SPIO nanoparticles were efficiently encapsulated into the core of PLG microspheres, and the resulting microspheres had similar size and morphology when compared to PBS-loaded “control” microspheres (). Scanning electron microscopy demonstrated that SPIO-encapsulated microspheres and control microspheres each had a spherical morphology (), which is consistent with previous studies using the water-in-oil-in-water emulsion technique. The size distribution of the SPIO-encapsulated microspheres closely mirrored that of the control micro-spheres, with the majority of microspheres <10 μm in diameter. The average diameters of the SPIO-encapsulated microspheres (6.1 ±0.1 μm) and control microspheres (5.4 ±0.2 μm) were not significantly different (p >0.05) (). Taken together, these results indicate that SPIO encapsulation does not significantly influence the size or morphology of PLG microspheres.
Figure 1 (A): SPIO encapsulated microsphere fabrication modeled with a schematic; (B) size and spherical morphology is maintained with FeREX encapsulation, visualized on scanning electron microscopy, scale bar =3 μm; (C) PLG size distribution with and (more ...)
SPIO-encapsulated microspheres included into an agarose gel phantom produced a signal loss on T2
-weighted MRI in a concentration dependent manner (). Previous results have shown that dispersed iron oxide particles injected into a phantom or tissue results in low T2
and small amounts of signal. Similarly, when SPIO-encapsulated microspheres were dispersed in solution, we were unable to detect a signal on MRI. However, when SPIO-encapsulated microspheres were injected into an agarose phantom in a concentrated solution, MRI pixel area was directly dependent on the microsphere mass (, R2
=0.9694). Therefore, SPIO-encapsulated microspheres could produce MRI contrast in an MRI phantom above a critical concentrated mass of ≈0.5 mg. To determine whether SPIO-encapsulated microspheres would be likely to lose MRI contrast over time due to hydrolytic microsphere erosion, we also measured release of the encapsulated SPIO nanoparticles in vitro. 26.40 ±3.18% of the encapsulated iron was released from the microspheres during a 45-d incubation in a simulated body fluid (, Figure S1 in Supporting Information
), indicating that the majority of SPIO particles are not lost in the short term. These data were supported by daily iron release measurements (Figure S1
), which demonstrate that a significant amount of iron was released during each of the first 3 d of incubation (8.42 ±0.85% of cumulative iron release), but the amount of iron released during each subsequent day of release was below the detectable range of the assay (previously shown to be 0.2 nmol).[9b]
Taken together, these data indicate that minimal iron was released during the 45 d incubation in simulated body fluid, and significant iron release only occurred during the first 72 h. These data suggest that the majority of SPIO nanoparticles remained encapsulated within the delivery vehicle over multiple weeks.
In order to characterize the utility of SPIO-encapsulated microspheres for localized, in vivo tracking, we next injected varying concentrations of microspheres into two static muscle tissue models. Injections of SPIO-encapsulated microspheres and free SPIO particles were each detectable on MRI from the base to the apex of a static (non-beating) in vivo pig heart – post MI – just prior to animal sacrifice, while control PLG microspheres were not visible (). Thus, SPIO-encapsulated microspheres of at least 3 mg · mL−1
per injection were detectable in static pig cardiac muscle. SPIO-encapsulated microspheres were also detectable in explanted skeletal muscle at concentrations of 15 mg · mL−1
(Supplementary Figure 2
) and 16.67 mg · mL−1
in a pig heart (Figure S2, S3, Supporting Information
). This successful detection of injected SPIO-encapsulated microspheres in static tissues motivated further studies to track retention and distribution of microspheres in dynamic musculoskeletal tissues.
Interestingly, imaging of SPIO-encapsulated micro-spheres in dynamic tissue environments indicated that a substantial portion of injected PLG microspheres were localized to the site of injection for at least 28 d. We analyzed and compared images of SPIO-encapsulated microspheres injected into the rat lower limb, specifically into rat gastrocnemius muscle, MCL or Achilles tendon. These are highly vascularized tissues that undergo routine motion, and were therefore an attractive test-bed for microsphere tracking. SPIO-encapsulated microspheres were detectable in these tissues at dosage ranges similar to those described above for static explanted muscle models. In vivo injections of 10 mg · mL−1 SPIO-encapsulated microspheres into the rat gastrocnemius muscle, MCL or Achilles tendon resulted in a detectable MRI signalin each of the injected areas, while there were no detectable signals present in contra lateral control tissues injected with the same mass of “blank” PLG microspheres ().
Figure 3 In vivo analysis of SPIO encapsulated microspheres injected into a dynamic tissue environment (A): SPIO encapsulated micro-spheres injected into three tissue types (muscle, ligament and tendon) and tracked over 4 weeks on MRI. Contra lateral control leg (more ...)
Quantification of MRI pixel area from each of the three tissue types over time demonstrated a decrease of MRI signal between day 0 and day 5 (), suggesting that individual microspheres are not entirely retained in dynamic, vascularized tissue. This initial decrease in microsphere pixel area is likely due to low level SPIO nanoparticle release (see , Figure S1
) or due to transport of microspheres away from the injection site rather than microsphere erosion, as erosion of this PLG composition has been shown to take place over weeks to months in vivo,
and would not be significant during a 5 d period. Interestingly, there was no significant decrease in the MRI pixel area between day 5 and 28 in any of the injection sites tested (p
-values =0.36, 0.60. 0.47 for the gastrocnemius, MCL, and Achilles, respectively), and >45% of the MRI pixel area was retained at 28 d for each of the tissue types tested. These data indicate that SPIO-encapsulated microspheres that were retained during the first 5 d after injection tended to remain at the initial injection site long term, even in highly vascularized tissues undergoing motion.
It is noteworthy that the changes in the average pixel density when comparing time points may also be partially attributable to the subtle variations in the imaging protocol at each time point. Subtle differences in the orientation angle or thickness of the MRI slices taken through the rat limbs, and a difference in relaxivity of the background tissue between bone and surrounding tissue may have some impact on the detectable pixel area. However, normalizing all experimental regions to the day 0 injection for each animal and using the contra lateral leg as a negative control likely allowed for consistent measurements across days in this study.
Our results are consistent with some portions of previous studies, which have incorporated iron oxide particles within PLG delivery vehicles to remotely control their location using an external magnetic field. Specifically, our results in are consistent with the previous findings of Patel et al.
that SPIO-loaded microspheres are visible in T2
images on MRI in static tissue. These previous studies indicate that SPIO-encapsulated microspheres may be useful not only to track microspheres, but also to induce microsphere movement in vivo.