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. 2013 Oct 28;171(2):184-92.
doi: 10.1016/j.jconrel.2013.07.008. Epub 2013 Jul 18.

Drug-loaded sickle cells programmed ex vivo for delayed hemolysis target hypoxic tumor microvessels and augment tumor drug delivery

Se-woon Choe  1 , David S Terman, Angela E Rivers, Jose Rivera, Richard Lottenberg, Brian S Sorg
Affiliations

Drug-loaded sickle cells programmed ex vivo for delayed hemolysis target hypoxic tumor microvessels and augment tumor drug delivery

Se-woon Choe et al. J Control Release. .

Abstract

Selective drug delivery to hypoxic tumor niches remains a significant therapeutic challenge that calls for new conceptual approaches. Sickle red blood cells (SSRBCs) have shown an ability to target such hypoxic niches and induce tumoricidal effects when used together with exogenous pro-oxidants. Here we determine whether the delivery of a model therapeutic encapsulated in murine SSRBCs can be enhanced by ex vivo photosensitization under conditions that delay autohemolysis to a time that coincides with maximal localization of SSRBCs in a hypoxic tumor. Hyperspectral imaging of 4T1 carcinomas shows oxygen saturation levels <10% in a large fraction (commonly 50% or more) of the tumor. Using video microscopy of dorsal skin window chambers implanted with 4T1 tumors, we demonstrate that allogeneic SSRBCs, but not normal RBCs (nRBCs), selectively accumulate in hypoxic 4T1 tumors between 12 and 24h after systemic administration. We further show that ex vivo photo-oxidation can program SSRBCs to postpone hemolysis/release of a model therapeutic to a point that coincides with their maximum sequestration in hypoxic tumor microvessels. Under these conditions, drug-loaded photosensitized SSRBCs show a 3-4 fold greater drug delivery to tumors compared to non-photosensitized SSRBCs, drug-loaded photosensitized nRBCs, and free drug. These results demonstrate that photo-oxidized SSRBCs, but not photo-oxidized nRBCs, sequester and hemolyze in hypoxic tumors and release substantially more drug than photo-oxidized nRBCs and non-photo-oxidized SSRBCs. Photo-oxidation of drug-loaded SSRBCs thus appears to exploit the unique tumor targeting and carrier properties of SSRBCs to optimize drug delivery to hypoxic tumors. Such programmed and drug-loaded SSRBCs therefore represent a novel and useful tool for augmenting drug delivery to hypoxic solid tumors.

Keywords: Blood; Cancer; Carrier; Chemotherapy; Particle; Photosensitizer.

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Figures

Fig. 1
Fig. 1
Accumulation of sickle cells in 4T1 mouse mammary adenocarcinomas grown in dorsal skin window chambers. (A) Fluorescence from tumor regions normalized to adjacent tissue regions 12 hours after systemic administration of erythrocytes (mean ± standard deviation, * = p < 0.05, ANOVA followed by Bonferroni’s post-hoc test). (B) Number of immobile cells from tumor regions normalized to adjacent tissue regions for video analysis method 12 hours after systemic administration of erythrocytes (mean ± standard deviation, * = p < 0.05, ANOVA followed by Bonferroni’s post-hoc test).
Fig. 2
Fig. 2
Histological analysis of SSRBC accumulation in liver (n=6), spleen (n=8), and 4T1 tumors (n=17). Measurement of the difference in fluorescence signal (ΔFluorescence, arbitrary units) from labeled wild-type RBCs from C57BL/6 mice and SSRBCs from Knock-in mice show enhanced accumulation of SSRBCs in the tumor (≥4.75x fluorescence signal of liver and spleen). Values are given as mean ± standard deviation and * = p < 0.05 (ANOVA followed by Bonferroni’s post-hoc test).
Fig. 3
Fig. 3
Brightfield (left) and hemoglobin saturation (right) images of a 4T1 tumor 11 days after implantation in a mouse dorsal skin-fold window chamber. The colorbar to the right of the hemoglobin saturation image indicates the percent hemoglobin saturation of the corresponding pixels. The image field-of-view is about 3.6mm high × 4.9mm wide
Fig. 4
Fig. 4
In vitro delayed photolysis of mouse red blood cells. Hemoglobin release was measured by optical absorption spectroscopy at 415nm to track the rate of photolysis. The data points show the percent photolysis (mean ± standard deviation ). (A) Delayed photolysis of wild-type C57BL/6 red blood cells. The red blood cells were photosensitized by incubation with 50μM PpIX, and photoactivation was by halogen lamp for the times indicated at 0.33W in a 24°C degree solution with dark incubation at 37°C. Control samples were not photosensitized or irradiated. Delayed photolysis was measured from the start of dark incubation. (B) Delayed photolysis of sickle cells from Knock-in mice. The sickle cells were photosensitized by incubation with 25μM PpIX, and photoactivation was by halogen lamp for the times indicated at 0.08W in a 24°C degree solution with dark incubation at 37°C.
Fig. 5
Fig. 5
Optical microscopy and flow cytometry analysis of calcein loading of sickle cells from Knock-in mice. (A) Image of calcein fluorescence from loaded sickle cells overlaid on the corresponding transmitted light image. (B) Flow cytometry data of unloaded (native sickle cells). Calcein fluorescence is plotted on the abscissa and cell size estimate from scattering is plotted on the ordinate. (C) Flow cytometry data of sham loaded sickle cells. (D) Flow cytometry data of calcein loaded sickle cells.
Fig. 6
Fig. 6
Calcein delivery to tumors in vivo using sickle cells with ex vivo photoactivation for delayed photolysis controlled release. The data in the graph are the fluorescence signal from the collected microdialysis samples (mean ± standard deviation, n=4 mice per group). Two microdialysis probes were placed in each animal during dialysate collection, one in the tumor (“Tumor”) and one in the quadriceps muscle of the opposite leg (“Normal”). Photoactivated sickle cells enhance deposition of calcein in tumors relative to reference normal tissue. Calcein delivery to tumors was enhanced for photoactivated sickle cells (SS-RBC+PpIX) compared to non-photosensitized sickle cells (SS-RBC), photoactivated normal red blood cells from wild-type C57BL/6 mice (RBC+PpIX), and free calcein, with statistically greater delivery seen at 12 (†) and 24 (*) hours (ANOVA followed by Bonferroni’s post-hoc test, p<0.05) compared the other methods at the same time points (data at 48 hours not available for the other groups).
Fig. 7
Fig. 7
In vitro delayed photolysis release of cisplatin from sickle cells from Knock-in mice compared to calcein and hemoglobin release. The sickle cells were photosensitized by incubation with 25μM PpIX, and photoactivation was by halogen lamp for 1 minute at 0.08W in a 24°C degree solution with dark incubation at 37°C. Delayed photolysis was measured from the start of dark incubation. Hemoglobin release was measured by optical absorption spectroscopy at 415nm, calcein release was measured using a fluorescence plate reader, and cisplatin release was measured using ICP-AES. The data points show the percent photolysis (average ± standard deviation) with n=4 for each data point.
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