Surface display of SARS-CoV-2 proteins on nanoparticle vaccines produces robust immunity to COVID-19.

• In this work, we evaluated the best way to deliver the protein that is found on the outside of SARS-CoV-2 known as the Receptor Binding Domain or RBD.
• (a) We designed polymer nanoparticle vesicles (polymersomes) that display RBD on their surface (RBD_surf) and compared these to polymersomes that encapsulate RBD within their shell (RBD_encap) and to RBD on its own (RBD_free). As an adjuvant or danger signal to stimulate the immune system we used polymersomes containing a lipid derived from bacteria (MPLA PS).
• (b) Our primary finding is that only mice vaccinated with RBD_surf are able to produce antibodies that neutralize SARS-CoV-2. That is, these antibodies prevent the virus from infecting cells in vitro. And they work as good as or better than antibodies from people who have contracted and recovered from COVID-19.
• What does this mean for future nanoparticle vaccine design? Making vaccines that “look” like viruses by displaying proteins on their surface may be an effective formulation strategy.

Check out our 2021 publication in ACS Central Science for the full story.

Electrostatic complexes of insulin and a polycation provide glucose-responsive insulin delivery.

• Positively charged polymers such as polyethyleneimine (PEI) have been used to deliver negatively charged nucleic acids for decades through the formation of electrostatic complexes. We hypothesized that we could use a similar technique to deliver insulin in a glucose-dependent manner.
• Insulin has a slight negative charge under physiological conditions, so we thought it could form complexes with polycations. To test this hypothesis, we performed molecular dynamics (MD) simulations between insulin and PEI as a model polycation.
• (a) The simulations provide a theoretical basis for the formation of our complexes: we found that insulin and PEI interact throughout the simulation and that a single amino acid, the glutamic acid residue at position 21 on insulin’s B chain, is primarily responsible for these interactions.
• (b) When we form complexes that contain the enzyme glucose oxidase (GOx), we can create a glucose-responsive insulin delivery system. GOx converts glucose to gluconic acid and thus lowers the pH near the complexes. At pH < 5, insulin develops a positive charge and is repelled from the PEI. Therefore, insulin is released in conditions of high glucose.
• Importantly, these complexes are comprised over half insulin, so we can limit the amount of extra material injected in each dose. These complexes are able to provide glycemic control in diabetic mice that is similar to glucose profiles of healthy mice.

Read our 2021 publication in the Journal of Controlled Release for more details.

A co-formulation of nanoparticles provides both rapid and extended self-regulated insulin release.

• (a) We developed a glucose-responsive insulin delivery system comprised of pH-sensitive acetalated-dextran (Ac-Dex) nanoparticles (NPs) encapsulating glucose oxidase (GOx), catalase, and insulin. GOx converts glucose to gluconic acid, translating a change in glucose concentration to a change in pH. Catalase aids in this process by turning the harmful byproduct of this reaction into a useful starting reagent. Therefore, when glucose levels are high, the pH is reduced, Ac-Dex degrades under these acidic conditions, and insulin is released on-demand.
• (b) When we combine two types of Ac-Dex NPs, one that degrades quite rapidly in response to glucose and one that degrades more slowly, we can achieve both rapid and extended insulin release. This means that the insulin will start working right away from the rapid release NPs and will last the entire day from the extended release NPs.
• (c) We show in healthy mice that the NPs release insulin only after an injection of glucose known as a glucose tolerance test (GTT). If the glucose-sensing enzymes or the glucose are absent, no increase in insulin release is observed. This experiment provides direct evidence of glucose-responsive insulin release in animals.
• (d) In diabetic mice, our NPs provide better control over blood glucose levels than free insulin or a commercial long-acting insulin at the same insulin dose. After two GTTs, mice receiving our NPs remain in the normal glycemic range while mice receiving regular insulin or long-acting insulin have high blood glucose levels.

Read about more experiments we performed in our 2020 ACS Nano publication.

Encapsulating glucose-responsive nanoparticles in microgels prolongs glycemic control in diabetic mice.

• (a) Here, we encapsulate the glucose-responsive nanoparticles (NPs) that we previously developed into alginate microgels to extend their functional lifetime. The microgels are porous such that when glucose diffuses in, it is enzymatically converted to gluconic acid, the local pH decreases, the NPs degrade, and insulin is released.
• The insulin release kinetics of microgel-encapsulated NPs are similar to free NPs but when subcutaneously injected into healthy mice the microgel-encapsulated NPs remain at the injection site significantly longer. Thus, the lifetime of the NPs can be extended by encapsulating them in microgels.
• (b) In a diabetic mouse model, two doses of microgels 6 days apart provide over 3 weeks of glycemic control, defined as blood glucose levels less than 200 mg/dL. This means fewer injections and better control compared to free NPs which are dosed daily.
• (c) We give the mice large doses of glucose (glucose tolerance tests, GTTs) on days 3, 6, and 9. The areas under the blood glucose curves (AUCs) provide a measure of how the mice respond to these tests. Mice treated with our microgel system respond similarly to healthy mice, while mice that received empty microgels with no NPs have a much worse response.

Find out more about this work in our 2021 paper published in Biomaterials.

Core-shell microgels have structurally distinct compartments that enable loading and delivery of two different classes of drugs.

• (a) We form core-shell droplets in a double T-junction microfluidic device. In the first T-junction, there is liquid-liquid phase separation of polyethylene glycol (PEG) and dextran. In the second T-junction, these aqueous phases enter into a stream of oil containing surfactant that results in the formation of water-in-oil droplets.
• (b) Using an ultra high-speed camera coupled with a microscope, we can actually visualize these droplets forming in the device.
• (c) Due to the phase separation of PEG and dextran, the droplets contain a core and a shell. The droplets are about 50 μm in diameter and are highly reproducible in size.
• We also add the protein lysozyme into both aqueous phases. While soluble under physiological conditions, this protein aggregates to form ordered nanofibrils upon heating. Thus, after incubation the droplets gel to form a stable core-shell structure.
• (d) We studied the formation of these nanofibrils in the two phases using biophysical characterization techniques to elucidate the core-shell structure on the nano- and micro-scales. Additionally, cryo-scanning electron microscopy (SEM) shows that the core and shell phases are morphologically distinct with different pore sizes.
• Finally, we found that two different classes of drug-like small molecules can be encapsulated in the different phases for use in co-delivering combination therapeutics.

This work was published in 2014 in the Journal of Materials Chemistry B. Click to read more about our biophysical characterization including AFM, SAXS, and WAXS measurements.