Using flexible microparticles as drug carriers to shuttle nanoparticles to the vascular wall

Using flexible microparticles as drug carriers to shuttle nanoparticles to the vascular wall
Material properties of NP-loaded hydrogel MPs. (A) Schematic and representative confocal microscopy fluorescence images of hydrogel MPs evaluated, having varied modulus and NP loading. Red is MP hydrogel, green is 50-nm PS NPs, and the two are overlaid to show colocalization of NPs and hydrogel MPs. Scale bar, 5 μm. Swollen shear moduli for (B) 15% PEG and (C) 50% PEG hydrogels showing the influence of adding NPs to bulk material rheometry. Statistical analyses were performed using one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test, where (***) indicates P < 0.001 in comparison to the nonloaded hydrogels. N = 3. Error bars plot SE. Credit: Science Advances, doi: 10.1126/sciadv.abe0143

Drug carriers that target the vascular endothelium must adhere to the endothelial vessel wall to achieve clinical stability. The particle size is a critical physical property to prescribe particle margination within biological blood flows and those conducted in-lab. While microparticles are optimal for margination, nanoparticles are better for intracellular delivery. In a new report now on Science Advances, Margaret B. Fish and a research team in chemical engineering, pharmacology and cardiovascular medicine and engineering at the University of Michigan, Ann Arbor U.S., tested flexible hydrogel particles as carriers to transport nanoparticles to a diseased vascular wall. Based on the microparticle modulus, nanoparticle-loaded poly (ethylene glycol)-based hydrogel microparticles delivered more than 50-nm nanoparticles to the vessel wall, when compared to freely injected nanoparticles to achieve more than 3000 percent increase in delivery. The work showed the benefit of optimizing the efficiency margination of microparticles to enhance transport of nanocarriers to the vascular wall.

Designing drug carriers

Drug carriers that target the vascular wall are usually made of polymeric particles engineered to adhere to sites of disease and accumulate via markers on the vessel wall for localized drug delivery. The physical properties of drug carriers can determine the circulation time, biodistribution, vascular adhesion and immune interactions. Efficient vascular wall adherence is vital for the accurate release of their drug payload to the diseased endothelium tissue. Although nanoparticles (20 to 80 nm in diameter) are an appealing drug carrier candidate, only less than 1 percent reach the intended site. Comparatively, microparticles with 2- to 3-micrometer-diameter appear to be optimal drug carriers. Fish et al. therefore examined the possibility of loading nanoparticles into vascular-targeted flexible microparticles to overcome the existing limits with free nanoparticles. Using nanoparticle-loaded microparticles, the team showed the comparatively effective delivery of nanoparticles to the vascular wall. This outcome provides an avenue to increase the clinical use of nanoparticle drug carriers to treat common diseases.

Using flexible microparticles as drug carriers to shuttle nanoparticles to the vascular wall
Adhesion of NP-loaded hydrogel MPs to an inflamed HUVEC monolayer at 200 s−1 WSR. (A) Schematic detailing “fixed MP concentration” in vitro flow experiments. Quantified (B) adhesion for anti–ICAM-1–coated hydrogel MPs dosed in blood at a fixed MP concentration and scaled to (C) the corresponding number of NPs delivered by the adherent hydrogel MPs in (B). (D) Schematic of the free NP in vitro flow experiments. (E) Number of NPs delivered to the vascular wall by free anti–ICAM-1–coated PS NPs dosed at 3 × 107 NPs/ml or based on (F) the adhesion of hydrogel MPs dosed in blood to carry a fixed three times lower NP cargo of 1 × 107 NPs/ml. For all, adhesion was quantified after 5 min of laminar blood flow over an IL-1β–activated HUVEC monolayer. N ≥ 3 human blood donors per particle condition. Statistical analysis of adherent density was performed using one-way ANOVA with Fisher’s LSD test, where (*) indicates P < 0.05, (**) indicates P < 0.01, (***) indicates P < 0.001, and (****) indicates P < 0.0001 versus the first bar in each plot. Error bars plot SE. Credit: Science Advances, doi: 10.1126/sciadv.abe0143
Developing and testing nanoparticle (NP)-loaded microparticles (MPs).

The scientists first fitted the hydrogel carriers with polymeric nanoparticles as cargo. To accomplish this, they chose polystyrene (PS) NPs due to their uniform size distribution and the consistency of NP loads across different MP formulations. The team then tested parameters of particle adhesion to understand how rigid polystyrene nanoparticles with an elastic modulus of about 2 GPa affected the bulk modulus of the hydrogels. For this, they loaded the 50-nm polystyrene NPs into hard microparticles and noted no significant increase in the bulk shear modulus, as well as considerable flexibility. Then, Fish et al. tested the capacity of NP-loaded hydrogel microparticles to bind to an activated human umbilical vein endothelial cell (HUVEC) monolayer during human blood flow in a parallel plate flow chamber, in the lab. Using the test assay, they quantified the number of nanoparticles and microparticles trafficked to the vessel wall. The team further studied the loaded hydrogel MPs relative to free NPs on a plate-reader. The results showed how drug carriers with higher NP loading delivered a significantly higher NP payload to the wall. Based on the constitution of diverse drug carrier prototypes, Fish et al. noted the 50 percent polyethylene glycol (PEG) constituting microparticles to have delivered the most nanoparticles. Compared to free NPs alone, the hydrogel microparticle delivery quantitatively achieved a 1550 percent increase in the number of nanoparticles to reach the vessel wall.

Using flexible microparticles as drug carriers to shuttle nanoparticles to the vascular wall
Adhesion of NP-loaded hydrogel MPs to an inflamed HUVEC monolayer at 1000 s−1 WSR. Quantified (A) adhesion for hydrogel MPs dosed in blood at a fixed MP concentration and scaled to (B) the corresponding number of NPs delivered by the adherent hydrogel MPs in (A). (C) Number of NPs delivered to the vascular wall by free anti–ICAM-1–coated NPs dosed at 3 × 107 NPs/ml or based on (D) the adhesion of hydrogel MPs dosed in blood to carry a fixed three times lower NP cargo of 1 × 107 NPs/ml. For all, adhesion was quantified after 5 min of laminar blood flow over an IL-1β–activated HUVEC monolayer. N ≥ 3 human blood donors per particle condition. Statistical analysis of adherent density was performed using one-way ANOVA with Fisher’s LSD test, where (*) indicates P < 0.05, (**) indicates P < 0.01, (***) indicates P < 0.001, and (****) indicates P < 0.0001 versus the first bar in each plot. Error bars represent SE. Credit: Science Advances, doi: 10.1126/sciadv.abe0143

Nanoparticle (NP) vessel wall binding dynamics

Based on several control experiments, Fish et al. next confirmed how the difference between NPs delivered to vessel walls via MPs versus free NPs, did not merely rely on the free NPs binding to blood cells or being phagocytosed by blood leukocytes. To accomplish this, they performed flow cytometry experiments of blood samples collected after flow assays and found an insignificant number of leukocytes bound by NPs. In addition to that, when they incubated free NPs in static blood setups in the lab, only a very minimal number of blood cells bound NPs in static assays. The team therefore credited the low NP adhesion to be due to a failure to bind to the vessel wall, and not due to their clearance via phagocytosis, nor due to their non-specific binding to blood cells. They then conducted clinical tests to compare the adhesion of NP-loaded MPs vs. free 50 nm NPs in the mesentery veins of mice. They chose the mesentery with acute inflammation to visualize particle adhesion using intravital microscopy. The hydrogel MPs were significantly more efficient at delivering 50 nm polystyrene nanoparticles to an inflamed mesentery in the biological model, compared to free NPs, regardless of the quantity of free NPs loaded.

Using flexible microparticles as drug carriers to shuttle nanoparticles to the vascular wall
Delivery of NPs to an inflamed mesentery endothelium as a function of loading into hydrogel MPs. (A) Representative bright-field and fluorescence images of particle adhesion to inflamed mesentery. n/a, not applicable. (B) Quantified adhesion density of three different particle conditions, 15% PEG, low loading hydrogel MPs, 15% PEG, high loading hydrogel MPs, and free NPs. Particles were dosed by equivalent NP payload. (C) Data scaled to the number of NPs delivered by adherent hydrogel MPs to show the efficiency of NP delivery by each VTC system. N = 3 mice per group, and statistical analysis was performed using one-way ANOVA with Fisher’s LSD test, where (**) indicates P < 0.01 and (***) indicates P < 0.001 compared to the low NP–loaded 15% PEG. Error bars plot SE. Scale bar, 50 μm. Credit: Science Advances, doi: 10.1126/sciadv.abe0143

Sustained adhesion of particles in time.

While nanoparticles are known to maintain longer circulation times when compared to micro-sized particles, it is assumed that 50 nm polystyrene particles would outperform MPs across time. To understand this, the team assessed targeted particle binding duration by investigating and comparing three flexible particle types directly to the 50 nm polystyrene particles. They then captured particle adhesion in five distinct locations of the mesentery vein every five minutes for one hour. During the hour-long frame, the hydrogel NPs did not match or surpass the hydrogel MPs in targeted adhesion efficiency. The team next investigated a longer targeting window with an acute lung injury model and noted an extended presence of targeted flexible adhesion of the hydrogel MPs in vivo.

Using flexible microparticles as drug carriers to shuttle nanoparticles to the vascular wall
Behavior of targeted hydrogel particles in mice with acute lung injury. Accumulation of PEG-based (A) 2-μm MPs and (B) 500-nm NPs in lung injury mouse lungs 2, 4, 8, and 24 hours after particle injection. (C and D) Blood circulation profile over time in lung injury mice showing the concentration of PEG-based particles remaining in the bloodstream of lung injury mice minutes after particle injection. Plots are shown for both ICAM-1 targeted (T) and untargeted (U) particles. Bars represent the SE for N = 4. Statistical analysis was performed using one-way ANOVA with Fisher’s LSD test, where (*) indicates P < 0.05 compared to the untargeted particle at that time point. Credit: Science Advances, doi: 10.1126/sciadv.abe0143
Outlook

In this way, Margaret B. Fish and colleagues showed how loading (NPs) into hydrogel microparticles (MPs) had excellent influence on improving the delivery of smaller NPs for a variety of clinical situations suited for targeted drug delivery. Due to their highly tuneable flexibility, the team could design the hydrogel carriers to ensure easy transport through the vasculature with low risk of vessel occlusion on binding, much like the native white blood cells. When compared to free NPs, the soft hydrogel MPs offered significantly stronger and sustained adhesion, during all experiments. This work demonstrated a massive advantage of trafficking NPs to the vessel wall via the strategy of loading NPs into hydrogels and the outcome can be optimized for clinical applications across regenerative medicine and bioengineering.


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More information: Fish M. B. et al. Deformable microparticles for shuttling nanoparticles to the vascular wall, Science Advances, DOI: 10.1126/sciadv.abe0143

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