CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 62/949,288 filed December 17, 2019, which is incorporated herein in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] The present invention was made with Government support under Grant No. R01CA199668 awarded by the National Institutes of Health/National Cancer Institute, and Grant No. R01HD086195 awarded by the National Institutes of Health/National Institute of Child Health and Human Development. The Government has certain rights in the invention.

BACKGROUND

[0003] Distance-dependent magnetic resonance tuning (MRET) technology has many advantages over fluorescence-based Forster resonance energy transfer (FRET) in sensing/imaging biological targets. These include deeper tissue penetration and fewer undesirable interactions with the surrounding biological environment. However, the contrast enhancement and stability of current Ti-based MRET technology needs significant improvement before it can achieve broadly in vivo applications. Herein is a new two-way magnetic resonance tuning (TMRET) nanoprobe with dually activatable T1&T2 magnetic resonance signals coupled with dual-contrast enhanced subtraction imaging (DESI) to dramatically enhance contrast in targeted tissues and suppress the background signal from normal tissue. It was demonstrated that this integrated platform could quantitatively image molecular targets in tumours and sensitively detect very small intracranial brain tumours (-0.75 mm ³ ) in patient-derived xenograft models with a tumour-to-normal-tissue ratio (TNR) >10. The TMRET nanoprobes and complementary DESI technology with ultra-high TNR will offer new opportunities for enhanced molecular diagnostics and image-guided biomedical applications.

[0004] Nanometer-scale distance-dependent physical processes are invaluable for imaging and sensing a wide range of biological targets. These have significantly advanced the fundamental understanding of living systems and facilitated the development of new molecular medicines for better patient care. Several technology platforms based on distance- dependent physical processes have been proposed for such applications, and each of them encompasses both distinct advantages and limitations. For instance, fluorescence-based Forster resonance energy transfer (FRET) is one of such physical process by which excitation energy is absorbed by a molecular fluorophore (the donor) and then transferred to a fluorophore (the acceptor) in close distance. It is a highly sensitive technique for investigating a variety of biological phenomena that produce changes in molecular proximity. However, FRET suffers from low and fluctuating signal intensities from single fluorophores and short observation times due to photobleaching. Nanoplasmonic sensing and imaging techniques are based on distance variations between noble metals (e.g. gold and silver nanoparticles) to generate a surface plasmon resonance (SPR) absorption wavelength shift and subsequent colour changes. It can overcome the limitations of organic fluorophores and provide a fast and convenient platform for mapping biological activities such as nucleic acid-protein interactions. However, together with FRET, the broad applications of these optical techniques for in vivo sensing and imaging may be hampered by their intrinsic low tissue penetration and undesirable photon interactions with the complicated biological environments in the body.

[0005] Most recently reported is the pioneering work on magnetism-based nanoscale distance-dependent magnetic resonance tuning (MRET). MRET opens new possibilities for non-invasive investigation of a variety of biological processes in vivo because magnetic resonance imaging (MRI) can provide high-resolution functional and anatomical imaging without being limited by the tissue penetration depth. In MRET, T i relaxation is modulated by controlling the distance between a paramagnetic T i enhancer and a superparamagnetic quencher. The distance-dependent T i MRI signal was demonstrated as a nanoscale ruler to provide quantitative sensing and imaging of molecular interactions (e.g., cleavage, binding & conformational changes). Furthermore, proof-of-concept distance-dependent MR imaging studies have been performed in vivo to demonstrate the matrix metalloproteinase-2 (MMP-2)- induced cleavage of MRET probes at the tumour site. However, despite their ability to generate a positive T i MRI contrast for quantitative analyses of biological events, the use of T i contrast agent in MRET may be compromised by its lack of sensitivity and intrinsic low MR relaxivity. The first MRET probes were administrated intratumourally, while the size, stability and surface properties of these probes need to be further optimized for broad in vivo applications by systemic injection in which these probes are exposed to the bloodstream. Furthermore, ¹ 11 MRI typically suffers from low contrast enhancement at the target sites with high background noise from normal tissue, due to the interference from intrinsic ¹ 11 signals in the body. This often makes the interpretation of the acquired images difficult.

[0006] Herein is a new two-way magnetic resonance tuning (TMRET) nanotechnology with dually activatable T1&T2 signals for the first time. It is designed to significantly improve the MR contrast and reliability as well as suppress the background signal of MRET. The TMRET nanotechnology is enabled by a new nanostructure, constructed by the concurrent encapsulation of a two-way MRET pair, a paramagnetic porphyrin-manganese (II) chelate (P- Mn) and a superparamagnetic iron oxide nanoparticle (SPIO) into a nanoscale micelle with structure-dependent stability and stimuli-responsiveness. Mn ²⁺ serves as both an ‘enhancer’ in Ti MRI signal and a ‘quencher’ in T2 MRI signal, while the SPIO nanoparticle acts as an ‘enhancer’ in T2 MRI signal and a ‘quencher’ in Ti MRI signal. When the two-way MRET pair is “locked” within the nanoscale micelle core at a close distance with an optimal Mn ²⁺ /SPIO ratio, T1&T2 MRI signal could be dually turned “OFF”. Upon interaction with biological stimuli (the “key”, FIG. 1), T1&T2 MRI signals could be dually turned “ON” depending on the increased distance between “Mn ²⁺ ” and “SPIO”, which is controlled by the integrity of the micelles in response to these stimuli. Furthermore, a new and complementary post- imaging processing and reconstruction method named “dual-contrast enhanced subtraction imaging (DESI)” is introduced for better implementation of the dual T1&T2 MRI signal changes. It was further evaluated that this TMRET nanotechnology platform integrated with DESI for non-invasively and quantitatively imaging biological targets within the tumour like glutathione (GSH), the key sensing/imaging molecule for redox-responsive nano platforms. This strategy can be applied to different micellar nanostructures with SPIO and P- Mn pair and engineered to respond to other biological markers, such as acidic tumor pH. The proposed TMRET nanoprobe with DESI technique allowed us to detect very small tumours deeply embedded in the mouse brain by intravenous administration of the nanoprobes.

BRIEF SUMMARY OF THE INVENTION [0007] In one embodiment, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising: a plurality of amphiphilic compounds that self- assemble to form the nanocarrier; and a first magnetic resonance imaging (MRI) contrast agent and a second MRI contrast agent in the interior; wherein one MRI contrast agent is paramagnetic, and the other MRI contrast agent is superparamagnetic, and the first MRI contrast agent and the second MRI contrast agent are configured within the interior to substantially cancel the magnetic signal of each contrast agent. [0008] In another embodiment, the present invention provides a method of imaging, comprising: administering to a subject to be imaged an effective amount of a nanocarrier of the present invention, wherein, upon exposure to a stimulus, the nanocarrier disassembles such that the first MRI contrast agent and the second MRI contrast agent are released; and detecting the first MRI contrast agent and the second MRI contrast agent.

[0009] In another embodiment, the present invention provides a method of detecting a disease, comprising: administering to a subject an effective amount of a nanocarrier of the present invention, wherein, upon exposure to a stimulus, the nanocarrier disassembles such that the first MRI contrast agent and the second MRI contrast agent are released, and detecting the first MRI contrast agent and the second MRI contrast agent, thereby detecting the disease in the subject.

[0010] In another embodiment, the present invention provides a compound with the following structure of Formula (II):

R'

L' R'

X ² ~L'

PEG— X. ¹

X ² -L'

L' i R'

R' (H) wherein: each L’ is a linker comprising boron; PEG polymer has a molecular weight of 1-100 kDa; each R’ is a porphyrin; X ¹ is a diamino carboxylic acid; and each X ² is independently a bis(hydroxymethyl)propionic acid derivative.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 shows schematic illustration of the TMRET nanotechnology & dual-contrast enhanced subtraction imaging (DESI).

[0012] FIGs. 2A-20 shows TMRET nanoprobes and the T1/T2 dual-quenching properties as well as the mechanism. FIG 2A shows size distributions and FIG. 2B shows TEM micrograph of DCM@P-Mn-SPIO. FIG. 2C shows size distributions and FIG. 2D show TEM micrograph of disassembled DCM@P-Mn-SPIO treated with 20 mM GSH and 3 mg/mL SDS for 24 h. FIG. 2E shows the release profiles of Pa from DCM@P-Mn-SPIO in the presence of different GSH concentrations (0, 5, 10, 20 mM). FIG. 2F shows GSH- responsive release profiles of Pa from DCM@P-Mn-SPIO upon the addition of GSH (20 mM) at a specific time (4 h). Values reported as mean ± s.d. (n=3). FIG. 2G shows Ti- weighted images and the colour-coded Ti map and FIG. 2H shows T2-weighted images and the colour-coded T2 map of DCM@P-Mn-SPIO before and after the payload release (triggered by GSH). The changes in 1/Ti values (DI/Ti) (FIG. 21) and I/T2 values (DI/T2) (FIG. 2J) of DCM@P-Mn-SPIO at different concentrations before and after incubation with GSH (0, 5, 10, 20 mM) and SDS. The changes in 1/Ti values (DI/Ti) (FIG. 2K) of DCM@P- Mn and 1, I/T2 values (DI/T2) of DCM@SPIO at different concentrations before and after incubation with GSH and SDS. FIG. 2M shows T2 relaxivity values of free SPIO (75.04 mM V ¹ ), DCM@SPIO (88.97 mM V ¹ ) and DCM@P-SPIO (86.31 mMV ¹ ). FIG. 2N shows electron paramagnetic resonance (EPR) spectroscopy studies ofDCM@P-Mn, DCM@SPIO, DCM@P-Mn-SPIO in the absence and presence of GSH and SDS. FIG. 20 shows Magnetic moment of DCM@SPIO, DCM@P-Mn-SPIO and DCM@P-Mn-SPIO with SDS and GSH, measured over the same sample volume with background subtracted by vibrating-sample magnetometer (VSM).

[0013] FIG. 3. shows illustration of the mechanism of the Ti and T2 quenching and recovery in TMRET nanoprobe. The black circle denotes SPIO and the golden star stands for P-Mn. The red and green arrow on SPIO stand for their magnetization; Different lengths represent the magnetization strength. OFF state: Ti and T2 contrast agents are coloaded into DCM with specific concentration ratios. The spin fluctuation of T 1 contrast agent is slowed, the effective magnetic field from T2 contrast agent is weakened and the diamagnetic field further reduces the effective dipole field. This leads to quenched Ti and T2 MRI signals. ON state: Ti and T2 contrast agents are separated apart. Fast spin fluctuation of Ti contrast agent and strong magnetic field from T2 contrast agent would help relax water protons effectively. This leads to enhanced Ti and T2 MRI signals.

[0014] FIGs. 4A-4E show the dual-responsiveness of T1&T2 MR signals of DCM@P-Mn- SPIO compared to different levels of GSH in cells. FIG. 4A shows T 1 WI and FIG. 4B shows T2WI MR images and the corresponding quantitative analysis of PC-3 cells treated without and with GSH inhibitor (FBS), incubated with DCM@P-Mn-SPIO for different time points. The normal prostate cells (RWPE-1) were employed as control. FIG. 4C shows Ti and FIG 4D shows T2 colour coded map and the changes in relaxivity (ARi and AFG) of DCM@P- Mn-SPIO in PC-3 cells treated with various concentrations of FBS. The GSH concentrations in cells were measured by using ThiolTracker™ Violet. FIG. 4E shows TEM of PC-3 cells after 24 h incubation with DCM@P-Mn-SPIO. [0015] FIGs. 5A-5C show the relationship between the MRI signal and the concentrations of the molecular target of TMRET. FIG. 5A shows quantitative MRI visualization of GSH in tumours by using DCM@P-Mn-SPIO as a t-MERT nanoprobe. Ri and R2 mapped images of PC-3 tumour-bearing mice with different levels of GSH (6.15, 6.80, 8.10, 11.44 mM) quantitatively determined by using ThiolTracker™ Violet. Plot of ARi (FIG 5B) and AFV (FIG. 5C) of tumours versus GSH concentration in tumours. ARi and AFG increased linearly with GSH concentration in the tumours. Pearson’s test was used for correlation analysis.

[0016] FIGs. 6A-6I show in vivo MRI of tumours by using DCM@P-Mn-SPIO. FIG. 6A shows T1&T2WI MR images of PC-3 tumour-bearing mice (n=3) after injection of DCM@P- Mn-SPIO. FIG. 6B shows SNR and the applicable DESI area of DCM@P-Mn-SPIO in the tumour. The shaded area of the curves represents the effective Ti and T2 DESI area. FIG. 6C shows the T2 star and colour coded T2 map images of DCM@SPIO and DCM@P-Mn-SPIO. FIG. 6D shows R2 star (*) and FIG. 6E shows R2 of the tumour treated by DCM@SPIO and DCM@P-Mn-SPIO at different timepoints. DESI subtraction images of FIG. 6F shows DCM@P-Mn-SPIO, FIG. 6G DCM@P-Mn and FIG. 6H shows DCM@SPIO groups at 12 h after the probe administration. The arrows point out the tumours. FIG. 61 shows TNR of DCM@P-Mn-SPIO treated tumours. DCM@P-Mn and DCM@SPIO were employed as control groups ns, not significantly; *, p<0.05; **, p<0.01.

[0017] FIGs. 7A-70 show In vivo applications of TMRET probe with DESI technique on orthotopic brain tumours. FIG. 7A shows T1&T2 WI and T1&T2 mapped MRI images of the mice (n=3) treated with DCM@P-Mn-SPIO. FIG. 7B shows Ti WI and Ti mapped MRI images of the mice (n=3) treated with DCM@P-Mn. FIG. 7C shows T2 WI and T2 mapped MRI images of the mice (n=3) treated with DCM@SPIO. FIG. 7D shows T1&T2 SNR of DCM@P-Mn-SPIO mediated MRI on orthotopic brain tumour bearing mice. FIG. 7E shows Ti SNR of DCM@P-Mn mediated MRI on orthotopic brain tumour bearing mice. FIG. 7F shows T2 SNR of DCM@SPIO mediated MRI on orthotopic brain tumour bearing mice. FIG. 7G shows DESI processing of the T1&T2WI MRI images in FIG. 7A. FIG. 7H shows DESI processing of the T 1 WI MRI images in FIG. 7B. FIG. 71 shows DESI processing of the T2WI MRI images in FIG. 7C. Histopathology of the whole-brains of DCM@P-Mn-SPIO treated mice (FIG. 7J), DCM@P-Mn treated mice (FIG. 7K), and DCM@SPIO treated mice (FIG. 7L). The scale bar is 1 mm. FIG. 7M shows TNR of DCM@P-Mn-SPIO, DCM@P- Mn and DCM@SPIO treated tumours based on DESI. Prussian blue showed DCM@P-Mn- SPIO (FIG. 7N) and DCM@SPIO (FIG. 70) accumulation in orthotopic brain tumour tissue.

**, p<0.01.

[0018] FIGs. 8A-8S show application of TMRET nanotechnology and DESI on a pH responsive nanoprobe (POP@P-Mn-SPIO). The size distribution and morphology of POP@P-Mn-SPIO in FIG. 8A, and POP@P-Mn-SPIO in FIG. 8B treated with acidic pH (5.5). FIG. 8C shows Ri and FIG. 8D shows R2 quenching behaviours of POP@P-Mn-SPIO. The Ri and R2 can be recovered under stimulation of acidic pH (5.5). FIG. 8E shows T1&T2WI and T1&T2 mapped MRI images of the mice (n=3) treated with POP@P-Mn-SPIO. FIG. 8F shows TiWI and Ti mapped MRI images of the mice (n=3) treated with POP@P- Mn. FIG. 8G shows T2WI and T2 mapped MRI images of the mice (n=3) treated with POP@SPIO. FIG. 8H shows T1&T2 SNR of POP@P-Mn-SPIO, FIG. 81 shows Ti SNR of POP@P-Mn, FIG. 8J shows T2 SNR of POP@SPIO mediated MRI on orthotopic brain tumour bearing mice. FIG. 8K shows DESI processing of the T1&T2WI MRI images in FIG. 8E. FIG. 8L shows DESI of the TiWI MRI images in FIG. 8F. FIG. 8M shows DESI processing of the T2WI MRI images in FIG. 8G. Histopathology of the whole-brains of POP@P-Mn-SPIO (FIG. 8N) treated mice; POP@P-Mn treated mice (FIG. 80); and POP@SPIO (FIG. 8P) treated mice. The red arrows denote the orthotopic brain tumours. The scale bar is 1 mm. FIG. 8Q shows TNR of POP@P-Mn-SPIO treated tumours based on DESI. POP@P-Mn and POP@SPIO were employed as control groups. FIG. 8R shows T2* and FIG. 8S shows R2 of the tumours treated by POP@SPIO and POP@P-Mn-SPIO at different timepoints. **, P<0.01; ns, not significantly.

[0019] FIGs 9A-9B show optical measurements determined the chelation of Mn ²⁺ to pheophorbide a (P). FIG. 9A shows the absorbance spectra of pheophorbide a (P) before and after chelating manganese (II) (Mn ²⁺ ) ions. FIG. 9B shows fluorescence emission spectra of pheophorbide a (P) before and after chelating Mn ²⁺ . Excitation: 412 nm. The fluorescence quenching and disappearance of UV absorbance of Pa after the chelation of Mn ²⁺ confirmed the successful synthesis of P-Mn. Three experiments were repeated independently with similar results.

[0020] FIG. 10 shows T1&T2- weighted images of DCM@P-Mn-SPIO with different P-Mn to SPIO ratios (1 :0.006, 1 :0.013, 1 :0.025, 1 :0.050) before and after 24 h after incubation with GSH (20 mM) in the presence SDS. The concentration of P-Mn was fixed at 1 mg in 1 mL micelle solution. Only the DCM@P-Mn-SPIO nanoprobe with a P-Mn/SPIO ratio of 1:0.025 (red box) showed obvious dual quenching and recovery in T1&T2 signals without and with incubation with GSH and SDS. The same volume of water was added to other samples if SDS + GSH solution were introduced to one sample. Three experiments were repeated independently with similar results.

[0021] FIGs. 11A-11B shows TEM micrographs of DCM@SPIO (FIG. 11A), and DCM@P-Mn (FIG. 11B). The DCM@SPIO showed similar spherical morphology with a diameter of ~80 nm, in which clusters of small SPIOs were encapsulated. The DCM@P-Mn was spherical, and the size was around 14 nm. Five experiments were repeated independently with similar results.

[0022] FIG. 12A-12B shows Ti relaxivity rates of DCM@P-Mn and T2 relaxivity rates of DCM@SPIO were measured on a 7.0 T MRI scanner. FIG. 12A shows the Ti relaxivity value of DCM@P-Mn was calculated to be 5.2 mM V ¹ . FIG. 12B shows the T2 relaxivity value of DCM@SPIO was calculated to be 88.8 mM V ¹ . Three experiments were repeated independently with similar results. Pearson’s test was used for correlation analysis.

[0023] FIG. 13 shows T2 relaxivity values in various formulations. High and low T2 signals of the DCM@SPIO, and free SPIO were mapped with colour, respectively. Three experiments were repeated independently with similar results.

[0024] FIG. 14 shows the magnetic moment of DCM@P-Mn, measured by vibrating- sample magnetometer (VSM) over the same sample volume with background subtracted. The inset is TiWI of DCM@P-Mn and DCM@P-Mn+SDS+GSH. H2O was employed as a control to demonstrate the Ti signal enhancement. To make the concentrations of all samples consistent, the same volume of water to other samples was added if SDS+GSH solution were introduced to one sample. Three experiments were repeated independently with similar results.

[0025] FIG. 15 shows double integration of EPR spectra of DCM@P-Mn (value is 320102.57), DCM@SPIO (value is 12040202), DCM@P-Mn-SPIO (value is 8444116) and DCM@P-Mn-SPIO+GSH+SDS (value is 13070774). The double-integration value of the EPR spectrum of DCM@P-Mn-SPIO which corresponds to the total number of free, unpaired electrons in the system was lower than that of the probe with only SPIO (DCM@SPIO). In the presence of GSH and SDS, the integral recovers to a similar level to that of DCM@SPIO. Three experiments were repeated independently with similar results. [0026] FIGs. 16A-16B show in vitro cytotoxicity of DCM@P-Mn-SPIO, DCM@P-Mn, and DCM@SPIO by MTS assay (n=3). FIG. 16A shows the PC-3 cells were treated with nanoprobes for 24 h for cytotoxicity evaluation. The TMRET nanoprobes (DCM@P-Mn- SPIO) and the control probes (DCM@P-Mn and DCM@SPIO) all showed less than 12 % reduction of cell viability at an extremely high concentration of 5 mg/mL of nanoprobes. FIG. 16B shows the PC-3 cells were treated with fully released nanoprobes for 24 h for cytotoxicity evaluation. The SDS used to destroy the micelle is a strong detergent that will break the cell membrane and cause severe cytotoxicity. To avoid SDS-induced toxicity, the fully released nanoprobes (DCM+P-Mn, DCM+SPIO and DCM+P-Mn+SPIO) were prepared by mixing the components of each nanoprobe together without going through any process of nanoparticle preparation. The concentrations of all materials was calculated based on DCM amounts. Values presented as mean ± s.d.

[0027] FIG. 17 shows TiWI MRI images and Ti mapped images of tumour bearing mice (n=3) at pre-injection and different time points (1 and 12 h) post- injection of DCM@P-Mn. The mean Ti -weighted signal intensities were measured for each tumour at different times (0 h, 1 h and 12 h). Ti map showed gradual color change at the tumor site, from red (that is, low Ri) to blue (that is, high Ri), indicating increased Ri after injection of DCM@P-Mn at 1 h and 12 h.

[0028] FIG. 18 shows T2WI MRI images and T2 mapped images of tumour bearing mice (n=3). The mean T2-weighted signal intensities were measured for each tumor at different times (0 h, 1 h and 12 h). T2 map showed gradual color change at the tumor site, from red (that is, low R2) to blue (that is, high R2), indicating increased R2 after injection of DCM@SPIO at 1 h and 12 h.

[0029] FIG. 19 shows H&E staining determined the systemic toxicity of PC-3 tumour bearing mice that were treated with DCM@SPIO, DCM@P-Mn and DCM@P-Mn-SPIO respectively. Three experiments were repeated independently with similar results. The scale bar is 200 pm.

[0030] FIGs. 20A-20B show concentration-dependent Ti and T2 MR signal of DCM@P- Mn-SPIO. The nanoprobes were broken down with 20 mM GSH and SDS. FIG. 20A shows Ti and T2 weighted MRI images. FIG. 20B shows quantitative analysis demonstrating the linearity of the relationship between the Ti and T2 MR signal and the nanoprobe concentration. MRI signal could be detected with probe concentrations as low as 0.003 mM. Ti (R 0.98) and T2 (R=-0.97) signals changed linearly with the concentrations of DCM@P- Mn-SPIO. Pearson’s test was used for correlation analysis. Three experiments were repeated independently with similar results.

[0031] FIGs. 21A-21B show concentration-dependent MRI signal variations of DCM@P- Mn-SPIO in mouse muscle. FIG. 21A shows different concentrations of DCM@P-Mn-SPIO (P-Mn concentrations ranged from 0.03 to 1.0 mM) were injected subcutaneously into the flank of living mice ( n = 3) and scanned with MRI. FIG. 21B shows quantitative analysis of the Ti and T2 signal intensities. For DESI analysis, the concentration limit of DCM@P-Mn- SPIO should be above 0.06 mM. At concentrations less than 0.06 mM, reverse Ti and T2 signal readouts were observed, i.e. the T2 signals became higher than the Ti signals. Data analysis was conducted by Graphpad Prism 7.00. Values presented as mean ±s.d. Two-tailed Student’s t-test was employed for statistical analysis. *p< 0.05; ** p<().() 1 ; *** p<().()() 1.

[0032] FIGs. 22A-22C show Ti and T2 signal variations and the DESI processing of DCM@P-Mn-SPIO in PC-3 prostate cancer cells (FIG. 22A); PC-3 cells treated with a GSH inhibitor (L-BSO, 10 mM) (FIG. 22B); normal prostate cells (RWPE-1) (FIG. 22C). By DESI processing, PC-3 showed decent DESI area (shadow); PC-3 cells with GSH inhibitor exhibited smaller DESI area, indicating that GSH plays a key role in MR signal recovery; the normal cells showed reversed MR signals, which meant the Ti and T2 signals were both in the quenched state. All experiments were conducted in triplicates. Values presented as mean ±s.d.

[0033] FIG. 23 show enlarged DESI images from FIG. 5. The black arrows pointed out the tumor sites. Three experiments were repeated independently with similar results.

[0034] FIG. 24A shows Ri and FIG. 24B shows R2 elevations in orthotopic brain tumour bearing mice (n=3) after the administration of DCM@P-Mn-SPIO. FIG. 24C shows Ri gradually decreased in orthotopic brain tumour bearing mice (n=3) after the administration of DCM@P-Mn. FIG. 24D R2 gradually decreased in orthotopic brain tumour bearing mice (n=3) after the administration of DCM@SPIO. Data analysis was conducted by Graphpad Prism 7.00. Values presented as mean ± s.d. Two-tailed Student’s t-test was employed for statistical analysis n.s., not significant; *, p<0.05; **, p<0.01; ***, pO.001.

[0035] FIG. 25 shows enlarged DESI images from FIG. 7. The black arrows pointed out the tumor sites. [0036] FIGs. 26A-26B show TMRET nanotechnology on a DCM@P-Mn-SPIO nanoprobe measured by 3.0 T MRI. FIG. 26A shows Ri and FIG. 26B shows R2 quenching and recovery behaviours of DCM@P-Mn-SPIO (n=3). The signal model probes were employed as controls to demonstrate the non-quenched status of the Ri and R2. The nanoprobe was treated with SDS+GSH to dissociate the nanostructure. To make the concentrations of all samples consistent, the same volume of water to other samples was added if SDS + GSH solution were introduced to one sample. Values presented as mean ± s.d.

[0037] FIGs. 27A-27B show TMRET nanotechnology on a DCM@P-Mn-SPIO nanoprobe measured by 9.4 T MRI. FIG. 27A shows RI and FIG. 27B shows R2 quenching and recovery behaviours of DCM@P-Mn-SPIO. The signal model probes were employed as controls to demonstrate the non-quenched status of the Ri and R2. The nanoprobe was treated with SDS+GSH to dissociate the nanostructure. To make the concentrations of all samples consistent, the same volume of water to other samples was added if SDS + GSH solution were introduced to one sample. Values presented as mean ± s.d. [0038] FIGs. 28A-28D show TMRET nanotechnology on a DSPE-PEG@P-Mn-SPIO nanoprobe that was constructed based on an FDA-approved polymer (DSPE-PEG). FIG. 28A shows the size distribution and morphology (inset) of the DSPE-PEG@P-Mn-SPIO. Scale bar is 50 nm. FIG. 28B shows the size distribution of DSPE-PEG@P-Mn-SPIO that has been treated with SDS. FIG. 28C shows Ri and FIG. 28D shows R2 quenching behaviors of DSPE-PEG@P-Mn-SPIO. The signal model probes were employed as controls to demonstrate the non-quenched status of the Ri and R2. The nanoprobe was treated with SDS to dissociate the nanostructure. Values presented as mean ± s.d.

[0039] FIG. 29 shows synthetic routes of PEG-OHs.

[0040] FIG. 30 shows synthetic routes of PPBA. [0041] FIG. 31 shows the synthetic route and the chemical structure of the formation of

PEGsooo-OHs-PPBA (POP).

[0042] FIG. 32 shows mass spectrometry demonstrated that the molecular weight of PEG- OHs was consistent with the theoretical value. Three experiments were repeated independently with similar results. [0043] FIG. 33 shows mass spectrometry demonstrated that the molecular weight of PPBA was consistent with the theoretical value. Three experiments were repeated independently with similar results.

[0044] FIG. 34 shows enlarged DESI images from FIG. 8. The black arrows pointed out the tumor sites.

DETAILED DESCRIPTION OF THE INVENTION

I. GENERAL

[0045] The present invention provides a porphyrin dendrimer comprising a boron crosslinker and bis(hydroxymethyl)propionic acid derivatives, which can form nanocarriers. The present invention provides also provides a nanocarrier comprising two MRI contrast agents which are configured within the interior to substantially cancel out the magnetic signal of each contrast agent. The nanocarrier comprises a plurality of amphiphilic compounds is a dendrimer, a phospholipid or derivatives thereof. The nanocarriers can be used for imaging and detecting a disease.

II. DEFINITIONS

[0046] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

[0047] “A,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0048] “Nanoparticle” or “nanocarrier” refers to a micelle resulting from aggregation of the dendrimer conjugates of the invention. The nanocarrier has a hydrophobic core and a hydrophilic exterior. [0049] “Amphiphilic” refers to a compound having both hydrophobic portions and hydrophilic portions. For example, the amphiphilic compounds of the present invention can have one hydrophilic face of the compound and one hydrophobic face of the compound. Amphiphilic compounds useful in the present invention include, but are not limited to, cholic acid and cholic acid analogs and derivatives.

[0050] “Imaging” refers to using a device outside of the subject to determine the location of an imaging agent, such as a compound of the present invention. Examples of imaging tools include, but are not limited to, fluorescence microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT) and x-ray computed tomography (CT).

[0051] “Contrast agents” or “imaging agents” refer to a compound which increases the contrast of structure within the location of the cell or body for imaging methods including, but not limited to fluorescence microscopy, MRI, PET, SPECT, and CT. Contrast agents can emit radiation, fluorescence, magnetic fields or radiowaves. Contrast agents include, but are not limited to paramagnetic compounds, superparamagnetic compounds, radiometal chelators, and fluorophores.

[0052] “Paramagnetic” refers to a compound wherein at least one electron is unpaired. Paramagnetic compounds are attracted to magnetic fields due to the unpaired electron(s). Paramagnetic compounds are useful for imaging techniques which use magnetic fields such as MRI. Examples of paramagnetic compounds useful in the present invention includes, but is not limited to, Gd ³⁺ , Mn ²⁺ , Dy ³⁺ and Cu ²⁺ .

[0053] “Superparamagnetic” refers to ferromagnetic and ferromagnetic compounds wherein the magnetization can flip direction due to changes in temperature. Superparamagnetic compounds are useful for imaging techniques such as MRI. Examples of superparamagnetic compounds include but are not limited to cobalt nanoparticles, ferromagnetic nanoparticles, superparamagnetic iron oxide (SPIO) and superparamagnetic iron platinum particle (SIPP). Superparamagnetic iron oxides include, but are not limited to, iron oxides with the formula Fe304 and Fe203. Superparamagnetic iron platinum particles include, but are not limited to phospholipids conjugated to SIPPs, such as DSPE-SIPP, and superparamagnetic iron platinum nanoparticles.

[0054] “Pendrimer” and “dendritic polymer” refer to branched polymers containing a focal point, a plurality of branched monomer units, and a plurality of end groups. The monomers are linked together to form arms (or "dendrons") extending from the focal point and terminating at the end groups. The focal point of the dendrimer can be attached to other segments of the compounds of the invention, and the end groups may be further functionalized with additional chemical moieties.

[0055] “Telodendrimer” refers to a dendrimer containing a hydrophilic PEG segment and one or more chemical moieties covalently bonded to one or more end groups of the dendrimer. These moieties can include, but are not limited to, hydrophobic groups, hydrophilic groups, amphiphilic compounds, and organic moieties. Different moieties may be selectively installed at a desired end group using orthogonal protecting group strategies.

[0056] “Cholic acid” refers to (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 12S, 13R, 14S, 17R)-3, 7, 12-trihydroxy- 10, 13-dimethylhexadecahydro- 1 H- cyclopenta[a]phenanthren- 17- yl)pentanoic acid. Cholic acid is also known as 3 a, 7a, 12a- trihydroxy-5 b-cholanoic acid; 3- a,7-a, 12-a-Trihydroxy-5-cholan-24-oic acid; 17-b-(1 - methyl-3 -carboxypropy l)etiocholane- 3a, 7a, 12a-triol; cholalic acid; and cholalin. Cholic acid derivatives and analogs, such as but not limited to, allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, chenodeoxycholic acid, are also useful in the present invention. Cholic acid derivatives can be designed to modulate the properties of the nanocarriers resulting from telodendrimer assembly, such as micelle stability and membrane activity. For example, the cholic acid derivatives can have hydrophilic faces that are modified with one or more glycerol groups, aminopropanediol groups, or other groups.

[0057] “Porphyrin” refers to any compound, with the following porphin core: wherein the porphin core can be substituted or unsubstituted. Examples of porphyrins useful in the present include, but are not limited to, pyropheophorbide-a, pheophorbide-a, chlorin e6, purpurin, and purpurinimide

[0058] “Linker” refers to a chemical moiety that links one segment of a dendrimer conjugate to another. The types of bonds used to link the linker to the segments of the dendrimers include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas. One of skill in the art will appreciate that other types of bonds are useful in the present invention.

[0059] “Diamino carboxylic acid” refers to a compound which comprises two amine functional groups and at least one carboxyl functional group.

[0060] “Bis(hydroxymethyl)propionic acid” refers to a compound which has a propionic acid backbone, with two hydroxyl methyl substituents on the second carbon.

[0061] “Administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0062] “Subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.

[0063] “Effective or sufficient amount or dose” refer to a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0064] “Detecting a disease” refers to identifying or discovering the presence of a disease, using techniques such as, but not limited to imaging methods, immunoassays, and nucleic acid detection assays. Imaging methods useful in the present invention include, but is not limited to MRI and fluorescence microscopy.

[0065] “Subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

III. COMPOUNDS OF FORMULA II

[0066] In some embodiments, the present invention provides a compound with the following structure of Formula (II): wherein: each L’ is a linker comprising boron; PEG polymer has a molecular weight of 1-100 kDa; each R’ is a porphyrin; X ¹ is a diamino carboxylic acid; and each X ² is independently a bis(hydroxymethyl)propionic acid derivative.

[0067] The L’ linker can be any suitable boron comprising linker known by one of skill in the art. In some embodiments, each L’ is independently phenylboranes, carboxyphenylboranes, 3-carboxy-5-nitrophenylboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 4-

(hydroxymethyl)phenylboronic acid, 5-bromo-3-carboxyphenylboronic acid, 2-chloro-4- carboxyphenylboronic acid, 2-chloro-5-carboxyphenylboronic acid, 2-methoxy-5- carboxyphenylboronic acid, 2-carboxy-5-pyridineboronic acid, 6-carboxy-2-fluoropyridine-3- boronic acid, 5-carboxy-2-fluoropyridine-3-boronic acid, 4-carboxy-3-fluorophenylboronic acid, 4-(bromomethyl)phenylboronic acid, or derivatives thereof. In some embodiments, each L’ is a linker with the following structure:

[0068] The PEG polymer can be any suitable molecular weight known by one of skill in the art. In some embodiments, the PEG polymer has a molecular weight of 1-100 kDa. In some embodiments, the PEG polymer has a molecular weight of 1 to 50 kDa. In some embodiments, the PEG polymer has a molecular weight of 1 to 25 kDa. In some embodiments, the PEG polymer has a molecular weight of 1 to 10 kDa. In some embodiments, the PEG polymer has a molecular weight of about 5kDa. In some embodiments, the PEG polymer is PEG ^5k .

[0069] Any suitable porphyrin can be used for the compound of the present invention. In some embodiments, the porphyrin is protoporphyrin IX, octaethylporphyrin, tetraphenyl porphyrin, pyropheophorbide-a, pheophorbide-a, chlorin e6, purpurin and purpurinimide. In some embodiments, the porphyrin is pyropheophorbide-a, pheophorbide-a, chlorin e6, purpurin and purpurinimide. In some embodiments, the porphyrin is pheophorbide-a.

[0070] In some embodiments, X ¹ is diamino carboxylic acid. Any suitable diamino carboxylic acid can be used for the compound of the present invention. In some embodiments, the diamino carboxylic acid can be 2,3 -diamino propanoic acid, 2,4- diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine, 3-amino-2-aminomethyl propanoic acid, 3-amino-2- aminomethyl-2-methyl propanoic acid, 4-amino-2-(2-aminoethyl) butyric acid or 5-amino-2- (3-aminopropyl) pentanoic acid. In some embodiments, the diamino carboxylic acid is lysine. In some embodiments X ¹ is lysine.

[0071] In some embodiments, each X ² is independently a bis(hydroxymethyl)propionic acid derivative. Any suitable bis(hydroxymethyl)propionic acid derivative can be used for the present invention. The bis(hydroxymethyl)propionic acid derivative can be substituted at the hydroxyl and or carboxyl groups with other functional groups such as carboxylic acids, amines, amides, peptides, and halogens. In some embodiments, the bis(hydroxymethyl)propionic acid derivative is 2,2- bis(hydroxymethyl)propionic acid, 2,2- bis(hydroxymethyl)propionic acid substituted with carboxylic acids, or 2,2- bis(hydroxymethyl)propionic acid substituted with peptides. In some embodiments, the 2,2- bis(hydroxymethyl)propionic acid substituted with carboxylic acids. In some embodiments, the 2,2- bis(hydroxymethyl)propionic acid is substituted with two 2,2- bis(hydroxymethyl)propionic acid moieties. In some embodiments, X ² is a bis(hydroxymethyl)propionic acid derivative with the following structure:

[0072] In some embodiments, the present invention provides a compound of Formula II wherein: each L’ is a linker with the following structure:

PEG is PEG ^5k ; each R’ is a pheophorbide-a; X ¹ is lysine; and each X ² is a bis(hydroxymethyl)propionic acid derivative with the following structure:

IV. NANOCARRIERS

[0073] In some embodiments, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising: a plurality of amphiphilic compounds that self-assemble to form the nanocarrier; and a first magnetic resonance imaging (MRI) contrast agent and a second MRI contrast agent in the interior; wherein one MRI contrast agent is paramagnetic, and the other MRI contrast agent is superparamagnetic, and the first

MRI contrast agent and the second MRI contrast agent are configured within the interior to substantially cancel the magnetic signal of each contrast agent.

[0074] Paramagnetic MRI contrast agents useful in the present invention include any suitable paramagnetic agent known by one of skill in the art. In some embodiments, the paramagnetic agent is Copper-Gad (CG), dysprosium oxide nanoparticles, gadopentetate dimeglumine, gadoterate meglumine, gadodiamide, gadoteridol, gadobutrol, gadofoveset, gadoversetamide, gadobenate dimeglumine, gadoxetic acid disodium, gadophostriamine trisodium, Mn ²⁺ chelated pheophorbide a, Mn ²⁺ dipyridoxal diphosphate (DPDP), Mn ²⁺ diethylene triamine pentaacetic acid (DTP A), or Mn ²⁺ ethylenediamine tetraacetic acid (EDTA).

[0075] Superparamagnetic MRI contrasting agents useful in the present invention include any suitable superparamangnetic agent known by one of skill in the art. In some embodiments, the superparamagnetic agent is cobalt nanoparticles, ferromagnetic nanoparticles, ferromagnetic nanoparticles, superparamagnetic iron oxide (SPIO), or a superparamagnetic iron platinum particle (SIPP). [0076] When the first and second MRI contrast agents are configured within the interior of the nanocarrier, the magnetic signals of each contrast agent are substantially canceled. “Substantially canceled magnetic signal” refers to the minimizing the magnetic dipole moments of the two contrast agents to zero or substantially close to zero, and can occur when two contrast agents are in close proximity, such as encapsulated in a nanoparticle. The proximity of the two contrast agents can affect each other’s dipole moment to cancel out the signal. “Substantially canceled” refers to having a dipole moment of about 20% or less of the original dipole moment with the contrast agents are not affected by the magnetic field of other contrast agents. In some embodiments, the substantially canceled magnetic signal is measured by the relaxivity time described below.

[0077] In some embodiments, the first MRI contrast agent is a Ti contrast agent. A T i contrast agent measures the T i spin-lattice relaxation time for MRI as known by one of skill in the art. In some embodiments, the T i agent is a paramagnetic contrast agent.

[0078] Any suitable paramagnetic contrasting agent can be useful for the present invention. In some embodiments, the paramagnetic contrasting agent comprises Gd ³⁺ , Dy ³⁺ , or Mn ²⁺ . In some embodiments, the paramagnetic contrasting agent further comprises a chelator. Any suitable chelator can be used for the present invention. In some embodiments, the chelator is porphyrin, ethylenediamine tetraacetic acid (EDTA), 1,4,7,10-Tetraazacyclododecane-

1.4.7.10-tetraacetic acid (DOTA), diethylenetriaminepentacetate (DTP A), ethoxybenzyl diethylenetriamine pentaacetic acid (EOB-DTPA), 2-[bis[2-(carboxylatomethyl- (methylcarbamoylmethyl)amino)ethyl] amino] acetate (DTPA-BMA), 8,11- bis(carboxymethyl)- 14-[2-[(2-methoxyethyl)amno]-2-oxoethyl]-6-oxo-2-oxa-5, 8,11,14- tetraazahexadecan-16-oato(3-) (DTPA-BMEA), dipyridoxal diphosphate (DPDP), DTPA- DPDP, 4-carboxy-8,l l-bis(carboxylatomethyl)-5-(carboxymethyl)-l-phenyl-2-oxa-5, 8,l 1- triazatridecan- 13-oate (BOPTA), 10-[( lSR,2RS)-2, 3-dihydroxy- 1 -hydroxymethylpropyl]-

1.4.7.10-tetraazacyclodecane-l,4,7-triacetic acid (BT-D03A), and 1,4,7-triscarboxymethyl-

1.4.7.10-tetraazacyclododecane (HP-D03 A).

[0079] In some embodiments, the first MRI contrast agent comprises Gd ³⁺ or Mn ²⁺ . In some embodiments, the first MRI contrast agent is gadopentetate dimeglumine, gadoterate meglumine, gadodiamide, gadoteridol, gadobutrol, gadoversetamide, gadobenate dimeglumine, gadoxetic acid disodium, gadophostriamine trisodium, Mn ²⁺ chelated pheophorbide a, Mn ²⁺ dipyridoxal diphosphate (DPDP), Mn ²⁺ diethylene triamine pentaacetic acid (DTP A), or Mn ²⁺ ethylenediamine tetraacetic acid (EDTA). In some embodiments, the first MRI contrast agent is Mn ²⁺ chelated pheophorbide a.

[0080] In some embodiments, the second MRI contrast agent is a T2 contrast agent. A T2 contrast agent measures the T2 spin-spin relaxation time for MRI as known by one of skill in the art. In some embodiments, the T2 contrast agent is a superparamagnetic agent.

[0081] Superparamagnetic agents useful in the present invention include any suitable superparamagnetic agent known by one of skill in the art. In some embodiments, the superparamagnetic agent comprises cobalt nanoparticles, ferromagnetic nanoparticles, ferromagnetic nanoparticles, superparamagnetic iron oxide (SPIO), or a superparamagnetic iron platinum particle (SIPP).

[0082] In some embodiments, the second MRI contrast agent comprises superparamagnetic iron oxide (SPIO) or a superparamagnetic iron platinum particle (SIPP). In some embodiments, the second MRI contrast agent is superparamagnetic iron oxide (SPIO).

[0083] Amphiphilic compounds can be any suitable amphiphilic compound known by one of skill in the art. In some embodiments, each amphiphilic compound is a protein, a phospholipid, or a dendrimer. In some embodiments, each amphiphilic compound is a phospholipid, or a dendrimer. Dendrimers are branched conjugates with repetitive end branches. The end branches of the dendrimer can comprise cholic acids, porphyrins, or derivatives thereof. [0084] In some embodiments, each amphiphilic compound is a dendrimer comprising at least two cholic acids, a dendrimer comprising at least two porphyrins, a phospholipid, or derivatives thereof that have both a hydrophobic face and a hydrophilic face, and the dendrimers or phospholipid self-assemble in an aqueous solvent to form the nanocarrier such that a hydrophobic pocket is formed in the interior of the nanocarrier by the orientation of the hydrophobic face of each cholic acid, porphyrin, phospholipid, or derivative thereof towards each other. In some embodiments, the amphiphilic compound comprises a polyethylene glycol (PEG) polymer.

[0085] In some embodiments, the amphiphilic compound is a dendrimer. In some embodiments, each amphiphilic compound has the structure of Formula I: wherein: each L is a linker; PEG polymer has a molecular weight of 1-100 kDa; each R is cholic acid or derivative thereof; each X is a diaminocarboxylic acid; and each Y ² is a thiol bearing group. [0086] Polyethylene glycol (PEG) polymers of any size and architecture are useful in the present invention. In some embodiments, PEG has a molecular weight of 1 - 100 kDa. In some embodiments, PEG has a molecular weight of 1-50 kDa. In some embodiments, PEG has a molecular weight of 1 -20 kDa. In some embodiments, PEG has a molecular weight of 1 - 10 kDa. In some embodiments, PEG has a molecular weight of about 10 kDa, about 9 kDa, about 8 kDa, about 7 kDa, about 6 kDa, about 5 kDa, about 4 kDa, about 3 kDa, about 2 kDa, or about 1 kDa. In some embodiments, PEG polymer is PEG ^5k . One of skill in the art will appreciate that other PEG polymers and other hydrophilic polymers are useful in the present invention. PEG can be any suitable length.

[0087] R can be any suitable cholic acid or cholic acid derivative as known by one of skill in the art. Cholic acid derivatives and analogs include, but are not limited to, allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, and chenodeoxycholic acid. Cholic acid derivatives can be designed to modulate the properties of the nanocarriers resulting from telodendrimer assembly, such as micelle stability and membrane activity. For example, the cholic acid derivatives can have hydrophilic faces that are modified with one or more glycerol groups, aminopropanediol groups, or other groups.

[0088] In some embodiments, each R is independently taurocholic acid, taurochenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, cholic acid, allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, or chenodeoxycholic acid. In some embodiments, each R is independently cholic acid, allocholic acid pythocholic acid, avicholic acid deoxycholic acid, or chenodeoxycholic acid in some embodiments, each R is cholic acid. [0089] In some embodiments, L is a linker. The linker can be any suitable linker known by one of skill in the art. In some embodiments, the linker is a Ci-20 alkylene, C2-20 alkenylene, C2-20 alkynylene, a PEG polymer, peptide, or an Ebes linker. In some embodiments, each L is a Peg polymer or Ebes linker. In some embodiments, each L is a linker Ebes having the formula:

[0090] X can be any suitable diamino carboxylic acid as listed above. In some embodiments, the diamino carboxylic acid can be 2,3 -diamino propanoic acid, 2,4- diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine, 3-amino-2-aminomethyl propanoic acid, 3-amino-2- aminomethyl-2-methyl propanoic acid, 4-amino-2-(2-aminoethyl) butyric acid or 5-amino-2- (3-aminopropyl) pentanoic acid. In some embodiments X is lysine.

[0091] Alternatively, X can be any suitable adihydroxy carboxylic acid or hydroxyl amino carboxylic acid as known by one of skill in the art. In some embodiments, each dihydroxy carboxylic acid can be glyceric acid, 2,4-dihydroxybutyric acid, 2,2-

Bis(hydroxymethyl)propionic acid, 2,2-Bis(hydroxymethyl)butyric acid, serine or threonine. In some embodiments, each hydroxyl amino carboxylic acid can be serine or homoserine.

[0092] In some embodiments, Y ² is a thiol-bearing group. The thiol-bearing group can be any suitable thiol group known by one of skill in the art. In some embodiments, Y ² is a peptide comprising a thiol-containing group. In some embodiments, Y ² is glutathione, dithiothreitol, dithioerythritol, 3-mercaptopropane-l,2-diol, 2-mercapto- 1 -propanesulfonic acid, dimercaptosuccinic acid, methionine or cysteine. In some embodiments, Y ² is glutathione, dimercaptosuccinic acid, methionine or cysteine. In some embodiments, Y ² is methionine or cysteine. In some embodiments, Y ² is cysteine. [0093] In some embodiments, the present invention provides a nanocarrier comprising a plurality of amphilic compounds, wherein each amphiphilic compound has the structure of Formula (I), wherein: each L is a linker Ebes, PEG polymer is PEG ^5k , each R is cholic acid, each X is lysine, and each Y ² is cysteine; and comprising the first magnetic resonance imaging (MRI) contrast agent and the second MRI contrast agent in the interior; wherein the first MRI contrast agent is Mn ²⁺ chelated pheophorbide a, and the second MRI contrast agent is superparamagnetic iron oxide (SPIO), and the first MRI contrast agent and the second MRI contrast agent are configured within the interior to substantially cancel the magnetic signal of each contrast agent.

[0094] Each amphiphilic compound can also be any compound of Formula (II) described in Section III. In some embodiments, each amphiphilic compound has the structure of Formula (II):

R’

L' R'

X ² -|_'

PEG— X. ¹ 1

X ² - L' R'

R' (P) wherein: each L’ is a linker comprising boron; PEG polymer has a molecular weight of 1-100 kDa; each R’ is a porphyrin; X ¹ is a diamino carboxylic acid; and each X ² is independently a bis(hydroxymethyl)propionic acid derivative.

[0095] Each amphilic compound of Formula II can be any suitable compound as described in the section above. For example L’ can be any suitable boron linker described above. In some embodiments, each L’ is a linker with the following structure: [0096] PEG can be any suitable molecular weight as described above. In some embodiments, the PEG polymer is PEG ^5k .

[0097] The porphyrin of the present invention can be any suitable porphryin as described above. In some embodiments, the porphyrin selected from the group consisting of pyropheophorbide-a, pheophorbide-a, chlorin e6, purpurin and purpurinimide. In some embodiments, the porphyrin is pheophorbide-a.

[0098] X ¹ can be any suitable diamino carboxylic acid as described above. In some embodiments, X ¹ is lysine.

[0099] X ² can be any suitable bis(hydroxymethyl)propionic acid derivative as described above. In some embodiments, X ² is a bis(hydroxymethyl)propionic acid derivative with the following structure:

[0100] In some embodiments, the present invention provides a nanocarrier comprising a plurality of amphiphilic compounds, wherein each amphiphilic compound has the structure of Formula (II) wherein: each L’ is a linker with the following structure:

PEG is PEG ^5k ; each R’ is a pheophorbide-a; X ¹ is lysine; and each X ² is a bis(hydroxymethyl)propionic acid derivative with the following structure: wherein the first MRI contrast agent is Mn ²⁺ chelated pheophorbide a, and the second MRI contrast agent is superparamagnetic iron oxide (SPIO).

[0101] In some embodiments, the amphiphilic compound is a phospholipid or a derivative thereof. In some embodiments, each amphiphilic compound is glycerophospholipids, POPC (l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DMPE (l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine), DPPC (l,2-Dipalmitoyl-sn-glycero-3-phosphocholine), DPPE ( 1 ,2- Dip almitoy 1- sn-gly cero- 3 -pho spho ethano lamine) , DSPC (l,2-distearoyl-sn-glycero-3- phosphocholine), DSPE (l,2-distearoyl-sn-glycero-3-phosphoethanolamine), or derivative thereof. The phospholipids of the present invention can be substituted with other polymers such as PEG.

[0102] In some embodiments, each amphiphilic compound is DSPE or a derivative thereof. In some embodiments, each amphilic compound is DSPE-PEG. PEG can have any suitable molecular weight as described above. In some embodiments, each amphilic compound is DSPE-PEG ^5k , DSPE-PEG ^2k , DSPE-PEG ^lk or a combination thereof. In some embodiments, each amphiphilic compound is DSPE-PEG ^2k .

[0103] In some embodiments, the nanocarrier comprises a plurality of amphiphilic compounds wherein each amphiphilic compound is DSPE-PEG, and wherein the first MRI contrast agent comprises Mn ²⁺ , and the second MRI contrast agent comprises superparamagnetic iron oxide (SPIO) or a superparamagnetic iron platinum particle (SIPP).

In some embodiments, each amphiphilic compound is DSPE-PEG ^2k , and wherein the first MRI contrast agent is Mn ²⁺ chelated pheophorbide a, and the second MRI contrast agent is superparamagnetic iron oxide (SPIO).

[0104] In some embodiments, upon exposure to a stimulus, the nanocarrier disassembles to release the first MRI contrast agent and the second MRI contrast agent. The stimulus can be any suitable stimulus known by one of skill in the art. In some embodiments, the stimulus is a change in pH, a reducing agent, or an enzyme.

[0105] In some embodiments, the stimulus is a change in pH. In some embodiments, the stimulus is a reduction of pH. In some embodiments, the pH is reduced to less than 6.5. In some embodiments, the pH is reduced to about 5.5.

[0106] In some embodiments, the stimulus is a reducing agent. In some embodiments, the reducing agent is glutathione, 2-mercaptoethanol, Dithiothreitol (DTT), bis(2- mercaptoethyl)sulfone (BMS) or [N,N’-dimethyl-N,N’-bis(mercaptoacetyl)hydrazine (DMH). In some embodiments, the reducing agent is glutathione.

[0107] In some embodiments, the stimulus is an enzyme. In some embodiments, the enzyme is a gamma-interferon-inducible lysosomal thiol reductase, thioredoxin, protease, MMP-2, MMP-9, caspase-3, caspase-7, or cathepsin B. In some embodiments, the enzyme is a protease, MMP-2, MMP-9, caspase-3, caspase-7, or cathepsin B.

[0108] The relaxivity of the contrast agents can have any suitable relaxivity, and is measured after exposure of the nanocarrier to the stimulus to release the contrast agents. Relaxivity is measured in mM^sec ¹ and is measured for each contrast agent. The Ri relaxivity refers to the first contrast agent, and the R2 relaxivity refers to the second contrast agent.

[0109] In some embodiments, upon exposure to the stimulus, the Ri relaxivity of the contrast agents increases from about 0.2 to about 50 mM^sec ¹ after 24 hours. In some embodiments, the Ri relaxivity of the contrast agents increases from about 0.2 to about 10 mM^sec ¹ after 24 hours. In some embodiments, the Ri relaxivity of the contrast agents increases from about 0.2 to about 5 mM^sec ¹ after 24 hours. In some embodiments, the Ri relaxivity of the contrast agents increases from about 0.6 to about 2.5 mM^sec ¹ after 24 hours. In some embodiments, upon exposure to the stimulus, the Ri relaxivity of the contrast agents increases from about 10% to about 500% after 24 hours. In some embodiments, the Ri relaxivity of the contrast agents increases from about 20% to about 400% after 24 hours. In some embodiments, the Ri relaxivity of the contrast agents increases from about 20% to about 300% after 24 hours. In some embodiments, the Ri relaxivity of the contrast agents increases from about 50% to about 200% after 24 hours.

[0110] In some embodiments, upon exposure to the stimulus, the R2 relaxivity of the contrast agents increases from about 2 to about 150 mM^sec ¹ after 24 hours. In some embodiments, the R2 relaxivity of the contrast agents increases from about 2 to about 100 mM^sec ¹ after 24 hours. In some embodiments, the R2 relaxivity of the contrast agents increases from about 2 to about 80 mM^sec ¹ after 24 hours. In some embodiments, R2 relaxivity of the contrast agents increases from about 12 to about 60 mM^sec ¹ after 24 hours. In some embodiments, upon exposure to the stimulus, the R2 relaxivity of the contrast agents increases from about 20% to about 1000% after 24 hours. In some embodiments, the R2 relaxivity of the contrast agents increases from about 20% to about 700% after 24 hours. In some embodiments, the R2 relaxivity of the contrast agents increases from about 50% to about 500% after 24 hours. In some embodiments, the R2 relaxivity of the contrast agents increases from about 100% to about 500% after 24 hours.

V. METHODS

[0111] In some embodiments, the present invention provides a method of imaging, comprising: administering to a subject to be imaged an effective amount of a nanocarrier of the present invention, wherein, upon exposure to a stimulus, the nanocarrier disassembles such that the first MRI contrast agent and the second MRI contrast agent are released; and detecting the first MRI contrast agent and the second MRI contrast agent.

[0112] In some embodiments, upon exposure to a stimulus, the nanocarrier disassembles to release the first MRI contrast agent and the second MRI contrast agent. The stimulus can be any suitable stimulus known by one of skill in the art. In some embodiments, the stimulus is a change in pH, a reducing agent, or an enzyme.

[0113] In some embodiments, the stimulus is a change in pH. In some embodiments, the stimulus is a reduction of pH. In some embodiments, the pH is reduced to less than 6.5. In some embodiments, the pH is reduced to about 5.5. In some embodiments, the stimulus is a reducing agent. In some embodiments, the reducing agent is glutathione, 2-mercaptoethanol, Dithiothreitol (DTT), bis(2-mercaptoethyl)sulfone (BMS) or [N,N’-dimethyl-N,N’- bis(mercaptoacetyl)hydrazine (DMH). In some embodiments, the reducing agent is glutathione. In some embodiments, the stimulus is an enzyme. In some embodiments, the enzyme is a gamma-interferon-inducible lysosomal thiol reductase, thioredoxin, protease, MMP-2, MMP-9, caspase-3, caspase-7, or cathepsin B. In some embodiments, the enzyme is a protease, MMP-2, MMP-9, caspase-3, caspase-7, or cathepsin B.

[0114] The imaging techniques useful in the present invention are any suitable techniques known by one of skill in the art. In some embodiments, the imaging technique is positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), x-ray computed tomography (CT), echocardiography, or functional near-infrared spectroscopy. In some embodiments, the imaging technique is MRI. In some embodiments, the imaging technique is MRI and further comprises dual-contrast enhanced subtraction imaging. In some embodiments of imaging comprises a dual-contrast enhanced subtraction imaging (DESI) method). [0115] Contrast agents are useful for the method of imaging of the present invention. Any suitable contrasting agent can be used as described above. In some embodiments, the contrast agents can be paramagnetic or superparamagnetic. Paramagnetic agents imaging agents that are magnetic under an externally applied field. Examples of paramagnetic compounds useful in the present invention includes, but is not limited to, Gd ³⁺ , Mn ²⁺ , Fe, and Fe ³⁺ . Examples of superparamagnetic compounds include but are not limited to superparamagnetic iron oxide (SPIO) and superparamagnetic iron platinum particle (SIPP). Other contrast agents useful in the present invention include, but are not limited to, ³ H, ⁿ C, ¹³ N, ¹⁸ F, ¹⁹ F, ⁶⁰ Co, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, ⁸² Rb, ⁹⁰ Sr, ⁹⁰ Y, "Tc, ^99m Tc, ^m In, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I, ¹³⁷ Cs, ¹⁷⁷ Fu, ¹⁸⁶ Re, ¹⁸⁸ Re,

²¹¹ At, Rn, Ra, Th, U, Pu and ²⁴¹ Am.

[0116] Detecting the first and second MRI contrasting agent can be performed at any suitable time know by one of skill in the art. In some embodiments, the detecting of the first MRI contrast agent and the second MRI contrast agent occurs at least about 6 hours to about 36 hours after administering the nanocarrier. In some embodiments, the detecting of the first MRI contrast agent and the second MRI contrast agent occurs at least about 10 hours to about 24 hours after administering the nanocarrier. In some embodiments, the detecting of the first MRI contrast agent and the second MRI contrast agent occurs at least about 12 hours to about 24 hours after administering the nanocarrier.

[0117] The imaging method of the present invention is useful for detecting and imaging a higher tumor-to-normal tissue ratio (TNR) compared to traditional methods. High TNR is useful in determining early detection of diseases and the two-way magnetic resonance tuning (TMRET) technology in combination with dual-contrast enhanced subtraction imaging provides new opportunities for molecular diagnostics and image-guided biomedical applications. Previous methods can have low contrast enhancement with high background noise from normal tissue, which may result in lower tumor-to-normal tissue ratio, making it difficult for detection. TNR can be measured by imaging techniques such as MRI, DESI, or a combination thereof. For example, the TNR can be measured by the MRI signal intensity or relaxation rate (Ri and R2) at the tumor site divided by the corresponding MRI signal intensity or relaxation rate in normal tissue. In some embodiments, detecting the first MRI contrast agent and the second MRI contrast agent shows a tumor-to-normal tissue ratio greater than 4. In some embodiments, detecting the first MRI contrast agent and the second MRI contrast agent shows a tumor-to-normal tissue ratio greater than 6. In some embodiments, detecting the first MRI contrast agent and the second MRI contrast agent shows a tumor-to-normal tissue ratio greater than 10. In some embodiments, the tumor- to- normal tissue ratio about 4 to 20. In some embodiments, the tumor-to-normal tissue ratio about 6 to 10.

[0118] In some embodiments, the present invention provides a method of detecting a disease, comprising: administering to a subject an effective amount of a nanocarrier of the present invention, wherein, upon exposure to a stimulus, the nanocarrier disassembles such that the first MRI contrast agent and the second MRI contrast agent are released, and detecting the first MRI contrast agent and the second MRI contrast agent, thereby detecting the disease in the subject.

[0119] In some embodiments, the disease is a solid tumor, cancer, inflammation, infection, autoimmune disorders, immunodeficiency, or allergies. In some embodiments, the disease is a solid tumor or inflammation.

[0120] In some embodiments, the disease is a solid tumor. In some embodiments, the solid tumor is sarcomas, carcinomas, and lymphomas. In some embodiments, the solid tumor is basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, adenocarcinoma, Hodgkin’s lymphoma, Burkitt’s lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, multiple myelomas, brain cancer, gastric cancer, bladder cancer, melanoma, pancreatic cancer, prostate cancer, breast cancer, lung cancer, liver cancer, spleen cancer, or kidney cancer. In some embodiments, the solid tumor is brain cancer, gastric cancer, bladder cancer, melanoma, pancreatic cancer, prostate cancer, breast cancer, lung cancer, liver cancer, spleen cancer, or kidney cancer. In some embodiments, the solid tumor is brain cancer or prostate cancer.

[0121] In some embodiments, the disease is an inflammation. In some embodiments, the inflammation is acute inflammation, chronic inflammation, appendicitis, dermatitis, pancreatitis, prostatitis, pharyngitis, gastritis, nephritis, enteritis, encephalitis or arthritis. In some embodiments, the inflammation is gastritis, nephritis, enteritis, encephalitis or arthritis. I. FORMULATIONS

[0122] The nanocarriers of the present invention can be prepared in a wide variety of oral, parenteral and topical dosage forms. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. The nanocarrier of the present invention can also be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compositions described herein can be administered by inhalation, for example, intranasally. Additionally, the compositions of the present invention can be administered transdermally. The compositions of this invention can also be administered by intraocular, intravaginal, and intrarectal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see Rohatagi, ./. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75: 107-111, 1995). Accordingly, the present invention also provides pharmaceutical compositions including a pharmaceutically acceptable carrier or excipient and the nanocarrier of the present invention.

[0123] For preparing formulations from the nanocarriers of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0124] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5% or 10% to 70% of the nanocarier the present invention.

[0125] Suitable solid excipients include, but are not limited to, magnesium carbonate; magnesium stearate; talc; pectin; dextrin; starch; tragacanth; a low melting wax; cocoa butter; carbohydrates; sugars including, but not limited to, lactose, sucrose, mannitol, or sorbitol, starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins including, but not limited to, gelatin and collagen.

If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[0126] Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active nanocarrier (i.e., dosage). Pharmaceutical preparations of the invention can also be used orally using, for example, push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push- fit capsules can contain the nanocarrier of the present invention mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the nanocarrier of the present invention may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0127] For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the nanocarrier of the present invention is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

[0128] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0129] Aqueous solutions suitable for oral use can be prepared by adding he nanocarrier of the present invention in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

[0130] Also included are solid form preparations, which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0131] Oil suspensions can be formulated by suspending the nanocarrier of the present invention in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical formulations of the invention can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono- oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.

[0132] The nanocarriers of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be formulated for administration via intradermal injection of drug- containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routes afford constant delivery for weeks or months.

[0133] In another embodiment, the nanocarriers of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a body cavity or lumen of an organ. The formulations for administration will commonly comprise a solution of the nanocarriers of the present invention dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the nanocarriers of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol.

[0134] In another embodiment, the formulations of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the nanocarriers of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995;

Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).

II. ADMINISTRATION

[0135] The nanocarriers of the present invention can be delivered by any suitable means, including oral, parenteral and topical methods. Transdermal administration methods, by a topical route, can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0136] The preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the nanocarriers of the present invention. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0137] The nanocarrier of the present invention can be present in any suitable amount, and can depend on various factors including, but not limited to, weight and age of the subject, state of the disease, etc. Suitable dosage ranges for the nanocarrier of the present invention include from about 0.1 mg to about 10,000 mg, or about 1 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable dosages for the nanocarrier of the present invention include about 1 mg, 5, 10, 20,

30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg.

[0138] The nanocarrier of the present invention can be administered at any suitable frequency, interval and duration. For example, the nanocarrier of the present invention can be administered once an hour, or two, three or more times an hour, once a day, or two, three, or more times per day, or once every 2, 3, 4, 5, 6, or 7 days, so as to provide the preferred dosage level. When the nanocarrier of the present invention is administered more than once a day, representative intervals include 5, 10, 15, 20, 30, 45 and 60 minutes, as well as 1, 2, 4,

6, 8, 10, 12, 16, 20, and 24 hours. The nanocarrier of the present invention can be administered once, twice, or three or more times, for an hour, for 1 to 6 hours, for 1 to 12 hours, for 1 to 24 hours, for 6 to 12 hours, for 12 to 24 hours, for a single day, for 1 to 7 days, for a single week, for 1 to 4 weeks, for a month, for 1 to 12 months, for a year or more, or even indefinitely.

[0139] The nanocarrier can also contain other compatible therapeutic agents. The nanocarriers described herein can be used in combination with one another, with other active agents known to be useful in modulating a glucocorticoid receptor, or with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent.

[0140] The nanocarrier of the present invention can be co-administered with another active agent. Co-administration includes administering the nanocarrier of the present invention and an active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of each other. Co administration also includes administering the nanocarrier of the present invention and an active agent simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15,

20, or 30 minutes of each other), or sequentially in any order. Moreover, the nanocarrier of the present invention and the active agent can each be administered once a day, or two, three, or more times per day so as to provide the preferred dosage level per day.

[0141] In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single formulation including both the nanocarrier of the present invention and the active agent. In other embodiments, the nanocarrier of the present invention and the active agent can be formulated separately.

[0142] The nanocarrier of the present invention and the active agent can be present in the formulations of the present invention in any suitable weight ratio, such as from about 1 : 100 to about 100:1 (w/w), or about 1:50 to about 50:1, or about 1:25 to about 25:1, or about 1:10 to about 10:1, or about 1 :5 to about 5:1 (w/w). The nanocarrier of the present invention and the other active agent can be present in any suitable weight ratio, such as about 1:100 (w/w),

1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1 or 100:1 (w/w). Other dosages and dosage ratios of the nanocarrier of the present invention and the active agent are suitable in the formulations and methods of the present invention.

III. EXAMPLES

[0143] Materials. Unless otherwise described, the solvents and chemicals were purchased from commercial sources and used without purification. The pheophorbide a (Catalog no. sc- 264070B) was purchased from Santa Cruz Biotechnology, Inc. The SPIO (Catalog no. 700320-5mL) and Manganese (II) chloride (Catalog no. 244589-10G) were purchased from MilliporeSigma. The DSPE-PEG2000 (N-(carbonyl-methoxypolyethylene glycol 2000)- 1,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt) was purchased from Laysan Bio Inc. All other chemicals were purchased from MilliporeSigma.

Example 1. Compounds of Formula I [0144] Synthesis of thiolated telodendrimers. The synthesis of thiolated telodendrimers for making disulfide crosslinked micelles was well-established, and detailed synthesis procedures can be found in previous publications (Li, Y. et al, Nature Communications 5, 4712 (2014); Kato, J. et al, Molecular Pharmaceutics 9, 1727-1735 (2012); Xiao, K. et al, Biomaterials 67, 183-193 (2015); Li, Y. et al., Biomaterials 32, 6633-6645 (2011)). Example 2. Compounds of Formula II

[0145] Synthesis of PEG-OHs-PPBA (POP) telodendrimer - Synthesis of PEG-OHs.

Synthesis of acetonide protected bis-MPA. 2 g of 2,2-Bis(hydroxymethyl)propionic acid (bis- MPA) (15 mmol), 280 pL of acetone dimethyl acetal (22.4 mmol) and 142 mg of p- toluenesulfonic acid (0.746 mmol) were dissolved in 10 mL of acetone in a 25 mL flask and the mixture was stirred for 6 h at room temperature. Then, 120 pL of triethylamine (0.88 mmol) was added to neutralize the residual acid. A white crystalline product was obtained after the solvent was moved by rotavap.

[0146] Synthesis of chlorination of acetonide-2,2-bis(methoxy) propanoic anhydride. The acetonide-2,2-bis(methoxy) propanoic anhydride obtained as described above was chlorinated by refluxing in thionyl chloride. Chlorination of acetonide-2,2-bis(methoxy) propanoic anhydride was obtained as a colorless viscous liquid and used in the subsequent reaction immediately.

[0147] Synthesis of PEG-K. 500 mg of CH3O-PEG5000-NH2, 68 mg 6-Chloro-l- hydroxybenzotriazole (6-Cl-HOBT), 236 mg Fmoc-Lys (Fmoc)-OH and 62 pL diisopropylcarbodiimide (DIC) were dissolved in 5 mL DMF in a 10 mL flask and the mixture was stirred for 24 h at room temperature. Then, the mixture was precipitated in diethyl ether and the precipitate was dried under vacuum at room temperature for 24 h to obtain PEG-K-(Fmoc)2. Finally, 5 mL of piperidine (20% in DMF) was used to deprotect the Fmoc to get PEG-K with washing by ether three times. [0148] Synthesis of PEG-K-bis-MPA. PEG-K-(Fmoc)2 (200 mg, 0.039 mmol) was dissolved in 2 mL of dry DMF and stirred for 20 min. Acetonide-2,2-bis(methoxy) propanoic anhydride (Ac-MAP 54.34 mg, 0.312 mmol), DIC (48 pF) and Cl-HOBT (52.8 mg, 0.312 mmol) were added. The mixture was stirred at room temperature for 24 h. The flask was opened, and the product filtered off and precipitated in diethyl ether. The precipitate was dried under vacuum at room temperature for 24 h to obtain PEG-K-bis-MPA. Finally, the PEG-K-bis-MPA was dissolved in the Dowex H+/methanol mixture to get the PEG-OEEt.

[0149] Synthesis of PEG-OEL. PEG-OEEt (1 g), Cl-2,2-bis(methoxy) propanoic acid (290 mg) and DMAP (0.2 equivalent mass) were dissolved in 15 mL dichloromethane (DCM).

The mixture was stirred in an ice bath for 1 h and reacted at room temperature for 24 h under N2 atmosphere protection. The product was filtered and precipitated in cold ether. The precipitate was dried under vacuum at room temperature for 24 h. Then, the reaction system was dissolved in the Dowex 11 /methanol mixture to get PEG-01 E

[0150] Synthesis of porphyrin-PBA (PPBA). Pheophorbide a (237 mg, 0.4 mmol), EDC (306.6 mg, 1.6 mmol) andNEIS (184.0 mg, 1.6 mmol) were dissolved in 4 mL of anhydrous DMF and stirred for 20 min. Then the 3-aminophenylboronic acid monohydrate (248.0 mg, 1.6 mmol) was added into the solution. The mixture was stirred at room temperature for 24 h. The product was then filtered and extracted by water/DCM (2:1) for three times. The organic phase was added with 500 mg anhydrous sodium sulfate to remove the residual moisture and evaporated to obtain PPBA by rotavap.

[0151] Preparation of PEG-OHs-PPBA (POP). 10 mg PEG- OI Is and 1 mg PPBA was dissolved in 2 mL MeOEI and 2 mL DCM mixture solution and stirred under room temperature for 30 min. The POP monomer can be formed by coupling of PEG-01 Is and PPBA through boronate ester bonds.

[0152] Synthesis of Mn ²⁺ chelated pheophorbide a (P-Mn). Based on the method described, pheophorbide a (59.3 mg, 100 pmol) and MnCb (63 mg, 500 pmol) were dissolved in a mixed solution of methanol (8 mL) and pyridine (0.8 mL). The reaction system was refluxed under 60 °C for 2 h. Free Mn ²⁺ was removed by extraction (DCM against water). The P-Mn was dissolved in organic solvent, and the free Mn ²⁺ was washed off by water. The P-Mn was then aliquoted and dried on a rotavapor. Example 3. Nanocarrier

[0153] Preparation of DCM@P-Mn-SPIO. In order to prepare DCM@P-Mn-SPIO, P- Mn, SPIO and thiolated telodendrimers (20 mg) were dissolved in tetrahydrofuran (200 pL), and dripped into 1 mL deionized water with vigorous stirring overnight at 37 °C. The ratios ofP-Mnto SPIO were tuned from 1: 0.006, 1: 0.013, 1: 0.025 and 1: 0.05. After that, 4 pL of H2O2 were added to oxidize the thiol groups to form intra-micellar disulfide cross-linkages. DCM@P-Mn with single Ti contrast and DCM@SPIO with single T2 contrast were prepared by using similar procedures with identical amounts of P-Mn or SPIO, respectively. For the following in vitro and in vivo studies, the ratio of P-Mn to SPIO in DCM@P-Mn-SPIO was fixed to 1: 0.025.

[0154] Preparation of DSPE-PEG@P-Mn-SPIO and POP@P-Mn-SPIO. The preparation of these two nanoprobes followed the same procedures to the DCM@P-Mn- SPIO. Briefly, 20 mg of DSPE-PEG or POP, P-Mn and SPIO were dissolved in tetrahydrofuran (200 pL) and dripped into 1 mL deionized water with vigorous stirring overnight at 37 °C. The ratio between P-Mn and SPIO was set as 1 :0.025.

[0155] Characterization of the TMRET probes and the control probes with single contrast agents. The TMRET probes included DCM@P-Mn-SPIO, DSPE-PEG@P-Mn- SPIO and POP@P-Mn-SPIO; the probes with single contrast agents included DCM@P-Mn, DCM@SPIO, DSPE-PEG@P-Mn, DSPE-PEG@SPIO, POP@P-Mn and POP@SPIO. The size distributions of the nanoprobes (1.0 mg/mL) were measured by a dynamic light scattering instrument (DLS, Nano ZS, Malvern). The morphology was observed by transmission electron microscopy (TEM, Talos, L120c, FEI). The samples were made by directly dripping the aqueous nanoparticle solution (1.0 mg/mL) onto copper grids, and placed at room temperature to dry naturally. The UV-vis absorbance was measured by a UV- vis photospectrometer (UV-1800, Shimadzu), and the fluorescence spectra was obtained by fluorescence photospectrometer (RF6000, Shimadzu). For the optical measurement, including UV-vis and fluorescence spectra, the cuvette width was 1 cm.

[0156] GSH concentration-related MR relaxivity changes of DCM@P-Mn-SPIO, DCM@P-Mn and DCM@SPIO. The Ri and R2 values of the DCM@P-Mn-SPIO were measured on a 7.0 T MRI Scanner (Bruker Biospec, USA) at 37 °C. Different concentrations of DCM@P-Mn-SPIO were treated with 100 pL GSH (0, 5, 10 and 20 mM) before MRI. In DCM@P-Mn-SPIO, the concentrations of Mn ²⁺ varied from 0.1 to 0.6 mM, and Fe ³⁺ from 2.5 to 15 mM. The Ri and R2 values of the DCM@P-Mn-SPIO were measured from 0 h to 24 h after incubation with GSH in the presence of SDS. The acquisition parameters were set as: TiWI: TR=200 ms, TE=15 ms, slice thickness=l mm, slice spacing- 1 mm. A 100-mm square field of view (FOV) was used with an image matrix of 256 x 256. Ti map images: TR=100- 2000 ms, TE=14 ms, slice thickness=l mm, slice spacing=l mm. FOV= 10 x 10 cm, matrix = 256 x 256. T2WI: TR=1000 ms, TE=100 ms, slice thickness- 1 mm, slice spacing-1 mm. A 100 cm ² FOV was used with an image matrix of 256 x 256. T2 map images: TR=1000 ms, TEM 5-225 ms, slice thickness- 1 mm, slice spacing- 1 mm, FOV- 10 x 10 cm, matrix = 256 x 256. Quantitative Ti and T2 relaxation maps were reconstructed from datasets using Paravision 4 software. The same method was applied for the relaxivity calculation of DCM@P-Mn (0.1 to 0.6 mM, Mn ²⁺ ) and DCM@SPIO (0.1 to 0.6 mM, Fe ³⁺ ).

[0157] Synthesis and characterization of TMRET nanoprobe. TMRET nanoprobes were fabricated by co-encapsulation of a two-way MRET pair, the paramagnetic Mn- porphyrin chelates (P-Mn) and the superparamagnetic SPIOs, into a disulfide cross-linked micelle (DCM). The hydrophobic P-Mn was synthesized by chelating a paramagnetic metal ion, manganese (II) (Mn ²⁺ ), to a porphyrin derivative (pheophorbide a, Pa). The quench in fluorescence and disappearance ofUV absorbance of Pa after the chelation ofMn ²⁺ (FIG. 9) suggested the successful synthesis of P-Mn. Then, P-Mn and SPIO (5 nm) were co-loaded into the DCM, forming the TMRET nanoprobes (DCM@P-Mn-SPIO). The hydrodynamic size of DCM@P-Mn-SPIO was around 81 nm as measured by dynamic light scattering (DFS) (FIG. 2A). Under the observation of transmission electron microscopy (TEM), the morphology of DCM@P-Mn-SPIO was spherical in shape with a cluster of small SPIOs evenly constrained within the nanoconstructs (FIG. 2B). Upon the addition of glutathione (GSH, a biological target and reducing agent for cleavage of the disulfide bonds in DCM) and sodium dodecyl sulfate (SDS, a strong ionic detergent for disruption the integrity of micellar nanoparticles), the DCM@P-Mn-SPIO disassembled and its hydrodynamic size decreased from 81 nm to 12 nm (FIG. 2C). TEM image also supported that DCM@P-Mn-SPIO was completely dissociated into the small SPIOs in the presence of GSH and SDS (FIG. 2D). The payload release upon micelle disassembly was further investigated by monitoring the accumulated release of Pa in the presence of GSH. As shown in FIG. 2E, the DCM@P-Mn- SPIO exhibited a GSH concentration-dependent payload releasing pattern. FIG. 2F also showed that DCM@P-Mn-SPIO could be triggered to accelerate the release of their payload at a specific time (e.g. 4 h) by the addition of GSH. All these results supported that the DCM@P-Mn-SPIO can be responsively dissociated by its molecular target, like GSH.

[0158] TMRET and mechanisms. Next it is demonstrated DCM@P-Mn-SPIO possessed unique T1&T2 two-way magnetic resonance tuning property. The MRI quenching and recovery of a series of DCM@P-Mn-SPIO nanoprobes with different ratios of P-Mn to SPIO was first investigated. The nanoprobe with a particular ratio of P-Mn to SPIO (1 : 0.025 by mass) was chosen for the subsequent studies because this ratio showed the most substantial quenching and recovery in T1 and T2 signals (FIG. 10). Then, the T1 and T2 dual-quenching effect of the TMRET nanoprobes was explored. DCM@P-Mn with a single T1 contrast and DCM@SPIO with a single T2 contrast were employed as controls (FIG. 11). The

DCM@SPIO showed similar spherical morphology with a diameter of ~80 nm, in which clusters of small SPIOs were encapsulated. The DCM@P-Mn was spherical, and the size was around 14 nm. For the TMRET nanoprobe, T1 -weighted imaging (T1WI) and a colour-coded T1 map both showed obvious T1 quenching effects (FIG. 2G) when compared with that of DCM@P-Mn (control) at an identical concentration of P-Mn (0.1 mM). A similar MR quenching phenomenon in T2-weighted imaging (T2WI) and T2 maps was also observed (FIG. 2H). When the Ti and T2 contrast agents were co-loaded in DCM@P-Mn-SPIO, the Ti relaxation rate (Ri) reduced significantly to 1.23 mM V ¹ , and the T2 relaxation rate (R2) decreased dramatically to 11.7 mM V ¹ (Table 1). By comparison, DCM@P-Mn showed a Ri of 5.2 mM V ¹ and the R2 of the T2 contrast probe (DCM@SPIO) was 88.8 mM V ¹ (FIG. 12, Table 2). The changes of Ri and R2 further confirmed that dual T1&T2 quench occurred in the TMRET nanoprobe. The dual Ti & T2 signal recovery of the TMRET nanoprobe was then investigated. When the TMRET nanoprobe was dissociated, its Ti and T2 signal was recovered as the distance between P-Mn and SPIO increased (FIGs. 21, 2 J). The Ti and T2 signal recovery of DCM@P-Mn-SPIO was dependent on GSH concentration. In contrast, DCM@P-Mn and DCM@SPIO did not exhibit ‘on’ and ‘off switchable MR signals that corresponded to the integrity of the nanoprobes (FIG. 2K,2L).

Table 1. R1&R2 relaxivity changes of TMRET nanoprobe.

Ri (mM V

GSH R ₂ (mM V ¹ )

NPs ^v ) ARI A(rate) AR ₂ A(rate)

(mM)

0 ^~ h 24 h O h 24 h

0 ^~ L23 1.22 0.01 -0.82% 11.69 12.40 0.71 6.07%

DCM@P-Mn- 5 1.32 2.36 1.04 78.8% 12.33 36.32 23.99 194.6% SPIO 10 1.34 3.06 1.72 128.4% 13.07 45.42 32.35 247.5%

20 1.32 3.61 2.29 173.5% 13.19 67.80 54.61 414.0%

Table 2. R1&R2 relaxivity changes of DCM@P-Mn and DCM@SPIO nanoprobes.

DCM@P-Mn DCM@SPIO

GSH _

Ri (mM V ¹ ) R2 (mM V ¹ )

(mM) _ ARI A(rate) _ DGT A(rate)

O h 24 h O h 24 h

0 5.2 5.5 0.30 (5.8%) 88.8 88.6 -0.23 (-0.2%)

5 5.3 5.6 0.29 (5.5%) 87.9 86.1 -2.04 (-2.3%)

10 5.3 5.6 0.28 (5.3%) 86.6 84.2 -2.42 (-2.8%)

20 5.4 5.6 0.27 (5.0%) 85.4 82.6 -3.21 (-3.9%) [0159] The mechanisms of Ti and T ₂ quenching in the TMRET nanoprobe were investigated. There have been extensive reports on the quenching effect of T 1 relaxivity by strong magnetization of T ₂ contrast materials such as SPIO. The T ₂ quenching effect, on the other hand, is poorly understood. It was found that the 1/T ₂ relaxation time (R ₂ ) of DCM@SPIO increased slightly («15%) compared with that of disperse SPIO nanoparticles (88.8 mM V ¹ vs 75.0 mM V ¹ , FIG. 2M and FIG. 13), which ruled out the possibility that T ₂ quenching was induced by SPIO aggregation. Two possible mechanisms are proposed that could underlie the T ₂ quenching effect. First, the dipole field experienced by water from the DCM@P-Mn-SPIO can be approximated as the sum of the fields from SPIO and P-Mn. The SPIO possesses an average magnetic dipole field along the magnetic field direction, whereas P-Mn was measured to be strongly diamagnetic either when encapsulated in the DCM or when released (FIG. 14); P-Mn is composed of a diamagnetic pheophorbide a and a paramagnetic Mn ²⁺ , and the latter makes it an effective Ti contrast agent (inset of FIG. 14). For a range of magnetic fields and P-Mn to SPIO ratios, the net field components can match the diamagnetic field from the surrounding water. This would make the micelle ‘invisible’ to the protons and thus quench the T2 relaxation, although to achieve an exact cancellation in practice is unlikely. Second, attaching molecules to the surface of SPIO has been reported to decrease the net moment of the particle. It is known that Mn and Fe have a negative exchange interaction, which results in a non-collinear antiferromagnetic configuration in their binary alloy. In this case, the Mn ²⁺ in P-Mn may interact with SPIO, which reduces the net moment of SPIO. Both mechanisms result in a reduced effective magnetic moment of DCM@P-Mn- SPIO, which suppresses the dipole fields. These weakened dipole fields would extend the transverse relaxation time of the surrounding water protons to produce an increase in the T2 relaxation time. Once the DCM@P-Mn-SPIO is treated with GSH + SDS, the P-Mn and SPIO become dispersed and both mechanisms cease, and the T2 contrast would recover (FIG. 2H).

[0160] In support of these hypotheses, dynamic X-band electron paramagnetic resonance (EPR) and quasi-static magnetization versus field measurements were performed (FIG. 2N, FIG. 20 and FIG. 15). The EPR measurements probe the local field environment and field distributions specifically within the SPIO and P-Mn, as well as the density of ‘free’ un-paired electrons. A control sample DCM@P-Mn showed a typical EPR spectrum of Mn ions (Mn ²⁺ ) while DCM@SPIO exhibited the characteristic peaks in EPR similar to that of reported ultra small superparamagnetic iron oxide (FIG. 2N, black and red lines). Interestingly, upon co encapsulation of P-Mn and SPIO, forming DCM@P-Mn-SPIO, the characteristic peaks of both Mn ions and SPIO in EPR spectra decrease dramatically (FIG. 2N, green line). After the addition of GSH and SDS to DCM@P-Mn-SPIO, dispersing the contrast agents, the EPR intensity is recovered (FIG. 2N, blue line). The double-integration value of the EPR spectrum of DCM@P-Mn-SPIO which corresponds to the total number of free, unpaired electrons in the system was lower than that of the probe with only SPIO (DCM@SPIO). In the presence of GSH and SDS, the integral recovers to a similar level to that of DCM@SPIO (FIG. 15). The changes in the EPR intensity indicate that some of the electrons in the DCM@P-Mn- SPIO are no-longer free to rotate, potentially being constrained by the magnetic exchange interaction; the recovery of the EPR spectrum after GSH+SDS treatment indicates that any corresponding bonding between the P-Mn and SPIO is weak and breaks upon dispersion. These results are consistent with that of the dual Ti & T2 signal quenching and recovery as shown in FIG. 2G-2J and Table 1. The quasi-static magnetization curves for the DCM@SPIO, DCM@P-Mn-SPIO and DCM@P-Mn-SPIO+GSH+SDS are shown in FIG. 20. These measurements show a reduction in the magnetization upon encapsulation, and subsequent recovery after the GSH+SDS treatment, which is consistent with the Mn-ion inducing a non-collinear or antiferromagnetic spin texture in the SPIO (the second mechanism). These results are also consistent with the difference between Pa versus P-Mn, in that Pa is not expected to interact with the SPIO magnetically. However, the diamagnetic contribution is necessary to explain the concentration dependence (the first mechanism), which shows increased quenching even after the SPIO is fully coated. Typically, the diamagnetic field is much smaller than the ferromagnetic one, but in this case, due to the combined effects of the large concentration ratio (40: 1, P-Mn to SPIO), sizable diamagnetism induced at a 7 T field, and the reduced moment on the SPIO, they are able to balance. In this way, the two proposed mechanisms work synergistically to suppress the dipole fields from the DCM@P-Mn-SPIO and quench the Ti and T2 relaxation.

[0161] The mechanism of Ti and T2 quenching and activation is illustrated in FIG. 3. Ti contrast agents help relax protons in the surrounding water through a fast spin fluctuation, ¹⁵ while T2 relaxation is mainly affected by the dipole-dipole interaction between water protons and SPIO or micelle nanoprobes when SPIO and P-Mn are encapsulated. In the “OFF” state, the magnetization measurements show that while the contrast agents are in close proximity, the SPIO moment is reduced; in the large (7 T) fields of the MRI, the diamagnetic response from the P-Mn is also quite significant. Between these two effects, the dipole field from the nanoprobe is small, making it ineffective in reducing the transverse magnetization of water protons, resulting in a longer T2. Simultaneously, the Ti contrast agent has slow spin fluctuation due to the magnetic field from the T2 contrast agent (SPIO), including the effective field from any interactions with the surface of the SPIO, and is thus also ineffective in relaxing water protons.

[0162] In the “ON” (activated) state, the two contrast agents are separated and dispersed in solution as DCM is broken apart by the molecular targets. Once dispersed, the surface interactions between the P-Mn and SPIO are removed, and the magnetic moments influence the water in their local environment rather as a collective unit. Once the Mn is no-longer influenced by the SPIO it regains its fast spin fluctuation, decreasing Ti, and the SPIO recovers its magnetic moment, which helps relax surrounding water protons. This leads to enhancements in both Ti and T2 imaging after dispersion. Example 4. Imaging Detection

[0163] Electron paramagnetic resonance spectroscopy (EPR). The samples (DCM@SPIO, DCM@P-Mn, DCM@P-Mn-SPIO) for EPR characterization were prepared using the same procedures as described above, with the concentration of P-Mn of 1.5 mg/mL and SPIO of 0.0375 mg/mL. The X-band (9.43 GHz) continuous-wave (CW) EPR spectra were recorded on a Bruker (Billerica, MA) Biospin EleXsys E500 spectrometer equipped with a super-high Q resonator (ER4122SHQE). All CW-EPR data were acquired under non saturating conditions, with an excitation microwave frequency = 9.87 GHz, microwave power = 0.6325 mW, and modulation frequency = 100 kHz.

[0164] Magnetometry. The samples (DCM@SPIO, DCM@P-Mn-SPIO, DCM@P-Mn- SPIO+SDS+GSH) for magnetic characterization were prepared using the same procedures as described above, with the concentration of P-Mn of 0.5 mg/mL and SPIO at 0.0125 mg/mL.

In addition, reference samples were made for control groups without SPIO (DCM, DCM@P- Mn, DCM@P-Mn+SDS+GSH), whose concentrations were kept the same as the samples containing SPIO. Same volume of each type of sample was loaded into the same liquid sample holder with a cap from Lake Shore Cryotronics, filling it up to capacity before the measurement. The sample holder was sonicated by water and acetone and dried before changing samples. Thus each sample had nominally the same amount of SPIO, due to the identical sample volume and concentration. Hysteresis loops were measured at room temperature using a vibrating sample magnetometer from Princeton Measurements Corporation. After measurements, the background was subtracted using the reference samples. Lor example, the SPIO signal shown in FIG. 20 was obtained by subtracting the measured signal of the DCM reference sample from DCM@SPIO, removing background, including sample holder, water and the DCM from the measured signal. Similarly, DCM@P- Mn-SPIO, DCM@P-Mn-SPIO+SDS+GSH measurements were calibrated against DCM@P- Mn and DCM@P-Mn+SDS+GSH reference samples, respectively. Data from different samples were plotted together in the measured magnetic moment in FIG. 20. Additionally, DCM@P-Mn was calibrated against a DCM reference sample.

[0165] Cell uptake of DCM@P-Mn-SPIO evaluated by TEM. The cellular uptake of DCM@P-Mn-SPIO was further evaluated by TEM (Talos, L120c, PEI) with an accelerating voltage of 80 kV. In brief, the cells were seeded at a density of 1 x 10 ⁵ cells per well into an 8- well permanox slide for 24 h, reaching confluency of 80%. Then the cells were treated with DCM@P-Mn-SPIO for 2 h at 37°C. The embedded cells were sectioned (75 nm) and mounted onto 200-mesh copper grids after washing with phosphate buffer.

[0166] In vitro MRI on PC-3 and RWPE-1 cells. In vitro MRI was performed on PC-3 cells with and without GSH inhibitor (L-Buthionine sulfoximine, LBS) as well as on normal prostate cells (RWPE-1) (l x l 0 ⁶ ). The cells were incubated with DCM@P-Mn-SPIO (P-Mn concentration was 40 pg/mL) for 2 h, the cells were washed three times with PBS, then collected at various time point (2, 5, 12, 24, 36, 48 h) and fixed in agarose (1 mL, 1.0 %) in Eppendorf tubes. MRI was performed on a 7.0 T MR system. TiWI images were obtained using the following parameters: TR/TE (250 ms/ 14 ms); T2WI: (1000 ms/100 ms), slice thickness =1 mm; slice spacing- 1 mm; matrix =256 x 256; FOV =10 cm x 10 cm. The Ti and T2 signal intensities were measured within the region of interest (ROI).

[0167] In vitro MRI was further performed on PC-3 cells that were incubated with different concentrations of GSH inhibitor from 0 to 50 mM for 24 h. After incubation with P-Mn (40 pg/mL) for 2 h, cells were washed three times with PBS, cells were digested with 0.25% trypsin at different time points (2, 5, 12, 24, 36, 48 h), centrifuged for 3 min, and resuspended in agarose (1 mL, 1.0 %) in Eppendorf tubes. MR imaging was performed on a 7.0 T MR system. Ti map images: TR= 100-2000 ms, TE=14 ms, slice thickness=l mm, slice spacing=l mm. FOV= 10 x 10 cm, matrix = 256 x 256. T2 map images: TR=1000 ms, TE=15-225 ms, slice thickness=l mm, slice spacing=l mm. A 10 cm x 10 cm FOV was used with an image matrix of 256 x 256.

[0168] In vivo MRI. PC-3 tumours and orthotopic 12FLR glioma-bearing mice (n=3) were scanned on a 7.0 T MRI Scanner (Bruker Biospec, USA.), with a high-resolution animal coil. The mice were i.v. administrated with 100 pL of the nanoprobes and then subjected to MRI at different timepoints: DCM@P-Mn-SPIO (0, 1, 12, 24, and 48 h), DCM@P-Mn (0, 1 and 12 h) and DCM@SPIO (0, 1 and 12 h). The concentrations of P-Mn and SPIO were kept at 15 mg/mL and 0.25 mg/mL, respectively. All mice were imaged under the TiW (TR/TE=300/14 ms) & T2W spin-echo sequences (TR/TE= 1000/100 ms); Timap:

(TR/TE= 100-2000/ 14 ms) and T2 map image (TR/TE = 1000/15-225 ms) (slice thickness=l mm, slice spacing=l mm, FOV= 10 x 10 cm, matrix = 256 x 256). The mean Ti and T2- weighted signal intensities (Smcan) were measured for each tumor. Quantitative Ti and T2 maps were reconstructed from datasets using Paravision 4 software. Ti and T2 relaxation time were calculated with the Paravision 4 software. Then, the relative signal-to-noise ratio (SNR = Smcan/N SD (standard deviation of the background signal) was calculated based on a previously reported method.

[0169] T2 star measurement. For T2* images, the MRI parameters were TR=(1500 ms), TE = (4.0-61.5 ms), FOV = 8 x 8 cm and matrix = 256 x 256. Quantitative T2* maps were reconstructed from datasets using Paravision 4 software. Ri, R2 and R2* is defined as 1/Ti, I/T2 and I/T2* relaxation time.

[0170] DESI of the MRI (T1-T2). When the MRI study was performed using a 7.0 T MRI Scanner (Bruker Biospec, USA.), MR images were acquired by Ti and T2 -weighted sequences with identical MRI geometrical parameters to ensure that the image slices were consistent. The MR images were co-registered to ensure Ti-weight images correspond to the corresponding point of T2-weight images. Then the images of head skull and surrounding soft tissue of the mouse were removed using ImageJ software. The subtraction imaging obtained in MATLAB software by using the following commands:

A = imread ('D:T1WI');

B = imread (T>: T2WF);

C = imsubtract (A, B);

J = imcomplement c;

J : Subtraction images of the Tl and T2- weight imaging.

The final step involved anti-phase processing of subtraction images using MATLAB software.

[0171] H&E stain and Prussian blue stain. After humanely sacrificing the mice, the major organs were collected, and fixed them in 4% paraformaldehyde. The organs were then sliced and stained by hematoxylin and eosin (H&E) to evaluate the systemic toxicity of the nanoprobes. Prussian blue staining was performed as described ⁹ to detect iron-positive cells.

[0172] Statistical analysis. All data analyses were shown as mean ± standard deviation (s.d.). Analysis was performed using SPSS 19.0 software. MRI signal intensity, ARi & AFU versus GSH concentration, were compared and analyzed using univariate Analysis of Variance. SNK test was used for binary comparison. Pearson’s test was used for correlation analysis. » values < 0.05 were considered statistically significant for all analysis.

[0173] Evaluation of the Imaging Platform in the Biological System. In vitro evaluation of TMRET nanoprobes. The performance of TMRET nanoprobes in in vitro and in vivo biological systems was evaluated. Prior to the applications in these systems, the biocompatibility of the nanoprobes was evaluated. As shown in FIG. 16, DCM@P-Mn- SPIO, DCM@P-Mn and DCM@SPIO did not exhibit obvious cytotoxicity against PC-3 cells, even at a high concentration of 5 mg/mL (the concentration of DCM).

[0174] The development of innovatively imaging strategies based on stimuli-responsive TMRET nanoprobes with unique dual Ti & T2 two-way magnetic resonance tuning properties, enabled accurate imaging of biological targets. One of the most extensively investigated biological stimuli was the redox potential difference induced by concentration changes of a reducing agent (GSH) across different intra/extracellular regions, which enables redox-responsive nano-platforms to be promising cancer diagnostic probes and effective therapeutic delivery systems. It was previously reported that DCM was one of such stimuli- responsive nano-platforms with excellent responsiveness to GSH level. DCM has been demonstrated to retain its structural stability and minimize the payload release in blood circulation and allow for efficient release triggered by GSH at tumour sites. The TMRET nanoprobes were then incubated with PC-3 tumour cells, PC-3 cells treated with GSH inhibitor (L-Buthionine sulfoximine, LBS) and normal prostate cells (EWPE-1), and tested the T1&T2 signal recovery temporally. This experiment allowed us to evaluate the responsiveness of the nanoprobe to different GSH levels in cells. As shown in FIGs. 4A and 4B, DCM@P-Mn-SPIO exhibited time-dependent activation of both Ti and T2 MRI signals when incubated with PC-3 cancer cells at a high GSH concentration (11.5 mM) quantitated by using ThiolTracker™ Violet (Glutathione Detection Reagent). In contrast, DCM@P-Mn- SPIO did not show obvious T1&T2 signal recovery in normal prostate cells (EWPE-1) that contained a lower GSH concentration (1.89 mM). To verify if the MRI signals of DCM@P- Mn-SPIO specifically responded to GSH, a GSH inhibitor, LBS, was incubated with PC-3 cells to suppress the intracellular GSH level. The DCM@P-Mn-SPIO in the LBS treated PC- 3 cells (GSH concentration was measured as 2.3 mM after 24 h inhibition) showed significantly less MR signal responsiveness in both T 1 and T2 signals compared to that in untreated PC-3 cells, indicating that the GSH was the key factor for the dissociation of nanoprobes and activation of MR signals.

[0175] Furthermore, PC-3 cells were obtained with different concentrations of GSH after treatment with various concentrations of LBS. As shown in FIGs. 4C and 4D, the ARi and AR2 (defined as the difference in the Ti relaxation rate (Ri = 1/Ti) and T2 relaxation rate (R2 = I/T2), respectively) of the TMRET nanoprobe were measured to be 0.18, 0.31, 0.49, 0.82, 1.27 s ¹ (ARi) and 1.96, 3.97, 5.89, 11.20, 18.90 s ^_1 (AR2), respectively (Table 3), which showed an excellent linear relationship to the intracellular GSH level. It offers the possibility for quantitative measurements of GSH in biological systems. To confirm the cellular uptake of the TMRET nanoprobes, nanoprobes were incubated with PC-3 cells, and observed their intracellular distributions by TEM. As shown in FIG. 4E, obvious SPIO cluster can be found inside of the cells, indicating that the TMRET nanoprobe is internalized into tumour cells and able to enhance the tumour cell contrast.

Table 3. In vitro ARi and AR2 of the nanoprobes in PC3 cells containing various concentrations of GSH.

GSH (mM) ARi (s ¹ ) AR ₂ (S ¹ )

23 0.18± 0.09 1.96 ± 0.78

4.5 0.31 ± 0.09 3.97 ± 1.24

6.2 0.49 ± 0.08 5.89 ± 1.35

7.6 0.82 ± 0.08 11.20 ± 1.07

11.5 1.27 ± 0.06 18.90 ± 1.65

[0176] In vivo evaluation of TMRET nanoprobe. As proposed in FIG. 1, the two-way MRET pair (Mn ²⁺ / SPIO) is “locked” in 50 nm micelle core with cleavable crosslinkers and stays silent (the “OFF” state) with very low background T1&T2 MRI signal due to their very close proximity under normal physiological conditions (e.g. blood circulation) (FIG. 1).

Upon the preferential accumulation of TMRET nanoprobes at tumor sites “passively” through the enhanced permeability and retention effect, the crosslinkers will be cleaved by intrinsic stimuli within tumours, e.g. reducing agents (e.g. GSF1), acidic pFl, or proteases (the “key”, FIG. 1). The nanoprobes then dissociate and the MRET pair (Mn ²⁺ and SPIO) are separated, resulting in fewer magnetic interactions between them and thus significantly enhancing both T1&T2 MRI signals (the “ON” state) at tumor sites. Therefore, TMRET nanoprobes can suppress the background signal from normal tissue and enhance the MR contrast at the tumour site, and the activation of the T1&T2 imaging functions can correlate with the level of local stimuli (molecular target) at the tumour sites.

[0177] Then, the in vivo performance of TMRET nanoprobes was evaluated. DCM@P-Mn- SPIO was i.v. administrated to PC-3 tumour-bearing mice. The single MRI contrast agents (DCM@P-Mn and DCM@SPIO) were introduced as control groups. As shown in FIG. 6A, both Ti and T2 MRI contrast enhancements in the DCM@P-Mn-SPIO group were not distinguishable within 1 h but showed an obvious increase at 12 h (Ti -weighted images became brighter while T2-weighted images turned darker). Such significant changes could be attributed to the accumulation and subsequent activation of TMRET nanoprobes in tumours. These results indicated that DCM@P-Mn-SPIO simultaneously provided both distinct positive Ti and negative T2 contrast enhancements in tumours. The dual-contrast enhancing effect of the nanoprobe enables the synergistic combination of the two MR relaxation effects for accurate diagnosis of vague tumour sites. The MRI contrast remained at a prominent level for another 12 h (24 h post-injection), then tapered off in 48 h. The MR images were further analyzed by drawing ROIs of whole tumour volumes to obtain quantitative and dependable results for signal-to-noise ratios (SNRs) and Ti & T2 relaxation rates. The Ti SNR increased sharply after administration of DCM@P-Mn-SPIO and gradually grew by 54.9% at 24 h post- injection, while the T2 SNR decreased 56.3 % at 24 h post- injection compared to the pre-injection level (FIG. 6B). In control groups (DCM@P-Mn and DCM@SPIO), the MRI contrast enhancement of tumours increased much faster than that of DCM@P-Mn-SPIO. The Ti MRI signal started to increase at 1 h after the injection of DCM@P-Mn and remained at a prominent level after 12 h post-injection (FIG. 17). The DCM@SPIO mediated T2 MRI signal showed a similar pattern to the Ti MRI (FIG. 18). The T2* of the DCM@P-Mn-SPIO that solely depended on the SPIO contents and particle concentrations could be considered as the baseline of SPIO accumulation at the tumor sites. The T2 map of the DCM@P-Mn-SPIO not only depended on the SPIO accumulation but was also affected by the activation of the TMRET pair. We, therefore, could investigate the accumulation and activation of the nanoprobe by combining T2 and T2* MR sequences. The T2 and T2* MR images of PC-3 tumour-bearing mice at different time points (0, 1 and 12 h) were acquired before and after the injection of DCM@P-Mn-SPIO and DCM@SPIO (FIG. 6C). R2* values showed no significant difference between two groups at the time points post- injection indicating the similar SPIO accumulation at the tumors sites (FIG. 6D). The R2 values (FIG. 6E) were significantly different between the two groups and reflected the initial T2 quenching of the DCM@P-Mn-SPIO (from 0 to 1 h) in response to GSH. These results indicated that the MRI signal enhancements of the TMRET probe could be activated by the stimuli at the tumour site upon the accumulation of the probes. The tumours and major organs were collected for haematoxylin and eosin (H&E) staining (FIG. 19) and no obvious abnormities were observed. [0178] The GSH-correlated dual-MR signal responsiveness in vivo was quantitatively investigated. The tumours were first injected with different concentrations of GSH to achieve an intratumoural concentration gradients. Then the DCM@P-Mn-SPIO was injected into the mice via tail vein (FIG. 5A). Five hours later, the ARi values at the tumour sites were measured to be 0.21, 0.27, 0.33, 0.37 s ¹ which corresponds to the concentrations of GSH from 6.15 to 11.44 mM (Table 4). The AR2 values at the tumour sites were 5.26, 7.03, 9.79, 10.4 s ¹ , corresponding to the concentrations of GSH from 6.15 to 11.44 mM (Table 4). It was found that the ARi and AR2 positively correlated with the intratumoural GSH concentrations, exhibiting a good linear relationship (FIG. 5B and 5C), indicating that the TMRET nanoprobes are potentially useful for in vivo quantitative analysis of molecular target, such as GSH, in a non-invasive manner.

Table 4. In vivo ARi and AR ₂ of the nanoprobes in the PC3 tumours containing various concentrations of GSH. 6.15 0.21 ± 0.03 5.26 ± 0.47 6.81 0.27 ± 0.05 7.03 ± 0.68 8.10 0.33 ± 0.04 9.79 ± 1.10 11.44 0.37 ± 0.04 10.40 ± 0.62

[0179] Dual-contrast enhanced subtraction imaging (DESI) technology. A new dual contrast enhanced subtraction imaging (DESI) technology as a post-imaging processing and reconstruction method to better implement the unique T1&T2 two-way magnetic resonance tuning property of TMRET nanoprobes was developed. DESI was carried out by quantitative subtraction of the positive Ti signal from the negative T2 signal that switched from “OFF” to “ON” to acquire a significantly enhanced MR contrast of targeted sites and minimized the background signal utilizing the mathematical simulation function of MATLAB. The feasibility of DESI was investigated by determining whether the nanoprobes could have a higher Ti signal intensity than T2 signal intensity in different model systems, such as aqueous solution, muscle of normal mice, cancer cell lines and tumour xenografts on nude mice. In an aqueous solution, both Ti & T2 MRI signals could be detected at probe concentrations as low as 0.003 mM and the T 1 signal intensity was always higher than the T2 signal intensity (FIG. 20). When injected into the muscles of normal mice, the concentration limit of DCM@P-Mn- SPIO should be above 0.06 mM in order to have higher Ti signal intensities for DESI (FIG.

21). In PC-3 cells, the Tl signal intensity of DCM@P-Mn-SPIO was greater than the T2 intensity (FIG. 22A). However, DCM@P-Mn-SPIO did not exhibit a higher Tl signal in PC-3 cells with LBS (FIG. 22B) or in normal prostate cells (FIG. 22C), due to their low GSH level. In nude mice bearing tumour xenografts, DCM@P-Mn-SPIO could be activated to generate MR images with higher Ti SNR and lower T2 SNR at 12 h and 24 h post injection, which was determined to be the optimal time window for DESI (FIG. 6B). DESI technology was utilized to processed the MR images in FIG. 6A. The subtraction imaging is shown in FIG. 6F, the tumour contrast was dramatically increased while the background signal from normal tissue was minimized. DESI was not applicable for processing images obtained with a single contrast agent. The DESI images acquired from mice treated with DCM@P-Mn (FIG. 6G) and DCM@SPIO (FIG. 6H) only exhibited dim tumour outlines. DESI technology can be utilized to significantly enhance the MRI contrast and suppress the background from normal tissue. As shown in FIG. 61, the tumour-to-normal-tissue ratio (TNR) in DESI of DCM@P-Mn-SPIO (TNR is 6.73±1.45) was dramatically higher than that of DCM@P-Mn (TNR is 1.04±0.18) and DCM@SPIO (TNR is 0.89±0.24). Such a tailored subtraction technique for TMRET nanoprobe showed exciting potentials for cancer diagnosis, especially for the detection of the tumours at the early developing stage.

Example 5. Disease Detection

[0180] Accumulated payload release of DCM@P-Mn-SPIO. DCM@P-Mn-SPIO solution was prepared to determine the payload release profile. The UV-vis absorbance of P- Mn was measured to determine the payload release. 1 mg/mL (0.5 mL) of DCM@P-Mn- SPIO PBS solution with various GSH concentrations (0, 5, 10 and 20 mM) were injected into dialyzed cartridges (Pierce Chemical Inc.) with a 3.5 kDa MWCO. The cartridges were dialyzed against 2 L PBS at 37 °C. In the stimulus-responsive release experiment, GSH (20 mM) was added to the release medium at a specific time (4 h). The P-Mn concentrations that remained in the dialysis cartridge at various time points were calculated by the standard curve. The payload release was performed in triplicate samples to calculate the mean values.

[0181] Cell viability assay. To evaluate the biocompatibility of DCM@P-Mn-SPIO, DCM@P-Mn, and DCM@SPIO, PC-3 prostate cancer cells were incubated with these nanoprobes and the cell viability was measured by MTT (methyl thiazolyl tetrazolium). PC-3 cells were seeded in 96-well plate with a density of 3x 10 ⁵ cells per well, and incubated for 24 h (37 °C, 5% CO2) until all cells completely attached. Then different concentrations of DCM@P-Mn-SPIO, DCM@P-Mn and DCM@SPIO were added, (all probe concentrations were calculated based on DCM concentrations, which corresponded to 0, 0.1, 0.5, 1 and 5 mg/mL). After incubation for 24 h, the media was aspirated and 150 pL of dimethyl sulfoxide (DMSO) was added to dissolve the MTT crystal. Absorbance at 490 nm was measured by using a microplate reader (SpectraMax M3, USA) to assess cell viability.

[0182] Tumor xenograft and orthotopic tumor animal models. Nude mice, 4-5 weeks of age, were obtained from Harlan (Livermore, CA). All animals were kept under pathogen- free conditions according to AAALAC guidelines, and were allowed to acclimate for at least 4 days prior to any experiments. All animal experiments were performed under the requirements of institutional guidelines and according to protocol No. 07-13119 approved by the Use and Care of Animals Committee at the University of California, Davis. PC-3 cells in a 200 pL mixture of PBS suspension and Matrigel (1:1 v/v) were subcutaneously injected into the right flank of nude mice. The tumor sizes for all nude mice were monitored and recorded weekly. Tumors that reached the longest dimension of 0.8-1.0 cm were used for in vivo MR imaging.

[0183] For orthotopic or intracranial implantation, 2.5 x 10 ⁵ 12FLR glioma cells, derived from patient samples, resuspended in 5 pL PBS were injected into the right striatum area of the nude mouse with the aid of a mouse stereotactic instrument (Stoelting). The tumor sizes for all nude mice were monitored and recorded by the bioluminescence signal of luciferase weekly.

[0184] Diagnosis of small intracranial brain tumour. High TNRs are critical to successful detection of early-stage cancer by imaging approaches. The TMRET nanoprobe was silent in blood circulation but visible within the tumour. This allowed both T1&T2 MRI signals to amplify upon preferential accumulation at the tumour site to increase the tumor contrast. The complementary DESI technology could further suppress the background signal and enhance tumour contrast. This integrated imaging platform with TMRET nanotechnology and DESI is expected to achieve the highest possible TNR, and is therefore particularly suitable for early cancer detection. The capability of this platform was evaluated to detect early-stage tumors that were deeply embedded in the brain in a patient-derived xenograft (PDX) mouse model of glioma (12FLR). DCM@P-Mn-SPIO was administrated as a TMRET nanoprobe in comparison to the non-tunable nanoprobes with a single MRI contrast agent, DCM@P-Mn and DCM@SPIO. In FIG. 7A, at 12 h after i.v. injection of DCM@P-Mn- SPIO, both Ti MR contrast (brighter) and T2 MR contrast (darker) in the small intracranial brain tumour were enhanced significantly. Ti and T2 SNR dynamic enhancements (FIG. 7D) from control groups elevated much faster than that of the TMRET nanoprobe and reached a plateau within a short time (e.g. 1 h). The TMRET nanoprobe showed a gradual increase in MR signal (increase in Ti, decrease in T2) due to the activation by GSH. Both the Ti & Ti relaxation rates (Ri and R2) at tumour sites also increased dramatically at 12 h post-injection (FIGs. 23A and 23B). In contrast, the probes with single contrast agent, including DCM@P- Mn (FIG. 7B) and DCM@SPIO (FIG. 7C), started the MR enhancements at 1 h and remained the signal for 12 h (FIGs. 23C and 23D). Ti SNR of DCM@P-Mn (FIG. 7E) and T2 SNR of DCM@SPIO (FIG. 7F) treated mice started to increase at 1 h, indicating a single Ti or T2 enhancement, and the MRI signal was in the “always ON” state. Then, the DESI technique was utilized to enhance the TNR and suppress the background signal from normal brain tissue. As shown in FIG. 7G, the subtraction imaging results showed the signal of normal brain tissue could be dramatically reduced, further highlighting the tumour area (FIG. 24). In the control groups (DCM@P-Mn and DCM@SPIO) with single MRI contrast enhancements, the DESI technique was not applicable. As shown in FIG. 7H and 71, the subtraction imaging of DCM@P-Mn and DCM@SPIO only showed vague tumour outline in the brain (FIG. 24). With DESI, the TNR of DCM@P-Mn-SPIO reached 11.6 while that of DCM@P-Mn and DCM@SPIO were only 1.25 and 1.09, respectively (FIG. 7M), which demonstrated that TMRET equipped with DESI is able to dramatically enhance the TNR. The H&E stained whole-brain sections confirmed the location of the tumours (FIG. 7J-7L). In the DCM@P-Mn-SPIO group, the tumour size in the cerebrum was approximately 0.75 mm ³ (the tumour volume is calculated by the formula (L*W ² )/2, L=1.5 mm, W=1 mm,) as measured in H&E slides (FIG. 7J). Prussian blue stain (indicated SPIO that is composed of ferric iron) further confirmed that the iron contained nanoparticles, including DCM@P-Mn- SPIO (FIG. 7N) and DCM@SPIO (FIG. 70), accumulated in orthotopic brain tumour tissue. The biocompatibility of DCM@P-Mn-SPIO, DCM@P-Mn and DCM@SPIO after systemic administration into mice was evaluated. The tumours and main organs of mice were collected for H&E stain. As shown in FIG. 25, no obvious abnormities were observed in these normal organs. [0185] To broaden the applications of the TMRET technique, it was also measured on the TMRET probe on a 3.0 T and 9.4 T MRI scanner, respectively. FIGs. 26 and 27 show that the T1 and T2 of DCM@P-Mn-SPIO can be quenched and readily recovered, which supports that the TMRET system is stable and can be broadly applied to MRI scanners with different magnetic fields. This increases the potential for TMRET nanotechnology to be translated into clinical use. To test if the TMRET technique is applicable to different nanocarriers and molecular targets, two new probes that can realize the Ti and T2 dual quench and recovery was developed. First, an amphiphilic polymer (1,2-distearoyl- phosphatidylethanolamine-methyl-polyethylene glycol 2000, DSPE-PEG2000) was employed to encapsulate the TMRET pair. The DSPE-PEG readily constrained the TMRET pair in a spherical nanostructure (FIG. 28A) and can be dissociated into smaller nanoparticles (FIG. 28B). The Ri and R2 of DSPE-PEG@P-Mn-SPIO were quenched when the nanostructure was intact and recovered in the presence of SDS (FIG. 28C-28D), which indicates that the TMRET pair is applicable to other micellar systems that constrain the hydrophobic contrast agents tightly. A similar telodendritic nanocarrier (PEG5000-OH8- PPBA (POP); PPBA, porphyrin phenylboronic acid) was developed, which could be responsively dissociated on exposure to acidic pH. The chemical structure and characterization of POP are shown in FIGs. 29-31 and in FIGs. 32 and 33, respectively. The TMRET pair was encapsulated into POP to form a new pH-responsive TMRET probe (POP@P-Mn-SPIO). The particle size changes (FIGs. 8A,9B) indicated that POP@P-Mn- SPIO can be broken down by acidic pH. The Ri (FIG. 8C) and R2 (Fig. 8D) of POP@P-Mn- SPIO can also be quenched and recovered on acidic pH stimulation. In a patient-derived xenograft mouse model with intracranial glioma, the POP@P-Mn-SPIO detected ultrasmall intracranial tumours with very high TNRs (FIGs. 8E-8Q and FIG. 34). The T2* acquisition (FIGs. 8R,8S) supported that the initial T2 quenching and recovery of the POP@P-Mn-SPIO were caused by the MRI signal activation.

[0186] Conclusions. Unique TMRET nanotechnology was designed and developed, that is, the first distance-dependent T1&T2 two-way magnetic resonance tuning platform. A DESI technique that is complementary to TMRET nanotechnology. This integrated imaging platform shows advantages in the quantitative imaging of a biological target (such as redox stress and acidic pH) in tumours, and ultrasensitive detection of very small intracranial tumours (~0.75mm ³ ) in mouse brains with a TNR over 10, which is an extensive improvement in comparison with conventional single model MRI contrast agents. [0187] Magnetic resonance tuning is a new sensing and imaging technique that allows biological targets in deep tissues and complicated biological environments to be imaged non- invasively with higher spatial resolution compared to most optical techniques, such as FRET and SPR. Flowever, the application of current Ti-based MRET probes may be limited by the lack of sensitivity and intrinsic low MR relaxivity of Ti-based MR contrast agents. Furthermore, the stability, size and surface properties of these probes need to be further optimized for broad in vivo sensing and imaging applications with systemic administration. Developing new MRET probes that integrate both Ti and T2 MRI contrast capabilities are highly desirable for advancing this distance-dependent MR technique. The two-way MRET probes presented in this manuscript may be able to overcome the limitations of individual T 1 and T2 imaging modalities by improving sensitivity and accuracy. The combined Ti and T2 dual-modal MRET also have double-checked, self-improved merits in practical diagnosis. Flowever, how to “quench” the strong superparamagnetic nature or magnetic moments of these nanoparticles, such as SPIO, remains elusive. A series of experiments were designed to elucidate the mechanism of T2 quenching in TMRET probe and found that the large amounts of manganese are able to hamper the magnetization of SPIO when they are very close and packed with each other. This mechanism may further inspire other scientists to develop other probes or even applications on physics area. T1&T2 two-way magnetic resonance tuning was achieved by optimizing the ratio of Mn chelate (Ti enhancer & T2 quencher) and SPIO (T2 enhancer & T 1 quencher) in nanosized micelles with structure-dependent stability (assembly vs dissociation) and stimuli-responsiveness (GSH and acidic pH). This TMRET pair is well applicable to micellar systems, allowing efficient magnetic interactions between the enhancers and quenchers. Typically, it has been difficult to implement the inherent negative (dark) contrast effects of T2 MRI agents in vivo. A new DESI technique was developed as a complement to TMRET nanotechnology. DESI is a post-imaging processing technique that is performed by the mathematic simulations of MATLAB software, which extensively highlights the tumours by eliminating the background signal of the surrounding tissues. Compared with current Ti-based MRET technology, the TMRET nanotechnology is much more sensitive and has lower background signals. Furthermore, the stability and surface properties TMRET nanoprobes have been optimized for a broad range of in vivo sensing and imaging applications upon exposure to the bloodstream. In comparison to the limited TNR achieved by Ti-based MRET technology on 100 mm ³ tumours by intra-tumoural injection, TMRET nanoprobes via systemic administration could reach a very high TNR (>10) on a tumour that was 100 times smaller in volume. [0188] The integrated imaging platform, TMRET probe equipped with DESI technique, makes MRIs much more sensitive and selective than the conventional techniques used in cancer diagnosis. This is due to the following advantages: i) preferential accumulation at the tumour site via the EPR effect, thus endowing MRIs with a high tumour selectivity; ii) activation by an intrinsic tumour stimuli, such as GSH and acidic pH, upon accumulation to further increase the TNR; iii) a dual-modal, subtraction-based, high sensitivity MRI for early- stage small lesions diagnosis; iv) the ultrahigh TNR may highlight the tumour margin which can significantly improve the accuracy of the MRI-guided surgical procedures. Therefore, the new technique may not only be used a research tool for molecular imaging in various intact preclinical systems such as cells or animal models, but also have enormous potential to be translated into precision imaging platforms in human patients for diagnosis of diseases at early stage, image-guided molecular therapy and non-invasive post-treatment assessment of the molecular responses to therapy.

[0189] The applications of TMRET nanotechnology may be further broadened by the development of reversibly crosslinked micelles with stimuli-responsive crosslinkers containing cleavable boronate ester bonds or particular peptide substrates for sensing and imaging of acidic tumour pH, caspase and tumour-associated proteases such as matrix metalloproteinases (MMPs) and cathepsin family, respectively. The improvements in the targeting properties of TMRET nanoprobes via targeting moieties for specific types of tumours will undoubtedly further increase the sensitivity and accelerate the translation of such technology platforms to the clinic. TMRET technology will further benefit from the development of better implementation approaches for better post-imaging processing and reconstruction.

[0190] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.