(M1430-01-04) Brain-Targeted Delivery of Plasmid DNA Encoding for Angiotensin II Receptor shRNA to Manage Neurogenic Hypertension

Purpose: According to the Centre for Disease Control and Prevention, nearly half of the adults in the United States are under antihypertensive therapy, yet two out of three still have uncontrolled blood pressure¹. Drug-resistant hypertension, also described as Neurogenic Hypertension (NH), is driven by the central actions of the peptide hormone angiotensin II (Ang II)². This fact is confirmed by several studies where reduced or inhibited Ang II activity within the brain has lowered blood pressure. Unfortunately, NH affects greater than 25% of the hypertension population globally. Yet, traditionally used antihypertensive formulations targeted towards Ang II fail to reach the brain in relevant therapeutic concentrations, primarily due to the protective effects of the blood-brain barrier. On the other hand, the lack of clinical translatability of surgical strategies (direct intracerebrovascular injections and renal arterial denervation) used to reduce or bypass Ang II activity limits the chances of finding a solution to this challenge³. However, the success of brain Ang II antagonism leads us to the hypothesis that the safe and brain-targeted delivery of Ang II shRNA into the brain will reverse NH. Therefore, our primary focus is to develop functionalized liposomes as a therapeutic tool to safely deliver plasmid DNA encoding Ang II receptor (AT₁r) shRNA (PEAS) to the brain and lower blood pressure. Our long-term aim is to develop a biocompatible, safe and clinically translatable method to treat NH, provide a sustainable solution towards NH management and improve the quality of life for the several people plagued by it.

Methods: Dual functionalized liposomes were prepared by the post-insertion technique. 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. The organic solvents were evaporated, and the thin lipid film hydrated with HEPES buffered saline pH 7.4 to form unfunctionalized liposomes. Mono functionalization was achieved by incorporating either DSPE-PEG ₍₂₀₀₀₎ coupled to transferrin or cell-penetrating peptide (penetratin or Tetanus toxin Fragment C (TTC)). Dual functionalized liposomes were formed by adding both DSPE-PEG conjugated penetratin and transferrin. PEAS electrostatically complexed to chitosan and loaded into the liposomes. Liposomes were characterized for size, PDI, zeta potential, and encapsulation efficiency. The biocompatibility (in bEnd3, astrocytes and neuronal cells) of the formulation, hemolytic ability, and gene transfection (in bEnd3 cells, human cerebral microvascular endothelial cell lines (hCMEC/D3), and neurons) were also evaluated. Efficacy of the formulation to reduce blood pressure was investigated in spontaneously hypertensive (SHR) rats.

Results: In this study, we synthesized and characterized dual-functionalized liposomes encasing PEAS. Liposomes were conjugated with transferrin and a cell-penetrating peptide (penetratin or Tetanus toxin Fragment C (TTC)) to improve brain targeting and trafficking of formulations across the BBB. All synthesized liposomes were positively charged, had an average hydrodynamic diameter of ~150nm and presented with a polydispersity of < 0.3 (Figure 1a). The size makes the liposomes suitable for BBB permeation, the cationic charge indicating ability to sufficiently interact with cells to allow uptake, and low PDI indicated that the formulations will be stable, and the observed size was uniformly distributed. All liposome formulations encapsulated more than 80% of plasmid DNA (Figure 1a) and protected the pDNA from degradation by DNase (Figure 1b). Functionalized liposomes showed good in vitro cellular compatibility and transfection ability in brain-derived cell cultures (Figure 2a-c) and erythrocytes (Figure 1d), providing confidence for their use in in vivo settings. Transfection of hypothalamic neuronal cultures with PEAS using dual functionalized liposomes resulted in a significant (p < 0.05) reduction of AT₁r gene expression by the third day and a prominently significant (p < 0.01) reduction by day seven (Figure 3a-b). In bEnd.3 cell cultures, dual functionalized PEAS liposomes showed superior ability to reduce AT₁r expression. When administered in vivo to SHR rats, dual functionalized PEAS liposomes showed a significant (p < 0.05) reduction in blood pressure when administered.

Conclusion: Dual functionalized liposomes were synthesized with desirable properties that can aid the transport of PEAS into the brain of SHR rats. The liposomes have shown good physicochemical properties and good cellular compatibility. Dual functionalized liposomes showed a superior ability to knock down the AT₁r gene in in vitro cell cultures as well as cause a significant reduction in mean arterial blood pressure of SHR rats. Dual functionalized liposomes can be a potential candidate for in vivo brain targeted delivery of therapeutics to manage NH.

References: 1. Prevention CfDCa. Hypertension cascade: hypertension prevalence, treatment and control estimates among US adults aged 18 years and older applying the criteria from the American College of Cardiology and American Heart Association’s 2017 Hypertension Guideline—NHANES 2013–2016. Atlanta, GA: US Department of Health and Human Services. 2019;
2. Osborn JW, Fink GD, Sved AF, Toney GM, Raizada MK. Circulating angiotensin II and dietary salt: converging signals for neurogenic hypertension. Current hypertension reports. 2007;9(3):228-235.
3. Carey RM, Calhoun DA, Bakris GL, et al. Resistant hypertension: detection, evaluation, and management: a scientific statement from the American Heart Association. Hypertension. 2018;72(5):e53-e90.


Figure 1. A. Table showing characterization of formulations for size, polydispersity, zeta, and encapsulation efficiency. B. DNase protecting effect of different polymeric micelles containing pVGF. Columns are described to the right.


Figure 2. Cytocompatibility studies of different formulations in (A.) primary astrocytes (B.) primary neurons (C.) b.End3 (D.) erythrocytes.


Figure 3 A and B. mRNA quantification following administration of PEAS loaded liposomes in primary neuron cultures. Cytocompatibility studies C and D. mRNA quantification of AT1r following administration of different formulations in b.End3 cell lines. E. Mean blood pressure measurements of Spontaneously hypertensive rats following treatment with PEAS.

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