A Manganese Porphyrin Platform for the Design and Synthesis of Molecular and Targeted MRI Contrast Agents

Magnetic resonance imaging (MRI) contrast agents, in contrast to the plethora of fluorescent agents available to target disease biomarkers or exogenous implants, have remained predominantly non-specific. That is, they do not preferentially accumulate in specific locations in vivo because doing so necessitates longer contrast retention, which is contraindicated for current gadolinium (Gd) agents. This double-edge sword implies that Gd agents can offer either rapid elimination (but lack specificity) or targeted accumulation (but with toxicity risks). For this reason, MRI contrast agent innovation has been severely constrained. Gd-free alternatives based on manganese (Mn) chelates have been largely ineffective, as they are inherently unstable. In this study, we present a Mn(III) porphyrin (MnP) platform for bioconjugation, offering the highest stability and chemical versatility compared to any other T1 contrast agent. We exploit the inherent metal stability conferred by porphyrins and the absence of pendant bases (found in Gd or Mn chelates) that limit versatile functionalization. As proof-of-principle, we demonstrate labeling of human serum albumin, a model protein, and collagen hydrogels for applications in in-vivo targeted imaging and material tracking, respectively. In-vitro and in-vivo results confirm unprecedented metal stability, ease of functionalization, and high T1 relaxivity. This new platform opens the door to ex-vivo validation by fluorescent imaging and multipurpose molecular imaging in vivo.


Introduction
Contrast agents are chemical species that boost contrast-to-noise and improve the sensitivity of detection across a broad range of medical imaging modalities. From highlighting tumors to identifying ischemic or inflammatory tissue, contrast agents are an indispensable facet in modern radiology. Different modalities utilize different mechanisms for contrast generation. In magnetic resonance imaging (MRI), gadolinium (Gd), a paramagnetic metal, is used to augment signal contrast [1]. With seven unpaired electrons, Gd induces very efficient relaxation of longitudinal relaxation, but it is toxic in free, ionic form and must be chelated for sequestration and safe elimination from the body. To minimize risk of demetalation in vivo, all clinically approved Gd agents are designed for rapid elimination, with a half-life under 1.5 to 2 h in healthy subjects [2]. Longer-circulating Gd agents have also been used clinically, with a recent example being Ablavar, a blood-pool agent with an affinity for albumin and an elimination half-life of 18.5 h [3]. However, in 2017, Ablavar was unexpectedly withdrawn from the market by Lantheus Medical, citing poor sales; interestingly, the parent compound of Ablavar, Gd-DTPA, was internationally restricted ceeds the kinetic stability of any known Gd-or Mn-based contrast agent. MnTPPS 3 NH 2 can then be functionalized with isothiocyanate, an electrophilic moiety ubiquitous in fluorescent bioconjugation, to form sodium Mn(III) 5-(4-phenylisothiocyanate)-10,15,20-(tri-4-sulfonatophenyl)porphyrin chloride (MnTPPS 3 NCS), for which we report the first successful synthesis and characterization. To demonstrate the conjugation versatility of our new Mn BFCA-MnTPPS 3 NCS, we use it to covalently label human serum albumin (HSA), a model protein, and collagen hydrogel, a common scaffolding material in tissue engineering applications.

Stability of MnTPPS 3 NH 2
Low toxicity of the MnTPPS 3 NH 2 precursor has previously been demonstrated on different cell lines [20,21], including human embryonic stem cells [22]. More generally, sulfonated porphyrins have been shown to remain intact in vivo and after elimination from the body in many animal studies [23,24]. The high thermodynamic stability of Mn 3+ porphyrins is well established, with high kinetic stability of MnTPPS 3 NH 2 specifically suggested in our previous experience in material labeling [18,20,25]. In this study, we aimed to confirm its kinetic stability before attempting to convert the single aromatic amine on a pre-metalated porphyrin to an isothiocyanate electrophile. In the following, we provide evidence in support of high stability: kinetic inertness was maintained at low pH, and trans-metalation (leading to Mn 2+ dissociation) was negligible even over long intervals in the presence of competitive metals, even at low pH.
To create the MRI bioconjugation platform, commercially available porphyrin TPPS 3 NH 2 was metalated with slight modification of an established procedure [18], which is outlined in Supplementary Scheme S1. The structure and purity of the resulting MnTPPS 3 NH 2 were determined by ultraviolet-visible (UV-VIS) spectra, high performance liquid chromatography (HPLC), flame atomic absorption spectroscopy (FAAS), and mass spectrometry (ESI-MS) (Supplementary Figures S1-S3). The kinetic inertness of MnTPPS 3 NH 2 was confirmed using an acid decomposition test (Figure 1), a method used to predict the stability of Gd contrast agents in vivo [26]. In control PBS solution, demetalation was absent over the 260-day study, a finding that places the stability of MnTPPS 3 NH 2 much higher than that of most Mn 2+ chelates, which decompose in PBS. No Mn 3+ release by MnTPPS 3 NH 2 was detectable when stored in PBS or HSA (0.6 mM, PBS) after 260 days. Stress testing in 0.1 (400× competitive [H + ] excess) and 0.5 M HCl (2000× competitive [H + ] excess) (note: measurements are typically performed at pH = 1, or 0.1 M HCl) [26] resulted in minimal Mn 3+ release: less than 1% after 112 days. Only at a 10-fold increase of HCl (at 1 M) was trace demetalation detected earlier by day 12. Linearly extrapolating the rate of decay suggests that the dissociation half-life (T 1/2 ) for MnTPPS 3 NH 2 exceeds 17, 15, and 10 years at HCl concentrations of 0.1, 0.5, and 1 M, respectively. By comparison, we found that Gadovist, the most common clinical MRI contrast agent, completely dissociated by 48 h when subjected to 0.1 M HCl at 37 • C ( Figure S4). This is consistent with most stable clinical Gd MRI agents, whose half-lives range between 7 to 24 h under equivalent conditions [27,28]. Faring even more poorly, a majority of Mn 2+ chelates cannot match the kinetic stability of many linear Gd chelates, with acid decomposition half-lives in the range of seconds [29,30]. Even the most kinetically stable Mn 2+ chelate for use as an MRI contrast agent demonstrated to date was found to have a decomposition half-life of 70 min when subjected to 0.1 M HCl (100× competitive [H + ] excess) at 25 • C [31].
Trans-metalation with endogenous metal ions (e.g., Zn 2+ , Cu 2+ , and Fe 3+ ) is another means by which the chelated metal is exchanged and released. Most attention is directed towards Zn 2+ due to its higher relative abundance in plasma (50 µM). Therefore, ZnCl 2 stress testing is critical for assessing kinetic stability under physiological conditions. Figure 2a confirms the high kinetic inertness of MnTPPS 3 NH 2 against trans-metalation. Incubation of MnTPPS 3 NH 2 in a 12× excess of ZnCl 2 (i.e., 3 mM ZnCl 2 in PBS) did not result in detectable decomposition even after 112 days. At a 40,000× excess of ZnCl 2 (10 M in DI water, Int. J. Mol. Sci. 2023, 24, 9532 4 of 16 pH 5.5), only 0.33% trans-metalation and 0.47% demetalation were detected by 112 days. A high resistance against trans-metalation was expected given that both trans-metalation and demetalation of porphyrins were previously reported to be primarily mediated by acid hydrolysis [13]. MnTPPS 3 NH 2 was demonstrated to maintain impressive resistance to decomposition in conditions of combined acid and ZnCl 2 stress in Figure 2b, where only 0.2% trans-metalation was detected at pH 1 after 12 days and none at a pH of 3. This is notable given that even the most trans-metalation-resistant Mn 2+ chelates lose this resistance in acid and rapidly degrade as the pH approaches 3 and their pendant metal coordinating bases become protonated [31]. Trans-metalation with endogenous metal ions (e.g., Zn 2+ , Cu 2+ , and Fe 3+ ) is another means by which the chelated metal is exchanged and released. Most attention is directed towards Zn 2+ due to its higher relative abundance in plasma (50 µM). Therefore, ZnCl2 stress testing is critical for assessing kinetic stability under physiological conditions. Figure 2a confirms the high kinetic inertness of MnTPPS3NH2 against trans-metalation. Incubation of MnTPPS3NH2 in a 12× excess of ZnCl2 (i.e., 3 mM ZnCl2 in PBS) did not result in detectable decomposition even after 112 days. At a 40,000× excess of ZnCl2 (10 M in DI water, pH 5.5), only 0.33% trans-metalation and 0.47% demetalation were detected by 112 days. A high resistance against trans-metalation was expected given that both trans-metalation and demetalation of porphyrins were previously reported to be primarily mediated by acid hydrolysis [13]. MnTPPS3NH2 was demonstrated to maintain impressive resistance to decomposition in conditions of combined acid and ZnCl2 stress in Figure 2b, where only 0.2% trans-metalation was detected at pH 1 after 12 days and none at a pH of 3. This is notable given that even the most trans-metalation-resistant Mn 2+ chelates lose this resistance in acid and rapidly degrade as the pH approaches 3 and their pendant metal coordinating bases become protonated [31].  Trans-metalation with endogenous metal ions (e.g., Zn 2+ , Cu 2+ , and Fe 3+ ) is another means by which the chelated metal is exchanged and released. Most attention is directed towards Zn 2+ due to its higher relative abundance in plasma (50 µM). Therefore, ZnCl2 stress testing is critical for assessing kinetic stability under physiological conditions. Figure 2a confirms the high kinetic inertness of MnTPPS3NH2 against trans-metalation. Incubation of MnTPPS3NH2 in a 12× excess of ZnCl2 (i.e., 3 mM ZnCl2 in PBS) did not result in detectable decomposition even after 112 days. At a 40,000× excess of ZnCl2 (10 M in DI water, pH 5.5), only 0.33% trans-metalation and 0.47% demetalation were detected by 112 days. A high resistance against trans-metalation was expected given that both trans-metalation and demetalation of porphyrins were previously reported to be primarily mediated by acid hydrolysis [13]. MnTPPS3NH2 was demonstrated to maintain impressive resistance to decomposition in conditions of combined acid and ZnCl2 stress in Figure 2b, where only 0.2% trans-metalation was detected at pH 1 after 12 days and none at a pH of 3. This is notable given that even the most trans-metalation-resistant Mn 2+ chelates lose this resistance in acid and rapidly degrade as the pH approaches 3 and their pendant metal coordinating bases become protonated [31].  The results in Figures 1 and 2 demonstrate the unmatched inertness of the MnTPPS 3 NH 2 complex to acid decomposition, ZnCl 2 stress test, and the combination of both, surpassing all current Mn 2+ and Gd-based chelates. A survey of the Gd contrast agent literature reveals that Gd-DOTA and other macrocyclic chelates are considered stable against transmetalation by Zn 2+ at physiological conditions when their pendant coordinating bases are unprotonated, but these studies were conducted within one week [32,33]. One study of Gd-DOTA reported a half-life of 100 h in the presence of 100× Zn 2+ [34], which suggests the agent would not stand up to the long interval of testing done in our study. These stability results support that MnTPPS 3 NH 2 may be ideal for applications requiring extended residence times in vivo. Furthermore, they support the notion that MnTPPS 3 NH 2 can be functionalized with isothiocyanate without risk of demetalation.

Electrophilic Functionalization of MnTPPS 3 NH 2
Scheme 1i illustrates our conversion of the amine on MnTPPS 3 NH 2 or TPPS 3 NH 2 to an isothiocyanate. This led to a near quantitative conversion of isothiocyanate-functionalized MnTPPS 3 NCS at high purity according to UV-VIS spectra, HPLC, FAAS, and ESI-MS and Fourier transform infrared spectroscopy (FTIR-ATR) (Supplementary Figures S5-S9a). The Mn-free TPPS 3 NCS was also synthesized and characterized as above with the addition of a 1 HNMR spectrum (Supplementary Figures S9b-S13).

Electrophilic Functionalization of MnTPPS3NH2
Scheme 1i illustrates our conversion of the amine on MnTPPS3NH2 or TPPS3NH2 to an isothiocyanate. This led to a near quantitative conversion of isothiocyanatefunctionalized MnTPPS3NCS at high purity according to UV-VIS spectra, HPLC, FAAS, and ESI-MS and Fourier transform infrared spectroscopy (FTIR-ATR) (Supplementary Figures S5-S9a). The Mn-free TPPS3NCS was also synthesized and characterized as above with the addition of a 1 HNMR spectrum (Supplementary Figures S9b-S13).
It is worth noting that the advantages of a pre-metalated conjugation approach were previously recognized. An isothiocyanate-functionalized Gd (1,4,7,10tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid) derivative (Gd-DOTA-NCS) was synthesized to achieve bioconjugation with proteins and macromolecules [10,11,35]. This functionalization was only possible because Gd-DOTA-based derivatives are the most inert commercial Gd chelates and are sufficiently stable under acidic conditions during their brief conversion to an isothiocyanate. In seeking a Gd-free alternative, other metal chelates, particularly Mn 2+ , are insufficiently stable in acid to undergo isothiocyanate functionalization. Other methods of electrophilic activation that do not require acidic conditions, such as carbodiimide and succinimides, are also not viable, because their pendant nucleophiles necessary for metal chelation are unprotected [36][37][38]. Without the use of a protective group, pendant bases are vulnerable to activation and conjugation in carbodiimide and succinimide chemistry. This leads to side reactions where these bases reduce the coordination number and stability of a fraction of chelates. The consequence is that purified conjugated macromolecules may contain different chelate species resulting from side reactions. It is worth noting that the advantages of a pre-metalated conjugation approach were previously recognized. An isothiocyanate-functionalized Gd (1,4,7,10-tetraazacyclododecane-N,N ,N ,N -tetraacetic acid) derivative (Gd-DOTA-NCS) was synthesized to achieve bioconjugation with proteins and macromolecules [10,11,35]. This functionalization was only possible because Gd-DOTA-based derivatives are the most inert commercial Gd chelates and are sufficiently stable under acidic conditions during their brief conversion to an isothiocyanate. In seeking a Gd-free alternative, other metal chelates, particularly Mn 2+ , are insufficiently stable in acid to undergo isothiocyanate functionalization. Other methods of electrophilic activation that do not require acidic conditions, such as carbodiimide and succinimides, are also not viable, because their pendant nucleophiles necessary for metal chelation are unprotected [36][37][38]. Without the use of a protective group, pendant bases are vulnerable to activation and conjugation in carbodiimide and succinimide chemistry. This leads to side reactions where these bases reduce the coordination number and stability of a fraction of chelates. The consequence is that purified conjugated macromolecules may contain different chelate species resulting from side reactions.

Conjugation to Human Serum Albumin (HSA) and MR Imaging
As proof-of-principle, HSA, a protein ubiquitously demonstrated in most bioconjugate techniques, was used to exemplify bioconjugation of MnTPPS 3 NCS to proteins. Scheme 1ii depicts the general scheme; conditions adopted were common for conjugating isothiocyanates to proteins. It is possible to conjugate more than one MnTPPS 3 NCS to a single HSA. This would be beneficial, for example, if one wishes to achieve a higher longitudinal (T 1 ) relaxivity for maximum detection sensitivity. Coupling efficiency was first ascertained at various stoichiometries, with reactions performed with 5×, 10×, 20×, and 40× excess of MnTPPS 3 NCS to HSA. To rule out the possibility of non-specific binding of porphyrin to HSA, concurrent reactions at all relevant equivalencies were performed between MnTPPS 4 and HSA. The resulting degree of labeling and tagging efficiencies for both MnTPPS 3 NCS and control MnTPPS 4 are shown in Table 1. MnTPPS 3 NCS conjugation to HSA produced a notably high tagging efficiency of 60-70% for the lowest tagging equivalences tested (5× and 10× excess of Mn), leading to 3.5 and 6 MnTPPS 3 per HSA, respectively. This tagging efficiency decreased to 48% at higher excesses, yielding 9.5 and 19 MnTPPS 3 per HSA. Non-specific binding from MnTPPS 4 resulted in negligible tagging. Our results can be compared to fluorescent and chelating platforms for HSA conjugation (see Tables S1 and S2 in the Supplementary materials). The efficiency of MnTPPS 3 NCS for achieving high protein loading is particularly important in applications where the biological target concentration falls below the limit of detection on MRI for small molecule T 1 contrast agents.
In-vitro MRI scans on a clinical 1.5-Tesla scanner confirmed a large reduction in T 1 relaxation times induced by the HSA-conjugated contrast agents ( Figure 3). Relaxation rate scaled linearly with the degree of tagging, producing a relaxivity of 15.4 mM −1 s −1 when using 0.05 mM of bound MnTPPS 3 (Supplementary Figure S14). These results suggest that MnTPPS 3 NCS is an exceptional platform for protein conjugation and can achieve a high degree of labeling to achieve high sensitivity.
conjugating isothiocyanates to proteins. It is possible to conjugate more than one MnTPPS3NCS to a single HSA. This would be beneficial, for example, if one wishes to achieve a higher longitudinal (T1) relaxivity for maximum detection sensitivity. Coupling efficiency was first ascertained at various stoichiometries, with reactions performed with 5×, 10×, 20×, and 40× excess of MnTPPS3NCS to HSA. To rule out the possibility of nonspecific binding of porphyrin to HSA, concurrent reactions at all relevant equivalencies were performed between MnTPPS4 and HSA. The resulting degree of labeling and tagging efficiencies for both MnTPPS3NCS and control MnTPPS4 are shown in Table 1. MnTPPS3NCS conjugation to HSA produced a notably high tagging efficiency of 60-70% for the lowest tagging equivalences tested (5× and 10× excess of Mn), leading to 3.5 and 6 MnTPPS3 per HSA, respectively. This tagging efficiency decreased to 48% at higher excesses, yielding 9.5 and 19 MnTPPS3 per HSA. Non-specific binding from MnTPPS4 resulted in negligible tagging. Our results can be compared to fluorescent and chelating platforms for HSA conjugation (see Tables S1 and S2 in the Supplementary materials). The efficiency of MnTPPS3NCS for achieving high protein loading is particularly important in applications where the biological target concentration falls below the limit of detection on MRI for small molecule T1 contrast agents.
In-vitro MRI scans on a clinical 1.5-Tesla scanner confirmed a large reduction in T1 relaxation times induced by the HSA-conjugated contrast agents ( Figure 3). Relaxation rate scaled linearly with the degree of tagging, producing a relaxivity of 15.4 mM −1 s −1 when using 0.05 mM of bound MnTPPS3 (Supplementary Figure S14). These results suggest that MnTPPS3NCS is an exceptional platform for protein conjugation and can achieve a high degree of labeling to achieve high sensitivity. In-vivo MRI scans of rats on a clinical 1.5-Tesla scanner injected intravenously with the blood-pool agents demonstrate a large T 1 reduction in the intravascular space ( Figure 4). (MnTPPS 3 ) 3.5 HSA and (MnTPPS 3 ) 6 HSA were compared for their contrast efficiency and time-course signal change. Both agents provided stable T 1 in blood for at least one hour. A greater T 1 reduction was seen for (MnTPPS 3 ) 6 HSA due to higher metal loading per HSA molecule. In-vivo MRI scans of rats on a clinical 1.5-Tesla scanner injected intravenously with the blood-pool agents demonstrate a large T1 reduction in the intravascular space ( Figure  4). (MnTPPS3)3.5HSA and (MnTPPS3)6HSA were compared for their contrast efficiency and time-course signal change. Both agents provided stable T1 in blood for at least one hour. A greater T1 reduction was seen for (MnTPPS3)6HSA due to higher metal loading per HSA molecule.

Stability of (MnTPPS3)6HSA
The stability of the protein-conjugated MnTPPS3NCS linkage was also tested. (MnTPPS3)6HSA was subjected to a series of acid and zinc stress tests at 37 °C: Distilled water, HCl solutions at pH = 1 and 3, and ZnCl2 solutions at 40,000× equivalence in either distilled water or HCl solutions (pH = 1 and 3). After 13 days, solutions were spin filtered to remove decoupled Mn porphyrin and free Mn. Figure 5 shows the cumulative total free and decoupled Mn porphyrin for various stress tests. No significant (MnTPPS3)6HSA decomposition was detected over the course of 13 days under any condition tested, which is consistent with our results for MnTPPS3NH2. This indicates that conjugates of MnTPPS3NCS remained inert to demetalation and trans-metalation. Furthermore, the thiourea linkage remained intact over 13 days in both physiological and highly acidic conditions.

Stability of (MnTPPS 3 ) 6 HSA
The stability of the protein-conjugated MnTPPS 3 NCS linkage was also tested. (MnTPPS 3 ) 6 HSA was subjected to a series of acid and zinc stress tests at 37 • C: Distilled water, HCl solutions at pH = 1 and 3, and ZnCl 2 solutions at 40,000× equivalence in either distilled water or HCl solutions (pH = 1 and 3). After 13 days, solutions were spin filtered to remove decoupled Mn porphyrin and free Mn. Figure 5 shows the cumulative total free and decoupled Mn porphyrin for various stress tests. No significant (MnTPPS 3 ) 6 HSA decomposition was detected over the course of 13 days under any condition tested, which is consistent with our results for MnTPPS 3 NH 2 . This indicates that conjugates of MnTPPS 3 NCS remained inert to demetalation and trans-metalation. Furthermore, the thiourea linkage remained intact over 13 days in both physiological and highly acidic conditions.

Labeling of Collagen
Targeting need not be limited to albumin or other endogenous entities. In fact, targeting materials has immense value in tissue engineering and regenerative medicine, and examples of material labeling with fluorescence abound. In contrast, material labeling with T1 agents is rare, and existing reports do not employ a covalent mechanism to permit equating signal loss to material degradation. In the following, we demonstrate the first covalent labeling of collagen for material tracking on T1-weighted MRI.

Labeling of Collagen
Targeting need not be limited to albumin or other endogenous entities. In fact, targeting materials has immense value in tissue engineering and regenerative medicine, and examples of material labeling with fluorescence abound. In contrast, material labeling with T 1 agents is rare, and existing reports do not employ a covalent mechanism to permit equating signal loss to material degradation. In the following, we demonstrate the first covalent labeling of collagen for material tracking on T 1 -weighted MRI.
Hydrogels composed of either 3 mg/mL or 10 mg/mL collagen were labeled in situ with MnTPPS 3 NCS. This procedure was repeated for both MnTPPS 4 and MnTPPS 3 NH 2 (two different controls) to account for possible retention of porphyrin due to non-specific binding. The retention of porphyrins in collagen was monitored over the course of 556 days ( Figure 6). Labeling with MnTPPS 3 NCS yielded much higher retention of MnTPPS 3 (56 ± 2% of total reacted MnTPPS 3 NCS) relative to controls MnTPPS 4 and MnTPPS 3 NH 2 , where the porphyrins were mostly removed after washing at day 0 ( Figure 6a). In fact, most MnTPPS 3 release occurred over the initial 5 days (Supplementary Figure S15), which is likely attributable to the release of residual free porphyrin. To further validate the results of MnTPPS 3 NCS binding, collagen samples were digested in nitric acid and assessed for Mn content by inductively coupled plasma-optical emission spectroscopy (ICP-OES) alongside their respective incubation and wash solutions (Figure 6b). ICP-OES results demonstrated the stability of MnTPPS 3 NCS collagen conjugates, with 3 mg/mL gels retaining 22 ± 3% (UV-VIS) and 26 ± 2% (ICP-OES), and 10 mg/mL gels retaining 46 ± 3% (UV-VIS) and 48 ± 3% (ICP-OES). Controls had no porphyrin retained, and the combined retention of MnTPPS 4 and MnTPPS 3 NH 2 was only −0.6 ± 4% (UV-VIS) and 2 ± 1.6% (ICP-OES).

Labeling of Collagen
Targeting need not be limited to albumin or other endogenous entities. In fact, targeting materials has immense value in tissue engineering and regenerative medicine, and examples of material labeling with fluorescence abound. In contrast, material labeling with T1 agents is rare, and existing reports do not employ a covalent mechanism to permit equating signal loss to material degradation. In the following, we demonstrate the first covalent labeling of collagen for material tracking on T1-weighted MRI.
Hydrogels composed of either 3 mg/mL or 10 mg/mL collagen were labeled in situ with MnTPPS3NCS. This procedure was repeated for both MnTPPS4 and MnTPPS3NH2 (two different controls) to account for possible retention of porphyrin due to non-specific binding. The retention of porphyrins in collagen was monitored over the course of 556 days ( Figure 6). Labeling with MnTPPS3NCS yielded much higher retention of MnTPPS3 (56 ± 2% of total reacted MnTPPS3NCS) relative to controls MnTPPS4 and MnTPPS3NH2, where the porphyrins were mostly removed after washing at day 0 ( Figure 6a). In fact, most MnTPPS3 release occurred over the initial 5 days (Supplementary Figure S15), which is likely attributable to the release of residual free porphyrin. To further validate the results of MnTPPS3NCS binding, collagen samples were digested in nitric acid and assessed for Mn content by inductively coupled plasma-optical emission spectroscopy (ICP-OES) alongside their respective incubation and wash solutions (Figure 6b). ICP-OES results demonstrated the stability of MnTPPS3NCS collagen conjugates, with 3 mg/mL gels retaining 22 ± 3% (UV-VIS) and 26 ± 2% (ICP-OES), and 10 mg/mL gels retaining 46 ± 3% (UV-VIS) and 48 ± 3% (ICP-OES). Controls had no porphyrin retained, and the combined retention of MnTPPS4 and MnTPPS3NH2 was only −0.6 ± 4% (UV-VIS) and 2 ± 1.6% (ICP-OES). The above results clearly support the value of covalent binding in tracking material degradation. It is also worth mentioning that our pre-metalation approach is necessary in material labeling for a simple reason: metalation and removal of free metal, as required in conventional post-metalation, is impractical-even impossible-for viscous solutions and solids.

Labeling Acid-Soluble Collagen and MR Imaging
Pre-labeled, acid-soluble collagen offers more versatility for in-situ labeling, expanding the range of applications to include cell-compatible gelation and injectable hydrogels. To further demonstrate the versatility MnTPPS 3 NCS, we synthesized MnTPPS 3 tagged acidsoluble collagen, adopting methods used in collagen labeling with fluorescence [39]. This T 1 reducing acid-soluble collagen can be stored and used later, similar to acid-soluble collagen, and can be blended with regular collagen. The method of labeling is akin to the in-situ conjugation procedure above, except that immediately after labeling of hydrogels, the labeled collagen is redissolved in 0.5 M acetic acid and dialyzed for 1 week to remove excess salt and free MnTPPS 3 ( Figure S39). Notably, this dialysis process requires extensive time at pH < 3, which would likely lead to significant demetalation in most other T 1 contrast agents. However, our stability results for both MnTPPS 3 NH 2 and (MnTPPS 3 ) 6 HSA in acid suggest that collagen-conjugated MnTPPS 3 is highly unlikely to demetalate in one week. To test the T 1 efficiency of MnTPPS 3 NCS-tagged, acid-soluble collagen, purified dry MnTPPS 3 NCS-tagged, acid-soluble collagen was blended with unlabeled collagen at a 25% (v/v) to produce T 1 -labeled hydrogels 5 and 60 days before imaging on a clinical 3-Tesla scanner. T 1 -and T 2 -weighted images and quantitative T 1 and T 2 relaxometry maps are shown in Figure 7. A significant reduction of T 1 and T 2 relaxation times could be detected for MnTPPS 3 NCS-labeled collagen hydrogels relative to unlabeled collagen, with both T 1 and T 2 remaining low after 60 days.
hydrogels. To further demonstrate the versatility MnTPPS3NCS, we synthesized MnTPPS3 tagged acid-soluble collagen, adopting methods used in collagen labeling with fluorescence [39]. This T1 reducing acid-soluble collagen can be stored and used later, similar to acid-soluble collagen, and can be blended with regular collagen. The method of labeling is akin to the in-situ conjugation procedure above, except that immediately after labeling of hydrogels, the labeled collagen is redissolved in 0.5 M acetic acid and dialyzed for 1 week to remove excess salt and free MnTPPS3 ( Figure S39). Notably, this dialysis process requires extensive time at pH < 3, which would likely lead to significant demetalation in most other T1 contrast agents. However, our stability results for both MnTPPS3NH2 and (MnTPPS3)6HSA in acid suggest that collagen-conjugated MnTPPS3 is highly unlikely to demetalate in one week. To test the T1 efficiency of MnTPPS3NCStagged, acid-soluble collagen, purified dry MnTPPS3NCS-tagged, acid-soluble collagen was blended with unlabeled collagen at a 25% (v/v) to produce T1-labeled hydrogels 5 and 60 days before imaging on a clinical 3-Tesla scanner. T1-and T2-weighted images and quantitative T1 and T2 relaxometry maps are shown in Figure 7. A significant reduction of T1 and T2 relaxation times could be detected for MnTPPS3NCS-labeled collagen hydrogels relative to unlabeled collagen, with both T1 and T2 remaining low after 60 days.

Stability of Acid-Stressed MnTPPS 3 NH 2 against De-Metalation
The stability of MnTPPS 3 NH 2 against demetalation was tested in neutral and increasingly acidic solutions. Stock solution of MnTPPS 3 NH 2 was mixed with HCl or water in two replicates each. Resulting solutions were composed of 0.25 mM MnTPPS 3 NH 2 with either PBS, PBS and 0.6 mM HSA, 0.1 M HCl, 0.5 M HCl, or 1.0 M HCl. Solutions were stored in an incubator at 37 • C for either 1, 12, 70, 112, or 260 days. The sample condition of PBS and 0.6 mM HSA was only tested at t = 0 and after 260 days. These samples were then diluted in 10% acetonitrile and immediately assessed by the Waters ACQUITY H-class UHPLC system with PDA detector and single quadrupole MS detector at the University of Toronto Department of Chemistry ANALEST facility. This was compared to a series of standards measured for MnTPPS 3 NH 2 , TPPS 3 NH 2 , and ZnTPPS 3 NH 2 ( Supplementary  Figures S22-S24). These three porphyrins were also mixed to ensure adequate separation (Supplementary Figure S25). Examples of UHPLC spectra for sample conditions are included in Supplementary Figures S26 and S27.

Stability of Zinc-Stressed MnTPPS 3 NH 2 against Trans-Metalation
The stability of MnTPPS 3 NH 2 against demetalation and trans-metalation by ZnCl 2 was tested in neutral and increasingly acidic solutions. Concentrated stock solutions of MnTPPS 3 NH 2 and ZnCl 2 were first mixed in either distilled water or PBS, resulting in n = 2 solutions of 0.25 mM MnTPPS 3 NH 2 with either PBS and 3 mM ZnCl 2 (12×) or distilled water and 10 M ZnCl 2 (40,000×). Acidic samples were similarly prepared from concentrated stock solutions before HCl was added, making samples comprising 10 M ZnCl 2 (40,000×) with either 0.1 M or 0.003 M HCl in two replicates each. The resulting solutions were stored in an incubator at 37 • C, where zinc-stressed samples were tested at 1, 12, or 112 days. These samples were diluted in 10% acetonitrile immediately before being assessed by UHPLC and compared to standards. Examples of UHPLC spectra for sample conditions are included in Supplementary Figures S28 and S29. 3.6. Conjugation of MnTPPS n NCS to HSA MnTPPS 3 NCS was added at different stoichiometric excess (0×, 5×, 10×, 20×, 40×, and 80×) to aqueous HSA solutions under vigorous stirring. Sodium carbonate buffer (0.1 M) solution was added to reaction until the pH reached 9 and was left for 24 h. Purification of (MnTPPS 3 ) n -HSA was performed with 50 mL ultra spin filters (Amicon Ultra Regenerated cellulose 50 kDa MWCO; Millepore, Darmstadt, Germany) for 5 min at 4000 rpm to remove unbound porphyrin. Reaction solutions were repeatedly spin filtered with PBS well past the point that the resulting filtrate was transparent (9×). Following this, 3× more filtrations were completed with deionized water to remove PBS salt. Reaction solutions where then lyophilized producing a green colored solid that could be dissolved and used directly as intravascular contrast agent. Matrix-assisted laser desorption/ionization (MALDI) was used to determine MnTPPS 3 NCS tagging efficiency by change in average molecular weight of HSA. As a control to account for possible non-specific absorption of porphyrin; this entire synthesis procedure was also performed with MnTPPS 4 .

Characterization of MnTPPS n NCS and MnTPPS 4 Labelled has
The degree of conjugation was determined by a Bruker Autoflex Speed matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF/MS) at the University of Toronto Department of Chemistry AIMS Mass Spectrometry Laboratory ( Supplementary Figures S30 and S31). MnTPPS 4 was used as a control for non-covalent absorption to HSA. Concentration and confirmation that free MnTPPS 3 was removed was determined with a AAnalyst 100 system (PerkinElmer, Waltham, MA, USA) with a Manganese Lumina Hollow Cathode Lamp at the University of Toronto Department of Chemistry ANALEST facility. (MnTPPS 3 ) 3.5 HSA was found to have a purity of 99.0% while (MnTPPS 3 ) 6 HSA had a purity of 97.4%.

Stability of (MnTPPS 3 ) 6 HSA
The stability of (MnTPPS 3 ) 6 HSA against acid hydrolysis, demetalation, and transmetalation was tested. Stock solution of (MnTPPS 3 ) 6 HSA was mixed into test solutions comprising distilled water and HCl or stock solution of ZnCl 2 , with two replicates each. For acid-stress testing, the resulting solutions contained 0.25 mM (MnTPPS 3 ) 6

In-Vitro MRI of (MnTPPS 3 ) n HSA
For in-vitro MRI measurements, 0.05 mM solutions of (MnTPPS 3 ) n HSA (n = 3.5, 6, 9.5, 19) were dispensed into borosilicate glass tube phantoms with 4 mm inner diameters. Solutions were prepared by weighing out lyophilized HSA with concentrations based upon average m/z determined by MALDI-TOF/MS. Tubes were placed in a 7 × 5 ultem resin circular holder with each solution being at least 5 mm in depth. Imaging was performed on a research 1.5 T MRI scanner (Siemens Aera, Siemens Healthineers, Erlangen, Germany) in the UHN STTARR facility using two flat surface coils. Quantitative T 1 relaxation times were measured using a two-dimensional (2D) inversion-recovery TSE sequence: inversion times (TI) = [25,50,75,100,200,250,500,750,1000,1250,1500,1750,2000, 2500] ms, repetition time (TR) = 3000 ms, echo time (TE) = 18.453 ms, field-of-view (FOV) = 60 mm, slice thickness (SL) = 2 mm, 0.4688 mm × 0.4688 mm in-plane resolution, and number of signal averages (NSA) = 1. Quantitative T 2 relaxation times were measured using multi-echo SE sequence: 11 echoes with TE spacing = 12 ms, TR= 2500 ms. MRI data were transferred to an independent workstation for quantitative analysis using in-house software developed in MATLAB (v.9.10, MathWorks). Calculations of T 1 and T 2 relaxation times were performed on a pixel-by-pixel basis as described previously [42,43]. T 1 and T 2 were averaged over all pixels within an area of interest and reported as mean values and standard deviation.

In-Vivo MRI of Rat Injected with (MnTPPS 3 ) 3.5 HSA and (MnTPPS 3 ) 6 HSA
All animal procedures were performed in compliance with a protocol approved by the University Health Network's Animal Resources Centre and associated animal care committee (protocol number: 6209). Two 8-weeks old Sprague Dawley female rats (Charles River Laboratories, Senneville, QC, Canada) were used as subjects. Rat 1 was injected via the tail vein with 0.65 mL of (MnTPPS 3 ) 3.5 HSA at 0.01 mmol/kg of Mn and imaged over the course of 40 min. Rat 2 was injected via the tail vein with 1 mL of (MnTPPS 3 ) 6 HSA at 0.02 mmol/kg of Mn and imaged over the course of 60 min. Imaging was performed on a research 1.5 T MRI scanner (Siemens Aera, Siemens Healthineers, Erlangen, Germany) in the UHN STTARR facility using two flat surface coils. Quantitative T 1 relaxation times were measured using a three-dimensional (3D) T1-weighted VIBE sequence with variable flip angles: TR = 25 ms, TE = 3.52 ms, flip angles (FA) = [2,10,20,25,35], FOV = 20 cm, SL = 3 mm, 0.7821 mm × 0.781 mm in-plane resolution, and NSA = 1.

Labelled Collagen Stability Experiments
Acid-purified bovine type 1 collagen (Advanced Biomatrix, Carlsbad, CA, USA) at concentrations 3 and 10 mg mL −1 was dispensed (1 mL) into 14 mL round-bottom falcon tubes on ice. Next, 0.25 mL of cold stock solutions of one of MnP-NCS, APO-NH 2 , MnTPPS 4 , or MnP-NH 2 was dispensed into tubes with collagen gel (n = 2) and mixed thoroughly. Next, to solidify gels, 0.25 mL of cold, freshly prepared 0.5 M Na 2 CO 3 was added and mixed with collagen gels. Gels were then left in the fridge for 24 h at 4 • C to solidify before being transferred to 50 mL falcon tubes filled with PBS for an initial rinse before being left in an incubator at 37 • C for 24 h in 25 mL PBS. The PBS solution was then removed from gels, and then, the gels were rinsed 2 times with 40 mL PBS and centrifuged to remove remaining free porphyrin. These solutions were put aside for quantification of free porphyrin by UV-VIS and ICP. Then, 40 mL of PBS and the antibiotic streptomycin was added to collagen gels before being put back into the incubator at 37 • C. Rinse solutions of PBS containing free porphyrin were each weighed before measurement by UV-VIS ( Figures S32-S34) to determine the amount of unbound collagen at day 0. Free porphyrin in sample solutions was measured periodically by UV-VIS (at 467 nm) over a month to determine the amount of porphyrin remaining inside collagen gels ( Figures S35 and S36). After 556 days, sample solutions were measured by UV-VIS before collagen gels were removed for digestion for ICP analysis. For digestions, collagen samples were added to falcon tubes containing 2 mL 70% nitric acid and left overnight. Samples were subsequently sonicated for an hour at 40 • C before distilled water was added to dilute to 4% nitric acid and filtered through a 0.4 µm PTFE filter (EMD Millipore-Merck, Darmstadt, Germany). Digested collagen samples and their corresponding consolidated solutions composed of both initial rinse solutions and long-term leached porphyrin solutions were weighed and sent for ICP analysis on a Thermo Scientific iCAP Pro ICP OES to determine the moles of Mn to compare to results from UV-VIS. The estimated moles of porphyrin per mole of collagen or gram of collagen are provided in Figures S37 and S38 and were calculated from % binding determined from UV-VIS and ICP.

Conclusions
This work demonstrates proof-of-principle for a pre-metalated conjugation platform that exploits a Gd-free T 1 contrast agent for conjugating proteins or labeling materials to achieve specific, targeted imaging. The stability of our starting material, MnTPPS 3 NH 2 , surpassed any current Mn 2+ or Gd-based contrast agents, resisting both demetalation and trans-metalation. Free from risk of demetalation, this compound was then converted to an electrophile, MnTPPS 3 NCS. Serving as a BFCA, MnTPPS 3 NCS achieved outstanding conjugation efficiency for the model human protein, plasma HSA, surpassing those recorded for previous chelates or fluorophores. The versatility and stability limits of MnTPPS 3 NCS conjugates were also tested with the labeling of collagen hydrogels. Labeled hydrogels produced substantial T 1 signal enhancement, with the conjugated porphyrin remaining stable for over a year. The proposed MnTPPS 3 NCS platform offers a simple and effective technique for labeling proteins for targeted molecular imaging and for specialized MRI applications such as biomaterial tracking in regenerative applications.