(W0930-04-21) Synthesis and Characterization of Functionalized Liposomes Encasing Angiotensin II Receptor shRNA for Management of Neurogenic Hypertension

Purpose: Neurogenic hypertension is chronically elevated blood pressure initiated and maintained by the brain. A major hallmark of neurogenic hypertension is an over-activated sympathetic nervous system. Elevated sympathetic nervous activity is mediated through the actions of angiotensin II on its receptors found within the brain. Angiotensin II plays a key role in blood pressure regulation both directly (on blood vessels) or indirectly (in the central nervous system). Control of sympathetic nervous activity mediated by Angiotensin II has been proposed and successfully used as a suitable strategy for the management of neurogenic hypertension. Techniques previously used to regulate sympathetic activity include renal denervation, baroreflex receptor activation, (in humans) and direct injection of angiotensin receptor antisense into the brain (in rats). Although successful, these techniques are highly invasive and will be difficult to translate clinically. Further, recent studies show that controlling sympathetic activity through angiotensin II receptor antagonism not only reduces blood pressure but also protects the brain from ischemia in spontaneously hypertensive rats. Given the success achieved in controlling blood pressure via angiotensin II receptor antagonism, we propose that the safe and targeted delivery of angiotensin II shRNA into the brain will reverse neurogenic hypertension. In this study, we develop dual-functionalized liposomes as a therapeutic tool to deliver plasmid DNA encoding angiotensin II receptor shRNA into the brain of spontaneously hypertensive rats. Our long-term aim is to develop a therapeutic method to treat drug-resistant hypertension (or neurogenic hypertension).

Methods: In this study, we developed dual functionalized liposomes encasing plasmid DNA encoding for angiotensin II receptor shRNA. The post-insertion technique was used to prepare dual functionalized liposomes. Briefly, the phospholipids, DOPE, DOTAP, cholesterol, and DSPE-PEG ₍₂₀₀₀₎ were dissolved in chloroform-methanol (2:1; v/v) according to the ratio 45:45:2:8 mole % respectively. Following evaporation of the organic solvent the thin lipid film was hydrated with HEPES buffered saline pH 7.4 to form unfunctionalized liposomes. To achieve mono functionalization, either DSPE-PEG ₍₂₀₀₀₎ coupled to penetratin, or Transferrin was added. Dual functionalized liposomes were formed by adding both DSPEPeg conjugated penetratin and transferrin. Liposomes were purified by passing through a Sephadex G-100 column. To aid visualization, Lissamine Rhodamine PE was added to the formulation at the organic phase. Plasmid DNA encoding Angiotensin II receptor shRNA was loaded into the liposomes. Liposomes were characterized for size, PDI, zeta potential, and encapsulation efficiency. The biocompatibility of the formulation, in cell lines and erythrocytes, was investigated.

Results: Liposomes encasing pDNA encoding for angiotensin II receptor shRNA were successfully synthesized. Liposomes were successfully functionalized with both penetratin and Transferrin with an average hydrodynamic diameter of ~150nm (Figure1). Liposomes were uniformly distributed and had a positive surface charge (Figure 1). All formulations were well tolerated in bEnd.3 cells, astrocyte, neuronal and hCMEC/D3 cell cultures from our cytotoxicity (Figure 2A-D) studies. Blood compatibility was also assessed in a Hemolysis study, and again the formulations were well tolerated (Figure 3E).

Conclusion: Dual functionalized liposomes were synthesized with desirable properties that can aid the transport of pDNA encoding for angiotensin II receptor shRNA into the brain. The liposomes have shown good cellular compatibility. The ability to target the angiotensin II receptors in the brain holds excellent prospects in managing neurogenic hypertension.

Acknowledgements: This research was supported by National Institutes of Health Grants R01 AG051574 and RF1 AG068034.


Figure 1. Table showing characterization of formulations for size, polydispersity, zeta, and encapsulation efficiency.


Figure 2 Cytotoxicity studies of different formulations in (A.) hCMEC/D3 (B.) b.End3 (C.) primary astrocytes. (D.) primary neurons. (E.) biocompatibility studies with erythrocytes.