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

Sanchita Patil, Swaliha Mulla, Prajakta Mali, Sayali Shendage, Sakshi Kolekar, Deepak Kare,

doi:10.5281/zenodo.16938975

Review Paper | Open Access
Volume 02 | Issue 08 | Article Id IJSRT/250308022

The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing by Their Lymph Node Transitivity and Distribution
Sanchita Patil* Swaliha Mulla Prajakta Mali Sayali Shendage Sakshi Kolekar Deepak Kare
Nootan College of Pharmacy, Kavathemahankal Sangli Maharashtra India

Abstract

Lymph node is a small bean shaped structure that is part of the body’s immune system. Lymph nodes filter substances that travel through the lymphatic fluid and they contain lymphocytes that help the body fight infection and disease. Lymph node is a critical component inducing immune responses against pathogens and cancer. The size and charge of the delivery system largely affect the transitivity and distribution with lymph nodes. Although PH sensitive lipid nanoparticles prepared by microfluidic mixing are the latest delivery system to be applied clinically, the effects of their size and charge on transitivity and distribution. LNs targeting LNPs as a significant approach to elicit immune responses fortify defence against pathogen invasion and treat certain diseases. LNPs for targeted nucleic acid delivery have thus offered a versatile technological foundation for vaccine and immunotherapy development. Lipid nanoparticles have emerged as a highly effective delivery system for nucleic acid-based therapeutics. However, the broad clinical transition of lipid nanoparticles-based drugs is hampered by the lack of robust and scalable synthesis techniques that can consistently produce formulation from only development to clinical application. Neutral or negatively charged particles (also small sized particles) are efficiently absorbed via lymphatic vessels and are transported to lymph nodes while positively changed nanoparticles are largely limited to direct lymphatic transport and are taken up by DCs at the injection site. A 30 nm sized lipid nanoparticle (30-LNPs) was efficiently translocated to lymph nodes and was taken up by CD8+ dendritic cells while the efficiency was drastically decreased in the cases of 100 and 200 nm sized lipid nanoparticles.

Keywords

Nanoparticles Microfluidic Mixing Lymph node, Transitivity Distribution Lipid Nanoparticles

Introduction

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:

An ionizable cationic lipid (whose positive change binds to negatively charged mRNA)
A PEGylated lipid (For stability)
A phospholipid (For structure)
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

Lipid nanoparticles are prepared by dissolving the lipids in an organic solvent before injecting them into an aqueous solution. After emulsification of the organic phase into the aqueous one, the solvent is evaporated to obtain the nanoparticles.

In this study, we investigated the effect of the size and charge of PH Sensitive LNPs prepared by microfluidic mixing on transitivity to LNs and their distribution in lymph nodes. Using a microfluidic device, we prepared 30, 100, and 200 nm sized PH Sensitive LNPs mainly composed of YSKOS, a PH Sensitive cationic lipid. A 30nm-sized LNP was efficiently translocated to LNS and was taken up by CD8+ Dendritic cells, while the efficiency was drastically decreased in the cases of 100 and 200 nm-sized LNPs. Furthermore, a comparative study between neutral, positively and negatively charged 30-LNP revealed that the negative 30-LNP moved to the LN more efficiently than the other LNPs, and the negative 30-LNP reached the deep cortex, namely the T-cell zone.^{(2), (3)}

Objective:

• To investigate the effect of size and charge on the distribution and transitivity of lipid nanoparticles (LNPs) within lymph nodes.

• The paper found that the negative 30-LNP reached the deep cortex, or T-cell zone, of the lymph nodes.

• The properties of LNPs are greatly influenced by their size and size distribution. Some of the findings of the paper include.

• Smaller LNPs with narrow size distribution are ideal for bio distribution.

• Smaller LNPs have an increased drug loading efficiency.⁽¹⁸⁾

Procedure:

Step 1: Lipid and Drug Preparation: Lipid molecules are selected based on their ability to form stable nanoparticles and encapsulate the desired drugs or nucleic acids. The lipids are mixed with the active ingredient in a controlled manner.

Step 2: Microfluidic Mixing: Lipid solutions are introduced into a microfluidic device, where they are mixed rapidly with an aqueous phase to form nanoparticles. The flow conditions (e.g., shear stress, flow rates) and concentrations are adjusted to control the size and charge of the resulting LNPs.

Step 3: Characterization: The size, zeta potential (charge), and morphology of the LNPs are measured using techniques like dynamic light scattering (DLS), transmission electron microscopy (TEM), or nanoparticle tracking analysis (NTA).

Step 4: In Vivo Testing: LNPs are administered to animal models (e.g., mice) via subcutaneous or intravenous injection. The fate of the nanoparticles is tracked using imaging techniques or by measuring the concentration of the drug or cargo in different tissues, especially the lymph nodes.

Step 5: Data Analysis: The lymph node accumulation, distribution patterns, and possible immune response are analyzed to determine the optimal size and charge properties of LNPs for efficient lymphatic transport.⁽⁵⁾

CONCLUSION:

The size and charge of lipid nanoparticles play a critical role in their lymphatic transitivity and distribution. Microfluidic mixing allows for precise control over these parameters, potentially leading to more effective targeting of lymph nodes for drug delivery or vaccine development. Tailoring these properties can significantly improve the therapeutic outcomes, especially for treatments targeting the immune system. Procedure for LNP Preparation and Lymphatic Transitivity Assessment:

1. Selection and Preparation of Lipid Materials:

Lipids: Choose appropriate lipids (such as phospholipids, cholesterol, or cationic lipids) to create stable lipid nanoparticles. These lipids should be selected based on their ability to encapsulate the drug or nucleic acid payload and ensure stability within the lymphatic system.

Surfactants: Use surfactants or stabilizing agents (like PEGylated lipids) to prevent particle aggregation and increase circulation time in vivo.

Payload: Prepare the payload (e.g., mRNA, small molecule drugs) to be encapsulated within the nanoparticles.

2. Microfluidic Mixing Setup:

Microfluidic Device Selection: Choose a microfluidic device with well-defined flow channels (e.g., a T-junction or Y-junction device) to facilitate rapid and controlled mixing of lipid and aqueous solutions. This ensures uniform particle formation.

Solution Preparation:

Prepare a lipid phase containing the selected lipids and any hydrophobic stabilizers in an organic solvent (e.g., ethanol).

Prepare the aqueous phase containing the payload and any hydrophilic stabilizers (e.g., PEG-lipids).

Flow Conditions:

Control the flow rates of the lipid and aqueous phases to determine the final size and uniformity of the LNPs.

Adjust the lipid-to-water ratio to optimize encapsulation efficiency and particle size.

3. LNP Formation:

Mixing Process: Introduce both phases into the microfluidic device, where they mix rapidly due to the laminar flow conditions. This leads to the formation of lipid nanoparticles via solvent evaporation or phase inversion techniques.

Particle Size Control: Vary parameters like flow rate, temperature, and lipid concentration to fine-tune the size of the LNPs (typically in the range of 50-200 nm).

Charge Control: Adjust the ratio of cationic lipids (if desired for enhanced cellular uptake) or include neutral/negatively charged lipids to control the zeta potential of the LNPs, optimizing their interaction with the lymphatic system.

4. Post-Production LNP Characterization:

Size and Morphology: Analyze particle size using Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA). For morphology, use Transmission Electron Microscopy (TEM) or Cryo-TEM.

Surface Charge: Measure the zeta potential using electrophoretic light scattering (ELS) to determine the surface charge and ensure that the LNPs have the desired charge for effective lymphatic uptake.

Encapsulation Efficiency: Perform centrifugation or dialysis to separate free payload and determine the encapsulation efficiency by measuring the drug or nucleic acid concentration in the supernatant.

5. In Vivo Injection:

Animal Model Selection: Use an appropriate animal model, such as C57BL/6 mice, for in vivo testing. Ensure the selected model is relevant to the lymphatic system (e.g., the mouse model allows for lymph node analysis).

Administration: Inject the LNPs into the animal via a subcutaneous (s.c.) or intradermal (i.d.) route to ensure nanoparticles reach the lymphatic system. Alternatively, intravenous injection can also be used to track systemic distribution.

Tracking LNP Distribution: Label the LNPs with a fluorescence dye (e.g., DiR or Cy5) or use radiolabeled LNPs for tracking their distribution. Monitor the LNPs’ movement through the lymphatic vessels and accumulation in regional lymph nodes using imaging techniques like fluorescence imaging, PET, or SPECT.

6. Lymph Node Accumulation and Distribution Analysis:

Sampling: After a set period (e.g., 1, -, 6, -, or 24 -hours post-injection), sacrifice the animals and dissect the lymph nodes (e.g., popliteal or inguinal).

Quantification: Measure the amount of LNPs in the lymph nodes using fluorescence or radioactivity assays. This can be quantified by homogenizing the lymph nodes and measuring the fluorescence intensity or counting radioactivity.

Histological Analysis: For detailed distribution, process the tissues for histology and analyse sections using confocal microscopy or immunofluorescence to observe the localization of the LNPs within lymphatic tissue and surrounding cells (such as dendritic cells or macrophages).

7. Evaluation of Immune Response (if applicable):

Immune Cell Activation: If the LNPs are intended for vaccine delivery, assess the immune response by isolating immune cells from the lymph nodes or spleen. Evaluate activation markers (e.g., CD86, MHC II) by flow cytometry.

Cytokine Release: Measure levels of cytokines (such as TNF-α, IL-6, or IFN-γ) in the serum or lymph node supernatant to gauge the inflammatory response triggered by the LNPs.

Antibody Response: If delivering an antigen, assess antibody levels (e.g., IgG or IgA) in serum or lymph node using ELISA or similar assays.

8. Data Analysis:

Analyze the LNP distribution in terms of size, charge, and lymph node accumulation. Correlate these factors with in vivo performance. Evaluate the impact of LNP characteristics on immune activation, drug release, and therapeutic efficacy (if applicable).

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