A needleless liquid jet injection delivery method for cardiac gene therapy: a comparative evaluation versus standard routes of delivery reveals enhanced therapeutic retention and cardiac specific gene expression

Abstract

This study evaluates needleless liquid jet method and compares it with three common experimental methods: (1) intramuscular injection (IM), (2) left ventricular intracavitary infusion (LVIC), and (3) LV intracavitary infusion with aortic and pulmonary occlusion (LVIC-OCCL). Two protocols were executed. First (n = 24 rats), retention of dye was evaluated 10 min after delivery in an acute model. The acute study revealed the following: significantly higher dye retention (expressed as % myocardial cross-section area) in the left ventricle in both the liquid jet [52 ± 4] % and LVIC-OCCL [58 ± 3] % groups p < 0.05 compared with IM [31 ± 8] % and LVIC [35 ± 4] %. In the second (n = 16 rats), each animal received adeno-associated virus encoding green fluorescent protein (AAV.EGFP) at a single dose with terminal 6-week endpoint. In the second phase with AAV.EGFP at 6 weeks post-delivery, a similar trend was found with liquid jet [54 ± 5] % and LVIC-OCCL [60 ± 8] % featuring more LV expression as compared with IM [30 ± 9] % and LVIC [23 ± 9] %. The IM and LVIC-OCCL cross sections revealed myocardial fibrosis. With more detailed development in future model studies, needleless liquid jet delivery offers a promising strategy to improve direct myocardial delivery.

Figure 1

Cardiac Gene Delivery Methods (A) IM Injection (B) Liquid Jet Injection Delivery (C) Intra left ventricular cavitary Infusion LVIC (D) Infusion featuring dual occlusion of aortic and pulmonary vessels LVIC-OCCL

Figure 2

Myocardial and Liver Cross Section Acute Study Results [4x magnification] By Group IM Injection, Liquid Jet Delivery, LVIC-OCCL and LVIC Infusion. Rhodamin B dye fluorescence (red-yellow intensity) demonstrated in myocardial cross sections (top) and systemic exposure in the liver sections (bottom). The delivery route had a significant impact on retention and the resultant intensity distribution profile immediately after 10 minutes.

Figure 3

Acute Model Results: (A) Liquid Jet and LVIC-OCCL demonstrate significantly greater retention versus both IM and LVIC groups * p<0.05 confidence level via Tukey’s test. (B) All groups resulted in systemic exposure (C) Direct methods, IM and Liquid Jet combined demonstrate lower overall exposure versus Infusion methods or LVIC-OCCL and LVIC combined, * p<0.05.

Figure 4

Left Ventricular Cross Section GFP Expression Distribution Per Delivery Method [4x magnification]. Top to Bottom, IM Injection, Liquid Jet, LVIC-OCCL and LVIC AAV groups. Immunohistochemistry revels EGFP (purple-brown) detection while (white-blue) regions are void. EGFP Expression profiles presented in the Middle and Basal LV sections (left to right) per group with 2D myocardial area coverage expressed as a %.

Figure 5

Masson’s Trichrome staining results indicating myocardial damage per delivery method, blue area indicates (+) LV fibrosis regions. The IM injection (A) and LVIC-OCCL (B) groups impose moderate left ventricular damage following delivery while the Liquid Jet (C) and LVIC (D) groups did not present with any indication of fibrosis.

Figure 6

Ex Vivo Ovine Methylene Blue Results for translational potential of needleless liquid jet injection (A) Liquid Jet entry points located on the epicardial surface demonstrate the jet’s local, yet dispersive profile along the adjacent surface contours and (B) robust transmural delivery retained per injection site within the endocardial surface.