Importance of thorough tissue and cellular level characterization of targeted drugs in the evaluation of pharmacodynamic effects

Abstract

Targeted nanoparticle delivery is a promising strategy for increasing efficacy and limiting side effects of therapeutics. When designing a targeted liposomal formulation, the in vivo biodistribution of the particles must be characterized to determine the value of the targeting approach. Peroxisome proliferator-activated receptor (PPAR) agonists effectively treat metabolic syndrome by decreasing dyslipidemia and insulin resistance but side effects have limited their use, making them a class of compounds that could benefit from targeted liposomal delivery. The adipose targeting sequence peptide (ATS) could fit this role, as it has been shown to bind to adipose tissue endothelium and induce weight loss when delivered conjugated to a pro-apoptotic peptide. To date, however, a full assessment of ATS in vivo biodistribution has not been reported, leaving important unanswered questions regarding the exact mechanisms whereby ATS targeting enhances therapeutic efficacy. We designed this study to evaluate the biodistribution of ATS-conjugated liposomes loaded with the PPARα/γ dual agonist tesaglitazar in leptin-deficient ob/ob mice. The ATS-liposome biodistribution in adipose tissue and other organs was examined at the cellular and tissue level using microscopy, flow cytometry, and fluorescent molecular tomography. Changes in metabolic parameters and gene expression were measured by target and off-target tissue responses to the treatment. Unexpectedly, ATS targeting did not increase liposomal uptake in adipose relative to other tissues, but did increase uptake in the kidneys. Targeting also did not significantly alter metabolic parameters. Analysis of the liposome cellular distribution in the stromal vascular fraction with flow cytometry revealed high uptake by multiple cell types. Our findings highlight the need for thorough study of in vivo biodistribution when evaluating a targeted therapy.

Fig 1. Targeted liposome synthesis.

Liposomes were synthesized with reverse phase evaporation, loading them with a 1 M Calcium Acetate Ca(OAc)₂ solution in preparation for remote loading with PPAR agonist tesaglitazar. Liposome buffer exchanges from Ca(OAc)₂ to saline, to 3.3 mg/mL tesaglitazar in HEPES, and then back to saline were performed with size exclusion chromatography. Targeting peptides were then added to the liposomes with the post insertion method by incubating liposomes with peptide micelles.

Fig 2. ATS-targeted tesaglitazar-loaded liposomes do not improve metabolic outcomes over untargeted tesaglitazar liposomes.

Male ob/ob mice were injected three times over the course of one week with liposomes that contained vehicle or tesaglitazar at a concentration of 1μmol of tesaglitazar/kg/day without peptide (NP drug) or with the ATS peptide (ATS drug) (A). Plasma isolated from blood harvested before and after treatment was utilized to measure circulating levels of insulin (B), triglycerides (C), and glycerol (D) and changes from pre-treatment to post-treatment were calculated. The body weight of each animal was also measured before and after treatment and the change in body weight was calculated (E). After treatment, whole epididymal (F) and subcutaneous (G) adipose depots were weighed and tissue weight was normalized to the post-treatment body weight of each mouse. RNA extracted from epididymal adipocytes was utilized to measure relative mRNA expression levels of Pdk4 (H). *p<0.05, **p<0.01, Kruskal-Wallis test.

Fig 3. Liposomal uptake is high in macrophages and endothelial cells independent of ATS targeting.

Ob/ob mice received one or three IV injections of DiD-labeled liposomes and their liposome uptake in adipose tissues was measured one day or one week later, respectively (n = 6). For the one-week study, tesaglitazar-loaded liposomes were used, while the one-day study used vehicle liposomes. At the end of each study, adipose tissues were harvested and ex vivo FMT scans were performed to assess liposome uptake (A,B). Whole tissues were also stained with BODIPY and CD31 and were examined with confocal microscopy (C). Manders overlap coefficient was calculated for the liposomes in the epididymal and subcutaneous adipose tissues with six images per comparison (48 total images) (D,E). A one-day flow cytometry study was also performed where the SVF was isolated from adipose depots 24 hours after ob/ob mice received IV injections of tesaglitazar loaded liposomes (F-I). **p<0.01, Kruskal-Wallis test.

Fig 4. ATS targeting increases uptake in the kidneys and causes no attenuation in uptake in the liver and bone marrow.

Ob/ob mice used in one-week and one-day studies of liposomal uptake, also had whole tissue ex vivo FMT scans performed on their kidneys, bone marrow, and livers (A,B). Bone marrow cells were analyzed by flow cytometry to quantify uptake of DiD-labeled liposomes (C,D). Livers of mice treated with three liposome injections over one week were sectioned and stained for Kupffer cell marker Clecsf13 to visualize cellular uptake of DiD-labeled liposomes (Scale Bar = 50 μm) (E). RNA was harvested from livers of mice treated for one week with untargeted vehicle-, untargeted drug-, or ATS-targeted drug-loaded liposomes to measure mRNA expression levels of Ehhadh (F) and Fabp3 (G). Kidney sections from mice treated for one week with ATS-targeted liposomes were stained with DAPI and αSMA to label nuclei and arteries, respectively (Scale Bar = 100 μm) (H). RNA was harvested from kidneys of mice treated for one week with untargeted vehicle-, untargeted drug-, or ATS-targeted drug-loaded liposomes to measure mRNA expression levels of Nos2 (I), Tgfb1 (J), and Serpine1 (K). Vehicle, vehicle-loaded liposomes; NP Drug, drug-loaded, untargeted liposomes; ATS Drug, drug-loaded, ATS-targeted liposomes. *p<0.05, **p<0.01, Mann-Whitney and Kruskal-Wallis tests.