RC T O RC/(T+O) Au20-PEG5000 0.35 0.75 0.17 0.38 Au50-PEG5000 12.88 4.68 3.11 1.66 Au80-PEG5000 2.29 2.24 1.23 0.66
Table 2.4: Areal density of Aux-PEG5000 NPs inside the kidney (# particles/mm2). Legend: RC = renal corpuscles; T = assorted renal tubules (e.g., proximal convoluted tubules, distal convoluted tubules, collecting tubules); O = other tissues (e.g., blood vessels, endothelial cells).
the renal corpuscles, immersed in a mesh of matrix are mesangial cells, phagocytic cells that can remove trapped residues and aggregated proteins from the basal lamina [128]. For our present system, Au50-PEG5000 NPs were entrapped by these mesangial cells (Fig. 2.8B1).
For Aux-PEG5000 NPs in the renal bloodstream to come into contact with these mesangial cells, they need to first exit the blood capillaries (known as glomerular tufts) through their porous endothelial membrane. Incidentally, this membrane separates the mesangium matrix (where mesangial cells lie) from the glomerular capillary bloodstream. Thus, Aux-PEG5000 NPs must be sufficiently small to penetrate through these pores of the glomerular membranes into the mesangium. Based on TEM measurements, such fenestrations have pore widths spanning from 71.4 to 95.2 nm (Fig. 2.8C1). The mean diameter cutoff is 83.3 nm. The gap between foot processes of podocytes is 59.5 nm by inspection (measured at the mean-height of the foot processes), in good agreement with previously reported values [129] [130] [131]. These size estimates support the notion that Au50-PEG5000 NPs (or smaller) are sufficiently small to pass through the pores and enter the mesangium.
This chapter seeks to optimize the delivery of submicron-sized nanoparticles used for cancer applications. To generate a viable strategy to maximize tumor targeting, mechanistic under- standing in the effect of each design parameter (e.g, particle size, surface charge, polymer coat- ing) on the in vivo behavior of nanoparticles (e.g., pharmacokinetics, distribution, toxicity) is crucial. Here, we investigate the effect of particle size on the in vivo distribution in mice bearing solid tumors, using PEGylated gold nanoparticles as model probes.
All gold nanoparticles contain of PEG5000 chains on their periphery, and are stable against salt-induced aggregation at physiological conditions. Changes in hydrodynamic size and ζ- potential of the particles before and after PEGylation match reasonably well with theoretical predictions. The PEG chain length of 5000 represent a careful balance between circulation time and diffusion. Particles coated with shorter PEGs may experience rapid binding by serum proteins and subsequent clearance by macrophages, failing to reach to the tumor in adequate amounts despite the EPR effect. Longer PEG coatings can prevent premature clearance by opsonization, but can add tremendous hydrodynamic drag to the nanoparticle in the tumor interstitium en route to its traversal to cancer cells within the tumor.
Passive tumor targeting
Aux-PEG5000 NPs do not contain any targeting ligands, but still reached the tumor after 24 h after their i.v. injection due to the EPR effect. From results given in Chapter 3, a threshold tar- geting ligand content is necessary for nanoparticles in the tumor interstitium to enter cancerous cells. Thus, passively targeted Aux-PEG5000NPs predominantly accumulated in the tumor inter- stitium and in the vicinity of leukocytes, and only entered Neuro2A cells at infrequent instances, independent of particle size. The only exception is the smallest particles (Au20-PEG5000 NPs), which localized inside vesicles within Neuro2A cells. This is consistent with observations made by Perrault et al. [132], who reported two size-dependent migration patterns of Aux-PEGy NPs
inside the tumor. 24 h after dosing, Au16-PEG2000 moved further away from blood vessels and accumulated in intracellular regions more frequently, when compared to their larger counterparts, Au33-PEG5000and Au84-PEG10000NPs. Moreover, based on pharmacokinetics measurements on submicron-sized Aux-PEGy NPs (see Chapter 4), larger particles have shorter blood circulation times than smaller particles. Thus, passive targeting is optimal when using nanoparticles smaller than 100 nm, for their deeper tumor penetration and longer circulation in blood.
Avoidance of RES clearance
Targeted delivery to biological destinations also requires the minimization of premature clearance.
The RES can serve as immunological barriers to the effective tumor targeting, with macrophages or monocytes sequestering injected nanoparticles. Kupffer cells (KCs) are an important RES cell type responsible for nanoparticle clearance. Tissue and cellular level data from this chapter show that KCs engulfed Aux-PEG5000 NPs in a size-dependent fashion. These data agree with the in vivo distribiution at the organ level, characterized by positive correlation between bulk nanoparticle uptake by the RES organs (liver and spleen) and particle size (see Chapter 4).
Ogawara et al. [133] reported biodistribution data of polystyrene microspheres of 50 and 500 nm in the liver. 59% of the 50 nm particles reached KCs, whereas 71% of the 500 nm particles arrived at KCs. Bogers et al. [134] discovered an elevated clearance for larger IgA polymers or aggregates compared to monomeric IgA, and showed the association of IgA (hydrodynamic size
∼11.36 nm) to KCs and encapsulation of IgA within vesicles in KCs. The degree of phagocytic response then translates into the speed of clearance. Sadauskas et al. [135] reported tremendous phagocytic activity at KCs even for 2 and 40 nm AuNPs, injected i.v. at similar concentrations to mice, although their AuNPs were not PEGylated to prevent salt-induced aggregation. Taking our data and existing literature into cosideration, the avoidance of RES clearance by KCs sets an upper limit of 100 nm for the nanoparticle size to optimize tumor targeting.
Renal accumulation
Unlike the tumor and liver, few studies in the nanomedicine field have reported submicron-sized particle accumulation in the kidney following an i.v. injection, because the kidney is regarded merely as a blood filtration organ that removes excess salts and metabolic wastes in the form of urine. Here, we observe size-dependent accumulation of Aux-PEG5000 NPs in the kidney, in which Au50-PEG5000 and Au80-PEG5000 NPs resided within renal corpuscles in more appreciable amounts than Au20-PEG5000 NPs. Foglieni et al. [136] reported the retention of locally injected lipopolyplexes (diameter ∼160 nm) in the renal corpuscles but not polyplexes (diameter ∼93 nm), suggesting the presence of a filtration mechanism that retains nanoparticles of 100 ± 25 nm in size within renal corpuscles (to be explained in Chapter 4).
In summary, passive targeting and avoidance of RES clearance dictate that the particle size for optimal tumor delivery should not exceed 100 nm. Armed with these results, the next step is to promote the engagement of nanoparticles accumulated in the tumor interstitium to cancerous cells in the tumor. In Chapter 3, we will explore the ability of Au50-PEG5000 NPs (size ∼ 70 nm) to internalize into cancerous cells via the surface attachment of targeting ligands (active targeting). Based on the kidney imaging data, an inevitable consequence of delivering Au50- PEG5000 NPs (with targeting ligands attached) to tumor bearing mice is their accumulation within renal corpuscles in the kidney.