Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1275—Lipoproteins or protein-free species thereof, e.g. chylomicrons; Artificial high-density lipoproteins [HDL], low-density lipoproteins [LDL] or very-low-density lipoproteins [VLDL]; Precursors thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1277—Preparation processes; Proliposomes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
  • B01F33/30—Micromixers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
  • G01N2405/04—Phospholipids, i.e. phosphoglycerides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
  • G01N2405/04—Phospholipids, i.e. phosphoglycerides
  • G01N2405/06—Glycophospholipids, e.g. phosphatidyl inositol
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• Nano-sized vesicles and particles have many different functions in biological systems, such as regulating molecular transport (e.g. small molecules, various proteins, nucleic acids, etc.) and serving as vehicles for communication between cells.
• Biomimetic systems that recapitulate and are representative of cell membranes can aid in elucidating transport mechanisms at the cellular interface.
• biomimetic systems can be developed to model systems such as extracellular vesicles (EVs), lipoproteins, or viruses by controlling and combining nucleic acid-protein-lipid compositions.
• EVs extracellular vesicles
• lipoproteins or viruses by controlling and combining nucleic acid-protein-lipid compositions.
• such transport models can be used for testing of the ability of small molecules to pass through a placenta.
• such transport models can include phospholipid carriers with lipid and protein composition representative of placental trophoblast cells, also noted herein as placental proteoliposomes (PPLs).
• the techniques described herein relate to a proteoliposome including: one or more phospholipid carrier and one or more protein embedded in the one or more phospholipid carrier; wherein the one or more phospholipid carrier includes a phospholipid composition with similar proportions of phospholipids as a naturally occurring cell ty pe and a phospholipid concentration of about 1-50 mM; and wherein the one or more protein includes a protein composition with similar proportions of proteins as the naturally occurring cell ty pe.
• a ratio of the one or more phospholipid carrier to one or more protein is about 1: 100.
• the one or more phospholipid carrier includes one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SPH) and, optionally, cholesterol.
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• PS phosphatidylserine
• SPH sphingomyelin
• the one or more phospholipid carrier includes a mixture of one or more of PC, PE, PI, PS, and SPH.
• the one or more protein includes one or more transmembrane protein.
• the one or more transmembrane protein includes one or more of ABCB1, ABCG2, SLC22A5, CD 9, 63, 81, or 82, integrms, Alix, TSG101, clathnn, Ubiquitin, HSP90, HSC90, HSP70, PD-L1, MHC, growth factors, lipoproteins, polymerases, and capsid proteins.
• the proteoliposome further includes extracellular matrix (ECM) to form a proteoliposome-ECM composition.
• ECM extracellular matrix
• the ECM is formed as a droplet.
• the ECM includes one or more of structural proteins, grow th factors, and cytokines.
• the one or more nucleic acid does not have modifications to one or more of nucleotides and end capping.
• the one or more nucleic acid have modifications to one or more of nucleotides and end capping.
• the one or more nucleic acid is an encoding or non-encoding RNA or DNA.
• the one or more nucleic acid is present at a concentration of 0-2 mg/mL.
• the one or more nucleic acid is present at a concentration of about 0.01-100 pg/mL.
• the one or more nucleic acid includes one or more of a chemical bond, nanoparticle, and a conjugation.
• the one or more nucleic acid includes RNA, DNA, or a combination thereof, [0022] In some aspects, the RNA includes one or more of mRNA, miRNA, siRNA, and saRNA.
• the miRNA includes one or more of 10, 21, 124, 125, 126, 130, and 132.
• the techniques described herein relate to a method including: (a) a first mixing of one or more phospholipid to form a phospholipid carrier solution; and (b) a second mixing of the phospholipid carrier solution with a protein solution to produce one or more proteoliposome, wherein the protein solution includes one or more protein.
• the second mixing includes a microfluidics approach, wherein the microfluidics approach includes flowing the phospholipid carrier solution and the protein solution through a microfluidic channel under at least one of laminar or turbulent flow.
• the second mixing includes an extrusion approach, wherein the extrusion approach includes extruding the phospholipid carrier solution and the protein solution through a porous membrane for a predetermined number of times.
• the second mixing includes a combination of a microfluidic approach and an extrusion approach, wherein the microfluidic approach includes flowing the phospholipid carrier solution and the protein solution through a microfluidic channel under at least one of laminar or turbulent flow, and wherein the extrusion approach includes extruding the phospholipid carrier solution and the protein solution through a porous membrane a predetermined number of times.
• the phospholipid carrier solution includes ethanol.
• the protein solution includes in buffer.
• the one or more phospholipid includes one or more of PC, PE, PI, PS, and SPH.
• the one or more protein includes one or more transmembrane protein.
• the one or more transmembrane protein includes one or more of ABCB1, ABCG2, SLC22A5, CD 9, 63, 81, or 82, integrins, Alix, TSG101, clathnn, Ubiquitin, HSP90, HSC90, HSP70, PD-L1, MHC, growth factors, lipoproteins, polymerases, and capsid proteins.
• the method further includes a third mixing of each of the one or more proteoliposome with ECM to form one or more proteoliposome-ECM.
• the ECM is formed as a droplet.
• the ECM includes one or more of structural proteins, growth factors, and cytokines.
• the third mixing includes bioprinting.
• the bioprinting includes one or more printhead configured for high- throughput.
• the techniques described herein relate to a method for screening pharmaceuticals, including: (a) incubating one or more of a placental proteoliposome (PPL) and a PPL-ECM with one or more molecule of interest to form an incubation product; (b) filtering the incubation product to produce a filtered product; and (c) quantifying the filtered product to assess transport of the one or more molecule of interest into one or more of the PPL and the PPL-ECM.
• PPL placental proteoliposome
• PPL-ECM placental proteoliposome
• the one or more molecule of interest includes one or more of a pharmaceutical composition, a nutrient composition and a toxin.
• a phospholipid composition of the PPL includes a mixture of one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidyl serine (PS), and sphingomyelin (SPH) and, optionally, cholesterol.
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• PS phosphatidyl serine
• SPH sphingomyelin
• FIGS. 1A-1B present schematics for making the proteoliposomes and nucleic acid- loaded proteoliposomes, respectively.
• FIG. 2 presents a schematic for bioprinting proteoliposomes into ECM droplets.
• FIGS. 3A-3B illustrate models of both proteoliposomes and proteoliposome-ECM droplets, respectively, in the presence of pharmaceuticals or nutrients.
• FIGS. 4A-4C present experimental results of PPLs using microfluidic mixing. Three technical replicates are overlaid.
• FIGS. 5A-5C present experimental results of PPLs using extrusion. Three technical replicates are overlaid.
• FIGS. 6A-6B provide measured absorbance spectra of the PPLs using Folic Acid and Amphotericin B as testing variables, respectively.
• FIGS. 7A-7B provide kinetic study results of the PPLS using Folic Acid and Amphotericin B as testing variables, respectively.
• FIGS. 8A-8B provide kinetic study results of the PPLS using Folic Acid and Adenosine triphosphate (ATP) showing the activity of the proteins as testing variables, respectively.
• ATP Folic Acid and Adenosine triphosphate
• FIGS. 9A-9B provide an example of hydrodynamic diameters of PPLs. Three technical replicates are overlaid.
• FIGS. 10A-10B present experimental results of isolated EVs in comparison to biomimetic EVs generated by the methods of this disclosure. Three technical replicates are overlaid.
• FIG. 11 presents experimental results of nucleic acids that are encapsulated within biomimetic proteoliposomes generated by the methods of this disclosure.
• This disclosure relates generally to a composition for mimicking various nanoparticle and membrane systems for use as a reference standard or alternative to the naturally occurring counterpart in analytics and research applications.
• the present disclosure provides various biomimetic compositions with the incorporation of lipids and proteins for applications in drug delivery', analytics, cell and animal interaction studies, as well as general industry and research.
• this disclosure relates generally to a composition for mimicking active transport in cells, a method of making compositions for mimicking active transport in cells, and a method of using compositions for mimicking active transport in cells.
• this disclosure addresses a need to develop compositions with lipid and protein compositions representative of different cell ty pes for enabling cell-free and scalable in vitro tools for high-throughput transport studies of small molecules.
• the compositions and methods of the present disclosure may provide additional advantages, such as use for any lipid-protein models for mimicking and assessing active transport of pharmaceutical, nutrient, toxin, or nucleic acid compositions.
• the compositions and methods of the present disclosure provide additional advantages such as use for any lipid-protein-nucleic acid models for mimicking cell derived nanoparticles for the use in analytics and research applications.
• FIGS. 1A-1B illustrate exemplary biomimetic nanoparticles (or proteoliposome) of the present disclosure, either without (FIG. 1A) or with nucleic acids encapsulated within the nanoparticle (FIG. IB)
• the proteoliposome comprises a composition that is biomimetic of various model systems.
• Proteoliposomes are examples of nanoparticles that include of a lipid bilayer with an integrated transmembrane protein. The incorporation of lipids, proteins, and/or nucleic acids with tuned parameters enables the development of model lipid membranes and vesicles with applications where only specific components of the full biological system are needed.
• Biomimetic proteoliposomes representative of membranes of cells can aid in elucidating transport mechanisms at the cellular interface, and may provide highly useful tools for enabling rapid screening studies at effective costs.
• the biomimetic proteoliposomes can be used to model nanoparticle systems such as liposomes, lipid membranes, extracellular vesicles (EVs), endosomes, lipoproteins, or viruses with combinations of nucleic acid-protein-lipid biomimetics.
• the proteoliposomes of this disclosure may have different structures including hollow proteoliposomes, i.e., vesicles, or proteoliposomes with solid cores.
• the biomimetic proteoliposomes comprise one or more phospholipid carrier and one or more proteins embedded in the one or more phospholipid carrier.
• the phospholipid carrier of the present disclosure may comprise a lipid bilayer.
• the lipid bilayer in various nanoparticle systems has many different functions in biological systems, such as regulating drug transport and providing structure for transmembrane proteins.
• the lipid bilayer is a planar bilayer.
• the one or more phospholipid carrier includes one or more phospholipids.
• the phospholipids of the same type may form the phospholipid carrier.
• the phospholipid carrier is formed by a mixture of different phospholipids.
• the biomimetic phospholipid carriers are produced from the mixture of different lipid species in similar proportions as a naturally occurring cell type.
• the one or more phospholipid are synthetically generated or extracted from cells so as to produce a biomimetic phospholipid carrier or composition.
• the one or more phospholipid may be naturally occurring or synthetic phospholipids.
• the one or more phospholipids may include, but not limited to, one or more naturally occurring phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SPH), phospatidylglycerol (PG), phosphatidic acids (PA) or PEGylated phospholipids.
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• PS phosphatidylserine
• SPH sphingomyelin
• PG phospatidylglycerol
• PA phosphatidic acids
• PEGylated phospholipids PEGylated phospholipids.
• such phospholipids may
• the one or more phospholipid may be further modified to have various desired functions.
• a PEGylated phospholipid may function to add steric bulk.
• a fluorescent phospholipid may allow for imaging or more in-depth analysis of the phospholipid carrier.
• an ionizable lipid may function to incorporate an nucleic acid.
• the synthetically generated phospholipid may further be conjugated to one or more of a protein or peptide.
• the one or more proteins embedded in the proteoliposomes of the present disclosure may comprise multiple proteins, of the same type or different types.
• the one or more protein comprises one or more transmembrane protein.
• the type of transmembrane protein may vary based on the type of nanoparticle system to be modeled.
• the one or more transmembrane protein may include P-sheet transmembrane proteins and a-helical transmembrane proteins.
• the one or more proteins comprise capsid proteins. In some embodiments, the one or more proteins comprise carrier proteins. Carrier proteins are integral proteins that transport chemicals across the membrane both down and up the concentration gradient.
• the composition of the proteoliposomes are designed to mimic transport through a particular cell membrane or to mimic EVs of various cells.
• the cells being mimicked are pluripotent stem cells, adult stem cells, mesenchymal stem cells, embryonic stem cells, cardiovascular stem cells (CDCs), placental stem cells, induced pluripotent stem cells and other stem cells such as somatic stem cells, hematopoietic stem cells, neural stem cells, osteoblasts, cancer stem cells, epithelial stem cells, and bone marrow stem cells.
• the cells being mimicked are kidney cells, cancer cells, epidermal cells, immune cells, placental cells, mucosal cells, fibroblasts, blood brain barrier cells and other cells.
• the proteoliposomes of this disclosure are biomimetic of an EV of the particular cell types described herein.
• many EV-based therapeutics use stem cells which are cultured continuously, with EVs being extracted from the culture media. During this culturing the exposure to growth factors and other variables may affect the differentiation and thus the therapeutic efficacy of the derived therapeutics. Because of the altering expression of the cells there is a need for a highly repeatable reference standard to allow for accurate and efficient manufacturing of therapeutics.
• the proteoliposomes of this disclosure may meet this need by providing scalable and customizable models that are biomimetic of each of a variety of EVs.
• the one or more proteins may comprise tetraspannins proteins - such as CD proteins including CD 9, 63, 81, or 82 - integrins, Alix, TSG101, clathrin, Ubiquitin, HSP90, HSC90, HSP70, PD-L1, MHC, growth factors, lipoproteins, and polymerases, so as to be biomimetic of the EVs.
• the proteoliposomes that are biomimetic of EVs may comprise any of the aforementioned proteins.
• the composition of the proteoliposomes is designed to mimic transport through a virus.
• the phospholipids of the proteoliposomes are selected such that the composition of the phospholipid carrier mimics the phospholipid composition of a virus.
• the one or more proteins of the proteoliposomes that are biomimetic of a virus comprises capsid proteins.
• the proteoliposomes that are biomimetic of viruses further comprise one or more nucleic acid encapsulated within the one or more phospholipid carrier.
• the one or more nucleic acid encapsulated within the proteoliposome may be an RNA (such as mRNA, miRNA, siRNA, saRNA, or other form of RNA) or DNA with or without modifications to nucleotides and/or end capping.
• RNA such as mRNA, miRNA, siRNA, saRNA, or other form of RNA
• the present disclosure provides methods of making proteoliposomes for mimicking active transport in a model cell or nanoparticle system.
• the methods of this disclosure may be used for obtaining proteoliposomes with greater efficiency and control over previous methods, allow for commercialization and scale-up.
• this disclosure employs a microfluidic and/or extrusion-based mixing platform that may be utilized to produce proteoliposomes with uniform size distributions, low poly dispersity, and batch to batch reproducibility.
• the methods of the present disclosure may produce proteoliposomes by mixing one or more solutions.
• the solutions may be mixed using microfluidic techniques, extrusion techniques, or both.
• Each of the first solution and the second solution may comprise an organic phase solution, an aqueous phase solution, or a combination thereof.
• the first solution may be an organic phase solution and the second solution may be an aqueous phase solution.
• the organic phase solution may include the one or more phospholipids, the one or more protein or both in a volatile liquid, such as, for example, ethanol.
• the aqueous phase solution may include the one or more phospholipids, the one or more protein or both in a buffer or saline.
• the one or more phospholipids may be a phospholipid mixture of multiple types of phospholipids.
• the mixing of the phospholipid mixture with the one or more proteins produce the proteoliposomes.
• the proteoliposomes may be formed by mixing an organic phase solution including the one or more phospholipid with an aqueous phase solution comprising the one or more proteins.
• the organic phase solution may also include one or more proteins, which may be the same or different than the proteins in the aqueous phase. The addition of proteins into the organic phase solution may more reliably integrate the protein into the resulting phospholipid bilayer. If protein is only added with the aqueous phase, this may increase the concentration of protein within the phospholipid bilayer, but may affect the protein folding and thus provide less control over the integration of the protein within the lipid bilayer.
• the methods of mixing the first and second solutions may comprise a microfluidic approach, an extrusion approach, or a combination thereof for manufacturing the proteoliposomes.
• the method of making proteoliposomes may comprise using a microfluidic approach follow ed by an extrusion approach, a microfluidic approach without an extrusion approach, or an extrusion approach without a microfluidic approach.
• the microfluidics approach for mixing the solutions comprises flow ing one or more of the first solution and the second solution through a microfluidic channel under laminar or turbulent flow.
• the microfluidic approach is performed using flow rates of each solution, e.g., an organic phase flow rate and an aqueous phase flow rate, from 0.2 mL/min to 20 mL/min.
• the organic phase flow rate and aqueous phase flow rate flow are at a predetermined ratio.
• the predetermined ratio correlates to Reynolds numbers of near zero to the turbulent regime.
• the extrusion approach comprises mixing one or more of the first solution and the second solution, and extruding the mixture through a membrane.
• the mixing in the extrusion approach may comprise forming an organic phase solution of a mixture of one or more phospholipids and/or proteins, applying gas to the mixture (e.g., nitrogen) to form a thin film. vacuuming the thin film to remove the organic phase, rehydrating with a buffer solution, and further mixing the rehydrated solution with agitation such as, for example, by vortexing. This may form larger vesicles in the mixture that may be extruded through the membrane.
• the membrane is porous, such that extrusion of the mixture through the membrane forms proteoliposomes allows for control over the sizing and uniformity of the proteoliposomes.
• a pore size of the membrane may range from about 1 nm to about 1 mm.
• the mixture of proteoliposomes may be extruded through the membrane any number of times to achieve a desired uniformity.
• the resulting proteoliposomes are diluted any number of times to achieve a desired concentration.
• the resulting proteoliposomes are stabilized by using a buffer exchange.
• the proteoliposomes will go through an additional extrusion or filtration with a bilayer which may or may not contain one or more protein. Additional extrusion or filtration with a bilayer containing one or more protein may provide further incorporation of the one or more protein into the proteoliposome. whereas without the protein will yield no further addition of protein into the proteoliposome.
• the mixing is performed using a range of ratios of aqueous phase solution to organic phase solution from 1: 1 and beyond.
• the aqueous phase solution is acidic.
• the aqueous phase solution is basic.
• the aqueous phase solution is polar.
• the organic phase solution is configured to dissolve the one or more phospholipid.
• a ratio of the one or more phospholipid to one or more protein is 1:100.
• a concentration of the one or more phospholipid is 1-50 mg/mL.
• a concentration of the one or more phospholipid is 1-50 mM.
• the proteoliposomes can either be concentrated or diluted to meet the desired concentration.
• each of the one or more phospholipid includes one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SPH), or cholesterol.
• the one or more phospholipid are synthetically generated or extracted from the target cells or cell membrane to be mimicked so as to produce a biomimetic phospholipid carrier specific to that cell type.
• the one or more proteins to be mixed may be one or more channel proteins of the ATP binding cassette (ABC), solute carrier (SLC) family or both, such as, for example, ABCB1, ABCG2, or SLC22A5.
• the one or more proteins to be mixed may be tetraspannins - e.g., CD 9, 63, 81, or 82 - integrins, Alix, TSG101, clathrin, Ubiquitin, HSP90, HSC90, HSP70, PD-L1, MHC, grow th factors, lipoproteins, and/or polymerases, so as to mimic EVs.
• the method of making the proteoliposomes may be modified for loading the proteoliposomes with nucleic acids.
• the phospholipid mixture is formed by an organic phase solution containing the one or more phospholipid in ethanol, with or without the one or more protein, mixed with an aqueous phase solution containing buffer, with the one or more protein and the one or more nucleic acid, thereby forming nucleic acid-loaded proteoliposomes.
• the phospholipid mixture may be formed by an organic phase solution containing the phospholipids with proteins in ethanol, mixed with an aqueous solution containing the proteins and the nucleic acids in the buffer.
• the phospholipid mixture may be formed by an organic phase solution containing the phospholipids in ethanol, w ithout the proteins, mixed with an aqueous solution containing the proteins and the nucleic acids in the buffer. Concentrations of nucleic acids or proteins can be increased or decreased through concentration within the proteoliposomes or the concentration of the proteoliposomes in the solution.
• the one or more nucleic acid is an RNA (such as mRNA, miRNA, siRNA, saRNA, or other form of RNA) or DNA with or without modifications to nucleotides and/or end capping.
• the one or more nucleic acid is an encoding or non-encoding RNA or DNA. Examples of the one or more nucleic acid include miRNA 10, 21, 124, 125, 126, 130, and/or 132.
• the one or more nucleic acid is present at a concentration of 0-2 mg/mL. In some embodiments, the one or more nucleic acid is present at a concentration of about 0.01-100 pg/mL.
• the nucleic acid utilizes a chemical bond, nanoparticle, or conjugation to increase an encapsulation of the nucleic acid.
• the present disclosure provides methods of making placental proteoliposomes (PPLs).
• the organic phase solution includes the one or more phospholipid placental lipid components in ethanol and the aqueous phase solution includes the one or more placental channel protein in a buffer. In some embodiments, this mixing is performed using a range of ratios of aqueous phase solution to organic phase solution from 1 : 1 and beyond.
• the aqueous phase solution is polar.
• the organic phase solution is configured to dissolve the one or more phospholipid.
• the microfluidic mixing is performed using flow rates of each solution, e.g., an organic phase flow rate and an aqueous phase flow rate, from 0.2 mL/min to 20 mL/min.
• the organic phase flow rate and aqueous phase flow rate flow at a predetermined ratio.
• the predetermined ratio correlates to Reynolds numbers of near zero to the turbulent regime.
• a ratio of the one or more phospholipid to one or more protein is 1: 100.
• a concentration of the one or more phospholipid is 1-50 mg/mL.
• a concentration of the one or more phospholipid is 1-50 mM.
• the proteoliposomes can either be concentrated or diluted to meet the desired concentration.
• the present disclosure provides a composition of one or more of the proteoliposomes provided herein within a medium.
• the medium comprises extracellular matrix (ECM).
• ECM extracellular matrix
• the medium comprises collagen.
• the medium comprises agar.
• the medium comprises alginate.
• the medium is in the shape of a cell- or tissuerepresentative droplet.
• the droplet is representative of placental cell or tissue.
• FIG. 2 presents a schematic for bioprinting the cell-free model to form a proteoliposome-ECM.
• the cell-free model comprises proteoliposomes bioprinted into a medium.
• the medium comprises ECM.
• the ECM comprises one or more of collagen, laminin, and fibronectin.
• the medium comprises ECM droplets.
• the ECM is configured to cross-link.
• the ECM is bioprinted as a droplet.
• bioprinting comprises multiple printheads configured for high-throughput.
• each of the multiple printheads comprises one or more of the NPs and the ECM.
• the bioprinting is automated.
• the ECM comprises one or more of structural proteins, growth factors, and cytokines.
• one or more PPL is bioprinted onto ECM to form a PPL-ECM.
• compositions and methods of the present disclosure relates to the development of proteoliposomes, in the absence of cells, to facilitate active transport screening of a wide range of pharmaceuticals by incorporating key transmembrane proteins that have been identified as important components for various cell transport mechanisms. These methods further allow for the understanding of how pharmaceuticals impact nutrient transport.
• compositions and methods of the present disclosure may be used for any proteoliposome or nucleic-acid loaded proteoliposome models for mimicking cell-derived nanoparticles in analytics and research applications.
• the proteoliposomes disclosed herein can be used as reference standards for analytical assays that provide concentrations or critical quality attributes of products.
• Such assays may be used, as provided herein, for evaluating an efficacy and function of the biomimetic proteoliposomes of this disclosure in comparison to cells or cell-derived nanoparticles, depending on the type of cell or nanoparticle.
• quality attributes can be assessed through cell- or animal-based assays, such as, for example, scratch tests or other wound healing studies, or other transfection efficiency studies.
• proteoliposomes may be applied include chemical assays such as immunoassays, Bicinchoninic acid (BCA) assays, or fluorescencebased nucleic acid quantitation assays, as well as PCR-based analytics such as capillary electrophoresis, mass spectrometry, chromatography, or SDS-page.
• chemical assays such as immunoassays, Bicinchoninic acid (BCA) assays, or fluorescencebased nucleic acid quantitation assays
• PCR-based analytics such as capillary electrophoresis, mass spectrometry, chromatography, or SDS-page.
• ECM extracellular matrix
• the proteoliposomes are bioprinted into cell- or tissue-representative ECM droplets. Again, this technology allows for automation and scale up in order for the proteoliposomes and proteoliposome-ECM compositions to be commercially translatable. This method enables the screening of transport of pharmaceuticals and nutrients using the proteoliposome-ECM droplet models.
• the second mixing includes an extrusion approach.
• the phospholipid solution includes ethanol.
• the protein solution includes in buffer.
• the one or more phospholipid includes one or more of PC, PE, PI, PS, and SPH.
• the one or more phospholipid includes a mixture of one or more of PC, PE, PI, PS, and SPH.
• the one or more protein includes one or more transmembrane protein.
• the one or more transmembrane protein includes one or more of ABCB1, ABCG2, and SLC22A5.
• the method further includes a third mixing of each of the one or more phospholipid carrier with ECM to form one or more carrier-ECM.
• the ECM is formed as a droplet.
• the ECM includes one or more of structural proteins, growth factors, and cytokines.
• the third mixing includes bioprinting.
• the bioprinting includes one or more printhead configured for high- throughput.
• each of the one or more printhead includes one or more of the one or more phospholipid carrier and the ECM.
• the bioprinting is automated.
• the techniques described herein relate to a method for screening pharmaceuticals, including: (a) incubating one or more of a phospholipid carrier and carrier- ECM with one or more molecule of interest to form an incubation product; (b) filtering the incubation product to produce a filtered product; and (c) quantifying the filtered product to assess transport of the one or more molecule of interest into one or more of the phospholipid carrier and carrier-ECM.
• the one or more molecule of interest includes one or more of a pharmaceutical composition, a nutrient composition and a toxin.
• the nucleic acid utilizes a chemical bond, nanoparticle, or conjugation to increase the binding, entrapment, and/or encapsulation of the nucleic acid.
• the nucleic acid is an RNA (such as mRNA, miRNA, siRNA, saRNA, or other form of RNA) or DNA with or without modifications to nucleotides and/or end capping.
• the overall particle is to mimic membranes and vesicles, such as extracellular vesicles (EVs), endosomes, lipoproteins, or viruses.
• EVs extracellular vesicles
• endosomes such as endosomes, lipoproteins, or viruses.
• the composition includes a nucleic acid, protein, or lipid is developed to be representative of the naturally occurring counterpart.
• the placenta plays an important role during pregnancy, yet it remains one of the least understood human organs.
• the main cell type composing the placenta trophoblast cells, have important functions including nutrient and waste transport, invading the endometrium to anchor the placenta, and remodeling vasculature for adequate blood flow.
• These placental trophoblast cells have great potential for use in developing in vitro models of the matemal-fetal interface. Current models for understanding placental transport are extremely limited.
• the placenta is the most species-specific organ, which creates challenges for assessing in vivo studies. Due to this challenge, emerging technologies, including placental microtissues and placenta-on-a-chip microfluidic devices are being studied. However, so far there are no commercially available cell-free models of the placenta to provide highly useful tools for enabling rapid screening studies at effective costs.
• FIG. 1A presents a schematic that is applicable for making the placental proteoliposomes (PPLs).
• PPLs are developed using previously identified lipid composition (Table 1) representative of placental trophoblast cells.
• Table 1 representative of placental trophoblast cells.
• one or more of three different transmembrane proteins important for understanding active transport across the placenta, including ATP-binding cassette proteins ABCB1 and ABCG2 and solute earner SLC22A5 (Table 2) are incorporated into the PPLs.
• the PPLs are incorporated into extracellular matrix (ECM). Placental ECM and fluid further impact pharmaceutical and nutrient transport.
• ECM extracellular matrix
• the ECM orchestrates a complex environment of structural proteins, growth factors, cytokines, among other agents that influence the trophoblast cell’s health and ability to migrate and invade the endometrium. Additionally, the cell’s environment impacts how molecules interact with the cell membrane and transport proteins through both the physical and chemical cues present.
• the developed PPLs are incorporated into ECM to form PPL-ECM.
• the ECM or PPL-ECM are formed as droplets.
• the ECM droplets contain ECM components such as structural proteins, growth factors, and cytokines (Table 3) that mimic the placental microenvironment.
• similar incubation studies are performed with the PPL-ECM droplets.
• an additional step is required to degrade the structural protein.
• the structural protein studied is collagen
• a solution of collagenase is added to the droplet.
• an ultracentrifuge filter is used to collect the PPLs and the amount of compound transported into the PPL is measured.
• controls of PPLs formed without the transport proteins are performed with both models.
• transport across the PPL and PPL-ECM models are compared to transport across trophoblast cells.
• the formulated PPLs can be made on demand with better reproducibility than their cell derived counterparts. Additionally, they can be formulated to assess each component of the native particles, a feature that is not readily available using cell derived standards.
• the size of the biomimetic EVs can be tailored to meet the needs of the application from 50 nm to 1 pm.
• FIGS. 8A-8B provide kinetic study results of cell-extracted PPLs and biomimetic PPLs, respectively, using Folic Acid and Adenosine triphosphate (ATP) showing the activity of the proteins as testing variables, respectively.
• FIG. 8A depicts cell-derived vesicles extracted from placental trophoblast cells
• FIG. 8B depicts fully biomimetic proteoliposomes. i.e., the PPLs.
• In blue shows the transport through the bilayer without channel protein activity, i.e.. just the Folic Acid.
• the active transport through the bilayer is depicted from experimental results using ATP to drive the active transport.
• PBS with no drug product is added as a negative control.
• the testing variables were measured using absorbance spectra above the UV range, e.g., 0-300 nm.
• the PPLs used in FIGS. 8A-8B comprise the lipid structure in Table 1 and ABCB1 channel proteins.
• FIGS. 9A-9B provide an example of hydrodynamic diameters of the PPLS, indicating comparable diameters to the cell- extracted PPLs to the biomimetic PPLs.
• cryo-TEM cryo transmission electron microscopy
• compositions for representing reference standards or for critical quality attribute (CQA) assessment of pharmaceuticals for mimicking extracellular vesicles and other cell derived nanoparticles The utility of the assay described is needed, as most EV therapeutics use stem cells which are cultured continuously, and EVs are extracted from the culture media. During this culturing the exposure to growth factors and other variables will affect the differentiation and thus the therapeutic efficacy of the derived therapeutics.
• CQA critical quality attribute
• FIGS. 10A-10B present experimental results of isolated, i.e., cell-extracted, EVs in comparison to biomimetic EVs generated by the methods of this disclosure.
• the isolated EVs were extracted from placental trophoblast cells.
• the biomimetic EVs were produced by mixing phospholipids with proteins and nucleic acids, where the proteins comprise one or more of tetraspannins such as CD 9, 63, 81, or 82, integrins, Alix, TSG101, clathrin, Ubiquitin, HSP90, HSC90, HSP70, PD-L1, MHC, growth factors, lipoproteins, and polymerase proteins.
• the nucleic acids include at least miRNA such as one or more of miRNA 10, 21, 124, 125. 126, 130, and/or 132, mRNA encoding fluorescent protein, and/or other nucleic acids.
• the results were obtained by encapsulating nucleic acids in a lipid nanoparticle then extruding the lipid nanoparticle through a biomimetic phospholipid bilayer with or without protein.
• the characterization techniques included assessing the particle hydrodynamic diameter, polydispersity, and zeta potential via dynamic light scattering as well as performing cryo transmission electron microscopy (cryo-TEM) for structural analysis.
• the size of the biomimetic EVs can be tailored to meet the needs of the application from 50 nm to 1 pm.
• FIG. 11 presents experimental results of encapsulated nucleic acids, in particular nucleic acids that are encapsulated within biomimetic EVs generated by the methods of this disclosure.
• the biomimetic EVs were produced by mixing phospholipids with proteins and nucleic acids, where the proteins comprise one or more of tetraspannins such as CD 9, 63, 81, or 82, integrins, Alix, TSG101, clathrin, Ubiquitin, HSP90, HSC90, HSP70, PD-L1. MHC, growth factors, lipoproteins, and polymerase proteins.
• the nucleic acids include at least miRNA such as one or more of miRNA 10, 21, 124, 125, 126, 130, and/or 132, mRNA encoding fluorescent protein, and/or other nucleic acids.
• the results were obtained by encapsulating nucleic acids in a lipid nanoparticle then extruding the lipid nanoparticle through a biomimetic phospholipid bilayer with or without protein.
• Encapsulation efficiency w as evaluated by comparing the amount of nucleic acids were present outside of the biomimetic proteoliposomes relative to the amount inside the proteoliposomes. Concentrations of nucleic acids or proteins can be increased or decreased through concentration in particles or concentration of particles.
• a '‘cell’ 7 refers to a biological cell.
• Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaea cell, a cell of a single-cell eukary otic organism, a protozoa cell, a cell from a plant, an algal cell, a fungal cell, a fungal protoplast cell, an animal cell, and the like.
• a cell is not originating from a natural organism, e.g.. a cell can be a synthetically made, sometimes termed an artificial cell.
• a quantitative characteristic e.g., largest lateral dimension
• this refers to the quantitative characteristic having a value between the smaller value and the larger value or equal to the smaller value of the larger value.
• the characterizing term “uniform” in referencing a quantity refers to a variation in that quantity by no more than 10% more or less than the stated value or an average of that quantity (e.g., no more than 5% more or less, no more than 1% more or less, no more than 0.1% more or less than the stated value or an average of that quantity).

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• 2023
  • 2023-11-13 EP EP23889825.8A patent/EP4615419A2/de active Pending

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