Isoprenoid CARTs: In Vitro and In Vivo mRNA Delivery by Charge-Altering Releasable Transporters Functionalized with Archaea-inspired Branched Lipids

Abstract

The delivery of oligonucleotides across biological barriers is a challenge of unsurpassed significance at the interface of materials science and medicine, with emerging clinical utility in prophylactic and therapeutic vaccinations, immunotherapies, genome editing, and cell rejuvenation. Here, we address the role of readily available branched lipids in the design, synthesis, and evaluation of isoprenoid charge-altering releasable transporters (CARTs), a pH-responsive oligomeric nanoparticle delivery system for RNA. Systematic variation of the lipid block reveals an emergent relationship between the lipid block and the neutralization kinetics of the polycationic block. Unexpectedly, iA21A11, a CART with the smallest lipid side chain, isoamyl-, was identified as the lead isoprenoid CART for the in vitro transfection of immortalized lymphoblastic cell lines. When administered intramuscularly in a murine model, iA21A11-mRNA complexes induce higher protein expression levels than our previous lead CART, ONA. Isoprenoid CARTs represent a new delivery platform for RNA vaccines and other polyanion-based therapeutics.

Figure 1.

Polycationic CART co-oligomers undergo consecutive O-to-N acyl shifts, resulting in their neutralization through the formation of biologically compatible diketopiperazine (DKP) and the release of their polycationic cargoes. Lipids for diblock co-oligomer D₁₃A₁₁ and triblock co-oligomer ONA CARTs are shown alongside the saturated isoprenoid lipids, the subjects of this work.

Figure 2.

(A) Synthesis of isoprenoid CART monomers. Compound 1 is fully saturated; therefore, it was not subjected to hydrogenation. 1 was esterified with 9 to yield cyclic carbonate monomer 2. (B) Synthesis of isoprenoid CART co-oligomers. Monomers 2, 4, 6, or 8 were each oligomerized via OROP with the DBU/TU catalyst system using a benzyl alcohol as the initiator. In the same vessel, the resulting isoprenoid oligocarbonate block then initiates the oligomerization of morpholinone 10. The resultant diblock co-oligomer is purified by dialysis and deprotected with trifluoroacetic acid to yield the active isoprenoid CART in two steps (DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene; TU = 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea). (C) Structures of isoprenoid lipid side chains and block lengths (n, m) for each transporter. (D) Isoprenoid CARTs were named and assigned groups based on their side chains and block lengths. 10:10 contains shorter co-oligomers with approximately equal lipid and cation block lengths. 17:17 contains longer co-oligomers with approximately equal lipid and cation block lengths. 24:12 contains transporters with approximately twice as many lipid repeat units as cationic repeat units.

Figure 3.

(A) ζ-potential time courses for three representative CART-mRNA complexes, ONA-, C13A11-, and P19A19-mRNA complexes (n = 3). Each time course was globally fit with a linear regression to determine k, t_neutral, and ζ₀. Each time course was performed in triplicate. (B) Schematic representation of the DKP-producing charge-neutralization process in the absence of cells. (C) Data for each transporter-mRNA complex (10:1 (+/−) charge ratio) in this study is tabulated. ^aThe value contained within the parentheses is the nanoparticle’s average polydispersity index. ^bThe value contained within parentheses represents the average standard deviation of the nanoparticle formulation’s initial ζ-potential. ^ct_neutral is defined as the x-intercept of the global linear regression of the ζ-potential’s time course for each transporter. ^dThe value contained within parentheses is the standard error associated with variable k as derived from the global linear regression. ^eThe value contained within parentheses is the standard error associated with determining the encapsulation efficiency.

Figure 4.

Bioluminescence of six cell lines (HeLa, A549, Jurkat, GM12878, NALM6, and SUP-T1) treated with Lipo2000-, isoprenoid CART-, or ONA-mRNA complexes (10:1 (+/-) charge ratio). Lipo2000- and ONA-mRNA complexes are used as controls. (A) Bioluminescence of adherent cell lines (HeLa and A549). (B) Bioluminescence of T-cell (Jurkat and SUP-T1) and B-cell (NALM6 and GM12878) cell lines. Bioluminescence results of all experimental groups are normalized to those of ONA-mRNA complexes. Error bars represent the SD of two experiments in quadruplicates. (*) A549 cells were not evaluated with these transporters.

Figure 5.

Codelivery of Cy5-labeled and functional eGFP mRNA with isoprenoid CART complexes. (A) Schematic representation of the codelivery assay. Red stars represent Cy5-labels. Data bars represent the mean fluorescence intensity (MFI) of the reporter (Cy5 or eGFP). Data labels represent the average %Cy5⁺ (*) or %eGFP⁺ (†) of (B) HeLa cells, (C) Jurkat cells, and (D) NALM6 cells treated with a 1:1 mixture of Cy5-eGFP mRNA and eGFP mRNA. Cy5MFI and eGFP MFI results of all experimental groups are normalized to those of ONA-mRNA complexes. Error bars represent the SD of two experiments in quadruplicate (n = 8).

Figure 6.

Whole-body luciferase imaging 6 h after CART-FLuc mRNA intramuscular injections and quantitation of the resulting luciferase signals. (A) Schematic of CART-mRNA complex administration and expression. (B) Best candidates of each isoprenoid lipid were evaluated at 3:1 (+/-) charge ratio for their transfection in vivo. (C) iA21A11-mRNA complexes were screened at four charge ratios from 3:1 to 30:1 (+/-) and (D) compared to ONA at its respective optimized charge ratio (n = 6, p < 0.01). Error bars represent standard deviation (n ≥ 3). Data shown in panel B was collected in a single assay. Data shown in panels (C) and (D) was collected side by side in a single assay.