among them, the diketopiperazine-containing ionizable lipid OF-Deg-Lin, consisting of four ester bonds and four unsaturated tails, mediated protein expression in B lymphocytes (Fig. 9C). By comparison with structural analogs, it was hypothesized that it was the electrophilic ester bond in OF-Deg-Lin that was more readily degraded in the liver than in other organs, resulting in a unique biodistribution profile.¹⁴² Subsequently, the group altered the joint length of OF-Deg-Lin to obtain OF-C4-Deg-Lin, which induced protein expression primarily in the spleen.¹⁴³

Introduction of negative charges. There is a strong correlation between negative charge and lymphoid organ targeting. In the SORT strategy, the additional negatively charged phospholipids introduced are difficult to incorporate into LNPs due to poor solubility. In a recent study, spleen-specific LNPs Si12-C10 configured with siloxane-incorporated lipidoids were designed by concurrently attaching negatively charged sulfonic group and amine head-alkyl tails to the cyclic siloxane structure to impart a negative charge to the LNPs (Fig. 9D).¹⁴⁴

5.1.2. Targeting lung.
Connecting bond. Differences in the junction portion of ionizable lipids greatly affect the in vivo distribution of LNPs. When the tail portion of ionizable lipids contains an ester bond, LNPs typically exhibit hepatic targeting.¹⁴⁵ Qiu et al. reported that changing the tail junction of lipidoid from an ester bond (called the O-series) to an amide bond (called the N-series) shifted the delivery specificity from the liver to the lungs. The highest enriched protein corona of these two classes of LNPs is albumin. However, the second most abundant protein in the N-series changed from ApoE to fibrinogen beta chain and fibrinogen gamma chain compared to the O-series (Fig. 10A).¹⁴⁶ This lung-targeting approach led to a different composition of the protein corona than the lung-targeted SORT LNPs, reflecting the importance of elucidating the target-driven mechanism of the protein corona. Similar conclusions were made in siloxane-incorporated lipidoids-configured LNPs (SiLNPs). Lipidoids with epoxide/ester tails transport mRNA to the liver, whereas lipidoids with amide tails can deliver mRNA to the lungs. In lung-targeted SiLNPs, vitronectin, which binds to the homodimeric receptor αvβ3 integrin that is highly expressed in the lung endothelium, was identified as the most highly enriched protein in the protein corona, which provides a plausible explanation for the lung-targeting mechanism. Notably, this is the first report of liver, lung, and spleen targeting achieved by simply changing the structure of lipidoids in a single lipid library (Fig. 10B).¹⁴⁴ This strategy of changing the structure of the connecting bond is critical for the development of lung-targeted ionizable lipids. However, the versatility of this strategy deserves further investigation.¹⁴⁷

Fig. 10 (A) Schematic illustration of different organ targetability of O- and N-series LNPs, interaction of LNPs with proteins in the blood vessel, and quantification of percentage of total proteins of the top three protein components in the protein corona of the 306-O12B LNPs and the 306-N16B LNPs. Reprinted from ref. 146, with permission from National Academy of Sciences, Copyright 2022. (B) SiLNPs were formulated with a siloxane-incorporated lipidoid, helper lipid (DOPE), cholesterol, and PEG-lipid (C14PEG2K). The resulting SiLNPs with different siloxane-incorporated lipidoid structures mediate in vivo tissue-specific mRNA delivery to the liver, lungs, and spleen. Reprinted from ref. 144, with permission from Nature, Copyright 2024.

Novel head groups. In addition to the common amine-based lipids, a lung-targeted delivery of LNPs by sLNPs constructed from sulfonium-based lipids has recently been reported, where the sulfonium lipids have a cationic nature and a high positive charge density (Fig. 11A).¹⁴⁸ Notably, the most abundant protein in the protein corona of sLNPs was fibrinogen alpha chain, a result that, together with the composition of the protein corona of the N-series of LNPs reported by Qiu et al.,¹⁴⁶ demonstrates the critical role of fibrinogen in lung targeting. Further studies on the role of fibrinogen in lung targeting of LNPs and its interaction mechanism with lung cells will provide a more theoretical basis for the treatment of lung diseases.^149,150 Alpha-1-antitrypsin (AAT) is also one of the major protein types enriched in the protein corona of lung-targeted sLNPs. It is also well worth investigating given its protective function in the lung and the fact that it can be internalized by lung endothelial cells through endocytosis.¹⁵¹ In addition to this, apolipoproteins, fibronectin, vitronectin, and complement proteins are shared among lung-targeted LNPs and their role cannot be ignored.^{111,144,152,153}

Fig. 11 (A) Schematic illustration of the chemical structures of sulfonium lipids (DHSEH and DOSEH), the targeted delivery of mRNA-loaded sLNPs (mRNA/sLNPs) to the lungs in mice and top 10 most abundant proteins on sLNPs protein corona. Reprinted from ref. 148, with permission from American Chemical Society, Copyright 2024. (B) Crown-like biodegradable ionizable lipids (CBILs) utilize metal-ligand chemistry to deliver mRNA to the lung and enable magnetic and bioluminescent dual-mode imaging of early lung cancer. Reprinted from ref. 155, with permission from American Chemical Society, Copyright 2024.

Although some progress has been made in attenuating liver accumulation and delivering nucleic acids to the lungs, LNPs in these studies have primarily targeted lung endothelial cells.^17,146 Lung epithelial cells, the primary site of most lung diseases including cystic fibrosis and non-small cell lung cancer, have been understudied in the field of cell-specific targeted delivery.¹⁵⁴ Recently, a novel crown ether-like biodegradable ionizable lipids (CBILs) were developed. CBILs utilize metal ion coordination to modulate the safe and effective targeting of LNPs to the lungs in vivo and outperform current state-of-the-art LNPs in lung epithelial cell delivery (Fig. 11B).¹⁵⁵

5.1.3. Targeting other organs or cells. Activated hepatic stellate cells (aHSC) are the main target for the treatment of liver fibrosis. Younis et al. reported the first platform of LNPs for selective delivery of mRNA to aHSC by library screening of the novel ionizable lipid CL15A6.¹⁵⁶

Delivery of nucleic acids to the brain using LNPs has been a major challenge due to the blood–brain barrier (BBB). Through library screening of amine headgroups, effective ionizable lipids have been identified to cross the BBB via adsorption-mediated transcytosis for brain tumor-targeted siRNA delivery and glioblastoma immunotherapy (Fig. 12A).¹⁵⁷ Taking advantage of the inherent ability of some neurotransmitters (NT) to cross the BBB, Ma et al. constructed neurotransmitter-derived lipidoids (NT-lipidoids) based on tryptamine (NT1), phenylethylamine (NT2), and phenylethanolamine (NT3) (Fig. 12B). Doping NT-lipidoids into lipidoid 306-O12B-3, which can efficiently deliver ASOs, endowed LNPs with the ability to traverse the BBB, providing a new strategy for the treatment of CNS disorders.¹⁵⁸ Utilizing a similar approach, Xue et al. combined the widely used bone-targeting ligand bisphosphonate (BP) conjugated to the amine core of ionizable lipids to construct BP-LNPs, which are used to achieve mRNA-specific bone targeting by ligating with calcium ions in the bone microenvironment (Fig. 12C).^159,160

Fig. 12 (A) Structure of BAMPA-O16B, an ionizable cationic lipid for brain tumor-targeted siRNA delivery and glioblastoma immunotherapy. (B) Structure of neurotransmitter-derived lipidoids (NT-lipidoids) NT1-O14B. (C) BP-LNPs coordinated with calcium ions (Ca²⁺) in the bone microenvironment to enable specific bone targeting. Ex vivo luminescence and fluorescence imaging of bones after delivery of LNPs encapsulating luciferase-encoding mRNA, where LNPs are labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR). Reprinted from ref. 160, with permission from American Chemical Society, Copyright 2022.

Selective delivery of nucleic acids to disease cells is also a major challenge. Taking advantage of the up-regulated reactive oxygen species (ROS) levels in cancer cells, Cai et al. integrated ROS-degradable thioketal motifs into lipid structures for selective mRNA delivery to tumor cells. Upon the entry of LNPs into tumor cells, abundant ROS triggered the oxidation and degradation of the thioketal structures, thereby promoting the release and selective expression of mRNA in tumor cells.¹⁶¹

In summary, a number of structure-specific ionizable lipids with targeted delivery potential have been screened by lipid libraries. However, targeting mechanisms based on structural alterations have been less elucidated, and how the structural characteristics of ionizable lipids affect their ability to target cells and organs still needs to be further explored.

5.2. Phosphatidylinositol

While most preclinical studies have evaluated how the structure of ionizable lipids affects delivery, auxiliary lipids can also influence the surface properties of LNPs and thus the biodistribution of LNPs.^162,163 To overcome the inflexibility of conventional phospholipids, ionizable phospholipids (iPhos) have been designed by integrating the advantages of ionizable amines and multiple alkyl chains into the phospholipid structure. The length of the iPhos hydrophobic tail affects the organ selectivity of the mRNA. Among them, short-chain (9–12 carbons) iPhos phospholipids are more likely to function in the liver, whereas long-chain (13–16 carbons) iPhos phospholipids are more likely to function in the spleen (Fig. 13A). Encouragingly, iPhos can also be used for SORT, synergizing with different types of auxiliary lipids to achieve spleen, liver, and lung targeting.¹⁶⁴ Conventional LNPs are usually formulated using phospholipids containing unconstrained alkyl tails. In another study, “constrained LNPs” containing adamantyl-constrained phospholipids have been developed to deliver mRNA to liver immune cells in vivo.¹⁶⁵

Fig. 13 (A) IPhos with a phosphoryl side alkyl chain length of 9–12 had the highest mRNA expression efficiency in the liver, and 13–16 had the highest mRNA expression efficiency in the spleen. Reprinted from ref. 164, with permission from Nature, Copyright 2021. (B) Structure of PEG-DSPE, tween 80 and tween 20. (C) Formulation of LNPs with 20α-OH cholesterol. Reprinted from ref. 176, with permission from Wiley-VCH, Copyright 2019. (D) Formulation of LNPs with Cholesteryl Oleate. Reprinted from ref. 177, with permission from American Chemical Society, Copyright 2018.

5.3. PEG-lipids

The addition of PEG-lipids is considered to be a “double-edged sword”:¹⁶⁶ PEG-lipids can prolong the circulation time of LNPs in vivo, allowing more opportunities for LNPs to reach the target tissues or cells to play a role in the accumulation of LNPs at the disease site. However, the hydrophilic coating formed by PEG-lipids may interfere with the interaction of LNPs with the cell surface and affect the efficiency of cellular uptake of LNPs.^167,168 The rate of PEG desorption from LNPs can be controlled by varying the molecular weight and tail length of the PEG-lipid. The larger molecular weight of PEG-lipids and longer lipid tail lengths result in longer cycling times and reduced cellular uptake of LNPs.^169,170 The short acyl chain PEG-lipids used in the Onpattro formulation allow for rapid dissociation of PEG and easier interaction of LNPs with hepatocytes, promoting the uptake of LNPs in hepatocytes. In contrast, long acyl chain PEG-lipids such as PEG-DSPE slow the desorption of PEG, which interferes with and reduces specific uptake in the liver and further prolongs the circulation time, enhancing the accumulation of LNPs in other tissues.^169,171 PEG-lipid improves extrahepatic accumulation while reducing cell uptake activity, which is called “PEG-dilemma”.¹⁷² Therefore, the choice of PEG-lipid type depends on the type of organ and cell targeted.

Polyethylene glycolization of LNPs with branched PEG is a viable method to achieve targeting. Replacing PEG-DSPE with tween 20 composed of saturated carbon tails with a branched PEG-like structure, the LNPs formed showed highly specific transfection in lymph nodes. In contrast, tween 80 with a longer tail formed LNPs targeting the spleen, but with lower efficiency (Fig. 13B).¹⁷³ In addition, PEG-lipids often serve as scaffolds for targeting ligands, and specific antibodies,⁷⁶ peptides,^90,174 or glycans^91,175 have been linked to PEG-lipids to direct LNPs for targeted delivery.

5.4. Cholesterol and its analogs

The chemical structure of cholesterol is an important factor influencing the targeting of LNPs, it has been shown to optimize the targeting of LNPs by rational selection of cholesterol variants. Delivery to non-hepatic cells in the liver is challenging, and oxidized cholesterol 20α-OH has been shown to improve targeted delivery to liver endothelial cells and Kupffer cells (Fig. 13C). It has also been observed that oxidative modification of the hydrocarbon tail is more effective than oxidative modification of the B cholesterol ring.¹⁷⁶ In addition, esterified cholesterol has also been used to improve mRNA delivery to hepatic endothelial cells in vivo (Fig. 13D).¹⁷⁷ It is hypothesized that the alteration of cholesterol structure alters targeting by changing the protein corona adsorbed by LNPs, but the exact mechanism remains to be further elucidated.

Recently, the cholesterol analog bile acids (deprotonated bile acids) have also been reported to reduce delivery to hepatocytes and improve delivery in a variety of other cell types (especially T cells and B cells), achieving splenic tropism.¹⁷⁸ The generalizability of this replacement strategy has been demonstrated. It is hypothesized that a synergistic mechanism of charge, alterations in protein corona, and morphological changes drive preferential uptake within the spleen. In addition, cholesterol can be used as a peptide¹³³ and glycan^94,179 modification target to enhance LNPs targeting.

The structure of lipid components is fundamental to the design of LNPs. Compared with systems with targeted ligands or additional components, the method of directly optimizing the structural characteristics of components to obtain targeted LNPs is a simple alternative. The approach of changing the orientation of LNPs by modulating the structure of individual components better reflects the characteristics of the LNPs themselves and is easier to implement at a lower cost. However, it is still difficult to predict what structures have better targeting properties, and further studies are needed to elucidate the mechanisms of how structures affect the in vivo distribution of LNPs.

6. Conclusions and outlook

Improving the targeting of LNPs is key to advancing the clinical application of nucleic acid drugs. This review summarizes the specific methods and research progress to improve the targeting of LNPs, including ligand modification, formulation optimization, and design of new lipid structures. Although some effective strategies have been proposed to deliver LNPs to the liver, lung, spleen, brain, eye, and bone, targeting optimization strategies for LNPs still need further improvement.

First, targeted delivery of LNPs still suffers from off-target delivery to other cell types and organs (especially the liver), suggesting the need for higher specificity. Second, the design of lipid structures with targeted properties mainly relies on library screening strategies, which increases the complexity of identifying effective lipid structures. Despite many efforts, the lipid structure-targeting relationship remains unclear. More combinatorial chemistry and the use of artificial intelligence (AI)-assisted approaches are needed to accelerate the development of next-generation lipids and provide clues to continuously optimize the structural design of targeted lipids. Third, the natural mechanisms of non-hepatocyte transport remain elusive. To further improve the design of targeted LNPs, in-depth studies of the interactions between LNPs with biomolecules (e.g., proteins, cells, and tissues) of different structures or compositions are needed to inform the customized design of future targeted LNPs. Fourth, the immunogenicity and toxicity of LNPs cannot be ignored. In addition to enhancing targeting, streamlining group fractions, developing alternatives to PEG-lipids, or adopting degradable lipids are effective strategies to enhance safety. To promote clinical translation, more comprehensive strategies need to be proposed for effective targeted nucleic acid delivery.

In conclusion, this study provides insights into the rational and predictable design of organ- and cell-targeted LNPs for the development of precise gene therapies through simple chemical approaches. By continuously improving the lipid structure, optimizing the formulation, and surface modification, it is expected that safe and effective organ- and cell-specific LNPs can be constructed, expanding the types of diseases for which gene therapy is applicable and enhancing the precision of treatment.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included, and no new data were generated or analysed as part of this review.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 22278306, 22078239, and 22278314), and the Tianjin Municipal Science and Technology Bureau (No. 23JCQNJC01870).

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Footnote

† L. Y. and X. G. contributed equally to this work.

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