DNA and RNA therapeutics are drug products composed of an active genetic component linked to an efficient delivery system, generally Lipid NanoParticles – LNP. Those treatments include both prophylactic vaccines that prevent an infection by triggering the patient’s immune system and therapeutic vaccines that will be used to cure a patient from a specific disease. Each therapy will require its own LNP formulation depending on the genetic material and the targeted tissue. To adapt the delivery system to the need, a wide variety of lipids that modulate the encapsulation efficiency and the LNP properties is needed for therapeutic success.
As an innovator in the delivery field, Polyplus developed a range of cationic and ionizable lipids used in LNP formulation named LipidBrick®. Those “active lipids” will protect nucleic acids such as DNA, mRNA and siRNA and deliver them to targeted cells. By adding positive charges to an LNP, LipidBrick® broadens the current applications spectrum in terms of potency and targeting, as LNP electric charge is known to impact biodistribution and expression of mRNA-LNPs. LipidBrick® modifies the overall charge of the LNP, allowing to adapt the biodistribution.
Polyplus’ goal is to support customers from R&D to commercialization. LipidBrick® has been patented by Polyplus to avoid any patent infringement that could set back therapy advance.
|101000172||LipidBrick® IM21.7c 250 mg|
|101000173||LipidBrick® IM21.7c 1 g|
Lipid Nano Particles – LNP are the non-viral delivery system of choice for DNA and RNA therapeutics. The lipidic structure enables delivery of nucleic acids into cells using several administration routes (systemic or local injection). LNPs offer a great stability over the time that is essential for a drug product.
LNP are composed of 3 types of molecules:
- Active lipids (cationic and/or ionizable lipids): positively charged lipids will interact with negatively charged nucleic acids
- Structural lipids (phospholipids): Provide LNP rigidity and stability
- PEG-lipids: Provide steric stabilization and prolong blood circulation
- Cholesterol: Increase membrane fluidity and stability, promote membrane fusion
The manufacturing process of LNP is made using microfluidic systems that will homogenize the nucleic acids (mRNA, DNA, siRNA, or other types) and the lipid mixture in a laminar flow. Cationic or ionizable lipids will first interact with nucleic acids. The intermediate nanostructures will make the hydrophobic tails available to interact with both structural lipids and modulators. The LNP structure will then be finalized by the formation of full functional vesicles able to both protect and deliver nucleic acids (Fig 1.).
Figure 1: LNP formulation using microfluidic systems.
A deep understanding of the formulation steps, and of the microfluidic mixing, leads to self-assemble of the lipids and nucleic acids into the desired structure, finally resulting in scalable, reproducible and high-quality LNPs production.
Proof of concept
The proof-of-concept objective was to develop a successful LNP formulation using LipidBrick® IM21.7c optimized for mRNA delivery. A characterization of the LNP has been conducted before testing the Polyplus cLNP in in vitro on Caco-2 cells and in vivo in mice using IV injection. This proof of concept shows the potential of using LipidBrick® IM21.7c in a drug formulation.
- LNP characterization using mRNA
The initial LNP formulation screening consists in defining the reference standard. Two references have been tested successfully for the delivery mRNA (in vivo-jetRNA®+) and siRNA (Dlin-MC3-DMA):
- in vivo-jetRNA+: a ready to use liposome-based formulation available through Polyplus
- LNP with Dlin-MC3-DMA: Onpattro® like LNP formulation using Dlin-MC3-DMA ionizable lipids (commercially available)
The second step consists in testing several LNP formulation using mRNA encoding for Luciferase. The Polyplus cLNP formulation using LipidBrick® IM21.7c (cationic) has been selected depending on three parameters: Particle size, Zeta potential and Encapsulation efficiency (Fig 2.). By modifying the repartition between active lipids, structural lipids and modulators compare to LNP with Dlin-MC3-DMA, Polyplus succeeded to find an LNP formulation with an acceptable size of nanoparticle (<200 nm), a cationic overall charge (around 20 mV) and a 100% mRNA encapsulation efficiency.
Fig 2: Polyplus cLNP characterization. A/ Size of liposomes with in vivo-jetRNA®+ using the recommended protocol or LNPs using mRNA 250 μg/ml after 1 hour of complexation stored at room temperature (RT) were measured with the dynamic light scattering (DLS). B/ Zeta potential of liposomes with in vivo-jetRNA®+ using the recommended protocol or LNPs using mRNA 250 μg/ml after 1 hour of complexation stored at room temperature (RT) were measured with the electrophoretic light scattering (ELS). C/ mRNA (encoding for luciferase) using in vivo-jetRNA®+ or LNPs were analyzed using RiboGreen kit.
- Efficiency and Biodistribution
The LNP characteristics affect both efficiency of delivery and organ targeting. By modifying the lipid structure (i.e. liposome or lipid nanoparticles) and the overall charge of the particle, we can adapt the biodistribution of the nucleic acids in mice using systemic injection (Fig. 3). LNP formed using ionizable lipids will mostly target the liver whereas LNP formed using cationic lipids (LipidBrick® IM21.7c) will target predominantly lung and spleen.
Fig. 3: LNP with LipidBrick® IM21.7c lead to efficient mRNA delivery in different organs using IV injection. mRNA encoding Luciferase was injected into mice using in vivo-jetRNA®+ or LNPs through intravenous injection (retro-orbital). In vivo-jetRNA®+ and LNP have been used with 7.5 µg of mRNA/mouse. Luciferase expression was assessed 24 h post-injection.
- LNP Stability
The stability of the particles is very important for the patient’s safety and drug efficiency. Degradation of LNPs or nucleic acids results in loss of drug efficacy which can be a deal-breaker for health authorities during clinical trials.
Polyplus tested its LNP formulation over 3 months using an in vitro mRNA model in CaCo-2 cells (Fig. 4) This study shows a very high LNP stability correlated with a good expression when the LNP/mRNA are stored at 4°C.
Fig. 4: LNPs with LipidBrick® IM21.7c are very stable over 3 months. mRNA encoding Luciferase was used with Polyplus cLNP in CaCo-2 cell line. LNPs have been stored during 13 weeks at 4°C and Luciferase expression was assessed at 5 weeks, 10 weeks and 13 weeks. in vivo-jetRNA+ liposomes were freshly prepared according to our standard protocol with the mRNA encoding for Luciferase.
- LNP Safety
Next, we wanted to evaluate the impact of LNP formulation with cationic lipids on the pro-inflammatory response compared to LNP formulation with ionizable lipids (Dlin-MC3-DMA). Commonly modulated blood cytokines with known association with pathological responses were included, with the goal of performing a comprehensive assessment of the predictive biomarkers of in vivo toxicity. In this study, we showed that the Polyplus LNP formulation has a comparable cytokine profile as LNP using ionizable lipids (Fig. 5) and triggers low to no pro-inflammatory response.
Fig. 5: Polyplus cLNP with IM21.7c triggers similar cytokine profile than LNP with Dlin-MC3-DMA. 200 µL of mRNA-LNP using 10 µg of mRNA encoding Luciferase at a mRNA/LNP ratio of 1:2 (µg mRNA:µL LNP) has been injected through intravenous injection (retro-orbital injection) in mice. 2 to 24 hours after injection, blood was collected and the level of IFN-γ, TNF-α, GM-CSF, IL-2, IL-4, IL-5, IL-10, IL-12, IL-17A, IL-23 was measured by MACSPlex and the level of IL-1α, IL-1β, IL-6 by ELISA. LPS (200 µg) and PBS have been administered into mice as respectively positive and negative controls. Cytokine classification per function is based on Turner et al. (Biochimica et Biophysica Acta (BBA) – Molecular Cell Research, 2014; https://doi.org/10.1016/j.bbamcr.2014.05.014.)
Polyplus’ aim is to provide a pathway from Research to Clinic trials by providing both ready to use solutions easy to use for both in vitro and in vivo proof of concept as well as optimized cationic lipids to make your own LNPs.
Our ready-to-use solutions have been optimized specifically for in vitro use in the case of jetMESSENGER® and in vivo use for in vivo-jetRNA®+. Both reagents contain the same cationic lipid which is LipidBrick® IM21.7c but in a ready-to-use formulation optimized for mRNA delivery. jetMESSENGER® and in vivo-jetRNA®+ do not require any formulation equipment nor expertise in LNP formulation.
Polyplus now provides a range of ionizable or cationic lipids for LNP formulation, LipidBrick®. These lipids will be able to modulate your LNP properties depending on your therapy needs.
For more information, please contact us at: email@example.com.
Each batch of IM21.7c is tested in-house to assess both identity and purity of the product. Results are reported in a Certificate of Analysis.
Polyplus-transfection® is ISO 9001 Quality Management System accredited since 2002; this level of certification assures global customers that the supplier has established reliable and effective processes for product development, manufacturing, sales and customer support.