Pro- organic radical contrast agents (“pro - ORCAs”) for real -time MRI of pro-drug activation in biological systems

Nitroxide-based organic-radical contrast agents (ORCAs) are promising as safe, next-generation magnetic resonance imaging (MRI) tools. Nevertheless, stimuli-responsive ORCAs that enable MRI monitoring of prodrug activation have not been reported; such systems could open new avenues for prodrug validation and image-guided drug delivery. Here, we introduce a novel “pro-ORCA” concept that addresses this challenge. By covalent conjugation of nitroxides and drug molecules (doxorubicin, DOX) to the same brush-arm star polymer (BASP) through chemically identical cleavable linkers, we demonstrate that pro-ORCA and prodrug activation, i.e. , ORCA and DOX release, leads to significant changes in MRI contrast that correlate with cytotoxicity. This approach is shown to be general for a range of commonly used linker cleavage mechanisms ( e.g. , photolysis and hydrolysis) and release rates. Pro-ORCAs could find applications as research tools or clinically viable “reporter theranostics” for in vitro and in vivo MRI-correlated prodrug activation. of the BASP, from nm light. Only light-triggered DOX prodrug activation induces significant cell death.

Toward addressing this challenge, we describe herein a novel "pro-ORCA" design concept wherein the release of covalently conjugated ORCAs from a BASP scaffold is shown to induce a large (~30-fold) change in magnetic relaxivity correlates with prodrug activation. Our proof-of-principle pro-ORCA system is based on BASPs that feature both nitroxide ORCAs (for MRI imaging) and the Top2 poison doxorubicin (DOX) covalently conjugated to the same BASP through chemically equivalent linkers that cleave in response to either external or endogenous triggers ( Figure 1). Linker cleavage, i.e., pro-ORCA and prodrug activation, causes the nitroxide and DOX to diffuse away from the BASP. The former leads to a ~30-fold change in transverse relaxivity, and a concomitant change in MRI contrast, while the latter leads to DOX-induced cell death, enabling MRIbased, real-time monitoring of prodrug activation in a simple, modular format. This basic pro-ORCA concept ( Figure 1) is shown to apply to multiple widely-used triggering mechanisms, variable release kinetics, and in vitro and in vivo models, offering a promising new concept for MRI-guided prodrug activation.  (for MRI) and a prodrug (therapeutic) conjugated through cleavable linkers are copolymerized with a non-cleavable fluorophore-conjugated MM via the brush-first ring-opening metathesis (ROMP) method. The resulting BASP, which carries both the pro-ORCA and prodrug on the same polymer, displays high transverse relaxivity (r2) enabling T2-weighted MRI. Moreover, the prodrug is therapeutically inactive. Upon cleavage of the linkers that connect the nitroxide and drug to the BASP, i.e., pro-ORCA and prodrug activation, the nitroxide relaxivity drops significantly leading to a change of MRI contrast while the drug becomes therapeutically active. Thus, MRI signal changes (in this case, return of negative T2 contrast back to baseline) can be correlated with prodrug activation. Meanwhile, the fluoroscence signal should not change significantly before and after nitroxide and drug release, providing a constant imaging handle to visualize the BASP.

Experimental Section
Relaxivity and in vitro measurements by MRI. Phantom MRI data were acquired in a 12 cm outer diameter birdcage transceiver for imaging in a 20 cm bore Bruker 7 T Avance III MRI scanner. For relaxivity determination, samples at varying concentrations in PBS buffer were used. For in vitro measurements, samples were prepared as described below, and loaded into a 384-well clear polystyrene plate (Thermo Scientific Nunc), which had been pre-cut in half to optimally fit the coil. Unused wells were filled with PBS buffer. 2 mm slices were imaged through the samples with the field of view of 5 x 5 cm; and, the data matrices were 256 x 256 points. Longitudinal (r1) and transverse (r2) relaxivity measurements were acquired using multi-spin multi-echo (MSME) sequences (flip angle=180°). r1; TE=12 ms, TR=300, 350, 400, 450, 500, 600, 800, 1000, 1200, 1500, 3000, 5000, 10000 ms. r2; TR=5000 ms, TE=12, 24,36,48,60,72,84,96,108,120,132,144,156,168,280,192,204,216,228,240,252,264,276, 288, 300, 312, 324, 336, 348, 360 ms. Custom routines written in Matlab (Mathworks, Natick, MA) were used to reconstruct the images and compute relaxation time constants by fitting image intensity data to exponential decay curves. For in vitro measurements, cells (A549, MM.1S, and KMS11) were plated at 10,000 cells per well in a 96 well plate and incubated overnight. PC2 (17 mg BASP/mL, 107 µM DOX) were then added, and cells were incubated for another 2 h. Excess PC2 were removed via media wash, and fresh media was added. Cells were then either exposed to UV for 30 min (+UV) or not (-UV). Cells were then incubated for pre-determined time points (2h, 6h, 24h, or 48h), then subject to either MRI or viability assay (CellTiter-Glo, following standard operating procedure). For cells that were imaged by MRI, the media was removed. The wells were then filled with PBS (50 uL), Please do not adjust margins Please do not adjust margins mixed with 1% Triton X-100, transferred to a 384 well plate, and imaged using the MRI protocol described above. Cell culture. Human multiple myeloma cells (MM.1S and KMS11, ATCC) were cultured in RPMI media (Thermo Fisher Scientific) and were supplemented with 10% fetal bovine serum (FBS, VWR), 1% penicillin/streptomycin (Thermo Fisher Scientific), and 1% glutamine (Thermo Fisher Scientific). Human lung adenocarcinoma cells (A549, ATCC) were cultured in RPMI media, which was supplemented with 10% FBS and 1% penicillin/streptomycin. Cell lines were authenticated by short tandem repeat DNA profiling and were confirmed to be mycoplasma negative, using the MycoAlert Mycoplasma Testing Kit (Lonza). All cells were housed in 5% CO2 humidified atmosphere at 37 °C. In vitro cell viability. MM.1S, KMS11, and A549 cells were plated at 10,000 cells per well overnight in a 96 well plate. The media was then replaced with fresh media containing BASPs at various concentrations. The plate was incubated for 48 h unless otherwise stated; and, cell viability was then determined using the CellTiter-Glo assay (Promega). For viability assays involving DOX-PC or chex-PC, cellls were plated at 10,000 cells per well in a 96 well plate and incubated overnight. Cells were incubated for 2 h with DOX-PC or chex-PC at various concentrations. Excess DOX-PC were removed via media wash, and fresh media was added. Cells were then either exposed to UV for 30 min (+UV) or not (-UV). Viability was evaluated at 48 h with CellTiter-Glo. In vivo therapeutic efficacy of DOX-MHC-BASP. A549 cells were cultured following the protocol described above to a final concentration of 20%. Cells were then harvested, mixed with Matrigel and sterile pH 7.4 PBS buffer (1:1), filtered through sterile 0.2 µm filters, and injected subcutaneously (2.0 x 10 6 cells) into the hind flank of NCR-NU mice. Tumor growth was monitored for 2-4 weeks until appropriate cumulative diameters (~ 1 cm, measured by a digital caliper) were achieved. Tumor-bearing mice were then randomized into groups of n = 3 and injected intratumorally with 50 µL of DOX-M at varying concentrations (1, 2.5, 5, 10 mg BASP/mL). Tumor growth was then accessed via caliper measurements for 25 days. In vivo MRI instrumentation. In vivo MRI was acquired using a Bruker 9.4 T Biospec MRI scanner using a cross coil volume transmitter and surface receiver configuration. Tumor region was localized in focal spot of surface coil, with animal restrained in order to allow unhindered breathing while minimizing motion around the hind leg and tumor. Axial T1 weighted images (T1WIs) were collected using a RARE pulse sequence with TR = 721.1 ms; TE(eff) = 11.8 ms; RARE factor = 4; FOV = 30×30 mm 2 ; 256×256 matrix and 4 averages over 12 slices of 1 mm thickness and 0 mm gap, with a total scan time of 2 min 18 sec. Axial T2 weighted images (T2WIs) were collected using a RARE pulse sequence with TR = 4000 ms; TE(eff) = 48 ms; RARE factor = 8; FOV = 30×30 mm 2 ; 256×256 matrix and 2 averages over 12 slices of 1 mm thickness and 0 mm gap, with a total scan time of 3 min 12 sec. Images were analyzed using either ImageJ or custom routines written in Matlab (Mathworks, Natick, MA). In vivo MRI in tumor-bearing mice. Tumor-bearing NCR-NU mice were generated as described above. MRI and NIRF images were acquired for each animal (n = 3 mice/group) before injections. BASPs were prepared, passed through a sterile 0.2 µm filter, and administered directly into the tumor via intratumoral injections (50 µL of 70 mg/mL BASPs, F, M, or S). Tumor imaging was done at predetermined time points. In vivo MRI data analysis. T2-weighted images were inverted before analysis was performed so that high T2 regions will appear bright in inverted images. Signal intensities pre-and post-injection were compared using only slices in which tumors and muscle were clearly visible. Using ImageJ software (v.1.52i), a region of interest (ROI) around each component was manually drawn. The average signal intensities and areas of the ROIs were measured; these data were then normalized against the signal intensity of the muscle tissue. Signal intensity was acquired by multiplying area and normalized. This process was repeated for all relevant slices for a given organ; the sum of these signal intensities was then calculated and divided for the total area, affording the volume-averaged signal intensity. SNR variations were acquired via normalization against the earliest time point. A detailed step-by-step analysis is included in the SI. Ex vivo Fluorescence Imaging. For the 24h time point, mice were euthanized and tumor harvested (n = 3 mice/group). Tumor slices were mounted with DAPI staining and imaged with an upright Carl Zeiss microscope with an HXP 120C light source at 20x magnification (DAPI λex/λem = 365/445 nm; Cy5.5 λex/λem = 640/690 nm; DOX λex/λem = 470/525 nm)

Results and discussion
We initially targeted photoresponsive linkers to establish the pro-ORCA concept for MRI-correlated prodrug activation. Photocleavable linkers based on ortho-nitrobenzyl (ONB) derivatives have been widely used in the context of protecting group chemistry, drug delivery, and materials applications, [73][74][75][76][77][78][79] and while such linkers may have translational limitations, they offer powerful research tools for triggering specific, light-induced biological responses. DOX was chosen as the therapeutic payloads due to its clinical utility, its established mechanisms of action (in both prodrug and free drug forms), and its inherent fluorescence, the latter of which provides a useful secondary imaging handle for our proof-of-concept studies. [59][60][61]67,80 We have previously shown that conjugation of DOX to BASPs via an ONB linker deactivates the drug (i.e., it is a prodrug), providing no detectable release or cytotoxicity in cell culture over 72 h in the absence of 365 nm light. [59][60][61]67 Here, we reasoned that this "on/off" nature of light-induced release would simplify our system by enabling a binary readout upon irradiation. A previously reported MM with DOX 59 and a new MM with chex conjugated via chemically identical ONB linkers (Scheme 1A, see SI for full synthetic details) were prepared; their structures were confirmed by electron paramagnetic resonance spectroscopy (EPR; Figure S1), matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-ToF MS; Figure S2), and nuclear magnetic resonance (NMR) spectroscopy ( Figure S3) where appropriate. Four BASPs comprising either 100% chex (chex-PC) or 100% DOX (DOX-PC) or different chex:DOX ratios (1:1 and 27:1; PC1 and PC2, respectively, Table 1) were synthesized via the brush-first ring-opening metathesis polymerization (ROMP) strategy using an acetal-based bisnorbornene cross-linker (AXL) and Grubbs 3 rd -generation bispyridine complex (G3) (Scheme 1B). [59][60][61][62]66 Leveraging the efficiency of ROMP and the branched MM design, 59-66 different chex:DOX ratios can be conveniently realized by altering the stoichiometry of MM feed ratios. Gel permeation chromatography (GPC) and dynamic light scattering (DLS) analyses confirmed high MM-to-brush and brush-to-BASP conversions and consistent sizes and size distributions for these four particles; the hydrodynamic diameters (Dh) were all ~22 ± 5 nm ( Figure S4a, Table 1), highlighting the payload-agnostic nature of BASP synthesis. A cyanine5.5-based MM (Cy-MM) was also incorporated into BASPs that were intended to be deployed for in vivo studies; the cy5.5 dye is non-releasable, providing an independent imaging handle for the BASP scaffold that enables validation of the pro-ORCA design (vide infra). EPR spectroscopy revealed the characteristic broadening associated with conjugation of chex to BASPs ( Figure S4b). [64][65][66] Moreover, large per-chex r2 relaxivity enhancements (~30-fold), similar to those reported previously upon conjugation of chex to BASPs, 64-66 were observed (Table 1). This result was encouraging, as it confirmed that chexconjugated BASPs display high r2 values when cleavable pro-ORCAs linkers are used (previous work has exclusively used non-releasable chex linkers) and in the presence of a second payload such as DOX bound to the same polymer. To examine light-induced pro-ORCA and pro-drug activation, DOX-PC, chex-PC, and PC1 (1:1 chex:DOX) were exposed to 365 nm light (see SI for details). Samples of the reactions were taken at various timepoints and were analyzed by liquid chromatography-MS (LC-MS). The release behaviors were consistent for the three BASPs ( Figure 2A) with maximal release occurring with ~30 min of light exposure regardless of the payload (i.e., DOX, chex, or 1:1 DOX:chex). As such, an exposure duration of 30 min was used for subsequent experiments.
To confirm that photoinduced DOX prodrug activation induces cell death, DOX-PC was incubated with suspended multiple myeloma (MM.1S and KMS11) and adherent lung adenocarcinoma (A549) cells for 3 h to provide sufficient time for uptake. Excess DOX-PC was then removed by media washing, and the cells were exposed to 365 nm light for 30 min. After incubation for an additional 48 h, cell viability was assessed using the CellTiter-Glo kit ( Figure 2B). Consistent with our expectations, significantly greater toxicity was observed with all cell lines following exposure to 365 nm light when compared to its absence; note that exposure of cells to 365 nm light in the absence of DOX-PC induced no observable toxicity (99+% viability in all 3 cell lines). Moreover, irradiation of cells exposed to chex-PC rather than DOX-PC under the same conditions led to no meaningful effects at ARTICLE Please do not adjust margins Please do not adjust margins Motivated by these results, we examined the use of PC2 for correlation of DOX prodrug activation with changes in MRI contrast in vitro. PC2, which has a chex:DOX ratio of 27:1, was designed to maximize the MRI signal at practical concentrations (i.e., high chex loading) and to still provide sufficient amounts of DOX to induce toxicity following 365 nm light exposure. Cells were incubated with PC2 (17 mg PC2/mL; 107 µM of conjugated DOX) following the same procedure as described above. The cells were imaged using a 7 T MRI scanner ( Figure 2C). MRI SNR was measured as a function of time. In support of our design concept, these SNR values correlated with cell viability ( Figure 2D). Upon irradiation, a diminished T2 MRI signal was observed as the molar transverse relaxivity of the nitroxide decreases by ~30-fold upon release (Table 1, r2 of water-soluble chex analogue 3-CP = 0.17 mM -1 s -1 compared to PC2, which has a per-chex r2 of 4.91 mM -1 s -1 ). Substantial SNR changes were observed in all three irradiated cell lines ( Figure 2D, black solid curves) when compared to non-irradiated cells ( Figure 2D, black dotted curves). For instance, in the case of MM.1S a 57 ± 3% decrease in the T2 signal was observed from irradiated cells as compared to 5 ± 2% for nonirradiated cells. Finally, the MRI SNR data correlated with cytotoxicity ( Figure 2D, red solid curves) at all time points up to 48 h, indicating that chex and DOX are cleaved and diffuse away from the BASP at similar rates and that the latter was therapeutically active. Altogether, these results demonstrate proof-of-principle for the pro-ORCA concept, showing that light-induced prodrug activation correlates with MRI signal in vitro ( Figure 2D). Having established proof-of-concept for our pro-ORCA design, we sought to further demonstrate its generally using more translationally-relevant ester-based prodrugs. 81 Three ester linkers were designed and used to prepare pro-ORCAs and DOX prodrugs (Scheme 2A): one that was previously reported to display excellent in vivo efficacy when conjugated to DOX 60,61 (referred to herein as "medium") and two novel linkers with steric and electronic properties tuned to afford relatively "fast" and "slow" hydrolysis kinetics in vitro. These linkers utilize the same release mechanism: hydrolytic cleavage of the aromatic ester (rate-determining step), followed by a 1,6-elimination 73 to release free DOX. Control over their hydrolysis rate is predictable and tunable using rational structural modifications. Using these 3 linkers  Figure S6-18), EPR spectroscopy ( Figure S6, S9, and S12), NMR spectroscopy ( Figure S7, S10, S13, S15 and S17), and MALDI-ToF MS ( Figure S8, S11, S14, S16, and S18) were deployed where appropriate.
Here, release of chex and DOX via ester hydrolysis leads to concomitant MRI and efficacy changes that correlate with the measured hydrolysis rates.
To confirm their relative release rates, the MMs were incubated in PBS (pH 7.4) buffer; the amounts of released DOX or chex were quantified as a function of time via LC-MS. The pseudo-first order rate constants for release of DOX from DOX-F-MM, DOX-M-MM, and DOX-S-MM were 21.26 × 10 -3 h -1 , 8.95 × 10 -3 h -1 , and 3.62 × 10 -3 h -1 , respectively ( Figure 3A and Table S1) while the pseudo-first order rate constants for release of chex from chex-F-MM, chex-M-MM, and chex-S-MM were 8.23 × 10 -3 h -1 , 4.31 × 10 -3 h -1 , and 1.63 × 10 -3 h -1 , respectively ( Figure S15 and Table S1). Although these rate constants are not identical across DOX and chex pairs due to differences in the physical properties (hydrophobicity) of chex and DOX, the DOX:chex release rate ratios were similar: 2.58, 2.08, and 2.22 for the F, M, and S pairs, respectively, which enables correlation of DOX prodrug and pro-ORCA activation (vide infra). We note that these in vitro release kinetics studies were conducted using MMs rather than BASPs to ease sample handling and analysis. Though release from BASPs is generally much slower than from MMs using the same linker, trends in release kinetics across different linkers are preserved. 62 Next, these 6 MMs were used to prepare 6 different singly-loaded BASPs (DOX-F, DOX-M, DOX-S, chex-F, chex-M, chex-S) (Scheme 2B). GPC and DLS results confirmed that the BASP sizes (~21 nm) were independent of the payload (DOX or chex) or linker (F, M, or S); EPR spectroscopy and MRI-measured per-chex r2 values for the three BASP pro-ORCA+prodrugs showed that the magnetic properties of chex were not meaningfully affected by the linkers ( Figure S20-22 and Table 1). Cell viability assays were conducted with the same 3 cell lines used above following 48 h incubation with these 6 BASPs. As expected, negligible toxicity was observed for the pro-ORCAs lacking DOX ( Figure S23). In contrast, the toxicity of the DOX prodrug BASPs increased with the rate of DOX release ( Figure 3B); in MM.1S cells, DOX-F, DOX-M, and DOX-S exhibited IC50 values of 3.1 µM, 17.4 µM, and 113.3 µM DOX, respectively. Given that these BASPs have similar sizes and compositions (Table 1) and, thus, expectedly similar cellular internalization rates, this toxicity trend is likely due to varying levels of DOX prodrug activation and release within the 48 h incubation period. Moreover, the DOX-F viability curve approaches that of free DOX (IC50 of 2.9 µM for MM.1S), suggesting the majority of DOX is released for this material ( Figure S24). To further confirm this hypothesis, viability assessments of MM.1S and A549 were performed as a function of exposure time to each DOX-containing BASP using a fixed dose of 0.5 mg/mL BASP (83-86 µM DOX; Figure  S25). Validating our prodrug linker design, the toxicity toward both cell lines followed the same trend: DOX-F > DOX-M > DOX-S. In order to inform subsequent studies of MRI-guided monitoring of prodrug activation, we studied the in vivo toxicity of DOX-M using a subcutaneous lung adenocarcinoma (A549) model. A549 cells were injected into the hind flank of BALB/c mice (4 groups of n = 3 mice).
Once the tumors reached ~1 cm in diameter, the mice were randomized and each received a single 50 µL intratumoral dose (day 0) of 0 (blank), 1, 5, or 10 mg/mL of DOX-M. Intratumoral administration was selected to maximize tumor MRI SNR and preclude variables of tumor accumulation and pharmacokinetics, thereby simplifying imaging studies for this proof-of-principle work (vide infra). It should be noted that intratumoral drug delivery is a clinically-viable strategy for cancer therapy; 82-85 ~200 ongoing clinical trials leverage this method of administration. Moreover, intratumoral administration of BASPs has not been demonstrated before. In our study, significant tumor growth inhibition was observed over the 25 d period of observation following the administration of DOX-M when compared to the control group ( Figure S26). Moreover, a dose-dependent response was observed; the 10 mg/mL (1.7 mM) DOX-M dose provided gradual tumor shrinkage that we reasoned could allow for facile comparisons between different ester linkers in a "reporter" theranostic study. Based on the above results, a new set of 3 pro-ORCA+prodrug BASPs -F, M, and S -that each contained both DOX and chex attached to the same polymer (DOX:chex ratio of 1:6 to maximize MRI sensitivity) conjugated via the respective fast-, medium-, and slow-release ester linkers, as well as 1 mol% of non-releasable cy5.5, were synthesized and characterized by GPC, DLS, EPR, and in vitro MRI (Table 1, Figure  S20-22). The same subcutaneous A549 murine model was employed to correlate DOX prodrug activation from F, M, and S with MRI SNR changes in vivo. Each mouse was intratumorally administered either a pro-ORCA-based BASP (F, M, or S) or a non-cleavable chexcontaining BASP 66 as control (C); note that every animal received 50 µL of a 70 mg/mL solution, which corresponded to ~1.7 mM of conjugated DOX, matching the dose used for DOX-M (vide supra). T2weighted MR images of the tumors were taken at pre-determined time points, beginning at 30 min after BASP injection ( Figure 4A). Inversion of image intensity was performed for subsequent analysis in order to aid visualization. In the inverted T2-weighted images, signal decreases upon pro-ORCA activation; the image appears brightest in the areas where the contrast agent (F, M, or S) concentration is the highest. Importantly, SNR variations that correlated with the release kinetics of chex from the corresponding MMs were observed while no changes were observed for C ( Figure  4B), which confirmed the ability of this approach to monitor pro-ORCA activation kinetics in vivo using MRI. For instance, 24 h postinjection decreases in inverse T2-weighted signal of 80 ± 7%, 49 ± 14%, and 9 ± 1% were observed for F, M, and S, respectively; the constant signal observed for non-cleavable BASP C suggests that Please do not adjust margins Please do not adjust margins these BASPs remain in the tumor environment and that there is an insignificant amount of BASP-nitroxide degradation (e.g., via reduction) throughout the timeframe of this study ( Figure 4B and Figure S27). 66 To support our findings that these MRI SNR changes correlated with DOX release, the tumors of mice that had been administered F, M, and S were extracted for ex vivo fluorescence imaging to correlate the intratumoral locations of DOX (via its inherent fluorescence) with those of the BASPs (via cy5.5 fluorescence) ( Figure 4C). We stress here that fluorescence is used here as a supporting tool to validate our MRI measurements for this proof-of-concept study; most drugs are not inherently fluorescent and fluorescence imaging is not typically conducted in the clinic, motivating our development of pro-ORCAs. Gratifyingly, the DOX (green) and BASP (red) signals from the micrographs of tumors treated with the slow-releasing BASP (S) displayed good signal colocalization, suggesting that DOX was still conjugated to the BASP in its prodrug form and in agreement with MRI SNR variations. In contrast, significant separations of DOX (green) and BASP (red) were observed for the fast (F) as well as the medium (M) BASPs, suggesting that DOX release had occurred at a sufficiently earlier time points with these constructs. It is, however, difficult to directly compare F and M, as the fluorescence of DOX is susceptible to alteration by several biological processes. 86,87 Nonetheless, these results support the fact that T2 MRI signal changes induced by pro-ORCA activation correlate with DOX prodrug activation in this system, establishing the first example of controlled nitroxide release for correlation of drug release in vivo.

Conclusions
Herein, we introduced design principles and provided in vitro and in vivo proofs-of-concept for stimuli-responsive pro-ORCAs that enable real-time, non-invasive monitoring of prodrug activation in biological systems using a clinically viable imaging technique (MRI). Specifically, chex ORCAs were conjugated to BASPs through cleavable linkers generating "pro-ORCAs." When attached to the BASP, chex has a large r2 value that, when combined with the high density of chex on each BASP, leads to MRI contrast on par with clinically used metal-based contrast agents; cleavage of the linker that connects chex to the BASP leads to a ~30-fold decrease in r2 and concomitant loss of MRI contrast. By combining this pro-ORCA concept with DOX prodrugs conjugated to the same BASP using the same linkers, simultaneous pro-ORCA and prodrug activation occurs, which leads to changes in MRI contrast that correlate with drug release. This concept was demonstrated using 4 different linkers: 1 photocleavable and 3 hydrolytically cleavable linkers with different rates of hydrolysis, in both in vitro and in vivo settings. Regarding the latter, real-time monitoring of drug release via changes in T2 MRI signals within a tumor was demonstrated. Altogether, this work establishes a promising platform technology for monitoring prodrug activation in vitro and in vivo. We note that while the subject of this study was the real-time visualization of prodrug activation in biological environments via MRI, the demonstrated linker "plug-and-play" concept can potentially be expanded in the future to reporters of cell apoptosis or immunogenic cell death. Moreover, this approach could potentially be expanded to other therapeutic entities and a range of imaging modalities, providing valuable research tools as well as clinically translatable systems for personalized medicine.

Conflicts of interest
There are no conflicts to declare.