Application Note
纳米发电机™纳米粒子合成系统
PreciGenome NanoGenerator™ 是用于纳米粒子合成的高性能仪器，如脂质纳米粒子、脂质体、PLGA 等，广泛用于药物输送、基因治疗、LNP 配制和制造等。
NanoGenerator™ 生成的纳米粒子具有更好的尺寸均匀性和更小的 PDI。它可从 0.1mL/样品筛选扩展到 1L（>10L 定制设计）大批量 GMP 生产
纳米粒子，尤其是脂质体和聚合物纳米粒子，由于其优异的性能，在药物递送、mRNA疫苗和生物传感等制药工业等各个领域显示出巨大的生物医学应用潜力。
通过微流控技术合成纳米颗粒比传统的批量合成工艺具有优势，因为它能够在尺寸和形状上具有更好的均匀性。例如，在药物递送领域，使用NanoGenerator™纳米粒子合成系统可以合成脂质纳米粒子（LNP）、脂质体、PLGA等多种纳米粒子。脂质纳米颗粒 (LNP)、脂质体和 PLGA 是最常用的可生物降解材料，用于输送亲水性和疏水性化合物。
1. Structural Basis of Targeted Delivery
LNPs are characterized by a core-shell structure, where a lipid envelope encapsulates nucleic acids or other therapeutic cargo. The core typically comprises ionizable lipids that electrostatically bind to negatively charged mRNA or siRNA, ensuring high encapsulation efficiency and protection from enzymatic degradation[1][2]. The shell consists of phospholipids, cholesterol, and PEGylated lipids, which stabilize the nanoparticle and reduce opsonization by immune cells[1][3]. This modular design allows for precise tuning of physical properties, including size, surface charge, and lipid packing density[2].
Fig 1. Essential components of LNPs
2. Passive Targeting Strategies
Passive targeting includes all methods which do not involve surface modification, instead leveraging the innate biophysical properties of LNPs to achieve tissue-specific accumulation. These properties include size, surface charge, or shape.
2.1 LNP Size
The size of LNPs could also affect their targeting of certain organs (such as liver and kidneys) owing to the organ’s fenestrated structure. Smaller LNPs (≤ 30 nm) preferentially extravasate into lymph nodes, while larger particles (80 – 150 nm) accumulate in the liver via sinusoidal fenestrations[4].
Fig 2. LNP size affects organ targeting. In the liver, small openings called fenestrations permit passage of LNPs about 100 nm large, while in the kidneys, LNPs about 400 nm can transcytose across endothelial cells. Image adapted from Chem Soc Rev (2023): 7579-7601.
2.2 Surface Charge
Surface charge also dictates interactions with cell membranes: cationic LNPs bind anionic proteoglycans on endothelial cells, whereas anionic variants avoid hepatic clearance and target splenic macrophages[4].
LNP protein coronas play an important role in organ-selective targeting. Various physicochemical properties of LNPs including surface charge will lead to the adsorption of different types and components of proteins on the surface, which may determine their interactions with specific organs/cells. The protein corona’s effect on selective targeting of brain, liver, lung, spleen, and kidneys has been reported, while its effect on targeting heart and bone remains elusive.
Fig 3. The protein corona has been known to affect organ selectivity depending on the corona's exact composition. Image adapted from Chem Soc Rev (2023): 7579-7601.
3. Active Targeting Strategies with Surface Ligands
Passive targeting approaches by themselves face limitations in how tissue-specific they can be. Instead, more advanced approaches use active targeting that bind ligands to the LNP surface. These in turn allow binding to receptors on the target cell, and include several key methods:
3.1 Antibody-Mediated Targeting
Antibodies or their fragments can be conjugated to LNPs to enable high-affinity binding to cell-specific receptors. These are typically attached to maleimide-functionalized PEG-lipids. Notable examples include anti-PD-L1 antibodies used to treat melanoma, or anti-CD4 antibodies used to treat HIV[4].
3.2 Peptide Functionalization
Peptides are another potential ligand which can be very stable and modular. Notable examples include RVG29 used to bind brain endothelial cells, or mApoE used to target LDLR-expressing hepatocytes[4][6][7].
3.3 Aptamer Functionalization
Aptamers, or folded oligonucleotides, offer programmable targeting with low immunogenicity. One example application is the use of CCR5-targeting RNA sequences which can facilitate receptor-mediated intake at the blood-brain barrier[4].
3.3 Aptamer Functionalization
Carbohydrates are another major class of ligand which may be incorporated to LNPs. Examples include mannose used for dendritic cells, or GalNAc (N-acetylgalactosamine) used for liver-targeted therapies[4][6][7].
3.5 Selective Organ Targeting (SORT) Lipids
The last major method of active targeting is SORT, a novel approach which incorporates supplemental lipids into the core formulation to redirect biodistribution. These tertiary lipids include positively charged, negatively charged, and short-tail ionizable lipids[8].
Fig 3. Overview of SORT mRNA delivery in liver and lung specific applications. Image from J. Control Release (2023), 361: 361-372
4. Surface Ligand Conjugation Methods
With a diverse array of surface ligands available, several conjugation methods may be employed depending on the type of ligand in question:
Fig 4. Assorted in-situ conjugation methods for attaching ligands to LNPs. Image from Biophys Rep. (2023), 9: 255-278
4.1 Covalent Conjugation
Covalent conjugation is widely used for antibody, peptide, and aptamer conjugation. In principle, the ligands of interest are covalently attached to various reactive groups of the PEGylated lipid on the LNP membrane. This is done by incorporating their own reactive groups complementary to those on the PEGylated lipid[9].
4.2 Click Chemistry (Azide-Alkyne Cycloaddition)
The most notable form of click chemistry is the copper-catalyzed azide-alkyne cycloaddition reaction, which may be used for bioorthogonal conjugation. By incorporating an azide- or alkyne-functionalized lipid, the LNP may be conjugated to a ligand that features the complementary group. Importantly, this reaction offers high specificity and efficiency while being possible post-formulation under mild conditions[9]. Similar results may be achieved with other click chemistry reactions, such as the thiol-maleimide reaction.
Fig 5. Using the azide-alkyne reaction, human Transferrin was conjugated to siRNA loaded LNPs. Image from Eur J Pharm Biopharm (2024), 198: 114242
4.3 Carbodiimide Chemistry (EDC/NHS Coupling)
Carboxyl-functionalized lipids on the LNP surface may be activated using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide), forming an active ester intermediate. Ligands with primary amines may then be covalently linked via stable amide bonds[9].
4.4 Non-covalent Adsorption and Affinity Interactions
Some ligands can be attached through non-covalent interactions, such as electrostatic adsorption (for charged peptides or proteins) or biotin-streptavidin affinity. For example, biotinylated LNPs can bind streptavidin-linked antibodies or peptides, providing a modular and reversible conjugation strategy[9].
4.5 Post-Insertion Functionalization
In this last method, the ligand-functionalized PEGylated lipids are incorporated into preformed LNPs by incubating them together. This allows the the ligand-bearing lipids to insert into the LNP membrane rather than including them in the initial formulation process. This method is particularly useful for sensitive ligands that may not withstand the mixing involved in LNP synthesis[9].
Fig 6. Comparison of pre- vs. post-insertion methods for incorporating ligands. Image from Biophys Rep. (2023), 9: 255-278
5. Basic LNP Preparation Workflow
Incorporating surface ligands allows LNPs to be used in various critical applications where high editing efficiency is needed and off-target effects are highly undesirable:
5.1 mRNA Vaccines
The COVID-19 vaccines are easily the most notable application of mRNA LNPs in recent years. In this case, attaching functional groups to the LNPs' PEGylated lipids is done to prevent rapid clearance and promote on-target trafficking.
5.2 Gene Editing
An increasingly important field of research, gene editing relies on LNPs to deliver CRISPR-Cas9 components with high tissue specificity. One solution involves using SORT technology for lung-specific editing, correcting mutations in cystic fibrosis.
5.3 Cancer Therapy
Antibody-conjugated LNPs may be used to target oncogenes for cancer therapy when combined with an appropriate ligand. For example, antibody-conjugated-siRNA LNPs may be used to target HSP47 in liver fibrosis, while positively charged LNPs with docetaxel may be used against breast cancer[10].
6. Challenges and Future Directions
While surface modifications offer a powerful tool to enhance target specificity, some challenges persist to their incorporation.
6.1 Protein Adsorption
Adsorbed serum proteins can form a layer known as the protein corona. This layer may alter LNP surface chemistry and negatively affect targeting efficacy. A common countermeasure is to incorporate various proteins to pre-coat LNPs.
6.2 Manufacturing Complexity
Ligand conjugation requires multi-step processes that further complicate LNP production, especially when scaled up. Simplified production methods such as SORT are therefore essential for integrating surface modification.
Fig 7. Five-layered hyaluronan-decorated LNPs. Two layers of Poly-L-Arginine (PLA) are included to facilitate endosomal escape. Image from Pharmaceutics (2024), 16(4): 563
6.3 Endosomal Escape Barriers
Even if LNPs successfully enter cells, they must first undergo endosomal escape to release their cargo into the cytoplasm. This step is especially crucial for mRNA LNPs, and may be addressed by incorporating ligands such as endosomolytic peptides[11].
6.4 Immune Activation
Despite their versatility in attaching ligands, PEGylated lipids also induce anti-PEG antibodies, which can complicate LNP delivery. Alternatives are under active investigation to mitigate this.
7. Conclusion
Surface modification has transformed LNPs from passive carriers into precision tools for drug delivery. By engineering size, charge, and ligand presentation, researchers can direct LNPs to previously inaccessible tissues. Technologies like SORT and aptamer conjugation exemplify the shift toward modular, scalable platforms capable of addressing diverse clinical needs. As understanding of lipid-protein interactions deepens, LNPs will play an increasingly pivotal role in mRNA vaccines, gene therapies, and personalized medicine.
References:
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Development of mRNA Lipid Nanoparticles: Targetign and Therapeutic Aspects - PMC
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Composition of lipid nanoparticles for targeted delivery: application to mRNA therapeutics - PMC
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The Future of Tissue-Targeted Lipid Nanoparticle-Mediated Nucleic Acid Delivery - PMC
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Surface Modification of Nanoparticles for Targeted Drug Delivery - Springer
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Ligand-Tethered Lipid Nanoparticles for Targeted RNA Delivery to Treat Liver Fibrosis - UPenn
