Targeted delivery of thymosin beta 4 to the injured myocardium using CREKA-conjugated nanoparticles

Abstract

Purpose: Thymosin beta 4 (Tβ4) has multiple beneficial facets for myocardial injury, but its efficiency is limited by the low local concentration within the infarct. Here, we established a Tβ4 delivery system for cardiac repair based on the interaction between the abundant fibrin in the infarct zone and the fibrin-targeting moiety clot-binding peptide cysteine-arginine-glutamic acid-lysine-alanine (CREKA).

Methods and results: CREKA and Tβ4 were conjugated to nanoparticles (CNP-Tβ4). In vitro binding test revealed that CNP-Tβ4 had a significant binding ability to the surface of fibrin clots when compared to the control clots (NP-Tβ4). Based on the validation of fibrin expression in the early stage of ischemia injury, CNP-Tβ4 was intravenously administered to mice with acute myocardial ischemia-reperfusion injury. CNP-Tβ4 revealed a stronger fibrin-targeting ability than the NP-Tβ4 group and accumulated mainly in the infarcted area and colocalized with fibrin. Subsequently, treatment with CNP-Tβ4 resulted in a better therapeutic effect.

Conclusion: CRKEA modification favored Tβ4 accumulation and retention in the infarcted region, leading to augmented functional benefits. Fibrin-targeting delivery system represents a generalizable platform technology for regenerative medicine.

Keywords: CREKA; cardiovascular; fibrin; ischemia reperfusion; targeting delivery; thymosin beta 4.

Figure 1

Generation of CNP–Tβ4 and in vitro binding properties. Notes: (A) A schematic diagram of the synthesis of NP–Tβ4. (B) TEM images of NPs, NP–Tβ4, and CNP–Tβ4. (C) Particle size, zeta potential, surface density of moiety, and conjugation efficiency of NPs (n=3). (D) Fluorescent microscopic images showing the binding of CNP–Tβ4 (DiD labeled) to FFP clots. Microscope magnification = 200×. Abbreviations: CONH, amido bond; CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; NP, nanoparticle; TEM, transmission electron microscope; DiD, 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine; Fluoro, fluorescence; FFP, fresh-frozen plasma; COOH, carboxyl; PEG, poly(ethylene glycol); PLA, poly(lactic acid); LM, light microscope; MAL, maleimide; MPEG, methoxy poly(ethylene glycol); CREKA, cysteine–arginine–glutamic acid–lysine–alanine; PBS, phosphate-buffered saline; Tβ4, thymosin beta 4.

Figure 2

Pharmacokinetics of CNP–Tβ4 in mice. Notes: Blood Tβ4 concentration (% ID/mL) versus time after intravenous injection of CNP–Tβ4, NP–Tβ4, and Tβ4. Data are presented as the mean ± sd of n=6 mice/point. Abbreviations: CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; Tβ4, thymosin beta 4; % ID, percentage of injected dose; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; sd, standard deviation.

Figure 3

Fibrin expression in mice infarcts. Notes: (A) Fibrin expression in the infarcted area after 3 h reperfusion under a fluorescence microscope. Blue: cell nuclei, green: cTnT, and red: fibrin. (B) Time course of fibrin deposition in mice infarcts. All data are presented as mean ± sd. Abbreviations: cTnT, cardiac troponin T; DAPI, 4′,6-diamidino-2-phenylindole; LV, left ventricle; sd, standard deviation.

Figure 4

Fibrin-targeted CNP–Tβ4 accumulation in infarct heart. Notes: (A) Ex vivo optical imaging of the hearts at different times points post i.v. injection of DiR-labeled nanoagents (12 h, 24 h, 3 days, 7 days) and semiquantitative results of optical imaging of heart. Statistically significant differences were obtained when compared to the corresponding values of the other groups at each time (P<0.05). (B) Ex vivo optical imaging of other major organs at 24 h after i.v. administration of DiR-labeled nanoagents and semi-quantitative analysis of radiant efficiency of the organs. (C) Fluorescent microscopic images confirming enhanced CNP–Tβ4 targeting to fibrin of the injured heart. CNP–Tβ4 (red) conjoined fibrin (green) in the infarct area. Microscope magnification =200×. Abbreviations: CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; i.v., intravenous; DiR, 1,10-dioctadecyl-3,3,30,30-tetramethylindo-tricarbocyanine iodide; PBS, phosphate-buffered saline; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; Min, minimum; Max, maximum; DAPI, 4′,6-diamidino-2-phenylindole; NP, nanoparticle.

Figure 5

CNP–Tβ4 improved myocardial function and altered scar formation after MI in mice. Notes: (A) LVEFs. (B) FS. (C) Left ventricular EDD. (D) ESD. (E–G) Masson’s trichrome stained for histological assessments of infarct size and infarct wall thickness. Infarct size was presented as a percentage of the left ventricular free wall circumflexion length, and infarct wall thickness was presented as a percentage of the thickness of septal wall. The following images are higher magnifications (400×) of the earlier, respectively. Collagen in scar is indicated in blue and myocytes in red. *P<0.001 when compared with any other groups. ^#P<0.001 when compared with the PBS group. Abbreviations: CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; MI, myocardial infarction; LVEFs, left ventricular ejection fractions; FS, fractional shortening; EDD, end-diastolic diameter; ESD, end-systolic diameter; PBS, phosphate-buffered saline; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; Tβ4, thymosin beta 4.

Figure 6

CNP–Tβ4 promoted myocardium survival and cell proliferation after MI in vivo. Notes: (A) Representative images of TUNEL-positive myocytes in the LV 1 week post MI and treatment. (B) Quantification of apoptosis, expressed as the proportion of TUNEL-positive cells. (C) Representative images of Ki67-positive cells in the LV 1 week post MI and treatment. (D) Quantification of Ki67-positive cells. Microscope magnification: (A) 400× and (C) 800×. *P<0.05 when compared with the PBS group, ^#P<0.001 when compared with any other groups. Abbreviations: cTnT, cardiac troponin T; CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; MI, myocardial infarction; LV, left ventricle; PBS, phosphate-buffered saline; Tβ4, thymosin beta 4; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 7

CNP–Tβ4 reactivated the quiescent adult epicardium after MI in vivo. Notes: Immunohistochemical analysis (A and B) and Western blot analysis (C and D) show increase in Wt-1 expression notably in the thickened epicardium after CNP–Tβ4 treatment when compared to any other groups. Microscope magnification =400×. *P<0.05 when compared with the PBS group. ^#P<0.001 when compared with any other groups. Abbreviations: cTnT, cardiac troponin T; CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; MI, myocardial infarction; Wt-1, Wilms tumor 1; PBS, phosphate-buffered saline; Tβ4, thymosin beta 4; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 8

CNP–Tβ4 promoted vasculogenesis in the border area after MI in vivo. Notes: (A) Representative images of immunofluorescence using SMαA. (B) Quantification of arteriole density, expressed as the number of SMαA+ vessels per mm². (C) Representative images of immunofluorescence using CD31. (D) Quantification of capillary density, expressed as the number of CD31+ vessels per mm². (E and F) Western blot analysis of CD31 and SMαA protein post 1 week MI and treatment. Microscope magnification: (A) 400× and (C) 200×. ^#P<0.001 when compared with any other groups. Abbreviations: cTnT, cardiac troponin T; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CNP–Tβ4, cysteine–arginine–glutamic acid–lysine–alanine and thymosin beta 4 conjugated to nanoparticles; MI, myocardial infarction; SMαA, smooth muscle α-actin; CD31, cluster of differentiation 31; PBS, phosphate-buffered saline; Tβ4, thymosin beta 4; NP–Tβ4, thymosin beta 4 conjugated to nanoparticles; DAPI, 4′,6-diamidino-2-phenylindole.