Atty. Docket No. NEX-13360
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`PROVISIONAL APPLICATION FOR PATENT
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`Cationic Peptide/Protein Modified Exosomes for Tissue
`Targeting for Applications in Drug Delivery
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`Government Support
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`This invention was made with government support under Grant Number EB028385
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`awarded by the National Institutes of Health. The government has certain rights in the invention.
`Overview
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`Exosomes are known to have intrinsic therapeutic potential and have recently been shown
`to be effective in tissue repair. Exosomes are emerging as a cell free regenerative therapy. Here
`we develop a new class of surface modified exosomes that are cationic in charge.
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`Exosomes have a negatively charged bilayer making it difficult to penetrate dense tissues
`like cartilage which is rich in aggrecan glycosaminoglycans. Similarly, a wide range of tissues
`exist that have negatively charged groups like proteoglycan, hyaluronic acid, anionic proteins
`etc. Some examples include musculoskeletal tissues like meniscus, tendon, ligaments,
`intervertebral discs, eye and tumors. Exosomes in their current form are ineffective in targeting
`these dense negatively charged tissues. We provide a method for easy modification of the surface
`of exosomes to make them cationic. The chemistry enables modular surface properties such that
`any peptide or protein of interest can be added to the surface of exosome for efficient tissue
`targeting.
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`Here we provide data using milk and mesenchymal stem cells (MSC) derived exosomes
`whose surface charge was neutralized or made slightly cationic enabling effective targeting of
`the mucosal membrane for oral drug delivery as well as for cartilage targeting for drug delivery
`applications. These exosomes are packed with genetic materials or anchored with protein drugs.
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`In certain aspects, the present invention provides a new class of cationic exosomes and
`methods for synthesizing these cationic exosomes.
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`The chemistry presented herein enables modulation of the surface of exosomes, such that
`the properties of the exosomes may be tuned according to desired applications. Additionally, the
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`data show excellent targeting and penetration of cartilage tissue which is a negatively charged
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`Atty. Docket No. NEX-13360
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`tissue that remains a challenge in the field of drug delivery. By contrast, native (anionic)
`exosomes cannot target cartilage. The present invention also provides application of the
`exosomes in targeting mucosal membrane for oral delivery of biologics (see appendix).
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`Drug delivery to cartilage remains challenging due to their rapid clearance from intra-
`articular joint space and hindered transport into cartilage deep layers due to its dense
`extracellular matrix (ECM) comprising of high density of negatively charged
`glycosaminoglycans (GAGs) and collagen II network. MSC derived exosomes could facilitate
`cartilage repair in OA animal models, but their large size (40~200 nm) and negatively charged
`lipid bilayer (-20~-25 mV) limited their penetration into deep layers of negatively charged
`cartilage. To solve these problems, for the first time, we reversed the net charge on anionic
`exosomes by anchoring their surfaces with cartilage targeting cationic peptide carriers (CPCs)
`and cationic glycoprotein Avidin. These cationic motifs were designed to effectively target
`cartilage based on its negative fixed charge density enabling ~100 - 400x higher uptake than
`their neutral counterparts, full-thickness penetration, and long-term intra-cartilage retention. We
`use the hydrophobic tail of amphipathic DSPE-PEG (2 kDa)-azide (DPA) for insertion into Exo
`lipid bilayer and the terminal azide for clicking cationic motifs enabling modular surface
`properties. About 300-500 cationic motifs were loaded per exosome resulting in reduced zeta
`potential of exosome from -25.4 + 1.3 mV to -2.5 = 1.5 mV. By making use of the charge
`interaction, these surface modified exosome showed fast penetration, high chondrocyte uptake
`and longer retention time in arthritis cartilages. Nucleus acids and proteins can be loaded in
`exosomes for intra-cartilage delivery.
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`As compared to prior technologies, the new class of neutral or cationic exosomes exhibit
`good thermal stability. Provided herein is a detailed method for synthesizing exosomes with
`varied net charge and storing the formulation long-term to avoid any aggregation issues.
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`Furthermore, the method laid out here enables synthesizing exosomes with varied net
`charge which is important for targeting a wide range of tissues with varying net negative fixed
`charge densities. This technique enables users (pharmaceutical/cell therapy companies) to make
`exosomes of any surface property and charge depending on their application and tissue target
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`As shown, the inventors functionalized cartilage targeting cationic peptides (designed in
`the inventors’ lab and showed be detailed in the patent) and proteins in different densities and
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`demonstrate that these cationic exosomes can penetrate through the full-thickness of cartilage in
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`high concentrations while unmodified exosomes cannot. This discovery has the potential to
`transform the therapeutic space of cartilage repair and osteoarthritis.
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`The inventors designed simple and effective modular surface modification techniques for
`exosome membrane where any peptides or proteins of interest can be clicked, providing safe, cell
`free natural lipid carriers with intrinsic therapeutic potential for targeted drug delivery to
`cartilage and other negatively charged tissues.
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`The inventors also developed milk exosome harvest techniques by applying casein
`chelation, differential ultracentrifugation and size-exclusion chromatography methods to obtain
`exosomes with high yield and high purity from the cheap, scalable resource.
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`This invention provides numerous advantages over known technologies, including
`enabling intra-cartilage targeting, reversing the net charge on anionic exosome, elevating
`chondrocyte uptake of surface modified exosome, enabling modular design of exosome surface
`using any peptides or proteins, enabling loading and delivery of nucleus acids, proteins and small
`molecular drugs, improving the stability of exosomes, and tuning a wide range of tissue targeting
`properties. The cationic exosomes described herein can target tissues due to electrostatic
`interactions. Native (negatively charged) exosomes cannot.
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`The invention has numerous applications, including in intra-cartilage targeting, cell free
`tissue repair therapy, and delivery of nucleus acids, proteins and small molecular drugs.
`Moreover, the drug delivery applications of the technology described herein can be extended to
`drug delivery in a wide range of negatively charged tissues like meniscus, intervertebral discs,
`mucosal membrane, and cancer tumors. The technology is also applicable to osteoarthritis
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`treatment and various oral administration applications.
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