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Enhanced adhesion of ligand-conjugated biodegradable particles to colitic venules

Harshad S. Sakhalkar^*, Justin Hanes^¶, Jie Fu^¶, Uruguaysito Benavides, Ramiro Malgor, Cheri L. Borruso^*, Leonard D. Kohn^,, David T. Kurjiaka and Douglas J. Goetz^*,1

^* The Department of Chemical Engineering,
The Department of Biological Sciences,
Edison Biotechnology Institute,
The Department of Biomedical Sciences, Ohio University, Athens, Ohio USA; and
^¶ The Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA

¹ Correspondence: Department of Chemical Engineering, 172 Stocker Center, Ohio University, Athens, OH 45701, USA. E-mail: goetzd{at}ohio.edu

SPECIFIC AIMS

There has been increasing effort to develop schemes that selectively deliver drugs to sites of pathological inflammation via endothelial cell adhesion molecules (ECAMs). We sought to determine whether biodegradable polymeric particles conjugated with ligands to VCAM-1, which has been shown to be up-regulated at sites of pathological inflammation, would exhibit augmented and avid adhesion to inflamed endothelium in an animal model of inflammatory bowel disease.

PRINCIPAL FINDINGS

1. Biodegradable particles conjugated with a mAb to VCAM-1 exhibit augmented adhesion to the colonic vasculature of DSS-induced colitic mice compared with control mice
We recently demonstrated that particles made from a biodegradable polymer conjugated with ligands to ECAMs that are up-regulated at sites of inflammation exhibit specific and augmented adhesion to inflamed endothelium relative to noninflamed endothelium in vitro and in vivo. We term these particles leukocyte-endothelial cell adhesive particles (LEAPs) to reflect the fact that their design is inspired by the biochemistry and biophysics that govern leukocyte adhesion to the endothelium. Inflammatory bowel disease (IBD) is a relentlessly debilitating ailment characterized by rapid and prolonged infiltration of leukocytes and involving an increased expression of VCAM-1. In this paper, we explored the behavior of LEAPs in dextran sulfate sodium (DSS)-induced murine colitis, a well-accepted model of human IBD.

For this study, we synthesized a biodegradable block copolymer consisting of biotinylated poly(ethylene glycol) (PEG) and poly(lactic acid) (PLA) blocks. Phase separation of PEG and PLA upon particle preparation using emulsion methods ensures that the particle surface is rich in biotinylated PEG, allowing facile linkage of targeting moieties to the particles. As revealed by flow cytometric analysis, we successfully coupled a VCAM-1 mAb to the PLA-PEG particles at a high density. We term these particles -V LEAPs. Confocal analysis of the -V LEAPs revealed that the bound mAb was localized to the periphery of the -V LEAPs.

We tested the adhesion of -V LEAPs in the DSS-induced (3% DSS, 7 days) murine model of colitis. Suspensions of rhodamine-loaded -V LEAPs or rat IgG PLA-PEG particles (negative control) were injected directly into the bloodstream of groups of mice (1x10⁷/mouse). As shown in Fig. 1 A, B, a significantly greater number of -V LEAPs were adherent in colitic mice than in control mice (noncolitic and colitic mice pretreated with the VCAM-1 mAb) and a significantly greater number of -V LEAPs (13-fold) were adherent in colitic mice vs. the number of IgG PLA-PEG particles adherent in colitic mice. The -V LEAPs appeared to bind directly to the vessel wall and exhibited a biphasic adhesive behavior wherein the -V LEAPs traveled at the free stream velocity; those that adhered abruptly adhered (the velocity of the particles went to zero within 1 video frame, 1/30th of a second) and remained firmly adherent with no rolling observed.

View larger version (37K): [in this window] [in a new window]   Figure 1. -V LEAPs exhibit selective and augmented adhesion to DSS-induced colitic vasculature. A) Segments of postcapillary venules from typical experiments. White spheres are rhodamine-loaded -V LEAPs or IgG particles that adhered to the walls of postcapillary venules. An image taken just before particles were injected (top panels) and another 2.5 min after injection (bottom panels). Ligand indicates which molecule was coupled to the particles: a mAb to murine VCAM-1 (-V) or rat IgG (IgG); DSS indicates whether postcapillary venule is in a mouse treated with DSS to induce colitis (+) or in a control mouse (–). B) -V LEAPs or IgG (negative control) particles were injected as a bolus into colitic and noncolitic mice (1x107/mouse) and the number of adherent particles observed in the postcapillary venules of the colon was noted. All adherent particles were firmly adherent (i.e., not rolling). mAb pretreatment indicates pretreatment of mice with VCAM-1 mAb (+) or no pretreatment (–). Ligand indicates which molecule was coupled to the particles: a mAb to murine VCAM-1 (-V) or rat IgG (IgG); DSS indicates treatment with DSS to induce colitis (+) or no treatment (–). n 3; *P < 0.05 compared with each of the right bars.

2. Selectivity and ligand efficiency are functions of the total number of particles injected
We next explored the relationship between the number of particles injected and targeting efficiency. We injected 5 x 10⁶, 1 x 10⁷, and 3 x 10⁷ -V LEAPs and IgG PLA-PEG particles into different sets of mice. The number of particles injected was varied by varying the concentration of particles in the suspension. We found that the number of -V LEAPs adherent in colitic or noncolitic mice was a function of the number of particles injected (Fig. 2 A–C). In contrast, the number of adherent IgG PLA-PEG particles in colitic mice and the number of adherent -V LEAPs in colitic mice pretreated with the VCAM-1 mAb was not a function of the number of particles injected. In each case the number of -V LEAPs adherent in colitic mice was significantly greater than the number of -V LEAPs adherent in control mice (i.e., noncolitic and colitic mice pretreated with the VCAM-1 mAb) and significantly greater than the number of IgG PLA-PEG particles adherent in colitic mice.

View larger version (14K): [in this window] [in a new window]   Figure 2. The number of particles injected affects the efficiency of targeting. A–C) -V LEAPs or rat IgG-conjugated particles (A, 5x106; B, 1x107, or C, 3x107/mouse) were injected at a volumetric flow rate of 40 µL/min into colitic and noncolitic mice and the number of adherent particles in the postcapillary venules of the colon was noted. All adherent particles were firmly adherent (not rolling). mAb pretreatment indicates pretreatment of mice with the VCAM-1 mAb (+) or no pretreatment (–). Ligand indicates which molecule was coupled to the particles: a mAb to murine VCAM-1 (-V) or rat IgG (IgG); injected indicates the total number of particles infused into the mice; Black bars indicate adhesion to the postcapillary venules in colitic mice; gray bars indicate adhesion to the postcapillary venules in noncolitic mice. n 3; *P < 0.05 compared with each of the other 3 bars in panel; ANOVA revealed that -V LEAP adhesion in colitic and noncolitic mice was a function of particle number whereas IgG particle adhesion in colitic mice and -V LEAP adhesion in colitic pretreated with the VCAM-1 mAb were not a function of particle number. D) Selectivity, defined as the ratio of the number of -V LEAPs that adhere to the colonic vasculature of colitic mice relative to the number of -V LEAPs that adhere to the colonic vasculature of noncolitic mice, was plotted vs. total number of -V LEAPS initially injected. ANOVA indicated that the selectivity was a function of number of particles injected (P<0.05). Selectivity at 1 x 107 was significantly greater than at 3 x 107, but not statistically different from the selectivity at 5 x 106. E) Ligand efficiency (ratio of the number of -V LEAPs relative to the number of IgG PLA-PEG particles that adhere to colonic vasculature of colitic mice) was plotted vs. total number of particles injected. Ligand efficiency was an ascending function of the number of particles injected and did not appear to have an optimal value. ANOVA indicated that ligand efficiency was a function of the number of particles injected (P<0.002).

We plotted the selectivity (ratio of -V LEAPs adherent in colitic mice to -V LEAPs adherent in normal mice) and ligand efficiency (ratio of -V LEAPs adherent in colitic mice to IgG PLA-PEG particles adherent in colitic mice) vs. the number of particles injected. Both selectivity and ligand efficiency were a function of the number of particles injected (Fig. 2D, E ). Note that the selectivity observed when 1 x 10⁷ particles were injected (3-fold) was significantly greater than that observed when 3 x 10⁷ particles were injected. The highest ligand efficiency was seen when 3 x 10⁷ particles were injected: 24-fold more -V LEAPs bound to colitic vasculature compared with the IgG PLA-PEG particles.

CONCLUSIONS AND SIGNIFICANCE

Since certain drugs commonly used to treat pathological inflammation (e.g., corticosteroids for the treatment of IBD) have significant adverse side effects, there is a strong interest in the development of delivery schemes that target drugs selectively to sites of pathological inflammation. Drug carriers made from biodegradable polymers are easily prepared, have a long shelf life, can carry several orders of magnitude more drug than a monoclonal antibody (mAb), can be designed to have well-defined drug-release rates, and so may be ideal vehicles around which to develop such a targeting scheme. ECAMs whose expression is increased at sites of pathological inflammation could serve as a target for directed drug delivery.

We previously developed biodegradable particles (LEAPs) that selectively and avidly adhere to inflamed endothelium in vitro and in vivo. The present work significantly advances this study by demonstrating that -V LEAPs exhibit enhanced and avid adhesion to inflamed endothelium in a disease model of pathological inflammation (i.e., a model of IBD). In line with other reports, we found evidence of a statistically significant 3-fold increase in VCAM-1 expression in the colon of DSS-treated mice vs. controls (data not shown). Thus, the 3-fold selectivity we observed may reflect the maximal selectivity that can be obtained in this model. The data presented in Fig. 2D also suggest that increasing the number of drug carriers injected will not necessarily help increase selective targeting. In contrast, it appears that increasing the number of particles has a dramatic, positive impact on ligand efficiency in that the binding of IgG particles does not increase with increasing particle number; adhesion of IgG particles appears to be saturated.

In the present study we used particles on the order of 1000 nm in diameter. Since these are too big to be endocytosed by the endothelium, the drug would be released in the flow stream adjacent to the endothelium and therefore be subject to the convective effect of blood flow, which could diminish the selective targeting. We point out, however, the routine observation that soluble mediators (cytokines and chemokines released by endothelial cells and leukocytes) can have a dramatic localized effect on cell behavior. Our laboratories are now working to develop smaller LEAPs (<200 nm) that can be endocytosed by endothelial cells and thus could release their payload within the inflamed endothelial cells. Altering particle size may also affect the nonspecific trapping/uptake of particles by the RES, an issue that needs to be systematically addressed in future studies.

In conclusion, we demonstrated that -V LEAPs exhibit significant enhanced and avid adhesion to inflamed endothelium in the DSS-induced model of colitis. In addition, we found that selectivity and ligand efficiency are both functions of the number of particles injected for the range of particle numbers tested. This study significantly extends our previous report, where we demonstrated that LEAPs exhibit selective and avid adhesion to inflamed endothelium, and represents a key step forward in developing an approach to target drugs selectively to sites of IBD.

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic representation of selective drug delivery that exploits the increased expression of ECAMs (in this study, VCAM-1) at sites of pathological inflammation (here, colitis). Black spheres represent a particle conjugated with a targeting ligand (LEAPs)—in this study, a ligand to VCAM-1 (-V LEAPs); gray spheres represent a particle conjugated with a control ligand—in this study IgG.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2668fje;

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