(M1130-04-21) Penetratin and Transferrin Functionalized Liposomes Can Deliver Losartan Potassium to the Brain of Wistar Kyoto Rats

Purpose: The brain's paraventricular nucleus and circumventricular organs acting via angiotensin II receptor activity contribute to high blood pressure. Control of sympathetic activity has been proposed and successfully demonstrated to effectively reduce blood pressure in brain-induced high blood pressure. Strategies successfully demonstrated include direct injection of therapeutics into the brain (in rats) and surgical destruction of the renal nerves (in humans) are highly invasive and undesirable for clinical application. In addition, recent studies show that controlling sympathetic activity through angiotensin II receptor antagonism reduces blood pressure and further protects the brain from ischemia in spontaneously hypertensive rats. Unfortunately, most clinically used antihypertensives that target angiotensin II activity rarely cross the blood-brain barrier. Given the success of controlling blood pressure via angiotensin II receptor activity regulation, we propose that targeted delivery of losartan potassium (an angiotensin receptor blocker) into the brain will reduce neurogenic hypertension. In our preliminary studies, losartan potassium (an angiotensin II antagonist) delivered intravenously did not cross the Blood-Brain Barrier. In this study, we develop dual-functionalized losartan liposomes as a therapeutic tool to deliver Losartan into the brain of wistar kyoto rats. We study the formulations for drug release, stability, in-vitro and in-vivo biocompatibility, and cellular uptake. Our long-term aim is to develop a therapeutic tool to treat neurogenic hypertension.

Methods: This study initially evaluated Losartan potassium's ability to cross the blood-brain barrier when administered in solution. Wistar Kyoto rats were dosed with 3mg/kg (i.v. tail vein injection) Losartan potassium in saline. Losartan concentration in the brain and cerebrospinal fluid was estimated using the USP method for Losartan potassium with slight modification. The post-insertion technique was used in follow-up studies to prepare dual functionalized liposomes. Briefly, the phospholipids, DOPE, DOTAP, cholesterol, and DSPE-PEG ₍₂₀₀₀₎ (45:45:2:8) were dissolved in chloroform-methanol (2:1; v/v). The solvent was evaporated and lipid rehydrated with HEPES buffered saline pH 7.4 to form unfunctionalized liposomes. To achieve mono functionalization, either DSPE-PEG ₍₂₀₀₀₎ coupled to penetratin (D-Pen) or Transferrin (D-Tf) was added. Dual functionalized liposomes were formed by adding both D-Pen and D-Tf. Liposomes were purified by passing through a Sephadex G-100 column. Lissamine Rhodamine PE was added to the formulation at the organic phase to aid visualization. To form Losartan Potassium loaded liposomes, losartan potassium was added to the organic phase of the formulation. Liposomes were characterized for size, PDI, zeta potential, and encapsulation efficiency. The stability of the formulation, drug release, hemolytic ability, in-vitro cellular toxicity (in bEnd3, astrocytes and neuronal cells), and cellular uptake (in bEnd3 cells, human cerebral microvascular endothelial cell lines (hCMEC/D3), and neurons) were also evaluated. All quantitative studies were performed using the HPLC method previously established.

Results: From our HPLC method, Losartan has a retention time of 7.9 minutes (Figure 1a). No peak at this retention time was observed in the rats' brain tissue and Cerebrospinal fluid when injected with Losartan solution (Figure 1b). Losartan liposomes were successfully functionalized with both penetratin and Transferrin entrapping 66.8 ± 1.445 of Losartan when loaded at 8%w/w. The particle size, polydispersity index, and zeta potential for the dual functionalized losartan liposomes were 143.1 ± 4.676 nm, 0.285 ± 0.051, and 40.4 ± 3.93 respectively (Figure 1c). The liposome formulations were stable over 15 day storage period (Figure 2a) and showed a burst release profile (Figure 2b). Formulations were well tolerated in bEnd.3 cells, astrocyte, and neuronal cultures from our cytotoxicity studies (Figure 2 c-f). Blood compatibility was also assessed in a Hemolysis study, and again the formulations were well tolerated (Figure 2g). After a 4-hour in-vitro cellular uptake study in different cell lines, dual functionalized liposomes showed a superior uptake profile than single functionalized and unfunctionalized liposomes. (Figure 3 a-d). Finally when injected into wistar Kyoto rats, ~1.5 % injected dose of losartan per gram body weight was detected in the rat brain.

Conclusion: Losartan potassium in solution was not detected in the brain upon intravenous injection. Dual functionalized losartan liposomes were synthesized with desirable properties that can aid the transport of Losartan into the brain. The losartan liposomes have shown good in-vitro and in-vivo characteristics. Dual functionalized liposomes can be used to deliver losartan potassium to the brain of rats.

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


Figure 1. a. HPLC chromatogram of a. Losartan potassium (retention time 7.9min), Thioridazine - internal Standard (retention time 9.4), and EXP3174 - active metabolite of Losartan (retention time 11.1min) in rat tissue. b. Brain tissue from a rat that received Losartan potassium (3mg/kg i.v. tail vein injection) shows that Losartan potassium was not detected in brain tissue of rats that received 3mg/kg Losartan potassium, i.v. Tail vein injection. c. Table showing characterization of formulations for size, polydispersity, zeta, and encapsulation efficiency. d. encapsulation efficiency studies at different loading to select optimum loading e. average size and polydispersity distribution at different loading ratios.


Figure 2 a. Formulation stability studies over 15 days. b. Drug release studies of different formulations. - Cytotoxicity studies of different formulations in hCMEC/D3 (c) b.End3 (d.) astrocytes (e.) and neuronal (f.) cell lines. g. Haemolytic compatibility studies of different formulations.


Figure 3. Cellular uptake studies of losartan loaded liposomal formulations (a) b.End.3 (b) hCMEC/D3 (d) primary neurons (e) primary astrocytes. (e) HPLC chromatogram showing losartan detected in rat brain following injection of dual functionalized liposomes (f) Losartan concentration as estimated from HPLC detection.