P-selectin targeting polysaccharide-based nano-gels for miRNA delivery

In recent years, non-coding microRNAs have been evaluated as they may offer great therapeutic potential to treat cancer, diabetes, cardiovascular diseases and genetic conditions. Non-coding miRNAs regulate gene expression but are sensitive to degradation and rely on suitable vehicles to target cells efficiently.

Nanogels are polymeric materials that can be used for the delivery of these microRNAs since they allow a high loading capacity, protecting the molecules against degradation. They can be natural or synthetic and contain more than one type of polymer.

Pullulan is a water-soluble homopolysaccharide produced by the fungus Aureobasidium pullulan. It’s a promising material for biomedical applications such as drug/gene delivery and tissue engineering. Cationic pullulan derivatives can form complexes with plasmid DNA and siRNA in cationized pullulan-based hydrogels and be used for in vitro miRNA delivery.

So, researchers at the Université de Paris (France), and Universidade do Mino (Portugal), proposed a simple drug delivery system with polysaccharides pullulan and fucoidan, which instantly and spontaneously self-assemble to form nanosized hydrogels.

Methods

Targeting assays

Chemical crosslinking is an approach to stabilizing polyelectrolyte complexes (PECs) and genipin is an alternative natural crosslinking agent. In this experiment, the researchers tested the interaction of genipin cross-linked NPs with P-selectin in vitro. This adhesion molecule is typically over-expressed at the surface of activated platelets and endothelial cells in atherothrombotic-related diseases.

For that, they coated Vena8 Fluoro+ chambers (Cellix) overnight with 50 µg/mL of collagen. Then, they washed them with NaCl 0.9%. After that, they perfused human whole blood labelled with DiOC6 (a green fluorescent dye) under arterial flow conditions for 5 minutes to induce platelet activation and aggregation on the collagen-coated channel.

Then, the researchers perfused rhodamine-labelled cross-linked PECs (G-PECs) at 1 mg/mL in NaCl 0.9% for 5 minutes to monitor their accumulation onto preformed platelet aggregates in real-time by fluorescence microscopy. They took images of each micro-channel after washing with NaCl 0.9%. G-PECs formulated with carboxymethyl-dextran were the negative control.

Results

G-PECs with fucoidan were able to target P-selectin expressed in activated platelets, Fig. 8.

Fig. 8. Evaluation of G-PECs binding on activated platelets under arterial flow conditions. Particles and platelets aggregates co-localization was evaluated by merged fluorescence microscopy images (right). G-PECs without fucoidan were used as control. Scale bars correspond to 50 µm.

Other experiments in this study

The researchers describe the synthesis of a cationic pullulan derivative by grafting primary amine groups. By adding genipin, they produced nanosized hydrogels with high colloidal stability.

Besides testing G-PEC’s ability to target P-selectin, the researchers also investigated their capacity to cargo nucleic acids, in particular, miRNA into endothelial cells.

Main findings of these experiments

G-PECs were able to promote miRNA delivery inside cells, as demonstrated by fluorescence microscopy images of labeled miRNA, Fig. 10.

Fig. 10. FAM-miRNA delivery. (A) Fluorescence microscopy images of HUVECs evidencing miRNA delivery. Left column: cells’ nuclei stained with DAPI (depicted in blue); middle column: FAM labelled miRNA (depicted in green); right column: merged images for cells treated with naked FAM-labelled miRNA and cells treated with G-PECs nanogels loaded with FAM-labelled miRNA; Treatments were performed for 2 h at 37 ◦C. Scale bar: 20 μm. (B) Mean fluorescence intensity (MFI) of fluorescein amidite (FAM) measured by flow cytometry analysis. Results are shown as mean ± SD from three experiments.

Conclusions

These results indicate that nanogels could be an interesting platform for miRNA-based therapeutics in atherothrombotic diseases due to their ability to target over-expressed P-selectin.

If you'd like to learn more, you can click here to access the complete study.

How to get started?

Are you considering running similar experiments in your lab? This is the minimum experimental setup you’ll need:

Vena8 Fluoro+ biochips — to mimic human blood vessels and model blood clots.
Mirus Evo pump — to control flow rates in the biochip. You may set the shear rate to model thrombosis in microcapillaries or other vessels.
Microenvironmental chamber — a temperature-controlled frame that keeps the biochip at 370C. The microenvironmental chamber sits on the microscope stage
Inverted microscope — we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option
Digital camera — to capture images and video recordings. We supply the Prime BSI Express with the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.
Image Pro Cell Analysis software — for your image and video analysis.

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. We have options that suit all budgets. You can check them out on our eShop.

References

Moraes, Fernanda C., et al. "P-selectin targeting polysaccharide-based nanogels for miRNA delivery." International Journal of Pharmaceutics 597 (2021): 120302.