In situ gelling silk-elastinlike protein polymer for transarterial chemoembolization

Abstract

Hepatocellular carcinoma annually affects over 700,000 people worldwide and trends indicate increasing prevalence. Patients ineligible for surgery undergo loco-regional treatments such as transarterial chemoembolization (TACE) to selectively target tumoral blood supply. Using a microcatheter, chemotherapeutics are infused followed by an embolic agent, or the drug is encapsulated by the embolic moiety; simultaneously inducing stasis while delivering localized chemotherapy. Presently, several products are used, but no universally accepted system is promoted because very disparate limitations exist. The goal of this investigation was to design and develop in situ gelling recombinant silk-elastinlike protein polymers (SELPs) for TACE. Two SELP compositions, SELP-47K and SELP-815K, with varying lengths of silk and elastin blocks, were investigated to formulate a new embolic that was injectable through commercially available microcatheters. The goal was to develop a composition providing maximal permeation of tumor vasculature while exhibiting effective embolic activity. The SELPs evaluated remain soluble until reaching 37 °C, when irreversible transition ensues forming a solid hydrogel network. SELP-815K formulated at 12% w/w with shear processing demonstrated acceptable rheological properties and clear embolic capability under flow conditions in vitro. A rabbit model showed feasibility of embolization in vivo allowing selective occlusion of lobar hepatic arterial branches.

Keywords: Embolization; Hepatocellular carcinoma; Recombinant polymers; TACE.

Fig. 1. Diagram of SELP-47K and SELP-815K protein polymers (single letter amino acid abbreviations are used)

The silk-like block, GAGAGS, is represented in red and the elastin-like block, GVGVP, is represented in blue. While the overall content of silk and elastin-like blocks between SELP-47K and SELP-815K are comparable (approximately 1:2), SELP-815K has twice the number of silk and elastin-like blocks per repeat. When in alignment between strands, the silk repeats form beta sheets via hydrogen bonding. Because the silk repeats are twice longer in SELP-815K, they have twice the potential for formation of hydrogen bonds within each repeat. Greater numbers of hydrogen bonds results in stronger network formation and a stronger gel.

Fig. 2. Viscosity traces of candidate polymer compositions

The maximum viscosity target of 150 centipoise (cP) was specified to permit a formulation to be injectable through a commercially available 2.8F microcatheter. Viscosities were measured between 18 and 37°C, representing the injecting temperature range. A) 16% w/w formulations of reconstituted lyophilized polymer non-sheared (NS) of both SELP-47K (47K) and SELP-815K (815K) remained below 150cP. B) 14% w/w sheared SELP-815K began to gel upon thawing the sample and the viscosity was above the specification. Reduction to 12% w/w sheared SELP-815K provided appropriate viscosity. C) Comparison of non-sheared and sheared 12% w/w SELP-815K. 12% sheared (S) SELP-815K showed increasing viscosity as a result of network formation but the viscosity remained in the injectable range. D) Comparison of 12% w/w sheared SELP-815K and SELP-47K. The lack of increasing viscosity with increasing temperature by 12% w/w sheared SELP-47K indicated slower network formation. For all traces n=3 ± SEM.

Fig. 3. Rheological characterization of candidate polymer compositions

The shear storage modulus, G′, and the shear loss modulus, G″, were measured and used to assess time to gel formation and final gel stiffness over 5hrs. Panels A–D provide G′/G″ traces of candidate compositions. Panel E compares G′ of sheared and non-sheared 12% w/w SELP-815K, showing the effect of shear processing on final gel stiffness. Panel F shows the difference in rate of gelation between 12% w/w sheared SELP-815K and 12% w/w sheared SELP-47K; the faster gelation by 12% w/w sheared SELP-815K finalized the selection of this candidate to move into in vitro testing. All traces n=3 ± SEM.

Fig. 4. Comparison of the rheological characteristics between candidates

Panel A presents a comprehensive comparison of the 5 hr stiffness of the formulations. Statistical significance is evident between all groups except 12% w/w sheared SELP-815K and 12% w/w sheared SELP-47K. Panel B compares the 5 min modulus between 12% w/w sheared SELP-815K and 12% w/w sheared SELP-47K, showing that the SELP-815K formulation gels significantly faster. Panel C presents a summary of the key parameters and their results used to identify the suitable candidate for an embolic, 12% w/w sheared SELP-815K. *p<0.05, **p<.01, ***p<.001.

Fig. 5. Schematic of the in vitro test setup

Setup used to evaluate the occlusive ability of the candidate formulation under flow conditions. The image shows occlusion of the microfluidics device by 12% w/w sheared SELP-815K.

Fig. 6. 12% w/w sheared SELP-815K tested in vivo in male New Zealand White rabbits

A) Contrast observed filling the hepatic arterial supply, with catheter tip in proper hepatic artery. B) Contrast angiography at 5 min post SELP injection shows hard stasis, no flow into hepatic branches. C) Contrast angiography at 10 min post SELP injection shows continued stasis. Below each panel, higher magnification of the image is shown for clarity.

Fig. 7. Histological analysis of hepatic tissue locating embolic material

Rows A) and B) provide histological comparison of control and test animal hepatic tissues showing evidence of SELP filling the hepatic arteriole. Both hematoxylin and eosin (H&E) stain and an immunohistochemistry (IHC) stain specific for SELP were used for visualization. The arrow indicates arterioles and the arrowheads indicate the portal venules, for reference. The presence of vacuolization in the hepatocytes of the test animal (encircled) provides more evidence of hypoxic damage induced by occlusion of the hepatic arterial vessels upstream. Images taken at 152X total magnification. Rows C–E) show evidence of SELP within hepatic arterial supply (indicated by the arrows) and no evidence of flow through into the venous drainage via the hepatic central veins (indicated by the arrowheads). Row C, images taken at 30X total magnification, showing a portal triad and its draining central vein. Rows D and E show the portal triad and central vein individually at 152X total magnification. SELP is evident in row D within the arteriole indicated by the arrow. SELP is not evident tracking to or entering the draining central vein in row E.

Fig. 8. Histological sections of rabbit lungs

Panels A and B are sections from a saline control animal. Panels C and D are sections from an SELP injected animal. No evidence of SELP in the lungs of test animals was found. Lungs did not show signs of vascular occlusion. Images taken at 76X total magnification.