Recently a novel supramolecular strategy has been developed to directly assemble small molecular anticancer drugs into discrete, stable, well-defined nanostructures with a high and quantitative drug loading^1,2. In this study, we used a similar strategy to construct a novel injectable nanofiber hydrogel that is composed of only anticancer drug Pemetrexed (Pem) and a short peptide. Most importantly, the inherent CEST MRI signal of Pem allowed the label-free monitoring the location and status of injected drug-peptide hydrogel, making such a drug delivery system inherently image-guided. The design of PemFE is illustrated in Figure 1. In brief, Pem was chemically conjugated to a hydrophilic peptide (FE) to generate an amphiphilic prodrug molecule (Figure 1a) that can self-assemble into nanostructure (Figure 1b).^1,2 Two glutamic acid (EE) residues were chosen for their negative charge; two phenylalanine (FF) residues were chosen to promote the self-assembly^3,4. A C12-hydrocarbon-conjugated FFEE peptide (C12FE) was used as control. The self-assembly of PemFE and C12FE were prepared and characterized as previously reported.^1,2 A GL261 orthotopic brain tumor model was used for in vivo demonstration. In brief, C57BL6 mice (female, 5-6 weeks, n=4) were stereotactically injected with 2x10⁴ GL261 cells. Twenty-five days after the inoculation of tumor cells, hydrogel was injected into the tumors using the same stereotactic settings and in vivo MRI was performed before and 2 and 96 hours after the injection. CEST MRI acquisition and data processed were performed as previously reported (B1= 3.6 µT and Tsat= 3 sec)⁵. CEST contrast was quantified by MTRasym=(SΔω – S+Δω)/ S0.As shown in the cryogenic Transmission Electron Microscopy (TEM) images (Figures 2a,b), PemFE and C12FE molecules self-assemble into cylindrical-shaped nanofibers under physiological condition, with a diameter of 9.1 ± 1.4 nm and 8.5 ± 0.9 nm respectively. The length of these nanofibers was in the range of micrometer, resulting in the entanglement of nanofibers at high concentration and subsequently, the formation of hydrogel (Figure 2c). The intrinsic shear-thinning property of self-assembling PemFE and C12FE hydrogel enables fluidic injectable delivery under shear stress and recovers back to hydrogel state after injection, making them suitable for intratumoral drug delivery. The CEST MRI signals of Pem, PemFE and C12FE (10 mM per peptide or drug unit, pH 7.4, and 37 ^oC) PBS solutions were examined. As shown in Figure 2d,e,f, Pem and PemFE exhibited strong CEST effects at both 5.4 ppm and 2.0 ppm, likely attributed to aromatic amines and secondary amines. Then we performed experiment to investigate whether the CEST MRI signal can be used to monitor the drug delivery of hydrogel that was intratumorally injected to treat brain tumors. Figure 3 shows the T2w anatomical images and CEST images (at 5.4 ppm) of a representative mouse brain acquired before injection, 2 hours and 4 days after the injection. The results clearly show a conspicuous hyper-CEST signal on the injection side (right hemisphere) as compared to that in left hemisphere or that of pre-injection. Compared to that at 2-hour post injection, the area of the region showing hyper-CEST signal was noticeable larger, but the intensity of CEST signal became weaker on the 4th day, likely due to the slow release of drug from hydrogel to its nearby tissues. This study clearly demonstrated the ability of using CEST MRI to monitor drug delivery of PemFE hydrogel in vivo. In summary, we successfully developed a novel CEST MRI detectable drug-peptide nanofiber hydrogel system. With the inherent CEST MRI signal carried by Pem at 5.4 ppm, the location, distribution and drug release of the injected PemFE hydrogel could be easily monitored by CEST MRI in a label-free manner.