The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing by Their Lymph Node Transitivity and Distribution

The size and charge of lipid nanoparticles (LNPs) significantly influence their ability to translocate to and distribute within lymph nodes. When lipid nanoparticles are prepared using microfluidic mixing, several key factors contribute to their behaviour in vivo, particularly concerning lymphatic transitivity and tissue distribution:

1. Particle Size:

Smaller Particles (10-100 nm): Smaller nanoparticles are generally more efficient at being taken up by the lymphatic system due to their ability to escape from the capillaries and enter lymphatic vessels. The size of LNPs can affect their ability to traverse the endothelial barriers, with smaller particles having a higher chance of being absorbed into the lymphatic system. Typically, LNPs in the 20–100 nm range are known to have improved lymphatic uptake.

Larger Particles (>100 nm): Larger particles may have a lower uptake into the lymphatic system because they might be too large to pass through the endothelial lining of lymphatic vessels or may get trapped in the interstitial space. However, they may accumulate in other tissues like the liver and spleen.

2. Surface Charge:

Positive Charge (Cationic LNPs): Cationic nanoparticles tend to interact more strongly with negatively charged cell membranes, which can increase their uptake by immune cells in the lymph nodes (e.g., dendritic cells). This positive charge can improve lymphatic targeting but may also induce undesirable aggregation or immune responses in vivo.

Negative Charge (Anionic LNPs): Anionic nanoparticles may have a lower affinity for cell membranes, which can result in less cellular uptake and reduced lymphatic transport. However, they often have reduced toxicity compared to cationic nanoparticles.

Neutral Charge: Neutral or slightly amphiphilic nanoparticles might offer a good balance between avoiding aggregation and being taken up efficiently by the lymphatic system.

3. Lymph Node Transitivity and Distribution Evaluation:

In vivo Imaging: LNPs are often tracked using fluorescent labels or radiolabeling to monitor their translocation through lymphatic vessels and accumulation in the lymph nodes.

Ex vivo Analysis: After administration, tissues can be harvested, and the distribution of nanoparticles can be evaluated using techniques like histological analysis, flow cytometry, or quantification of fluorescence/radioactivity in various organs.

Targeting Efficiency: The lymph node targeting efficiency can be quantified by measuring the amount of LNPs that accumulate in the draining lymph node (for example, after subcutaneous injection near a lymph node).

4. Effects of Microfluidic Mixing:

Controlled Size Distribution: Microfluidic mixing enables precise control over nanoparticle size and homogeneity, ensuring a consistent particle size distribution. This can enhance reproducibility and optimize the lymphatic uptake of the LNPs.

Improved Encapsulation: Microfluidic methods may also result in more efficient encapsulation of therapeutic agents or imaging probes, which can affect their distribution within the lymph nodes and their ability to reach target cells. The lymph node is small-bean shaped structure that is part of the body's immune system. It is a critical organ where immune response against pathogens and cancers begins for local defence. The lymph system is part of the body's immune system and is made up of tissues and organs that help protect the body from infection and disease. Lymph nodes are made up of lymph tissue and different types of cells including: -

• White blood cells (lymphocytes)

• B-cells                

• Macrophages

• T-cells                

• Plasma cells

• Dendritic cells

The most important role of the LN is to provide a specialised meeting place for immune cells. LN- resident DCs engulf antigens that have flowed in the lymphatic vessels. Lymph nodes work closely with two body systems including the immune system. The immune system protects your body from foreign invaders like bacteria and viruses to prevent infection, illness or diseases. LNP became more widely known in late 2020, as some COVID-19 Vaccines used RNA vaccine technology. LNPs use in mRNA vaccine for SARS-Cov-2 (The virus that causes COVID-19) are made up of four types of lipids:

1. An ionizable cationic lipid (whose positive change binds to negatively charged mRNA)
2. A PEGylated lipid (For stability)
3. A phospholipid (For structure)
4. Cholesterol (For structure)

because of rapid clearance by the immune system of the positively charge lipid, Neutral ionizable amino lipids were developed. Microfluidic mixture is a through and rapid mixing of multiple samples in a microscale device. The materials for microfluidic include rigid polymers, polydimethylsiloxane (PDMs), inorganics such as glass or si paper and combinations of these materials, including ones made by 3D printing (3DP) Using LNPs for drug delivery was first approved in 2018 for the SiRNA drug onpattro. The choice of route is greatly influenced by the size and surface nature of molecules or particles. Small molecules or less than 10nm sized macromolecules basically enter blood capillaries at the injection site. Larger than 10 nm sized macromolecules basically enter blood capillaries rather than the lymphatic capillaries at the injection site. Larger than 10 nm (16KDa) sized particles and macromolecules are mainly absorbed via lymphatic vessels. However, when the size exceeds 100 nm, the transport to LNs is substantially reduced. On the other hand, the larger particles (>200 nm) are mostly associated with DCs, and the DCs transport them to LNs. Neutral or negatively charged particles (also small sized particles) are efficiently absorbed via lymphatic vessels and are transported to LNs, while positively charged nanoparticles are largely limited to direct lymphatic transport and are taken up by DCs at the injection site. The positively charged nanoparticles appear to have a preference for interacting with extracellular components and DCs in the interstitial. Various factors involved in recent development in lipid nanoparticles (LNP) technology have been extensive, and small interfering RNA (SiRNA) loaded LNP, Parisian (ONPATTRO by Alnylam), was approved by the U.S. food and drug administration. (FDA) But more important factors include the use of a PH Sensitive cationic lipid and the method used to prepare the LNP.

• Synthesis of lipid nanoparticles