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Lipid Nanoparticles Introduction

Application Note

In recent years, lipid nanoparticle-based solutions have gained significant attention and research focus, primarily due to their pivotal role in the development and utilization of mRNA COVID-19 vaccines amid the pandemic. Over the past two decades, lipid nanoparticles have been a subject of growing interest, and they have achieved remarkable success in clinical applications, beginning with the approval of Doxil in 1995. Furthermore, lipid nanoparticles have showcased substantial potential in delivering nucleic acid drugs, exemplified by the approval of two RNA therapies and mRNA COVID-19 vaccines.

Lipid nanoparticles (LNPs) stand as the most advanced non-viral gene delivery system within clinical practice. They have proven their capability to safely and efficiently transport nucleic acids, addressing a significant obstacle that previously hindered the progress and utilization of genetic medicines. Genetic medicine encompasses various applications, including gene editing, the expedited development of vaccines, immuno-oncology, and the treatment of rare genetic and previously untreatable diseases. All of these applications have traditionally faced challenges due to inefficiencies in nucleic acid delivery.


What is Lipid Nanoparticle?

Lipid Nanoparticles (LNPs) are a class of nanoscale delivery systems designed to transport and protect therapeutic molecules, such as drugs and RNA, to specific target sites within the body. They have gained significant attention in the field of medicine and biotechnology due to their ability to enhance the bioavailability and efficacy of drugs by improving stability and ability to target points of interest for drug delivery. The basic structure of an LNP consists of a lipid bilayer surrounding a hydrophobic core. This structure allows the LNPs to encapsulate hydrophobic drugs or nucleic acids within the core while keeping the hydrophilic components on the surface, making them stable and compatible with the aqueous environment of the body.
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Schematic of oligonucleotide based LNP synthesis

LNPs can be tailored to suit specific applications and therapeutic needs. By modifying the composition of the lipid bilayer and the core, scientists can optimize factors like stability, drug-loading capacity, release kinetics, and target specificity. One of the most notable applications of LNPs is in the delivery of mRNA and siRNA. These molecules have great potential for treating a wide range of diseases, including genetic disorders, cancer, and infectious diseases. However, they face challenges when administered directly due to their susceptibility to degradation and difficulty crossing cell membranes. LNPs provide a method to bridge this gap by acting as a shield to protect the payload and deliver it to target cells. In addition to nucleic acid delivery, LNPs are also used to encapsulate hydrophobic drugs for various medical treatments. By using LNPs, researchers can improve drug solubility, increase drug circulation time, and achieve targeted delivery to specific tissues or organs. This targeted delivery minimizes off-target effects and reduces the required drug dosage, reducing adverse side effects and lowering production costs. Lipid nanoparticles have shown great promise in preclinical and clinical studies, and some LNP-based therapies have already been approved for medical use.

The development of LNPs represents a significant advancement in the field of drug delivery and holds the potential to revolutionize the treatment of various diseases, making them a key area of interest in modern pharmaceutical research.
What is LNP

How LNP works?

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The Figure above illustrated a typical process of nucleic acid LNPs synthesis and in-vitro gene delivery. First, an aqueous buffer containing nucleic acids (DNAs or RNAs) and a lipid formulation in ethanol solution were injected to the two inlet channels of CHIP-MIX-4 mixing cartridge in a NanoGenerator Nanoparticle Synthesis System. With the proprietary microfluidic mixing structure, two fluids were well mixed and formed homogenous nucleic acid LNPs in a controllable and reproducible manner. By changing the total flow rate (TFR) and flow rate ratio (FRR), users can conveniently control the size and N/P ratio of the resulted nucleic acid LNPs. 

Nucleic acid LNPs are an excellent non-viral vehicle for gene delivery. The lipid layer of LNP protected delicate nucleic acids from DNase or RNase, which improve the stability and bioavailability of nucleic acid payloads. LNP size is a critical factor for successful internalization by target tissues. Users can produce LNPs with desirable size using microfluidic synthesis system. After internalized or taken up by the cells, DNAs or RNAs were released from LNPs containing pH-sensitive lipids in cytoplasm with lower pH. Desired proteins or antibodies were then produced in-vivo.
How LNP works

How to make lipid nanoparticles?

Lipid nanoparticle (LNP) formulation is a method used to deliver various bioactive molecules, such as small molecule inhibitors and vaccine components, to targeted cells and tissues.

A typical LNP formulation is composed of several components:
  1. Cationic or Ionizable Lipids: These lipids interact with polyanionic-type RNA to improve delivery.
  2. Neutral Helper Lipids: These are often zwitterionic lipids like 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and/or sterol lipid (i.e., cholesterol).  They stabilize the lipid bilayer of the LNP and enhance mRNA delivery efficiency.
  3. Polyethylene Glycol (PEG)-Lipid: This improves the colloidal stability in biological environments by reducing aspecific absorption of plasma proteins and forming a hydration layer over the nanoparticles.
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These components form a lipid shell surrounding an internal core composed of reverse micelles that encapsulate and deliver oligonucleotides, like siRNA, mRNA, and plasmid DNA. The success of mRNA-based COVID-19 vaccines could not have been possible without decades of research on lipid-based drug delivery (LBDD) systems, a subset of which are LNPs.

Table  Lipid molar ratios for LNPs in FDA-approved agents

* Ionizable/cationic lipid : neutral phospholipid : cholesterol : PEGylated lipid

How make LNPs

LNP/liposome preparation protocol

Conventionally, solvent injection and thin film rehydration are two common methods for liposome synthesis. Due to the simplicity of equipment requirements, such as sonicators or rotary evaporators, solvent injection and thin film rehydration methods are widely adopted in both research and production process. However, these traditional methods meet the challenges of low homogeneity of resulted LNPs, hash processes for delicate biomolecules, such as DNAs, RNAs or proteins. To address these challenges, microfluidic mixing method has been rapidly developed in the past decade.
Microfluidic mixing synthesis system provides a convenient and robust way to prepare LNPs. Here describes a general protocol for a typical nucleic acid LNP synthesis. The preparation protocol is illustrated by using NanoGenerator instrument here.
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  1. Dissolve nucleic acid samples in acidic aqueous buffer, such as sodium acetate buffer, citric acid buffer etc., in a clean DNase/RNase free 15mL tube. Dissolve lipid formulation mixture in anhydrous pure ethanol in another clean 15mL tube.
  2. Load the nucleic acid aqueous sample and lipid formulation to the corresponding reagent slots in Nanogenerator. Place a new tube for product collection.
  3. Enter LNP synthesis parameters, such as product volume, total flow rate (TFR) and flow rate ratio (FRR), in the touch screen user interphase of Nanogenerator.
  4. Press Run. The nucleic acid carrying LNPs are ready within minutes.
  5. Remove ethanol through dialysis or buffer exchange. The resulted LNP product is ready for downstream analysis and application.
Preparatin Protocol

LNP v.s. Liposome

Liposomes and lipid nanoparticles are two main types of lipid-based nanoscale structures that are similar in appearance but different in the inner structure, composition and function. Both are excellent drug delivery vehicles that transport cargo of interest within a protective, outer layer of lipids.
Slide 4 LNP liposome comparison
Liposomes, a precursor to LNPs, are a highly adaptable nanocarrier platform due to their ability to carry both hydrophobic and hydrophilic molecules, including small molecules, proteins, and nucleic acids. They hold the distinction of being the first nanomedicine delivery platform to transition successfully from theory to clinical use. Several liposomal drug formulations have received approval and have been effectively incorporated into medical practice.

Conventional liposomes are characterized by one or more lipid bilayer rings enclosing an aqueous compartment, but not all LNPs possess a continuous bilayer that would categorize them as lipid vesicles or liposomes. Certain LNPs adopt a structure similar to micelles, sequestering drug molecules within a non-aqueous core.  LNPs, which is similar to liposomes, are specifically designed to encapsulate a wide range of nucleic acids (RNA and DNA), making them the most favored non-viral system for gene delivery.

Both liposomes and LNPs have applications in medicine, such as drug delivery and imaging.

Types of LNP and Liposome

 lipid nanoparticle and liposome types
In addition to basic liposomes (A) and drug-loaded liposomes (B) which encapsulate a payload within its core, targeted liposomes (C) and stealth liposomes (D) are alternative methods used for drug delivery and treatment. Targeted liposomes (C) have ligands attatched to their surfaces that allow for it to target and bind to specific receptors of cells. These categories of liposomes have beenused for a variety of therapeutic applications such as cancer therapy or against inflammatory diseases like Crohn's disease.  However, this comes with the limitation of phagocytes targeting and removing these types of liposomes. Stealth liposomes (D) aim to address this limitation; by coating the outer layer with polymers such as PEG, they are invisible to phagocytes and allow the liposome to avoid the body's natural immune system which can be extremely useful for drug delivery.

In response to the limitations of liposomes, such as their low encapsulation efficiency, new nanoparticle drug delivery systems were developed. Solid lipid nanoparticles (SLN) (F) and nanostructured lipid carriers (NLC) (E)were two types of nanoparticles designed to fill in these gaps. SLN consist of solid lipids, while NLC are formed with a mixture of solid and liquid-crystalline lipids. Both SLN and NLC have particle sizes ranging from 40 to ∼1000 nm and offer improved physical stability compared to liposomes. They also have higher drug loading capacities, better bioavailability, and can be more easily produced at a large scale without the need of organic solvents. Moreover, SLN and NLC can precisely control drug release due to reduced molecular mobility in their solid state.However, one limitation of SLNs is that they can expel drugs during long-term storage due to crystallization. NLC is able to bypass this obstacle by incorporating small amounts of liquid lipids to reduce lipid core crystallinity, resulting in enhanced drug-loading capacity and long-term stability. SLN and NLC are typically manufactured using organic solvent-free techniques, such as high-pressure homogenization, emulsion/solvent evaporation, and solvent injection, minimizing the issues asscoiated with liposome development.

What is the lipid nanoparticles toxicity?

Lipid nanoparticles are generally regarded as non-toxic and biocompatible. However, some research has shown that certain types of LNPs, particularly those containing ionizable or cationic lipids, can be highly inflammatory and possibly cytotoxic.
LNP toxicity

iScience doi: 10.1016/j.isci.2021.103479

In the context of mRNA vaccines, LNPs are used as carrier vehicles to protect mRNA molecules from degradation and aid in their intracellular delivery. Some side effects often linked to inflammation, such as pain, swelling, fever, and sleepiness, have been reported in human trials of mRNA-LNP-based vaccines3. These side effects were initially thought to be generated from the potent immune response to the vaccine. However, recent research suggests that the inflammatory nature of the LNPs could be partially responsible for these side effects.

It’s important to note that positively charged lipids, which are often used in LNPs, are inherently toxic. Companies have struggled for years before landing on formulations that were safe and effective. When injected intravenously, these particles invariably accumulate in the liver, and delivery to other organs is still an obstacle.

Therefore, while LNPs have proven to be a crucial component in the success of mRNA vaccines, their potential inflammatory properties and toxicity are areas of ongoing research.

Introduction Videos and Webinars:

LNP Introduction

NanoGenerator LNP Synthesis & Application Webinar with ProMab Biotechnologies

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