OBJECTIVES/SPECIFIC AIMS: This study aims to evaluate an injectable sulfonated reserve thermal gel (SPSHU-PNIPAM) for angiogenic growth factor delivery by examining the vascularization and cardioprotective properties of the polymer system. This study could lead to clinical translation by moving into larger animal studies and eventually clinical trials. The success of this study was determined by analyzing the results of echocardiography data on cardiac function (ejection fraction, fractional shortening, and left ventricle inner diameter) and assessment of histological staining on cardiac tissue (fibrotic tissue formation, infarct size, wall thinning, blood vessel cell counts, and vessel size quantification) after MI. Five groups were compared for this study: saline, VEGF, SPSHU-PNIPAM, SPSHU-PNIPAM loaded with VEGF, and no injection (sham). Significant statistical differences between control groups and polymer injection groups, when p < 0.05, indicates successful outcomes from this study. METHODS/STUDY POPULATION: SPSHU-PNIPAM Polymer Synthesis: SPSHU-PNIPAM was synthesized as previously described. Briefly, PSHU was synthesized with N-BOC serinol, urea, and HDI at 90 °C for 7 days. PSHU was deprotected in DCM and TFA at room temperature for 45 min. PNIPAM was conjugated to the deprotected PSHU using EDC and NHS at room temperature for 24 h. PSHU-PNIPAM was sulfonated with 1,3-propanesultone and potassium tert-butoxide at 60 °C for 3 days. Surgical Procedure: Male C57BL/6 mice weighing 24-28 g were anaesthetized using isoflurane and artificial ventilation provided. A small left thoracotomy incision was made at the left fourth intercostal space to expose the heart, and the proximal left anterior descending coronary artery was ligated for 45 min. The coronary artery was then released and 30 μl injections of saline, SPSHU-PNIPAM (1% w/v), bolus VEGF (200 ng), or SPSHU-PNIPAM + VEGF (1%, 200 ng) were injected intramyocardially at the infarcted site and the incision closed. Echocardiography and Histological Staining: Standard serial transthoracic echocardiography was performed while simultaneously recording ECG to assess cardiac morphology and left ventricular function. Immunohistochemistry and histology staining procedures were used to identify: fibrotic tissue formation, infarct size, wall thinning, blood vessel cell counts, and vessel size quantification. These were performed according to manufacturer instructions or by previously published criteria. Statistical Analysis: Two-tailed t-test assuming unequal variances was used to determine significant differences between two groups. Analysis of variance (ANOVA) was used to determine significant differences between three or more groups followed by Tukey-Kramer to determine significant differences between two groups as appropriate. Statistical significance was considered when p < 0.05. References: Lee, D. J., Rocker, A. J., Bardill, J. R., Shandas, R. and Park, D. (2018), A sulfonated reversible thermal gel for the spatiotemporal control of VEGF delivery to promote therapeutic angiogenesis. J Biomed Mater Res. doi:10.1002/jbm.a.36496. RESULTS/ANTICIPATED RESULTS: Echocardiography results: Ejection fraction improved for the SPSHU-PNIPAM groups compared to the saline, VEGF, and no injection controls (Figure 1). SPSHU-PNIPAM either loaded with or without VEGF seemed to have very similar treatment effects for ejection fraction and fractional shortening. This indicates that the more significant component of the cardioprotective effects of the hydrogel system is the biomaterial itself rather than the release of VEGF (Figure 1). However, the only statistically significant improvement for ejection fraction, fractional shortening, and left ventricular inner diameter that was observed compared to the saline, VEGF, and no injection controls was the SPSHU-PNIPAM + VEGF group (Figure 1). Histology Results: After analyzing Masson trichrome staining, SPSHU-PNIPAM + VEGF demonstrated the smallest infarct size after MI reperfusion injury and was statistically reduced compared to the saline, VEGF, and no injection controls (Figure 2). Furthermore, left ventricular wall thickness showed that the SPSHU-PNIPAM + VEGF treatment group reduced the wall thinning resulting from MI. The SPSHU-PNIPAM group without VEGF displayed a thicker ventricular wall as well, which may be attributed to the increased mechanical stability with the intramyocardial injection of the biomaterial (Figure 2). The immunohistochemical results for vascularization show that the SPSHU-PNIPAM + VEGF group significantly increased the number of functional vascular endothelial cells compared to the saline, VEGF, SPSHU-PNIPAM, and no injection controls (Figure 3). Additionally, the SPSHU-PNIPAM + VEGF group showed a significant increase in total vessel formation compared to the control groups, although there was no significant difference compared to SPSHU-PNIPAM without VEGF (Figure 3). The promotion of angiogenesis, without the delivery of VEGF, may be attributed to inflammation induced vascularization, including VEGF dependent vascularization that is initiated via signal transducer and activator of transcription 3 (STAT3) pathway that is induced by the pro-inflammatory cytokine interleukin 6. DISCUSSION/SIGNIFICANCE OF IMPACT: The SPSHU-PNIPAM loaded with VEGF was evaluated for therapeutic angiogenesis to protect cardiac function after MI. Treatment with SPSHU-PNIPAM showed improved cardiac function and vascularization; however, the additional delivery of VEGF showed inadequate additional therapeutic benefits. Further investigation will include optimizing VEGF release characteristics including both loading amount and release rate. The decline of ejection fraction and fractional shortening after MI were reduced, while left ventricular internal diameter showed reduced ventricular dilation. Both infarct size and left ventricular wall thinning decreased while an increase in the vessel formation was observed. These results demonstrate the SPSHU-PNIPAM biomaterial has cardioprotective and increased vascularization properties for the treatment of MI.