(T1230-02-13) Reduction of Blood Pressure in the Spontaneously Hypertensive Rats via the Nose-to-Brain Delivery of Plasmid DNA Encoding for Angiotensin II Receptor shRNA

Purpose: High blood pressure caused by angiotensin II receptor activity in the brain's paraventricular nucleus and circumventricular organs can be effectively reduced by controlling sympathetic activity. However, current strategies for achieving this, such as direct brain injections and renal nerve destruction, are invasive and not ideal for clinical use. With the demonstrated success of angiotensin II antagonism in reducing blood pressure there is a crucial need for less invasive strategies to introduce these therapies. Meanwhile nose to brain drug delivery is seen as a promising route to reach the brain in a less invasive way. Taking advantage of this pathway to overcome the challenge of NH, we propose using dual-functionalized liposomes to deliver plasmid DNA that encodes for angiotensin receptor shRNA (PEAS) through the nasal route to the brain. Our study involves the synthesis and characterization of dual functional liposomes and the examination of the in vitro safety and efficacy. We further investigate the extent of knockdown achieved within different brain regions involved in cardioregulation. Our ultimate goal is to develop a safe and effective therapeutic tool for treating neurogenic hypertension.

Methods: We created dual functionalized liposomes encasing plasmid DNA encoding angiotensin II receptor shRNA in this study. Dual functionalized liposomes were created using the post-insertion technique. Briefly, the phospholipids, DOPE, DOTAP, cholesterol, and DSPE-PEG (2000) were dissolved in chloroform-methanol (2:1; v/v) in the mole ratio 45:45:2:8. After the organic solvent was removed, the thin lipid film was hydrated with HEPES buffered saline pH 7.4 to form unfunctionalized liposomes. Monofunctionalization was achieved by incorporating either DSPE-PEG (2000) coupled to penetratin or Transferrin. Liposomes were purified using a Sephadex G-100 column. Liposomes were loaded with plasmid DNA encoding Angiotensin II receptor shRNA. The size, PDI, zeta potential, and encapsulation efficiency of liposomes were all measured. The formulation's biocompatibility was investigated in brain derived cell lines and erythrocytes. In vitro transfection efficiencies were investigated in brain derived cell lines. For qualitative evaluation of transfection efficiencies, liposomes were loaded with plasmid encoding for green fluorescent protein. In vivo blood effect on blood pressure was investigated after intranasal administration of PEAS-loaded liposomes.

Results: Surface-functionalized liposomes are safe and effective for targeted genetic delivery, which could be used to treat neurologic disorders. In this study, we synthesized dual-functionalized liposomes for nose-to-brain gene delivery and showed improved blood pressure control in NH. Liposomes were found to have mean diameters below 200 nm and polydispersities below 0.3, indicating particle homogeneity. Although liposome composition and size may affect plasmid DNA encapsulation, all synthesized liposomes entrapped over 88% of the plasmid and were able to protect it from DNase degradation. Dual functionalized formulations caused a significant (p < 0.05) decrease (over 70%) in AT₁r gene expression in brain derived cell lines. Microscopic observation of green fluorescence further confirmed the efficacies of liposomal nanoparticles to lead to transfection. Intranasal administration of PEAS-loaded liposomes resulted in a significant (p < 0.05) reduction of AT1r gene expression in all regions of the brain. Over 70% reduction in AT1r gene expression was observed in the hypothalamus region which was higher than the gene knockdown observed within the brainstem and rest of brain combined. In addition, intranasal administration to the spontaneously hypertensive rats led to an over 25 mmHg reduction in blood pressure, a reduction which was significant (p < 0.05) compared to the saline treated group.

Conclusion: Gene liposomes delivered intranasally have advantages over intravenous or intramuscular injections. Self-administered and non-invasive, it's convenient for patients. It bypasses the liver, reducing liver toxicity and allowing a lower therapeutic agent dose. Herein we synthesize dual-functionalized liposomes with plasmids for nose to brain delivery to manage NH. Liposomes functionalized with transferrin and TTC were found to have desirable sizes ( < 200 nm), loading capacities ( > 80%), and cargo protection. Trans-TTC liposomal nanoparticles transfected brain-derived cells better than mono- and unfunctionalized liposomes and had concentration-dependent toxicity. Dual-functionalized liposomes targeted the hypothalamus and brain stem and reduced AT1r gene expression in brain-derived cell lines (p < 0.001). Intranasal gene knockdown was greatest in the hypothalamus. This study shows that intranasal PEAS can treat NH for 4 weeks. This treatment's long-term effects and efficacy in humans need further study. This research advances new NH treatments and quality of life. Overall, functionalized liposomes could provide a promising tool for efficient and targeted genetic delivery for neurologic disorders.

Acknowledgements: This research was supported by National Institutes of Health Grants R01 AG051574, RF1 AG068034 and 3RF1AG068034-01A1S1.

Characterization of PEAS/pGFP-loaded liposomes for particle size, polydispersity index, and surface charge (zeta potential)

Assessment of in vitro transfection efficiencies of pGFP-loaded liposomes. a. Fluorescent microscopic images (primary astrocytes 100 nm, primary neuron 50nm) showing expression of green fluorescent proteins within the cells after treatment. b. Histograms of cell count vs FITC positive cells as recorded from 20000 ungated events.

Relative mRNA expression levels in different regions of the brain following in vivo administration of PEAS through the Intranasal and IV route. (side) Changes in blood pressure in SHR upon administration of PEAS-loaded liposomes. Data expressed as mean ± SEM. “**” and “***” indicates a significant (p < 0.01 and 0.001) reduction in mRNA expression compared to the respective PBS-treated group.