Endothelial targeting of semi-permeable polymer nanocarriers for enzyme therapies

Abstract

The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their labile nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable polymer nano-carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, an H(2)O(2)-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 247 kD), peroxidase (MW 42 kD) and xanthine oxidase (XO, MW 300 kD) into approximately 300 nm diameter PNC composed of co-polymers of polyethylene glycol and poly-lactic/poly-glycolic acid (PEG-PLGA) was in the range approximately 10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted enzymatic activity on their PNC diffusible substrates, H(2)O(2) and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell (EC) adhesion molecule-1 delivered active encapsulated catalase to ECs and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities.

Figure 1. Antibody coupling to PNC using modular streptavidin-biotin system

(A) ¹²⁵I-IgG/SA was incubated with solid core PNC and centrifuged to remove unbound protein. One centrifugation was sufficient to remove most of unattached IgG/SA from non-biotinylated PNC used as a control (closed bars), while subsequent centrifugations did not remove IgG/SA coupled to biotinylated PNC (gray bars). (B) Size (left scale, closed bars) and amount of IgG/SA bound per PNC (right scale, grey bars) after coupling of ¹²⁵I-IgG/SA to biotinylated (left) or non-biotinylated (right) solid PNC with initial diameter ~140 nm. (C) ¹²⁵I-IgG/SA, but not ¹²⁵I-IgG coupling to catalase loaded Biotin-PEG-PLGA PNC (initial diameter 420nm). (N=3, M±SD).

Figure 2. Fluorescent microscopy of anti-PECAM/PNC targeting to PECAM transfected or control REN cells

Anti-PECAM/PNC or uncoated PNC were incubated with REN cells or REN/PECAM cells for 1 hour, washed, fixed and permeabilized and stained with an Alexaflour® 488-fluorescent secondary goat antibody against mouse IgG.

Figure 3. Endothelial targeting of catalase encapsulated into PECAM-targeted stealth polymer nanocarriers (anti-PECAM/PNC)

(A) Solid core PNC, uncoated vs coated with anti-PECAM, were incubated with REN cells or REN/PECAM cells for 1 hour. Cells were then washed, fixed, permeabilized, and stained with a FITC-labeled secondary antibody. Quantification was obtained by averaging 10 fields each from 2 separate slides. Unless specified otherwise, data in this and other figures is shown as M±SD. (B). Binding to HUVEC of anti-PECAM/PNC (closed symbols) vs IgG/PNC (open symbols) labeled by a 5 wt% fraction of ¹²⁵I-IgG-SA as a tracer coupled to solid PNC with 180 nm diameter. N=4. (C) Binding of ¹²⁵I-catalase encapsulated into anti-PECAM vs IgG coated PNC with 450 nm diameter to endothelial cells. N=4. (D) Biodistribution of anti-PECAM/PNC (closed bars) vs control IgG/PNC (gray bars) traced by 5mol% ¹²⁵I-IgG-SA 30 min after IV injection in naïve anesthetized mice. (N=3, M±SE).

Figure 4. Endothelial targeting of catalase encapsulated into anti-PECAM coated stealth polymer nanocarriers protects against oxidative stress

Panel A: cell culture studies. Human endothelial cells were incubated with ¹²⁵I-catalase loaded IgG/PNC (black bars) or anti-PECAM/PNC (grey bars) for 1 hour, washed and treated with 5mM H₂O₂. The amount of catalase delivered was determined by iodine tracing. Cell death was measured by ⁵¹Cr-release after 5 hours (56.1±2.1% ⁵¹Cr was released from control cells treated with 5mM H₂O₂) and percent of protection was calculated. N=4. Panels B–E: animal studies. Anesthetized mice were injected with either anti-PECAM/PNC/catalase or catalase-free anti-PECAM/PNC and lungs were isolated from the animals 30 min later to test their capacity to decompose perfused H₂O₂ (see Methods). Anti-PECAM/PNC loaded with catalase suppressed both lipid peroxidation in the lung determined by TBARs level (B: N=4, M±SE) and DCF-fluorescence (C–E) induced by H₂O₂. Typical images of 2-photon fluorescent microscopy show DCF fluorescence in the vasculature in control lungs without and with H₂O₂ infusion (C and D, respectively), as well as in the lungs obtained from mice injected anti-PECAM/PNC/catalase and infused with H₂O₂ (E). Scale bar = 54μm. Asterisks denote statistical significance (*p<=0.5, **p<=0.01).

Figure 5. Anti-PECAM/PNC/catalase provides durable antioxidant protection

⁵¹Cr-labeled endothelial cells were incubated with anti-PECAM/PNC/catalase at the indicated time interval, washed 5 times, and exposed to a 5 mM H₂O₂ insult for 1 hour. Rate of H₂O₂ decay in the cell medium was monitored (A) to calculate the activity of catalase by fitting a first order decay equation to the data (B). The kinetics of catalase inactivation after delivery to the cells (B) has a profile similar to previously published kinetics of proteolytic inactivation of catalase encapsulated into PNC (dashed line). The duration of residual enzymatic activity of catalase encapsulated into anti-PECAM/PNC bound to cells greatly exceeded that of protein anti-PECAM/catalase conjugate bound to cells (B). Analysis of ⁵¹Cr release from endothelial cells showed that anti-PECAM/PNC/catalase provided significant (p<0.01) protection against H₂O₂-induced injury even 21 hours after delivery. N=4 in all panels.

Figure 6. Encapsulation of diverse enzymes into PNC: substrate permeability via polymer control residual activity of the enzymes protected against external proteolysis

(A) ¹²⁵I-labeled catalase, horseradish peroxidase (HRP), and xanthine oxidase (XO) showed similar efficiency of encapsulation into PNC. (B). Residual enzymatic activity (closed bars) and load of radiolabeled enzyme (gray bars) after a 4 hour incubation of PNC loaded with catalase, HRP or XO in a 0.2% pronase solution. (C) Analysis of substrate permeability (a product of partitioning and diffusivity) via PLGA film showed that H₂O₂ and OPD have ~100-times higher permeability then xanthine. (C, inset) There was poor agreement between the experimental data and the Renkin diffusion model that describes the sieving effects of membrane pore size on substrate permeability. This result suggests that other chemical properties (e.g., polarity, electrostatic interactions, aqueous solubility) of the substrates also greatly impacted the observed rates of diffusion.