Nanostructured lipid carriers and stable emulsions and uses thereof
Inventors
FOX, Christopher B. • Khandhar, Amit Praful • Van Hoeven, Neal • ERASMUS, Jesse H. • Lin, Susan S.
Assignees
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Publication Number
US-12201722-B2
Publication Date
2025-01-21
Expiration Date
Abstract
Provided herein are nanostructured lipid carrier compositions, and methods of making and using thereof. The compositions comprise a nanostructured lipid carrier (NLC), where the NLC comprises an oil core comprising a mixture of a liquid phase lipid and a solid phase lipid, a cationic lipid, a sorbitan ester, and a hydrophilic surfactant, and optionally a bioactive agent. The bioactive agent can be associated with the NLC. The compositions are capable of delivery of a biomolecule to a cell for the generation of an immune response, for example, for vaccine, therapeutic, or diagnostic uses. Compositions and methods related to making the compositions and using the compositions for stimulating an immune response are also provided.
Core Innovation
The invention relates to nanostructured lipid carrier (NLC) particles for delivery of a bioactive agent to a cell. The NLC particles comprise an oil core comprising a mixture of a liquid phase lipid and a solid phase lipid that is trimyristin, a cationic lipid, a sorbitan ester, a hydrophilic surfactant, and the bioactive agent. The formulation and relative amounts of these components are selected so that particular performance outcomes are obtained for bioactive agents that are replicating viral RNA (rvRNA).
The disclosure describes NLC compositions and performance characterization for rvRNA and immune-response outcomes in vivo. For rvRNA encoding secreted alkaline phosphatase (SEAP), the highest peak expression occurs at a N:P molar ratio of 15 as measured by an in vitro SEAP expression assay using a SEAP-expressing replicon and measured by relative luminescence units (RLUs). The document also characterizes NLC particles by z-average diameter, average polydispersity index (PDI), zeta potential, and stability over time using z-average retention.
The disclosure includes NLC compositions in which the liquid phase lipid is squalene and the cationic lipid is DOTAP, with sorbitan monostearate and polysorbate 80. For rvRNA encoding ZIKV prM/E from a Venezuelan equine encephalitis virus (VEEV) plasmid, the composition induces mean PRNT80 titers between about 1:110 and about 1:2560 when loaded with about 30 ng to about 100 ng of the rvRNA at a N:P molar ratio of about 15. The NLC particles also include configurations where the bioactive agent is not encapsulated and instead is associated with the surface of the NLC particles.
Claims Coverage
The independent claims identified in the provided material are four compositions of NLC particles for delivery of a bioactive agent to a cell. Across these independent claims, the inventive structure includes a trimyristin-containing oil core, a cationic lipid, a sorbitan ester, a hydrophilic surfactant, and formulation constraints tied to peak expression or immune-response outcomes, including a N:P molar ratio target.
NLC oil core with trimyristin and rvRNA/SEAP peak expression at N:P=15
An NLC composition comprising NLC particles having an oil core of a mixture of a liquid phase lipid and a solid phase lipid that is trimyristin, a cationic lipid, a sorbitan ester, a hydrophilic surfactant, and the bioactive agent, wherein relative amounts and specific components are selected so that when the bioactive agent is replicating viral RNA (rvRNA) encoding secreted alkaline phosphatase (SEAP), the highest peak expression occurs at a N:P molar ratio of 15 measured by an in vitro SEAP expression assay using a SEAP-expressing replicon and measured by relative luminescence units (RLUs).
DOTAP with squalene/trimyristin oil core, sorbitan monostearate, and polysorbate 80
An NLC composition comprising NLC particles having an oil core comprising a mixture of a liquid phase lipid that is squalene and a solid phase lipid that is trimyristin, a cationic lipid that is DOTAP, a sorbitan ester that is sorbitan monostearate, a hydrophilic surfactant that is polysorbate 80, and the bioactive agent, wherein the NLC particles comprise specified % w/v liquid phase lipid, % w/v solid phase lipid, % w/v cationic lipid, % w/v sorbitan ester, and % w/v hydrophilic surfactant.
Hydrophilic surfactant-to-cationic lipid molar ratio 0.2–1.5 with DOTAP/sorbitan monostearate/polysorbate 80
An NLC composition comprising NLC particles having an oil core comprising a mixture of a liquid phase lipid that is squalene and a solid phase lipid that is trimyristin, a cationic lipid that is DOTAP, a sorbitan ester that is sorbitan monostearate, a hydrophilic surfactant that is polysorbate 80, and the bioactive agent, wherein a hydrophilic surfactant to cationic lipid molar ratio is about 0.2 to about 1.5.
Formulation inducing mean PRNT80 titers in mice for ZIKV prM/E rvRNA loaded at N:P≈15
An NLC composition comprising NLC particles having an oil core comprising a mixture of a liquid phase lipid and a solid phase lipid that is trimyristin, a cationic lipid, a sorbitan ester, a hydrophilic surfactant, and the bioactive agent, wherein the composition is formulated such that when administered to mice in which the bioactive agent is rvRNA encoding ZIKV prM/E from a Venezuelan equine encephalitis virus (VEEV) plasmid, the composition induces mean PRNT80 titers of between about 1:110 and about 1:2560 when loaded with about 30 ng to about 100 ng of the rvRNA at a N:P molar ratio of about 15.
Across the independent claims, the inventive coverage is directed to NLC particles with a trimyristin-containing oil core plus a cationic lipid, sorbitan ester, and hydrophilic surfactant, with specific component selections and formulation constraints that achieve targeted expression and targeted immune-response outcomes.
Stated Advantages
Induces higher chemokines than other comparators in blood/plasma cytokine assays for a Span 60 (sorbitan monostearate)-based NLC/QG863 formulation.
Produces sustained neutralizing titers after single or prime/boost dosing compared with rvRNA alone.
Promotes ZIKV-specific CD8+ T-cell responses and low-dose protection with higher neutralizing antibody and CD8 titers.
Enables antibody expression/delivery in vivo, with higher ELISA titers for NLC-formulated rvRNA than naked RNA.
Enhances induction of chemokines and innate immune markers in human PBMC-derived dendritic cells and MM6 cells when TLR3 and RIG-I agonists are formulated with NLCs versus naked agonist.
Supports dose-sparing effects in additional mouse studies using oil-in-water squalene emulsions compared in the document.
Documented Applications
Delivery of replicon/nucleic-acid agonists using NLC formulations, assessed by blood/plasma cytokine assays and chemokine induction.
In vivo immunization in mice with NLC-formulated VEEV replicon rvRNA encoding ZIKV PrM/E, evaluated by neutralizing titers (PRNT) and immune responses including CD8+ T-cell responses and antibody responses.
In vivo antibody expression/delivery studies in which NLC-formulated rvRNA yields higher ELISA titers than naked RNA.
In vitro studies of human PBMC-derived dendritic cells and MM6 cells using TLR3 (poly(I:C) HMW) and RIG-I agonists formulated with NLCs to assess chemokine and innate immune marker induction.
Additional mouse studies comparing oil-in-water squalene emulsions (including Hiltonol®/SE or SLA-SE, nanoAlum) demonstrating antigen-specific antibody and recall cytokine responses and dose-sparing effects.
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