Methods and compositions for inducing long-term tolerance by hybrid nanoparticles are provided. Compositions and formulations comprising hybrid nanoparticles with inherent affinity for innate immune cells are provided.

A preparation method of adiposomes, and use thereof. Provided is a method for preparing adiposomes consisting of neutral lipids and a monolayer phospholipid membrane, comprising a1) vortexing phospholipid and neutral lipids in a buffer, centrifuging the resulting mixture, and collecting an upper liquid phase; a2) purifying the upper liquid phase twice or more by uniformly mixing the upper liquid phase with the buffer, layering the mixture, and collecting an upper liquid phase; and a3) uniformly mixing the upper liquid phase obtained in step a2) with the buffer, layering the mixture, and collecting a lower liquid phase in containing adiposomes. For the adiposomes prepared by the method, one or more resident proteins and/or functional proteins can be recruited to obtain artificial lipid droplets, and one or more apolipoproteins can be recruited to obtain artificial lipoproteins; and they all play important roles in preparing drugs and/or drug carriers.

The present invention is directed to a cargo-loaded cholesteryl ester nanoparticle with a hollow compartment (cholestosome) consisting essentially of at least one non-ionic cholesteryl ester and one or more encapsulated active molecules which cannot appreciably pass through an enterocyte membrane in the absence of said molecule being loaded into said cholestosome, the cholestosome having a neutral surface and having the ability to pass into enterocytes in the manner of orally absorbed nutrient lipids using cell pathways to reach the golgi apparatus. Pursuant to the present invention, the novel cargo loaded cholestosomes according to the present invention are capable of depositing active molecules within cells of a patient or subject and effecting therapy or diagnosis of the patient or subject.

In one embodiment, the present application discloses synthetic LDL nanoparticles comprising mixtures of components selected from the group consisting of phospholipids, triglycerides, cholesterol ester and free cholesterol; optionally further comprising an agent selected from the group consisting of natural antioxidants, ubiquinol and vitamin E, and methods for preparing the synthetic nanoparticles. The disclosed synthetic LDL nanoparticles are capable of selectively delivering lipophilic drugs and prodrugs to cellular targets expressing LDL receptors after intra venous injection.

Methods of loading extracellular vesicles with payload molecules via homogenization are disclosed herein.

Disclosed are biomimetic proteolipid nanovesicles that possess remarkable properties for targeting compounds of interest to particular mammalian cell and tissue types. In particular embodiments, drug delivery vehicles are provided composed of synthetic phospholipids and cholesterol, enriched of leukocyte membranes, and surrounding an aqueous core. These nanovesicles are able to both avoid the immune system, thanks to the presence on their surface of self-tolerance proteins, as CD-45, CD-47, and MHC-1, and target inflamed endothelium, thereby diffusing in the tumor microenvironment. These properties make the composition highly suited for targeted drug delivery to mammalian tumor cells in vitro and in situ.

A method of generating an internally fixed lipid vesicle, comprising: providing a precursor lipid vesicle that contains an aqueous interior enclosed by a lipid membrane, wherein the lipid membrane of the precursor lipid vesicle is non-permeable to a crosslinker; permeabilizing the lipid membrane transiently to generate a permeable vesicle; contacting the permeable vesicle with an inactive activatable crosslinker, whereby the inactive activatable crosslinker enters the permeable vesicle; allowing the permeable vesicle to return to a non-permeable vesicle; removing any extravesicular crosslinker; and activating the inactive activatable crosslinker to allow crosslinking to occur inside the non-permeable vesicle, whereby an internally fixed lipid vesicle is generated.

A modified Extracellular Vesicle (EV), preferably an exosome, comprising an albumin protein or fragment (e.g., an albumin domain) present on the surface of the EV membrane, preferably as part of a fusion protein (e.g., a transmembrane protein). The EV exhibits an improved circulating half-life, stability and shelf-life (both formulation and storage). Methods of making said EVs, as well as pharmaceutical compositions and their use.

The present invention is directed to a cargo-loaded cholesteryl ester nanoparticle with a hollow compartment (cholestosome) consisting essentially of at least one non-ionic cholesteryl ester and one or more encapsulated active molecules which cannot appreciably pass through an enterocyte membrane in the absence of said molecule being loaded into said cholestosome, the cholestosome having a neutral surface and having the ability to pass into enterocytes in the manner of orally absorbed nutrient lipids using cell pathways to reach the golgi apparatus. Pursuant to the present invention, the novel cargo loaded cholestosomes according to the present invention are capable of depositing active molecules within cells of a patient or subject and effecting therapy or diagnosis of the patient or subject.

Disclosed are methods for designing and manufacturing biomimetic proteolipid nanovesicles using a microfluidic approach, and in particular, a NanoAssemblr-based platform, which allows for the high-throughput, reproducible, and scalable production of nanoparticles, without affecting their pharmaceutical and biological properties. These nanovesicles, which are composed of synthetic phospholipids and cholesterol, enriched of leukocyte membranes, and surrounding an aqueous core, possess remarkable properties for targeting compounds of interest to particular mammalian cell and tissue types.