I GOT A NEW WAY TO WALK: NEW METHOD FOUND TO GET PROTEIN DRUGS INTO CELLS

Michelle Ehrenberger

Title: “DNA-Functionalized Metal-Organic Framework Nanoparticles for Intracellular Delivery of Proteins”

Authors: Wang, S., Chen, Y., Wang, S., Li, P., Mirkin, C.A., Farha, O.K.

Journal: J. Am. Chem. Soc

Year: 2019

https://pubs.acs.org/doi/full/10.1021/jacs.8b12705

Featured image and figures used with permission via ACS Editors’ Choice open access

When we think of medicine, our first thought is often about small molecules like the antibiotic penicillin or the analgesic acetaminophen. Proteins, however, can also be used to treat human disease such as when the peptide hormone insulin is used to treat diabetes. Since many of the jobs inside cells are completed by proteins, it could be very beneficial to be able to deliver specific protein drugs directly into cells, such as when certain cells don’t make as much of a protein as they should. Proteins can be fairly large molecules though, so it can be difficult for them to move from the blood stream into cells by crossing the cell membranes that are intended to keep foreign substances out. For that reason, scientists are developing new ways to trick cells into engulfing proteins, much like a dog’s medicine might be hidden inside food.

The basis for this recent strategy is to encompass many proteins—in this case, insulin—within the framework of many oblong nanoparticles (NPs). These nanoparticles are small and stable, so they can travel throughout the bloodstream, and they are porous so they can carry cargo like proteins within their pores. These NPs, like many in recent years, are made of Metal-Organic Frameworks (MOFs), meaning a combination of organic and metal (in this case, Zirconium) building blocks. This combination gives peptides like insulin a lot of sticky chemical groups to hold onto, making this a good delivery system. In order to find the best insulin-delivering NP, they tried using two different MOFs: one called NU-1000 (using the organic molecule H4-TBAPy, Fig. 1B) and the other called PCN-222 (using another organic molecule, H4-TCPP-H2, Fig. 1B).

Fig. 1 Scheme of insulin in DNA-MOF-NPs Nu-1000 and PCN-222. (A) Cartoon, (B) MOF scheme, (C) DNA attachment scheme.

However, these MOF NPs tend to aggregate (clump together) which hinders them from flowing freely through body fluids to reach their targets. They are also highly positively charged which makes it difficult for them to enter cells and can even cause death in the cells they interact with. Thus, the scientists Wang and colleagues recognized the need for a coating around these nanoparticles to combat these downsides. Luckily, there is a very common and easy to work with biomolecule with a net-negative charge: DNA.

DNA is made up of nucleotide bases attached to each other via a backbone of deoxyribose sugars and negatively charged phosphate groups (Fig. 1C). The negatively charged phosphate group can interact with the positively charged zirconium metal in the MOF NP. This leads to a hair-like coating of DNA strands surrounding the NP. To determine whether the insulin and DNA would both cling to the MOF NPs, they labeled insulin with a green fluorescent dye and DNA with a red fluorescent dye and looked at their MOFs using a confocal microscope. Indeed, they did see the DNA and insulin (Fig. 2A) in the same oblong shapes as the MOFs (Fig. 2C,D). Now that the NPs had been fully assembled, they need to be tested for their ability to remain in suspension (i.e. not aggregate), enter cells, and not cause cell death.

Fig. 2 Microscopy. (A) Confocal microscopy of DNA-NU-1000 with red DNA and green insulin fluorescent dyes. (B,C) Scanning electron microscopy of NU-1000 (B) and PCN-222 (C). Scale bars are 100nm.

After determining that the DNA-MOF-NPs did not have their predecessors’ aggregation problems, the scientists tested whether they could enter human ovarian cancer cells better than free insulin and free DNA could. In this experiment, they again labeled insulin with green fluorescent dye and DNA with red fluorescent dye and looked at their location within cells through confocal microscopy (Fig. 4A-C, right). In samples with only free insulin/DNA, there wasn’t much sign of either inside the cells, but the cells with either DNA-MOF-NP showed fluorescence from both DNA and insulin. Another method—flow cytometry—was used to quantify the amount of green and red fluorescence overlapping in many cells. This can be seen on the graphs as a heat map of fluorescence intensity, and the more of the heat map that lies in the upper-right quadrant of the graph, the more DNA-MOF-NP uptake there was (Fig. 4A-C, left). The scientists also tested the number of living and dead cells before and after DNA-MOF-NP treatment, and found no change for either DNA-NU-1000 or DNA-PCN-222 up to 1nM concentration.

Fig. 4 (A,B,C) Co-localization of red stained DNA and green stained insulin inside human ovarian cancer cells measured by flow cytometry (left) and confocal microscopy (right).

These DNA-coated Molecular Organic Framework Nanoparticles show a lot of promise as a potential delivery system to get protein drugs into human cells, and thus there is a lot of work still to be done. How well can other proteins besides insulin be transported? What are the potential side-effects, whether from the whole DNA-MOF-NPs or their degradation products? As more years of research find answers to these questions, DNA-coated MOFs may one day become the standard way to carry life-saving protein drugs into our cells.

15 Shares

Leave a Reply