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Nanoparticles for RNA delivery

​​​​​​​​​​​​​​​​​​Our group works in the area of Biomaterials and Controlled Release of therapeutic nucleic acids for different applications, e.g. Tissue engineering of bone, but also new treatment approaches for cancer.

In order to make RNAs available, we generally employ nanoparticles of different kind tailored to the needs of the type of RNA as well as the kind of application. For example, we use calcium phosphate nanoparticles (CaP-NP) for siRNA delivery especially for bone regeneration approaches. Here calcium phosphate​​ provides the major part of the inorganic part of the bone matrix. However, calcium phosphate would not be suitable for an application in the acidic environment of the upper intestinal tract including the stomach because it would simply dissolve. Here, extracellular vesicles from bovine milk can be helpful as they are optimized for the oral delivery of proteins and RNAs.

Lipid nanoparticle (LNP) systems came into focus for mRNA complexation with the vaccination against the corona viruses and build a focus for Christian Wölk's group. Here different administration routes are of interest, including systemic as well as local strategies​.

siRNA is the abbreviation for small interfering RNA. The double stranded non-coding short RNA binds specifically to the complementary sequence of mRNA which causes an enzymatic degradation of ideally one specific mRNA. Since the mRNA-complementary strand of the siRNA remains intact and bound to the RISC enzyme complex, the degradation of mRNA works in a catalytic way for some time. Keeping the native chemical RNA structure, this effect is transient and will fade in the time frame of days to weeks depending on the expression level of the protein to be silenced. This is helpful if a process needs to be controlled in a defined stage, e.g. during regeneration or if locally remaining cancer cells need to be addressed.​

Oligomer-stabilized calciumphosphate nanoparticles (CaP-NP)

CaP-NP are traditional transfection systems for nucleic acids. Rapidly occuring instability of the nanoparticles (particle growth, crystallization) prevented the system‘s use for quite some time.
More recently, however, our and other‘s work to stabilize CaP-NP by maleic acid containing polymers made this concept interesting again [1].

The resulting stabilized CaP-NP have a size of 50 to 60 nm and remain stable for a couple of hours after preparation.

Meanwhile, CaP-NP have been utilized to load siRNA in extracellular vesicles [2].

team

DDr. Franziska Mitrachuring her PhD studies, Dr. Franziska Mitrach developed oligomer-stabilized CaP-NP as complex-forming system for siRNA. Silencing of survivin was shown to be a successful strategy to kill F98 rat brain cancer cells [1]. A key issue for this concept is the stabilization of the NP via an oligomer consisting of maleic anhydride, polyethylene methacrylate and tetradecylacrylate synthesized in JProf. Michael Hacker‘s group​.​

literature

[1] Mitrach F , Schmid M , Toussaint M , et al. Amphiphilic anionic oligomer-stabilized calcium phosphate nanoparticles with prospects in sirna delivery via convection-enhanced delivery. Pharmaceutics. 2022;14(2):326. doi: 10.3390/pharmaceutics14020326

[2] Roerig J , Mitrach F , Schmid M , et al. S​ynergistic sirna loading of extracellular vesicles enables functional delivery into cells. Small Methods. 2022;6(12). doi: 10.1002/smtd.202201001

funding​


Extracellular vesicles (EVs)

Maximilian KieckhöferAll cells continuously secrete extracellular vesicles (EVs). They consist of lipid double layers with integrated proteins similar to membranes of the cells they orgin from. The membranes are enriched in cholesterol and sphingolipids which provide them with mechanical stability. EVs have a negative zeta potential but are still well taken up by cells through receptor mediated uptake. We could show this for milk-derived EVs that are taken up by the neonatal Fc receptor of polarized Caco-2 cells as a model for the intestinal barrier [3].

 figure taken from [2]

Sokiyna AlbustanjiAlthough all cells secrete EVs, it is difficult to isolate them and to make sure that the EVs are a product of the cultured cells. This is due to the EVs that are also present in fetal bovine serum as well as human sera added as supplement to cell cultures. As we want to use EVs as carriers for RNAs, we need large amounts of EVs. To produce a reasonable amount of EVs from cell cultures large scale cultures are required. We therefore decided to work with bovine milk derived EVs in order to develop loading strategies for nucleic acids [4].

team

Maximilian Kieckhöfer progresses the work of Dr. Josepha Roerig as a PhD student. Sokiyna Albustanji is interested in loading other RNAs to EVs and in the development of controlled release systems.

 

literature

[2] Roerig J , Mitrach F , Schmid M , et al. Synergistic sirna loading of extracellular vesicles enables functional delivery into cells. Small Methods. 2022;6(12). doi: 10.1002/smtd.202201001

[3] Roerig J , Schiller L , Kalwa H , et al. A focus on critical aspects of uptake and transport of milk-derived extracellular vesicles across the caco-2 intestinal barrier model. European Journal of Pharmaceutics and Biopharmaceutics. 2021;166:61-74. doi: 10.1016/j.ejpb.2021.05.026

[4​] Roerig J , Schulz‐Siegmund M Standardization approaches for extracellular vesicle loading with oligonucleotides and biologics. Small. 2023;19(40). doi: 10.1002/smll.202301763​

funding​​​​​


Liposomes and lipid nanoparticles (LNPs)

Dr. Christian WölkNucleic acid based medicines can provide new strategies to treat various diseases. Hence, nucleic acids are connected with biopharmaceutical problems, the formulation, especially the delivery system, is the key for successful administration of nucleic acid based medicines.In our group the focus is set on different types of lipids/lipidoids as functionality determining components for nucleic acid delivery systems.


Sebastian Ammenwerth

These fascinating class of amphiphilic molecules can self-assemble to supramolecular structures (e.g. liposomes, lipid nanoparticles) while the physical-chemical characteristics of the lipids/lipidoids can be used to tune the performance of the lipid nanoparticles as nucleic acid delivery system. We prepare lipid formulations from commercial available lipids and also design novel lipids as component for nucleic acid delivery systems. The novel lipids include ionisable lipids and co-lipids to tune nanoparticle characteristics. Additional info available at ​Konsortium MRNA​.

Vincent Kampik

Beyond biological activity, the understanding of chemical-physical properties of lipids and lipid formulations is of interest, to find parameters for efficacy correlations and improve the design of new lipids.

Giselbrecht J, Wiedemann S, Reddy Pinnapireddy S, et al. Nucleic acid carrier composed of a branched fatty acid lysine conjugate-Interaction studies with blood components. Colloids Surf B Biointerfaces 2019; 184: 110547. doi: 10.1016/j.colsurfb​.2019.110547​​

Maria KrabbesTassler S, Dobner B, Lampp L, et al. DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids-The Effect of the Co-Lipid on the Structure and DNA Binding Capacity. Langmuir 2019; 35: 4613–4625. doi: 10.1021/acs.langmuir.8b04139​

Wölk C, Drescher S, Meister A, et al. General synthesis and physicochemical characterisation of a series of peptide-mimic lysine-based amino-functionalised lipids. Chemistry 2013; 19: 12824–12838. doi: 10.1002/chem.201204529​

fund​ing

BMWE​​

Liposomes as model membranes and drug carriers

Dr. Christian Wölk​One key of life is the compartmentalisation via lipid bilayer membranes. Nanometer sized lipid bilayer vesicles are named as liposomes. Liposomes are of various scientific interest. It is possible to use them as drug delivery systems, encapsulating APIs. Further it is possible to use them as membrane model to understand biomembranes. We design lipid vesicles and investigate the lipid assembly using various physical chemical methods:

  • differential scanning calorimetry
  • dynamic light scattering
  • zeta-potential measurements
  • assays to determine the pKa value and critical micelle
  • monolayer techniques
  • x-ray techniques 

In close cooperation with:​


Schulze J, Schöne L, Ayoub AM, et al. Modern Photodynamic Glioblastoma Therapy Using Curcumin- or Parietin-Loaded Lipid Nanoparticles in a CAM Model Study. ACS Appl Bio Mater 2023; 6: 5502–5514. doi: 10.1021​/acsabm.3c00695​

Brito Barrera YA, Hause G, Menzel M, et al. Engineering osteogenic microenvironments by combination of multilayers from collagen type I and chondroitin sulfate with novel cationic liposomes. Mater Today Bio 2020; 7: 100071. doi: 10.1016/j.mtbio.2020.100071

Schulze J, Schöne L, Ayoub AM, et al. Modern Photodynamic Glioblastoma Therapy Using Curcumin- or Parietin-Loaded Lipid Nanoparticles in a CAM Model Study. ACS Appl Bio Mater 2023; 6: 5502–5514.​ doi: 10.1016/j.msec.2020.111116

literature - on liposomes as model membranes

Hoernke M, Shi S, Hubbard ATM, et al. Daptomycin membrane activity is modulated by the localized interplay between calcium ions and phospholipids in monolayers and bilayers containing a lysyl-phosphatidylglycerol analogue. Biochim Biophys Acta Biomembr 2025; 1867: 184452. doi: 10.1016/j.bbamem.2025.184452

Wölk C, Shen C, Hause G, et al. Membrane Condensation and Curvature Induced by SARS-CoV-2 Envelope Protein. Langmuir 2024; 40: 2646–2655. doi: 10.1021/acs.langmuir.3c03079

Rehal R, Barker RD, Lu Z, et al. Lipid domain formation and non-lamellar structures associated with varied lysylphosphatidylglycerol analogue content in a model Staphylococcal plasma membrane. Biochim Biophys Acta Biomembr 2021; 1863: 183571. doi: 10.1016/j.bbamem.2021.183571

Wölk C, Youssef H, Guttenberg T, et al. Phase Diagram for a Lysyl-Phosphatidylglycerol Analogue in Biomimetic Mixed Monolayers with Phosphatidylglycerol: Insights into the Tunable Properties of Bacterial Membranes. Chemphyschem 2020; 21: 702–706.​ doi: 10.1002/cphc.202000026

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