Research ArticlePharmaceutics, Drug Delivery and Pharmaceutical TechnologyEfficient LRP1-Mediated Uptake and Low Cytotoxicity of Peptide L57 In Vitro Shows Its Promise as CNS Drug Delivery Vector
Introduction
The delivery of therapeutics into the brain for the remediation of specific central nervous system (CNS) disorders is impeded by their suboptimal blood-brain barrier (BBB) penetration. Macromolecules, such as proteins and nucleic acids, are prevented from paracellular and transcellular transport by specialized CNS barriers such as brain capillary endothelial cells of the BBB. This formidable challenge is one key reason why these disorders remain largely untreated, despite the ready availability of effective candidate drugs to treat them.1 As such, there is a growing need for effective modes of delivery to efficiently transport therapeutics across the BBB.
Over the last decade, there has been a heightened interest in neuro-pharmaceutics for CNS disorders, as reviewed in.2,3 Potential advantages of neuropharmaceuticals include high specificity, potency, minimal cross-reactivity, low immunogenicity and relative ease of modification to optimize their pharmacokinetic properties. Consequently, the pharmaceutical industry has made great strides in the development of peptide therapeutics, with over 70 therapeutic peptides on the market and more than 150 in clinical studies.3 Therapeutics targeted to the brain can be delivered through various routes of entry such as intravenous or sub-cutaneous injection, or intranasal administration.2,3 However, not all peptides cross the BBB.2 More recent approaches have incorporated the use of targeted nanoparticles to deliver therapeutic peptides to the brain.4 Small cationic peptides, in particular, have been used in a Trojan horse-like approach to remediate brain disorders such as using a SynB1 protegrin derivative to deliver benzylpenicillin across the BBB5 and treating ischemic brain damage using Tat-NR2B9c and pTat-PDZ1-2.6, 7, 8
Two well-characterized modes of peptide transport across the BBB are adsorptive-mediated transcytosis (AMT) and receptor-mediated transcytosis (RMT). Via the AMT mode, poly-lysine and poly-arginine peptides are translocated as efficiently as Tat peptides.9 Kamei et al.10 concluded that efficient transduction is achieved for a peptide with eight sequential arginines (RRRRRRRR, R8) in a mouse microvascular endothelial model based upon bEnd.3 cells. Using bEnd.3 cells in vitro and rat brains in vivo, they concluded that R8 was safe and effective for the non-covalent delivery of certain hydrophilic macromolecules such as the peptide drug insulin, which led us to select this peptide as a positive control for our studies in the main cells of interest, rat brain microvascular endothelial cells (BMVECs).
As for the RMT mode, the cationic peptides Angiopep-2 (A2, TFFYGGSRGKRNNFKTEEY)11,12 and its analogue Angiopep-7 (A7, TFFYGGSRGRRNNFRTEEY) cross the BBB in mouse models.12,13 A2 and A7 are peptides derived from the Kunitz domain of the low-density lipoprotein receptor-related protein 1 (LRP1) endocytic receptor12 which is expressed in many cell types including brain capillary endothelial cells and is enriched at the lipid rafts. LRP1 is also implicated in numerous biological processes such as transcytosis of macromolecules such as receptor-associated proteins and matrix proteins.14,15 In 2008, the uptake of ANG1005, an A2-Paclitaxel drug conjugate, was found to exhibit high brain accumulation and anti-tumor efficacy in both in vitro and in vivo animal models of brain and lung cancer, compared to the unconjugated Paclitaxel drug.16 This has shown promising results in Phase II clinical trials.17 Two other A2 conjugates, ANG1007 and ANG1009, using the anticancer drugs doxorubicin and etoposide, respectively, have also been developed.18 These drugs were effective in inducing cytotoxicity against human cancer cell lines such as NCI–H460 lung carcinoma. A non-covalent mode of delivery has also been developed which carries immunoglobulins to the brain using the K16ApoE synthetic peptide.19
The success of A2 peptide-drug conjugates in preclinical studies opened the door to the discovery of the first artificial LRP1 ligand, the cationic peptide L57, which specifically binds to cluster 4 of the LRP1 receptor. This peptide, developed by Sakamoto et al.,13 is less basic than the well-studied cell-penetrating peptide R8; thus, there may be a lower risk of non-specific binding. In their study, L57 showed significant BBB permeability and higher LRP1 binding affinity over both A2 and A7. Moreover, A7 was chosen as the reference peptide for their in vivo studies as it exhibited better BBB permeability than A2. This factored into the decision to use A7 in our study to compare it against L57. Importantly, Sakamoto et al. did not study the toxicity of the A7 and L57 peptides in vitro (in a suitable BBB model such as BMVECs) or in vivo. Furthermore, they did not provide an assessment of the levels of cellular uptake of these peptides in various cells in vitro - which was addressed in the present study.
In this study, we used primary rat BMVECs to visualize the intracellular accumulation and localization of the fluorescent L57, A7 and R8 peptides in vitro through fluorescence microscopy. The principal reason we chose these cells was because LRP1 receptors are found to be highly expressed in brain microvessels in mouse pups.20 Furthermore, BMVECs are the brain's first structural barrier to the CNS; thus these cells provide a suitable in vitro model to compare uptake and potential cytotoxicity of peptide carriers. We quantified the uptake of various concentrations of these peptides and reinforced the observations made by Sakamoto et al.13 that L57 peptides exhibited better cell uptake than A7, with our observations being made in BMVECs compared to their brain perfusion assay in mice. We also compared the cellular uptake of these peptides in two other cell types, LRP1-expressing brain astrocytes (another key component of the BBB) and PEA 10 cells (LRP1-deficient cells),21 with varying, but still encouraging, results. We also conducted cytotoxicity testing, which was previously unstudied. Our results showed that L57 has greater biocompatibility than both A7 and R8 peptides in BMVECs and PEA 10 cells. In summary, our results suggest that the L57 peptide could be a promising new carrier to enable more effective delivery of CNS therapeutics to the brain.
Section snippets
Materials
The fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, Rink Amide AM resin, 1-hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) were purchased from Aapptec (Louisville, KY, USA). N,N-diisopropylethylamine (DIPEA) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein (disodium salt) and diethyl ether were purchased from Fisher Scientific
Internalization of FITC-Labeled Peptides
Cells incubated with Fl-L57 (Fig. 1, Fig. 2 and Fig. 1, Fig. 2) displayed diffuse staining throughout the cells for all three cell types. Incubation of both types of primary brain cells with 100 μM Fl-A7 exhibited high uptake in (Figs. 1g and 2g) with very low uptake by PEA 10 cells (Fig. 3g). In BMVECs, the fluorescence was saturated when incubated with 30 μM Fl-L57; therefore, these results were excluded from analysis for this cell type. BMVECs showed the highest uptake efficiency for the
Discussion
The primary goals of this study were to assess the efficiency of uptake and cytotoxicity of the novel artificial LRP1-binding peptide L57 in primary BMVECs, and to compare this to previously-studied cationic peptides, A7 and R8, which are known to cross the BBB.10,13 Cellular uptake and cytotoxicity of Fl-L57 were evaluated in vitro for the first time in BMVECs, which are crucial for uptake of this peptide due to their high expression of the LRP1 receptor and location in the microvasculature of
Acknowledgements
Funding was provided by the Edmonson/Crump Professorship [Louisiana Board of Regents Support Fund Endowed Professorships] to TAM, a Louisiana Board of Regents Grant [LEQSF(2018-21)-RD-A-13] and a Louisiana Biomedical Research Network Grant [NIH 5P20GM103424 LSU, Subcontract No. PO-0000002131] to SP, and a Louisiana Tech University, College of Engineering and Science Graduate Scholarship to JPR.
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Declarations of interest: none.
Author contributions: JPR, TAM, SP, NP, and MAD designed the experiments, JPR and NP conducted experiments, JPR and TAM analyzed data, and JPR, TAM, SP, NP, and MAD wrote the manuscript.
Availability of data: Data are available at https://drive.google.com/drive/folders/1pIyNNQIk74aHQDuNfvXJFTyAztkm2bSp?usp=sharing.