Current progress to improve the relaxivity of the contrast agents

Since relaxivity is a key determinant for dose efficiency of MRI contrast agents, several strategies have been applied to improve the relaxivity. One of the most used strategies currently is to increase τR, sincethe τR of small contrast agents is on the order of 100 ps, which is the limiting factor to increasing the relaxivity (Fig. 1-1B and C). Meade and coworkers have conjugated three to seven DOTA-Gd-alkyne complexes to azide to form a triazole ring with constrained local rotation of the Gd3+ complex. They found that the relaxivity of DOTA-Gd- alkyne increases along with molecular size and restricted rotation of Gd(III) chelates. Gd-DOTA- alkyne derivative with one Gd3+ has a relaxivity of 3.2 mmol-1 s-1 (at 60 MHz 37 ˚C). The per Gd3+ relaxivity with seven Gd-DOTA complex increases up to 12.2 mM-1 s-1 at 60 MHz 37 ˚C.66

Since τR increases with expanding molecular weight, macromolecular-based contrast agents have been generated either by covalent binding of small monomeric agents or by non- covalent binding to macromolecules such as proteins (e.g. serum albumin) and polylysine with an observed increase in relaxivity (Table 1-2).67-73 Nanosized candidates, such as dendrimers, micelles, liposomes, nanoparticle emulsion, lipid nanoparticles, viral capsid, and nanotubes (Ta- ble 1-2), have been conjugated with chelators such as DTPA or DOTA. Due to the increase in

molecular size, the per Gd3+ relaxivities of these contrast agents are increased several folds compared with current clinical contrast agents. Although the relaxivity values are much lower than the theoretical upper limit, the per particle relaxivity can be extremely high due to high amount of Gd3+ conjugation. For example, the per Gd3+ relaxivity of emulsion is 10.8 mM-1 s-1 at 60 MHz, which is approximately three-fold higher than that of Gd-DTPA. Raymond’s group con- jugates gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids which leads to a peak relaxivity of 41.6 mM-1 s-1 at 30 MHz74.

Unfortunately, it is not an easy task to optimize τR and τm for macromolecular-based contrast agents, as the macromolecular contrast agents face several limitations. First, the local motion of the Gd3+-1H vector is hard to optimize due to the flexible spacer between the carrier and the contrast agent. In addition, since r1 decreases when ω2τc2 > 1 (Equations 1-3 to 1-7), macromolecular contrast agents, such as nanoparticles that have a large τR, may exhibit a sharp drop in relaxivity at high magnetic field. Moreover, as our simulation shows in Fig. 1-1C, when macromolecules have optimized τR value (in range of 10 ns) in clinical magnetic fields, τm often becomes the limiting factor to significantly increasing the relaxivity. Macromolecule contrast agents face an additional size limitation because increased size results in low permeability in in vivo applications. Furthermore, the size and charge differences in these high payload MRI con- trast agents could influence blood distribution, which could potentially lower the efficient de- livery of the contrast agents to the desired targeting site.

Tight noncovalent aggregation between small Gd3+ contrast agents with protein deriva- tives can yield significantly higher relaxivity due to decreased local motion and increased τR.33 Binding to albumin, a 65 kDa protein in the serum, increases τR of MS-325, and consequently

increases the relaxivity of MS-325 from 6.9 to 42 mM-1 s-1 at 20 MHz 37 ˚C.75 Binding to albumin also increases the blood circulation of MS-325. Recently, several responsive contrast agents have been developed based on τR values that vary with a change in relaxivity due to either me- tabolite76 or zinc-triggered binding of small chelators to albumin.77 Proton relaxivity can also be increased by decreasing the internal motion of the contrast agents through multilocus binding.78 Caravan points out that targeting contrast agents to other proteins could potentially increase relaxivity by decreasing τR. For example, the relaxivity of EP-2104R, a fibrin-specific contrast agent with four Gd-DOTA molecules conjugated to a fibrin targeting peptide, increases from 10.1 mM-1 s1 at 60 MHz 37 ˚C in TBS buffer to 17.8 mM-1 s-1 in clotted plasma at 60 MHz 37˚C.79 Similarly, collagen targeted contrast agent, EP-353380, exhibits a comparable r1 increase

in the presence of plasma.

According to Equation 1-3, increasing q should significantly shorten the relaxation time of water protons and result in a higher relaxivity. However, increasing q for small chelators may decrease the stability of the metal chelator complex because of the risk of free Gd3+ release. Tweedle suggests that Gd3+ deposit in bones is correlated with instability of gadolinium contrast agents.21, 81, 82 In addition, when q increases from 1 to 2, Gd3+ has greater solvent accessibility and is able to interact with anions56, 57 which decreases the relaxivity. Therefore, most of the clinically approved contrast agents, except for ProHance with q = 1.3,81 have an inner sphere q value equal to 1.

Recently, Raymond’s group designed a series of contrast agents using hydroxypyridinone class compounds with increased q values. When q is equal to 2, r1 is

relaxivity is further increased up to 13.1 mM-1 s-1 at 20 MHz 25 ˚C. Interestingly, their primary stability constant pGd value is 18.7, which is similar to the stability constant of clinical contrast agents.85 However, the design of Gd3+ binding proteins with desired change of water coordination number is not reported. I will introduce my research on the optimization of relaxivity and metal stability of ProCA3 variants by manupulating of water number at the coordination shell (Chapter 4).

Table 1-2. The relaxivity of typical Gd3+- based MRI contrast agents. 61, 62, 72, 77, 78, 84-102 CA class Compounds r1 (mM-1 s-1) r2 (mM-1 s-1) B0 (T)

Designed proteins CA1 117 129 1.5

Small compound Gd-DTPA 5.4 8 1.5

Pyridine-N-oxide Analogues 4.54 0.47

HOPO derivatives Gd-TREN-1,2-HOPO 10.5 0.47

Gd-TACN-3,2-HOPO 13.1 0.47

Cell-permeable CAs Gd3+-DTPA-Arg8 7.8 1.5

Linear polymer Gd4(H2O) 4.5 9.4

Dendrimers Gadomer-17 13 1.5

Protein carriers Albumin 11.5 12.4 0.25

Poly-lysine 13 15 0.47

protein polymer contrast agents 14 1.5 High-density lipoprotein 41 55 0.47 Multimeric CAs 12.2 1.5 Targeting peptide conguated CAs EP-2104 24.9 0.47 17.8 27.7 1.5 EP-3533 27.9 0.47 15.6 32.5 1.5 EP-1084 27.7 0.47 Liposome ACPL 12 11 1.5

porous polymersomes porous polymersomes 7.2 1.5

Nanoglobular MRI CAs G3 nanoglobular MRI CA 10.0 3

Nanoparticle emulsion Gd-perfluorocarbon nanoparticles

34 50 1.5

Ca2+-protein α-lactalbumin 4.2 5 3

Nanoassembled Capsules NACs 24 0.54

Gold Nanoparticles Au@DTDTPA-Gdx 4.1 7

Y-DtNP ~60 0.7

Lipid Nanoparticles GdDOTA(GAC12)2 40 0.47

Hydroxypyridonate Viral capsid conjugates

Gd-TREN-bis-HOPO-TAM-CO2H 41.6 0.7

Dendrimer Nanoclusters dendrimer nanoclusters 12.3 1.5

Trimetallic Nitride Metallofullerene CA Gd3N@C80[DiPEG(OH)x] 79 2.4 Nanodiamond Nanodiamond 58.82 1.5 Paramagnetic nanoparticles Gd@C82(OH)22±2 61.1 4.7

1.3 DESIGN OF PROTEIN-BASED MRI CONTRAST AGENTS WITH HIGH RELAXIVITY