Heterogeneity in nanoparticles influences biodistribution and targeting

Abstract

Aim: A large fraction of the administered dose of nanoparticles (NPs) localizes into nontarget tissue, which could be due to the heterogeneous population of NPs.

Materials & methods: To investigate the impact of the above issue, we simultaneously tracked the biodistribution using optical imaging of two different sized poly(d,l-lactide co-glycolide) NPs, which also varied in their surface charge and texture, in a prostate tumor xenograft mouse model.

Results: Although formulated using the same polymer and emulsifier concentration, small NPs were neutral (S-neutral-NPs), whereas large NPs were anionic (L-anionic-NPs). Simultaneous injection of these NPs, representing heterogeneity, shows significantly different biodistribution. S-neutral-NPs demonstrated longer circulation time than L-anionic-NPs (t1/2 = 96 vs 13 min); accounted for 75% of total NPs accumulated in the tumor; and showed 13-fold greater tumor to liver signal intensity ratio than L-anionic-NPs.

Conclusion: The data underscore the importance of formulating nanocarriers of specific properties to enhance their targeting efficacy.

Figure 1. Spectral analysis of near infrared dyes and photostability

(A) Emission spectra of SDB5700 (red), SDA5177 (green), SDB5491 (blue) and SDB6825 (cyan). (B) Emission spectra of SDB5700 (red) and SDB5177 (green) showing distinguishable maximum emission wavelengths. (C) Photostability of near infrared dyes SDBA5177 and SDB5700 in comparison to Cy5.5, a conventionally used near infrared cyanine dye, and quantum dots.

Figure 2. Characterization of nanoparticles

(A) Hydrodynamic diameter of S-neutral- and L-anionic-NPs and a mixture of the two NPs, as determined by dynamic light scattering. (B) Morphology of S-neutral- and L-anionic-NPs as determined with atomic force microscopy. Small and large NPs demonstrate different surface characteristics, as evident from the phase images from atomic force microscopy. (C) Separation of signal of SDB5700 dye-loaded S-neutral-NPs and SDA5177 dye-loaded L-anionic-NPs. (D) Photostability of the dye-loaded NPs. SDB5700 in S-neutral-NPs results in no loss of signal, while SDA5177 in L-anionic-NPs results in <3% loss of signal after ten consecutive images. (E) Color coding for S-neutral- and L-anionic-NPs, showing increasing color intensity with increasing nanoparticle amount, and (F) S-neutral- and L-anionic-NPs show increasing signal intensity with nanoparticle amount. This relationship is linear with adjusted R² of 0.99 for L-anionic-NPs and 0.98 for S-neutral-NPs. The two near infrared dyes used also show similar fluorescent yield per µg of NPs. L-anionic-NP: Large anionic nanoparticle; NP: Nanoparticle; S-neutral-NP: Small neutral nanoparticle.

Figure 3. Biodistribution of small and large nanoparticles in tumor-bearing mice

Equal doses of L-anionic-NPs loaded with SDA5177 and S-neutral-NPs loaded with SDB5700 were mixed and injected intravenously in prostate tumor-bearing mice. (A) Unmixing of the signal from S-neutral-NPs and L-anionic-NPs injected subcutaneously in mice. (B) Biodistribution over time of S-neutral-NPs and L-anionic-NPs following their intravenous injection in mice. Arrows indicate tumor. L-anionic-NP: Large anionic nanoparticle; S-neutral-NP: Small neutral nanoparticle.

Figure 4. Quantification of in vivo signal of small- and large-sized nanoparticles

(A) Region of interest created over the anatomic location of tumor. (B) Tumor accumulation of nanoparticles. Intravenous injection results in rapid uptake of NPs, which gradually drain out of tumor. p = 0.03 at 4 h postinjection in tumor. Data are shown as mean ± standard error of the mean, n = 6. (C) Clearance of L-anionic- and S-neutral-NPs from blood. (D) Ex vivo imaging of tissues excised from mouse 48 h postintravenous injection of L-anionic-NPs and S-neutral-NPs. Mice were perfused with heparinized saline to remove NPs remaining in blood vessels before ex vivo imaging. Red indicates S-neutral-NPs, green L-anionic-NPs and white indicates autofluorescence. L-anionic-NPs show a greater uptake into organs of the RES and little accumulation in other tissues. S-neutral-NPs show a greater uptake into tumor, kidneys, lungs and heart. This difference is probably due to their small size and neutral charge, which allows them to bypass the reticuloendothelial system, resulting in increased half-life, allowing them to accumulate in other tissues. (E) Average signals from excised tissue after imaging with Maestro^™ Ex Optical Imaging System (Caliper Life Sciences [PerkinElmer], MA, USA) was determined by drawing regions of interests around each tissue. L-anionic-NPs show a fivefold accumulation in the liver compared with S-neutral-NPs. S-neutral-NPs show a threefold accumulation in tumor and also show increased uptake by kidney. Data are shown as mean ± standard error of the mean (n = 6). L-anionic-NP: Large anionic nanoparticle; S-neutral-NP: Small neutral nanoparticle. *p ≤ 0.03; **p ≤ 0.01.