Contact-facilitated drug delivery via VLA-4-targeted nanomicelles

Targeted delivery of a radionuclide and a drug is necessary to enable co-localization in the same or adjacent cell for subsequent activation and therapy. Once internalized by a target cell, the radionuclide which essentially behaves as a point source of photoelectronic energy, can excite or stimulate photoactive materials in its vicinity (Fig. 1a). Previous studies have demonstrated the modularity afforded by this approach in treating cancer cells10 or subcutaneous solid tumours or mice using different radionuclides and photosensitizer combinations11,12. Unlike subcutaneous solid tumour xenografts, which do not recapitulate the physiopathology of human cancer and are treatable by conventional PT, most disseminated tumour models present a different set of challenges because they are embedded in the complex and protective microenvironment of the bone marrow13. As a result, it would require more effective targeting and delivery strategies to maximize cell death. We selected multiple myeloma (MM), an incurable plasma cell dyscrasia that predominantly affects the bone marrow, spleen, and bones as the representative orthotopic disseminated tumour model (Fig. 1a)14. We also used PyMT-BO1 cancer cell line derived from transgenic PyMT cancer cells as a highly aggressive metastatic breast cancer model15 (see below).

Fig. 1 Orthogonal cancer targeting strategy using nanomicelles. a Schematic of the process of photoactivation of Titanocene in disseminated cancer cells in the bone marrow microenvironment. The various phases are numbered: 1. Administration of targeted NM-TC; 2. The targeted NM enter the bone marrow from the vasculature and bind to α4β1 receptor on the cancer cells and subsequently deliver the drug to the cell; 3. Administration of radiopharmaceuticals (18FDG), which is typically 1.5–2 h after phase (1); 4. 18FDG enters the cancer cells through the overexpressed Glut transporters on cancer cells; 5. Once the drug and radiopharmaceutical are co-localized in the cancer cells, the former is photoactivated by the latter through CR leading to cell death (6). Notice that since the other vital cells in the bone marrow, such as stem cells and stromal cells, do not express the combination of α4β1 and glut receptors essential for the treatment to work, they would largely remain unaffected causing minimal off-target toxicity. b Schematic of phospholipid NM with VLA-4 homing ligands. c TEM image of micelles alone. Scale bar, 100 nm. Inset: single micelle. Scale bar, 10 nm. d Schematic of phospholipid NM encapsulating TC with VLA-4 homing ligands. e TEM image of micelle incorporated with TC in the membrane. Scale bar, 100 nm. Inset: single NM-TC. Scale bar, 10 nm Full size image

Titanocene (TC) was used in this study as the photosensitizer for several reasons, including its UV light excitability and responsiveness to low radiance of CR11; biodegradability with significantly low cellular footprint post therapy; ease of human translation due to its safety profile in phase 2 clinical trials16; and small size and lipophilicity, allowing integration into lipid-based vehicles and incorporation into cell membranes post targeting. In addition to harvesting CR luminescence, the metal centre can also interact with radiation particles to further stress cells. However, two fundamental challenges to TC and similar photoactive drugs, transvascular delivery to tumour cells and cellular localization, have remained unaddressed in the context of CRIT. In our previous study, we used transferrin (Tf) to deliver TC to tumour cells11. Tf has only two binding pockets for TC17,18. In the docking process, the cyclopentadienyl (Cp) ligands of TC can be displaced, leaving the Ti(IV) ion alone as the predominant component that binds to the pockets17. Because both photoactivation of Ti(IV) ion and oxidation of Cp ligand to peroxyl radical contribute to the cytotoxicity of TC, Tf-mediated transport of TC would potentially lower the therapeutic efficacy of Tf-TC.

ROS-mediated damage to lipid membranes is a primary mode of action in PT19. Given the short half-lives and small diffusion distance of some ROS, the mode of delivery of the drug to the target cell and its proximity to the cell membrane are important considerations for effective therapy. There is also growing evidence that therapeutic efficacy of PT can be enhanced by selective delivery of hydrophobic photoactive drugs to the plasma membrane compared to receptor-mediated endocytotic uptake20. The contact-facilitated delivery of drugs to the plasma membrane by lipid vehicles serves this purpose efficiently. Although liposomal formulations can deliver drugs to cells through this mechanism, conventional liposomes have an average diameter of 100 nm (for unilamellar vesicles) and 0.5–5 μm (for multilamellar vesicles)21, which exceeds the physiologic upper limit of 60 nm pore size for transvascular transport of macromolecules to flow across capillary walls of bone marrow22. To deliver pristine TC to the plasma membrane of MM cells, we used nanoscale unilamellar phospholipid micelles, also known as nanomicelles (NM), as a carrier vehicle. The NM have an average diameter of ≤15 nm, which is ideal for targeting the bone marrow interstitial space23. The upregulation of a key adhesion molecule, VLA-4 (α 4 β 1 integrin), in MM provides an attractive target for precision imaging and therapy24,25. Human MM1.S cell line is widely used to study MM in rodents. Screening of the MM human cell line, MM1.S, using anti-CD49d (α4) and CD29 (β1) antibodies showed a ≥95% expression level of VLA-4 (Supplementary Figure 1). We loaded the NM with LLP2A (Supplementary Figure 2a), a small molecule peptidomimetic that binds VLA-4 with an exceptionally high affinity (IC50 = 2 pM)26. LLP2A was synthesized on a solid support, followed by conjugation to phospholipids (DSPE) engrafted with polyethylene glycol (PEG) chains to improve circulation in blood (see “Materials and Methods' section for details; Supplementary Figure 2b, c). The NM were generated as a microfluidized suspension containing LLP2A-PEG-DSPE and TC. Control NM that excluded the homing ligand LLP2A or TC were also prepared (Fig. 1b, c). An average size distribution of NM with and without TC was 14.7 ± 2 nm and 11.9 ± 0.5 nm, respectively, with an average polydispersity index of 0.2 (Table 1).

Table 1 Size distribution of the nanomicelles Full size table

We successfully loaded 0.19 mg mL−1 of TC in the NM (Table 2). Based on the full-width half-maximum of the NM size distribution (about 15 nm), the volume of NM, and the net concentration of TC per volume of NM using inductively coupled plasma optical emission spectrometry, we determined the average number of TC per NM as 3 (range, 2–5). The incorporation of TC in the lipid layer was confirmed by electron microscopy (Fig. 1d, e). The metallic titanium (Ti) centre in TC rendered the vesicles electron dense in contrast to the control vesicles without TC. Upon addition into the NM, TC incorporated in the interface between the lipid and the hydrophilic layers, as evidenced by electron microscopy (Fig. 1d, e). Probably, the hydrolysis of TC dichloride to the dihydroxyl derivative in aqueous medium17 created an amphiphilic structure, favouring the orientation of the two cyclopentadienyl and dihydroxyl moieties toward the hydrophobic core and the outer hydrophilic segment, respectively. Incorporation of LLP2A did not destabilize the NM and the presence of unnatural amino acids conferred protease resistance and high plasma stability on the nanosystem26.

Table 2 Metal (Ti) and TC content in nanomicelles and HSA Full size table

In vivo pharmacokinetics of VLA-4-targeted nanomicelles

In vivo pharmacokinetic (PK) profile of the NM-TC was studied in naive rats. A plasma half-life of 123 min was obtained after systemic administration (Fig. 2a). We performed the PK in rats instead of mice to obtain sufficient blood sample for serial measurements of TC concentration in the same animal. Otherwise, the small volume of blood in mice would require us to pool samples from different mice, masking inter-specimen variability. Although the PK value in mice are expected to be shorter than rats, the information allowed us to estimate half-life of TC in rodent blood. The NM in circulation remained intact in vivo, until cleared or destroyed. However, the micelles have a limited half-life and must reach their target early before elimination.

Fig. 2 Monitoring nanomicelles biodistribution and spread of multiple myeloma in vivo. a Pharmacokinetics of NM-TC in rats using coupled plasma optical emission spectrometry. Half-life is 123.4 min. b Comparison of biodistribution in mice of targeted NM-TC and pristine TC in vivo showing highest uptake and retention in bones and spleen, characteristic of multiple myeloma, 2 h post injection. 18FDG-PET images showing increased uptake of 18FDG in mouse forelimbs, spine, and hind limbs of mice with multiple myeloma (c, e, g) compared to naive mice (d, f, h, i). Comparison of standard uptake values (SUV) of 18FDG in multiple myeloma vs. naive mice in various bones. Values are means ± s.e.m. *P < 0.05, **P < 0.01. n = 5 mice for each of the pharmacokinetics study in rats; and biodistribution study in mice Full size image

VLA-4-targeted nanomicelles delivers TC to MM-avid organs

The selectivity of LLP2A to MM cells and the serum stability of the NM in delivering the TC was determined by in vivo biodistribution analysis. Using inductively coupled plasma optical emission spectrometry, we determined the Ti metal content ex vivo in organ samples from an orthotopic disseminated MM1.S/SCID model. We compared the biodistribution of NM-TC, Tf-TC and MKT4, a water soluble analogue of TC that was used in phase 1 and phase 2 clinical trials16,27 at 90 min post injection. The choice of 90 min time point is based on rat PK data, which showed a t 1/2 of 123 min in rats but the rate of clearance after 90 min approached stasis, probably representing the contribution of an intraversation process of drug from tissues to blood (Fig. 2a). Although this time point is expected to be shorter in mice, we chose 90 min for the mouse study to ensure that the blood concentration of TC is sufficiently low, to prevent potential systemic toxicity, but not too late when the amount in tumour tissue is small. In mice administered with NM-TC, the highest Ti concentrations were found in skeletal tissue and spleen, which typically house MM cells, with relative values of 115 ± 7 and 52 ± 9.5 μg g−1, respectively (Fig. 2b). In comparison, the uptake of MKT4 was lower in tumour sites, with values of 53 ± 9 and 16 ± 4 μg g−1, for skeletal tissue and spleen, respectively (Fig. 2b). Similarly, the accumulation of TC in these tissues for mice treated with Tf-TC was only 27.5 ± 6 and 14 ± 1 μg g−1, respectively. These results demonstrate the advantage of using NM to deliver TC to MM target organs. Previous studies have suggested that the cylopentadienyl rings in TC, which assists in stabilizing the Ti(IV) ion in a monomeric form, are lost in MKT4 and Tf-TC17,18. Thus, sequestration of TC in the hydrophobic region of NM may help stabilize the drug and minimize rapid loss from target tissues.

CRIT inhibits tumour growth in disseminated MM mouse model

We used an FDA approved and clinically employed radiopharmaceutical, 18FDG (t 1/2 = 109.8 min), as a source of photoelectronic energy28. The radiopharmaceutical, which is currently the gold standard for clinical imaging of MM29,30, targets metabolically active tumours via the glucose transporter (GLUT1) protein. By using an orthogonal-targeting GLUT1 and VLA-4 strategy to, respectively, deliver the 18FDG and NM-TC to the MM cells, we aimed to minimize the potential saturation or depletion of the targeted receptors. In healthy subjects, 18FDG uptake is low in the bone marrow and spleen, but significantly higher in malignancy, inflammation or after administration of hematopoietic growth factors31. Using small animal positron emission tomography (PET) of MM in mice, we found more than twofold uptake of 18FDG in bones compared to naive mice (Fig. 2c–i).

The performance of CRIT in a disseminated MM1.S/ SCID mouse model was tested. Based on the biodistribution data, sequential tail vein injections of NM and then 18FDG were spaced 90 min apart to activate TC in tumours. Treatment was repeated four times at an interval of 1 week, and the disease progression was monitored weekly by bioluminescence imaging (BLI; Fig. 3a). A week interval was chosen for treatment for several reasons that include the need to allow the mice to fully recover from the treatment; account for full decay cycle of 18FDG; consider logistical reasons such as tail vein recovery; and allow sufficient time for imaging time points between treatment sessions. In the control groups consisting of untreated mice or those treated with either NM or 18FDG alone (Fig. 3b, c), we observed an exponential increase in the BLI signal over several weeks, demonstrating the systemic progression of the disease and indicating the primary involvement of the spleen and skeletal tissues. In contrast, mice treated with NM-TC and 18FDG showed a conspicuous decrease in the disease progression, suggesting the effective targeting and response of MM1.S to CRIT. Survival studies revealed a significant advantage of the CRIT over the control groups with 50% surviving up to about 90 days compared to about 62 days for the control groups (Fig. 3d). Correlative 18FDG-PET imaging confirmed the lower tumour burden in CRIT-treated mice compared to the control groups (Fig. 3e, f). The mice were killed after they developed hind limb paralysis resulting from spinal cord and spinal vertebral involvement. The treated mice eventually succumbed to cancer due to the remnant MM cells that could not be completely eradicated by CRIT.

Fig. 3 Response of multiple myeloma to CRIT. a Timeline of treatment. b Bioluminescence imaging of representative multiple myeloma-bearing mice in different treatment groups—untreated, 18FDG, NM controls and CRIT. All images are dorsal images and on the same scale. The images of control groups appear saturated on week 6 in comparison to CRIT. c Change in bioluminescence intensity as a result of treatment compared to untreated control. The intensity consistently remains lower than untreated controls during the treatment and beyond. d Comparison of survival of different treatment groups showing a twofold increase in survival in treated mice compared to control groups. **P < 0.01. e 18FDG-PET images of MM mice before and after treatment showing lower tumour burden in the latter. F: frontal view, S: sagittal view. Boxes denote tumour region. f SUV values of the treatment group were lower than untreated controls. **P < 0.01. n = 15 mice for CRIT, n = 10 mice for untreated control and n = 5 mice for NM-TC alone and 18FDG alone treated mice Full size image

CRIT selects for α4-deficient multiple myeloma cells in vivo

Residual cancer cells that escaped treatment appeared focal and confined at random sites within the major bones, particularly the vertebrae (Fig. 3b, e). These localized cancer cells continued to grow, albeit at a slow rate. The surviving cancer cells were subsequently extracted from the mice and reintroduced into a fresh group of naive SCID mice to determine response to when treated with CRIT. However, BLI (Fig. 4a) and 18FDG-PET did not show noticeable differences between the treated and untreated groups, suggesting the cells were resistant to CRIT. These CRIT-resistant MM1.S (MM1.SCRIT-RES) cells were harvested and analysed for the expression levels of GLUT1, α4 and β1 integrins to determine whether uptake of 18FDG by GLUT1 or α4β1 binding of the NM were compromised. GLUT1 mRNA (Fig. 4b) or β1 cell surface expression (Fig. 4c, d) analyses did not demonstrate significant difference between the parental MM1.S and the MM1.SCRIT-RES cells. However, the MM1.SCRIT-RES cells expressed lower cell surface α4 than parental MM1.S cells (Fig. 4e, f). Flow cytometry analysis demonstrated that LLP2A-Cy5, which selectively binds VLA-4 with high affinity32, did not internalize in the MM1.SCRIT-RES cells compared to the parental MM1.S cells (Fig. 4g, h). These results suggest that the MM1.SCRIT-RES cells had downregulated the expression of α4 (CD49d), possibly impairing the binding of the LLP2A functionalized NM to some MM cells. Unlike in vitro studies where static incubation of nanoparticles can abrogate specific binding of receptor-targeted materials, the in vivo dynamics and the relatively small number of these resistance cells in the initial tumour population could have favoured the homing of NM-TC to the VLA-4 positive cells in mice. As a result, CRIT could have preserved a subclone of MM1.S with low α4 that was present at low frequency in the injected cells. Thus, targeting VLA-4-rich cancer cells selects for the subset of MM1.S cells with low CD49d expression levels and low levels of the activated conformation of VLA-4. A potential approach to achieving complete eradication of MM cells is to identify complementary biomarkers that allow the delivery of TC-loaded NM to all MM1.S cells or through the use of combination therapy that more effectively targets and eliminates both CRIT-responsive MM1.S and MM1.SCRIT-RES cells in vivo.

Fig. 4 CRIT selects for CD49d cells in MM model. a Bioluminescence intensity plot showing resistant nature of MM cells extracted from treated cohort (MM1CRIT-RES) upon rechallenging with CRIT in fresh mice. b No difference in GLUT1 mRNA expression was observed between parental MM1.S cells and resistant MM1.SCRIT-RES cells as assessed by qRT-PCR. ns not significant. c–h No difference in expression of CD29 was observed between MM1.S cells (c) or MM1.SCRIT-RES cells (d) following treatment with CRIT in vivo. MM1.S stopped responding to CRIT by downregulating expression of VLA-4 subunit CD49d (Resistance = 28.12% CD49d+) (f) relative to parental cells injected into mice at the beginning of the experiment (parental = 99.92% CD49d+) (e), resulting in reduced binding of the VLA-4-targeting ligand LLP2A on resistant cells (h) (LLP2A+ = 6.6%) compared to parental MM1.S (g) (LLP2A+= 84.15%). i No significant difference in colony-forming units of progenitor stem cells was observed between untreated, control and treated mice. j To determine if CRIT reduced engraftment of haematologic cells in vivo, we assessed BM repopulation following CRIT treatment. Bone marrow from treated mice or PBS-treated controls were mixed with congenic B6.CD45.1/2 at a ratio of 1:1 before infusion of 1 × 106 total BM cells into lethally irradiated (TBI 1100 cGy) B6.CD45.1 recipients. k Percentage of cells derived from treated donor BM (CD45.2) were calculated as a percentage of total donor BM (CD45.2 + CD45.1/2). BM from CRIT-treated mice effectively repopulated recipients (n = 5 per group) Full size image

CRIT preserves normal hematopoietic stem cells

An important consideration during CRIT is to preserve and sustain the long-term viability of the hematopoietic stem cells and progenitor cell population in the bone marrow. Clonogenic assays of normal bone marrow progenitor cells extracted from mice treated with CRIT did not reveal a significant change in colony-forming units (CFU) compared to the control groups (Fig. 4i). Competitive bone marrow repopulation experiments showed that there was no detrimental effect of CRIT on the primitive hematopoietic stem cell compartment33. The 2-month hematopoietic reconstitution of mice transplanted with bone marrow from wild-type vs. CRIT-treated mice was not significantly different, suggesting that there was no obvious reduction in engrafting hematopoietic stem cell population after treatment with CRIT (Fig. 4j, k).

HSA-TC nanoparticles deliver drug to metastatic breast cancer

We extended the use of CRIT in a metastatic breast cancer model. PyMT-BO1 cell line is a highly aggressive breast cancer cell line derived from transgenic PyMT breast cancer mice15. Previous reports have demonstrated that rapidly proliferating tumour cells actively internalize albumin for use as a source of nitrogen and energy, partially accounting for the formulation of drugs in albumin or its nanoparticles for drug delivery to tumours34,35,36. Thus, formulation of hydrophobic drugs, such as TC in albumin, will not only enhance solubilization in an aqueous medium, but also mediate delivery to tumours. We mixed TC (80 mg) in an aqueous solution (16 mL) containing 0.5% human serum albumin (HSA) for 6 h before lyophilizing the entire mixture (see 'Materials and Methods' section). We used a low concentration of HSA in this formulation to prevent potential immunogenic response in mice. The lyophilized product was reconstituted in 0.9% saline immediately before use. Using inductively coupled plasma mass spectrometry, we determined the concentration of TC in the reconstituted sample as 5.6 mg mL−1 (Table 2). The corresponding concentration of HSA in the formulation was 8.95 mg mL−1, as determined by using a protein assay. Dynamic light scattering (DLS) measurements indicate that the HSA-TC nanoparticles were fairly monodispersed with an average size of 12–15 nm (Supplementary Figure 3). About 1.5% of the particles formed large aggregate clusters of 100 nm, creating a bimodal distribution that skewed the z-average diameter (120 nm) and polydispersity index (0.278). Electron microscopy size measurement correlated with the DLS results, showing mostly monodispersed HSA-TC nanoparticles of 10–15 nm in diameter (Supplementary Figure 4), along with few aggregates of 30–90 nm. The HSA nanoparticles allowed us to load high concentration of the hydrophobic TC per volume of aqueous solution for subsequent delivery to metastatic breast cancer.

Biodistribution of HSA-TC in metastatic breast cancer model

Intracardiac injection of PyMT-BO1 cells stably transfected with GFP-firefly luciferase in mice-induced bone metastases, especially to the lower limbs and other major organs (Fig. 5a, b). By day 10, the tumour burden was very high, requiring immediate killing (Supplementary Figure 5). For the biodistribution study, we reconstituted the lyophilized HSA-TC in saline and administered 100 µL of 0.6 mg per 20 g mouse. Both the non-invasive in vivo (Fig. 5b) and the ex vivo (Fig. 5c) BLI analysis showed that the tumour cells disseminated to most major organs, including the vertebrae and lower limbs. Quantitative analysis of the Ti contents in tissue samples from blood and lower limbs by inductively coupled plasma mass spectrophotometry showed the highest accumulation of the metal in the limbs at 3 h post injection of HSA-TC (Fig. 5d). The concentration of Ti in the lower limbs decreased gradually over time (p < 0.05). At 24 h, the Ti content from the HSA-TC in the limbs was indistinguishable from the background content. We had to subtract the background Ti from untreated mice to determine the contribution of HSA-TC because most mouse feedstock contains Ti products.

Fig. 5 Dissemination of metastatic breast cancer cells and biodistribution of TC in mice. a In vivo BLI non-tumour-bearing C57BL/6J mice. b In vivo BLI of metastatic breast cancer 10 days post intracardiac injection of PyMT-BO1 GFP/Luc in C57BL/6J mice. c Ex vivo BLI of metastatic tumour burden 10 days post tumour initiation. The left panel are tissues obtained from a mouse and the right panel are tissues obtained from b mouse. d Inductively coupled plasma mass spectrometry analysis of Ti content in blood samples and the lower limbs, where tumour burden is high. The Ti content was background-corrected from untreated mice; *P < 0.05. Studies were performed with n = 5 mice per each group Full size image

HSA-TC inhibits growth of metastatic breast cancer in mice

We explored the feasibility of using CRIT to inhibit tumour growth in the highly metastatic PyMT-BO1 GFP/Luc breast cancer model in C57B6 mice. Our biodistribution data indicate that the accumulation of TC in the cancer-homing organs is highest at about 3 h post injection of HSA-TC. 18FDG (50–60 µL; 800 µCi per mouse) was administered intraperitoneally at 2 h after the intravenous administration of HSA-TC. We chose intraperitoneal instead of intravenous route for 18FDG injection to maintain consistency across the experiments because of the difficulty of finding viable tail veins in the same mouse for multiple injections of both HSA-TC and 18FDG. Comparison of different routes of 18FDG administration in mice determined that the SUV of 18FDG injected intraperitoneally in tumours is optimal at about 1 h post injection, and is similar to intravenous route at closer to this time point37,38. We hypothesized that administering the radionuclide at about 2 h after injection of the HSA-TC (100 µL of 0.6 mg per 20 g mouse) will achieve maximum accumulation of both CRIT effectors in tumours after 3 h. Three treatment cycles of HSA-TC and 18FDG were administered 2 days apart starting from day 2 after initiating the metastatic disease when the tumours are observable by BLI (Supplementary Figure 5). Groups with no treatment, treatment with HSA-TC alone and 18FDG alone served as controls. Whole-body luciferase activity from day 2 to day 9 were analysed for tumour cell proliferation. Compared to the untreated mice (Fig. 6a), a small decrease in BLI signal was observed in the HSA-TC (Fig. 6b) and 18FDG (Fig. 6c)-treated mice compared to the untreated cohort. However, the pattern of tumour growth in all the three control groups was similar. In contrast, the CRIT group showed significant tumour stasis, with a few focused cluster of tumour cells that did not respond to the therapy (Fig. 6d, e). The slow growth and focal nature of the CRIT-resistant cells suggests that this therapeutic method can transform metastatic cancer into a surgical disease. Whereas PyMT-BO1 model is an excellent model for the rapid evaluation of drugs or treatment methods, all the animals eventually died or were killed between day 9 and 12 due to the aggressiveness of this model. The fast death cycle prevents longitudinal evaluation of each animal, which is needed to obtain reliable survival plots. Future studies will explore different models and establish the mechanism of therapy resistance in this cancer cell line.

Fig. 6 Representative BLI of PyMT-BO1 GFP/Luc metastatic breast cancer cells in C57B6. a Untreated C57B6 mouse bearing highly metastatic PyMT-BO1 cancer. Accumulation in the lower limbs were predominant. b Mouse treated with 30 mg kg−1 of HSA-TC nanoparticles. c Mouse treated with 800 µCi of 18FDG. d Mouse treated with a combination of HSA-TC and 800 µCi 18FDG. e Quantification of whole-body luminescence in CRIT-treated mice compared to untreated, HSA-TC treated or 18FDG-treated controls (*P values are 0.038, 0.23 and 0.017 for CRIT, HSA-TC alone and 18FDG alone, respectively). BLI and data analysis were performed on day 9 after initiation of PyMT-BO1 metastasis in mice. Studies were performed with n = 5 mice per each group Full size image

In summary, we have successfully demonstrated the application of PT for treating disseminated malignancies using VLA-4-targeted NM and HSA-TC nanoparticles, activated by radiopharmaceuticals. Integral to this strategy is the availability of a wide range of radionuclides for clinical PET imaging and preclinical Cerenkov luminescence imaging to further monitor and guide treatment response39,40. We demonstrated a strategy to rescue abandoned light-sensitive drugs with poor therapeutic outcomes such as TC and some FDA-approved drugs with inherent photoactivity into precision phototherapeutics. In addition, clinical biochemistry parameters and histopathologic assessment of vital organs in the treated and untreated controls were similar (Supplementary Figure 6 and Supplementary Figure 7). The brain, heart, liver and kidneys were of particular interest because 18FDG naturally accumulates in these organs because of their high glucose utilization and elimination pathways. The absence of off-target toxicity to normal hematopoietic stem cells may favour the translation of this approach in the clinic as either a standalone therapy or as a combination with other therapies, including chemotherapy, where the suppression of the bone marrow and the risk of pancytopenia may not be anticipated to be greater than those patients receiving chemotherapy without CRIT. Our results suggest that the sequential administration of the NM-TC and radionuclide minimizes the association of both therapeutic components in vital organs. These findings expand the potential use of PT for treating previously PT-inaccessible metastatic, infectious and cardiovascular diseases.