WO2024249782A2 - Nanovésicules protéolipidiques biomimétiques pour l'administration d'acides nucléiques - Google Patents

Nanovésicules protéolipidiques biomimétiques pour l'administration d'acides nucléiques Download PDF

Info

Publication number
WO2024249782A2
WO2024249782A2 PCT/US2024/031899 US2024031899W WO2024249782A2 WO 2024249782 A2 WO2024249782 A2 WO 2024249782A2 US 2024031899 W US2024031899 W US 2024031899W WO 2024249782 A2 WO2024249782 A2 WO 2024249782A2
Authority
WO
WIPO (PCT)
Prior art keywords
molar
nanovesicle
biomimetic
proteolipid
biomimetic proteolipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/031899
Other languages
English (en)
Other versions
WO2024249782A3 (fr
Inventor
Manuela SUSHNITHA
Francesca TARABALLI
Assaf Yosef ZINGER
Chiara MANCINO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Methodist Hospital
Original Assignee
Methodist Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Methodist Hospital filed Critical Methodist Hospital
Publication of WO2024249782A2 publication Critical patent/WO2024249782A2/fr
Publication of WO2024249782A3 publication Critical patent/WO2024249782A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • NP complex biological milieu encountered by NP upon entry into the bloodstream poses significant biological barriers that thwart their ability to deliver their payload to the target tissue.
  • systemic administration of NP exposes them to rapid uptake and clearance by components of the mononuclear phagocyte system (MPS).
  • MPS mononuclear phagocyte system
  • these NP do not reach the target site and, thereby, do not exert their therapeutic effects.
  • Previous efforts to overcome these challenges have included the incorporation of polyethylene glycol (PEG) to improve circulation times and conjugation of targeting moieties, such an antibodies and peptides 5 , to facilitate preferential accumulation to disease sites.
  • PEG polyethylene glycol
  • Biomimetic NP represent an emerging class of NP that aim to address the current challenges faced by the field of nanomedicine through biomimicry of native cells. Work in this field encompasses a broad range of NP, ranging from those mimicking red blood cells to immune cells to even cancer cells. Use of these biomimetic approaches has shown how traditionally used NP platforms can now harness the features of native cells to achieve specific function while maintaining the superior delivery capabilities of a synthetic NP.
  • Examples of this include red blood cell membrane-coated polymeric NP that achieve longer circulation times for toxin removal in the Docket No.10063-098WO1 blood and chemotherapy-loaded NP cloaked with cancer cell membranes for homotypic targeting of tumor cells.
  • the syntheses of biomimetic NP have taken on two primary forms: top-down and bottom- up approaches. Isolation of whole cell membranes which are then applied in toto onto a synthetic NP core is an example of a top-down approach where the extracted component maintains the full biological complexity of the source.
  • bottom-up approaches utilize incorporation of ligands or other components as the building blocks to integrate into the final NP, such as the integration of membrane proteins into synthetic NP.
  • top-down approaches serve as a bridge between synthetic NP and source cells
  • bottom-up approaches offer more control in the tuning of the final NP formulation.
  • synthesis approach utilized, maintenance of key NP physicochemical and biological characteristics, both during and after the synthesis process, is a crucial component in the engineering of these platforms. Achievement of specific functionality using these complex biomimetic NP warrants the careful and rational tuning of parameters associated with the synthesis process. Parameters such as the ratio of NP to extracted cell membrane, temperature used during the synthesis steps and post-synthesis purification process are examples of factors that must be carefully considered.
  • the engineering of these design criteria has significant effects not only on the physicochemical properties of the NP, but also their biomimetic behavior under biological conditions.
  • Leukocyte-based biomimetic NP for targeting inflamed tissues i.e., Leukosomes
  • Leukosomes Leukocyte-based biomimetic NP for targeting inflamed tissues
  • Leukosomes Leukosomes
  • Leukosomes have demonstrated the ability to home to sites of inflammation and preferentially adhere to inflamed endothelia.
  • the feasibility of synthesizing these NP was demonstrated using two synthesis methods – thin layer evaporation and a microfluidic-based approach.
  • characterization of the NP verified their physiochemical properties while their biological functions were demonstrated in a local inflammation model.
  • this NP platform provides a very powerful tool for effective targeting and therapeutic cargo delivery. Furthermore, the tunability of this targeting is important for the tailoring of these NP to a specific disease condition. Building off this foundational work, this work aimed to demonstrate the tunability of this system within the context of delivery of nucleic acids while retaining leukosome’s tropism towards inflammation. In particular, this work focused on the engineering of the synthesis parameters by establishing key design criteria. These design criteria included thresholds on size and PDI, conservation of key leukocyte proteins, maintenance of the lipid bilayer structure and NP stability.
  • a biomimetic proteolipid nanovesicle including: an ionizable or cationic lipid; a phosphocholine-based phospholipid ; a cholesterol; a leukocyte membrane protein ; and an siRNA encapsulated by the biomimetic proteolipid nanovesicle; wherein the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of from about 1:65 to about 1:85 by weight.
  • a biomimetic proteolipid nanovesicle including: an ionizable or cationic lipid; a phosphocholine-based phospholipid; a cholesterol; a leukocyte membrane protein; and an mRNA encapsulated by the biomimetic proteolipid nanovesicle; wherein the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of from about 1:80 to about 1:100 by weight.
  • a method of making any of the disclosed biomimetic proteolipid nanovesicles including: a) dissolving a phosphocholine-based phospholipid, an ionizable or cationic lipid, and a cholesterol in an organic solvent to produce an organic lipid solution; b) dissolving a leukocyte membrane protein and an agent in water to produce an aqueous protein solution; and c) loading the organic lipid solution into an organic phase inlet of a microfluidic mixer, and loading the aqueous protein solution into an aqueous phase inlet of said microfluidic mixer; and d) adjusting flow rates of each inlet stream and a flow ratio between each inlet stream to produce the biomimetic proteolipid nanovesicles having a specified lipid-to- protein ratio by weight therefrom.
  • a method for the delivery of an agent into a cell including introducing into the cell any of the disclosed biomimetic proteolipid nanovesicles.
  • a method of treating a disease or disorder in a subject in need thereof the method including administering to the subject any of the disclosed biomimetic proteolipid nanovesicles.
  • FIG. 1A, FIG.1B, FIG.1C, and FIG.1D depict physicochemical properties of NPs synthesized with 3 lipid backbones.
  • Size (FIG. 1A), polydispersity index (PDI) (FIG. 1B), zeta potential (FIG.1C), and siRNA encapsulation efficiency (FIG.1D) characterization of liposomes synthesized from 3 different lipid backbones revealed maintenance of size less than 200nm and PDI less than 0.2 among all 3 formulations, while DOTAP NPs exhibited positive surface charge and lowest siRNA encapsulation efficiency.
  • n 3-6. Results are shown as means ⁇ SEM. One- way ANOVA followed by Tukey’s multiple comparison test was used to determine statistical significance.
  • FIG.2A and FIG.2B depict cytotoxicity of DAP and DLin-MC3 liposomes on 4T1 cells.
  • n 4. Results are shown as means ⁇ SEM. Docket No.10063-098WO1
  • FIG.3A, FIG.3B, FIG.3C, FIG.3D, and FIG.3E depict siRNA knockdown efficiency of DAP and DLin-MC3 liposomes on TNBC cells.
  • FIG.3A Knockdown efficiency of STAT3 mRNA expression following treatment with DAP (FIG.3A) and DLin-MC3 (FIG. 3B) liposomes revealed the latter induced almost 80% knockdown.
  • FIG.3C shows further evaluation of DLin- MC3 knockdown efficiency using heparinase siRNA-loaded liposomes corroborated the 80% knockdown efficiency of this formulation.
  • FIG.3D shows reduction of GFP-protein expression in MDA-MB-231-GFP cells by Lipofectamine and DLin-MC3 indicated similar levels of protein reduction by both systems for up to 72h.
  • FIG.3E shows quantification of the green signal demonstrated the reduction in both the Lipo and Lipofectamine treated groups.
  • n 3. Results are shown as means ⁇ SEM.
  • FIG.4A, FIG.4B, FIG.4C, FIG.4D, FIG.4E, and FIG.4F depict a comparison of NP properties of siRNA-liposomes and siRNA-leukosomes. Size (FIG.4A) and polydispersity index (PDI) (FIG.4B) measurements revealed significant differences between siRNA-liposomes and siRNA-leukosomes, especially after dialysis. Both NPs maintained an overall negative charge in their zeta potential (FIG.4C).
  • FIG.4D shows visual inspection of the collected samples further corroborated the differences between liposomes and leukosome prior to filtration.
  • FIG.4E shows quantification of siRNA loss demonstrated the significant reduction of siRNA in the leukosomes during the filtration step.
  • n 3. Results are shown as means ⁇ SEM.2-way ANOVA followed by Sidak’s multiple comparison test was used to determine statistical significance in A-B. Welch’s t- test was used to determine statistical significance in F. P value ⁇ 0.05 among means was considered as statistically significant.
  • FIG.5A, FIG.5B, and FIG.5C depict the effect of removing PEG on NP physicochemical properties.
  • FIG.5A results are shown as means ⁇ SEM. 2-way ANOVA followed by Sidak’s multiple comparison test was used to determine statistical significance. P value ⁇ 0.05 among means was considered as statistically significant.
  • FIG.6A, FIG.6B, FIG.6C, and FIG.6D depict the effect of increasing flow rate on NP physicochemical properties. Size (FIGS.6A-6B) and polydispersity (PDI) (FIGS.
  • FIG.7A and FIG.7B depict a cytotoxicity profile of siRNA-leukosomes with different PEG ratios on 4T1 cells.
  • FIG.7A Cell viability analysis following treatment of 4T1 cells with leukosomes of mid PEG percentage (FIG.7A) and high PEG percentage (FIG.7B) for up to 72h indicated reduction of viability in concentrations above 500 ⁇ M and 250 ⁇ M, respectively.
  • n 4. Results are shown as means ⁇ SEM.
  • FIG.9A, FIG.9B, FIG.9C, and FIG.9D depict a biodistribution profile of siRNA-NPs with varying PEG ratios.
  • FIG.11 depicts a schematic of the leukosome-LNPs.
  • FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D depict testing results of iteration 1 for siRNA encapsulation.
  • FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D depict testing results of iteration 2 for siRNA encapsulation.
  • FIG.14 depicts day 0 post synthesis of iteration 3 for siRNA encapsulation.
  • FIG. 15B, FIG. 15C, and FIG. 15D depict stability results of iteration 3 for siRNA encapsulation.
  • FIG.16A and FIG.16B depicts in vitro tests for siRPL39 Leukosomes-LNPs. Docket No.10063-098WO1
  • FIG. 17A, FIG. 17B, and FIG. 17C depict in vivo downregulation tests for siRPL39 Leukosomes-LNPs.
  • FIG.18A and FIG.18B depict in vivo targeting tests for siRPL39 Leukosomes-LNPs.
  • FIG. 19A, FIG. 19B, FIG. 19C, and FIG. 19D depict testing results of iteration 1 for mRNA encapsulation.
  • FIG. 20C, and FIG. 20D depict in vivo biodistribution of Leukosome-LNPs.
  • DETAILED DESCRIPTION It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination.
  • each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably.
  • the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.”
  • the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
  • the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
  • Docket No.10063-098WO1 reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein.
  • an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • a “biocompatible” material is a synthetic or natural material used to replace part of a living system or to function in intimate contact with living tissue. Biocompatible materials are intended to interface with biological systems to evaluate, treat, augment, or replace any tissue, organ, or function of the body. The biocompatible material has the ability to perform with an appropriate host response in a specific application and does not have toxic or injurious effects on biological systems.
  • biocompatible material can be a biocompatible ceramic.
  • biomimetic shall mean a resemblance of a synthesized material to a substance that occurs naturally in a human body and which is not rejected by (e.g., does not cause an adverse reaction in) the human body.
  • buffer includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions.
  • Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.
  • the term “carrier” is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert (s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.
  • an “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material.
  • an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity.
  • the specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.
  • the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts.
  • the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration.
  • single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • a response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • the amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • prophylactically effective amount refers to an amount effective for preventing onset or initiation of a disease or condition.
  • prevent or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
  • the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • subject can refer to a vertebrate organism, such as a mammal (e.g. human).
  • Subject can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
  • treating can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as an ophthalmological disorder.
  • treatment can include any treatment of ophthalmological disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its Docket No.10063-098WO1 symptoms or conditions.
  • treatment can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment.
  • Those in need of treatment can include those already with the disorder and/or those in which the disorder is to be prevented.
  • the term “treating” can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • dose can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.
  • nucleic acid can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.
  • nucleic acid and nucleic acid sequences refer to nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).
  • deletions refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence.
  • a deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides.
  • a deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5′-terminal or 3′-terminal truncation or both of a reference polynucleotide).
  • variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein.
  • a “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence.
  • a fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous Docket No.10063-098WO1 nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively.
  • a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and/or the C-terminal region of a polypeptide or the 5′-terminal region and/or the 3′ terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.
  • insertions or additions refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides.
  • An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides. Fusion polynucleotides also are contemplated herein.
  • a “fusion polynucleotide” refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3′ end of a first polynucleotide to a 5′ end of the second polynucleotide).
  • the fusion may be such that the encoded proteins are in-frame and results in a fusion protein.
  • the first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).
  • “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence Docket No.10063-098WO1 analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above). Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
  • a “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250).
  • a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. Docket No.10063-098WO1
  • a “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
  • recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • substantially isolated or purified nucleic acid or amino acid sequences are contemplated herein.
  • substantially isolated or purified refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
  • mismatched or mismatched target sequence refers to an off-target sequence that is not perfectly complementary to the first DNA sequence or the second DNA sequence of the chimeric deoxyribonucleic acid described herein.
  • the dual retargeted DNA may have at least one mismatch, but can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides to the off-target sequence.
  • a biomimetic proteolipid nanovesicle including: an ionizable or cationic lipid; a phosphocholine-based phospholipid; a cholesterol; a leukocyte membrane protein; Docket No.10063-098WO1 and an siRNA encapsulated by the biomimetic proteolipid nanovesicle; wherein the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of from about 1:65 to about 1:85 by weight.
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of at least about 1:50 (e.g., at least about 1:55, at least about 1:60, at least about 1:65, at least about 1:66, at least about 1:67, at least about 1:68, at least about 1:69, at least about 1:70, at least about 1:71, at least about 1:72, at least about 1:73, at least about 1:74, at least about 1:75, at least about 1:76, at least about 1:77, at least about 1:78, at least about 1:79, at least about 1:80, at least about 1:81, at least about 1:82, at least about 1:83, at least about 1:84, at least about 1:85, at least about 1:90, at least about 1:95, at least about 1:100) by weight.
  • lipid-to-protein ratio of at least about 1:50 (e.g., at least about 1:55, at least about 1
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of up to about 1:100 (e.g., up to about 1:95, up to about 1:90, up to about 1:85, up to about 1:84, up to about 1:83, up to about 1:82, up to about 1:81, up to about 1:80, up to about 1:79, up to about 1:78, up to about 1:77, up to about 1:76, up to about 1:75, up to about 1:74, up to about 1:73, up to about 1:72, up to about 1:71, up to about 1:70, up to about 1:69, up to about 1:68, up to about 1:67, up to about 1:66, up to about 1:65, up to about 1:60, up to about 1:55, up to about 1:50) by weight.
  • lipid-to-protein ratio of up to about 1:100 (e.g., up to about 1:95, up to about 1:
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of about 1:50, about 1:55, about 1:60, about 1:65, about 1:66, about 1:67, about 1:68, about 1:69, about 1:70, about 1:71, about 1:72, about 1:73, about 1:74, about 1:75, about 1:76, about 1:77, about 1:78, about 1:79, about 1:80, about 1:81, about 1:82, about 1:83, about 1:84, about 1:85, about 1:90, about 1:95, or about 1:100 by weight.
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can have a lipid-to- protein ratio of from about 1:65 to about 1:85 (e.g., from about 1:66 to about 1:84, from about 1:67 to about 1:83, from about 1:68 to about 1:82, from about 1:69 to about 1:81, from about 1:70 to about 1:80, from about 1:71 to about 1:79, from about 1:72 to about 1:78, from about 1:73 to about 1:77, from about 1:74 to about 1:76, from about 1:65 to about 1:75, from about 1:66 to about 1:74, from about 1:67 to about 1:73, from about 1:68 to about 1:72, from about 1:69 to about 1:71, from about 1:75 to
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of from about 1:50 to about 1:100 by weight. Docket No.10063-098WO1
  • the ionizable or cationic lipid can be selected from the group consisting of DLin-MC3-DMA, SM-102, ALC-0315, 1,2-dimyristoyl-3-dimethylammonium-propane (DAP), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), C12-200, 5A2-SC8, and any combination thereof.
  • the biomimetic proteolipid nanovesicle can further include at least about 40 molar % (e.g., at least about 42 molar %, at least about 44 molar %, at least about 46 molar %, at least about 48 molar %, at least about 50 molar %, at least about 52 molar %, at least about 54 molar %, at least about 56 molar %, at least about 58 molar %, at least about 60 molar % ) of the ionizable or cationic lipid.
  • at least about 40 molar % e.g., at least about 42 molar %, at least about 44 molar %, at least about 46 molar %, at least about 48 molar %, at least about 50 molar %, at least about 52 molar %, at least about 54 molar %, at least about 56 molar %, at least about 58 molar %
  • the biomimetic proteolipid nanovesicle can further include up to about 60 molar % (e.g., up to about 58 molar %, up to about 56 molar %, up to about 54 molar %, up to about 52 molar %, up to about 50 molar %, up to about 48 molar %, up to about 46 molar %, up to about 44 molar %, up to about 42 molar %, up to about 40 molar %) of the ionizable or cationic lipid.
  • up to about 60 molar % e.g., up to about 58 molar %, up to about 56 molar %, up to about 54 molar %, up to about 52 molar %, up to about 50 molar %, up to about 48 molar %, up to about 46 molar %, up to about 44 molar %, up to about 42 molar %, up
  • the biomimetic proteolipid nanovesicle can further include about 40 molar %, about 42 molar %, about 44 molar %, about 46 molar %, about 48 molar %, about 50 molar %, about 52 molar %, about 54 molar %, about 56 molar %, about 58 molar %, or about 60 molar % of the ionizable or cationic lipid. It is considered that the biomimetic proteolipid nanovesicle can further include an amount of the ionizable or cationic lipid ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can include from about 40 molar % to about 60 molar % (e.g., from about 42 molar % to about 58 molar %, from about 44 molar % to about 56 molar %, from about 46 molar % to about 54 molar %, from about 48 molar % to about 52 molar %, from about 40 molar % to about 50 molar %, from about 42 molar % to about 48 molar %, from about 44 molar % to about 46 molar %, from about 50 molar % to about 60 molar %, from about 52 molar % to about 58 molar %, from about 54 molar % to about 56 molar %) of the ionizable or cationic lipid.
  • the phosphocholine-based phospholipid can be selected from the group consisting of phosphatidylcholine, egg phosphatidic acid, 1,2-dioleoyl-sn-glycerophosphocholine (DOPC), 1,2-diolyl-sn-lycerophosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn- glycerophosphocholine (DPPC), 1,2-distearoyl-sn-glycerophosphocholine (DSPC), L- ⁇ - phosphatidylserine (PS), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), and any combination thereof.
  • DOPC 1,2-dioleoyl-sn-glycerophosphocholine
  • DOPE 1,2-diolyl-sn-lycerophosphoethanolamine
  • DOPE 1,2-dipalmitoyl-sn- glycerophosphocholine
  • the biomimetic proteolipid nanovesicle can include at least about 7 molar % (e.g., at least about 7.5 molar %, at least about 8 molar %, at least about 8.5 molar %, at least about 9 molar %, at least about 9.5 molar %, at least about 10 molar %, at least about 10.5 molar Docket No.10063-098WO1 %, at least about 11 molar %, at least about 11.5 molar %, at least about 12 molar %, at least about 12.5 molar %, at least about 13 molar %, at least about 13.5 molar %, at least about 14 molar %, at least about 14.5 molar %, at least about 15 molar %) of the phosphocholine-based phospholipid.
  • at least about 7 molar % e.g., at least about 7.5 molar %, at least about 8 m
  • the biomimetic proteolipid nanovesicle can include up to about 15 molar % (e.g., up to about 14.5 molar %, up to about 14 molar %, up to about 13.5 molar %, up to about 13 molar %, up to about 12.5 molar %, up to about 12 molar %, up to about 11.5 molar %, up to about 11 molar %, up to about 10.5 molar %, up to about 10 molar %, up to about 9.5 molar %, up to about 9 molar %, up to about 8.5 molar %, up to about 8 molar %, up to about 7.5 molar %, up to about 7 molar %) of the phosphocholine-based phospholipid.
  • up to about 15 molar % e.g., up to about 14.5 molar %, up to about 14 molar %, up to about 13.5 m
  • the biomimetic proteolipid nanovesicle can include about 7 molar %, about 7.5 molar %, about 8 molar %, about 8.5 molar %, about 9 molar %, about 9.5 molar %, about 10 molar %, about 10.5 molar %, about 11 molar %, about 11.5 molar %, about 12 molar %, about 12.5 molar %, about 13 molar %, about 13.5 molar %, about 14 molar %, about 14.5 molar %, or about 15 molar % of the phosphocholine- based phospholipid.
  • the biomimetic proteolipid nanovesicle can further include an amount of the phosphocholine-based phospholipid ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can include from about 7 molar % to about 15 molar % (e.g., from about 7.5 molar % to about 14.5 molar %, from about 8 molar % to about 14 molar %, from about 8.5 molar % to about 13.5 molar %, from about 9 molar % to about 13 molar %, from about 9.5 molar % to about 12.5 molar %, from about 10 molar % to about 12 molar %, from about 10.5 molar % to about 11.5 molar %, from about 7 molar % to about 11 molar %, from about 7.5 molar % to about 10.5 m
  • the biomimetic proteolipid nanovesicle can include at least about 30 molar % (e.g., at least about 30.5 molar %, at least about 31 molar %, at least about 31.5 molar %, at least about 32 molar %, at least about 32.5 molar %, at least about 33 molar %, at least about 33.5 molar %, at least about 34 molar %, at least about 34.5 molar %, at least about 35 molar %, at least about 35.5 molar %, at least about 36 molar %, at least about 36.5 molar %, at least about 37 molar %, at least about 37.5 molar %, at least about 38 molar %, at least about 38.5 molar %, at least about 39 molar %, at least about 39.5 molar %, at least about 40 molar %) of the cholesterol.
  • molar % e.g
  • the biomimetic proteolipid nanovesicle can include up to about 40 molar % (e.g., up Docket No.10063-098WO1 to about 39.5 molar %, up to about 39 molar %, up to about 38.5 molar %, up to about 38 molar %, up to about 37.5 molar %, up to about 37 molar %, up to about 36.5 molar %, up to about 36 molar %, up to about 35.5 molar %, up to about 35 molar %, up to about 34.5 molar %, up to about 34 molar %, up to about 33.5 molar %, up to about 33 molar %, up to about 32.5 molar %, up to about 32 molar %, up to about 31.5 molar %, up to about 31 molar %, up to about 30.5 molar %, up to about 30 molar %) of
  • the biomimetic proteolipid nanovesicle can include about 30 molar %, about 30.5 molar %, about 31 molar %, about 31.5 molar %, about 32 molar %, about 32.5 molar %, about 33 molar %, about 33.5 molar %, about 34 molar %, about 34.5 molar %, about 35 molar %, about 35.5 molar %, about 36 molar %, about 36.5 molar %, about 37 molar %, about 37.5 molar %, about 38 molar %, about 38.5 molar %, about 39 molar %, about 39.5 molar %, or about 40 molar % of the cholesterol.
  • the biomimetic proteolipid nanovesicle can further include an amount of the cholesterol ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can include from about 30 molar % to about 40 molar % (e.g., from about 30.5 molar % to about 39.5 molar %, from about 31 molar % to about 39 molar %, from about 31.5 molar % to about 38.5 molar %, from about 32 molar % to about 38 molar %, from about 32.5 molar % to about 37.5 molar %, from about 33 molar % to about 37 molar %, from about 33.5 molar % to about 36.5 molar %, from about 34 molar % to about 36 molar %, from about 34.5 molar % to about 35.5 molar %, from about 30 molar % to about 40
  • the biomimetic proteolipid nanovesicle can further include a PEGylated lipid.
  • the PEGylated lipid can be selected from the group consisting of DMG- PEG2000, ALC-0159, DSPE-PEG2000, DOPE-PEG2000, 18:1 PEG1000-PE, and any combination thereof.
  • the biomimetic proteolipid nanovesicle can further include at least about 1 molar % (e.g., at least about 1.5 molar %, at least about 2 molar %, at least about 2.5 molar %, at least about 3 molar %, at least about 3.5 molar %, at least about 4 molar %, at least about 4.5 molar %, at least about 5 molar %, at least about 5.5 molar %, at least about 6 molar %, at least about 6.5 molar %, at least about 7 molar %, at least about 7.5 molar %, at least about 8 molar %, Docket No.10063-098WO1 at least about 8.5 molar %, at least about 9 molar %, at least about 9.5 molar %, at least about 10 molar %) of the PEGylated lipid.
  • at least about 1 molar % e.g.
  • the biomimetic proteolipid nanovesicle can further include up to about 10 molar % (e.g., up to about 9.5 molar %, up to about 9 molar %, up to about 8.5 molar %, up to about 8 molar %, up to about 7.5 molar %, up to about 7 molar %, up to about 6.5 molar %, up to about 6 molar %, up to about 5.5 molar %, up to about 5 molar %, up to about 4.5 molar %, up to about 4 molar %, up to about 3.5 molar %, up to about 3 molar %, up to about 2.5 molar %, up to about 2 molar %, up to about 1.5 molar %, up to about 1 molar %) of the PEGylated lipid.
  • up to about 10 molar % e.g., up to about 9.5 molar %
  • the biomimetic proteolipid nanovesicle can include about 1 molar %, about 1.5 molar %, about 2 molar %, about 2.5 molar %, about 3 molar %, about 3.5 molar %, about 4 molar %, about 4.5 molar %, about 5 molar %, about 5.5 molar %, about 6 molar %, about 6.5 molar %, about 7 molar %, about 7.5 molar %, about 8 molar %, about 8.5 molar %, about 9 molar %, about 9.5 molar %, or about 10 molar % of the PEGylated lipid.
  • the biomimetic proteolipid nanovesicle can further include an amount of the PEGylated lipid ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can include from about 1 molar % to about 10 molar % (e.g., from about 1.5 molar % to about 9.5 molar %, from about 2 molar % to about 9 molar %, from about 2.5 molar % to about 8.5 molar %, from about 3 molar % to about 8 molar %, from about 3.5 molar % to about 7.5 molar %, from about 4 molar % to about 7 molar %, from about 4.5 molar % to about 6.5 molar %, from about 5 molar % to about 6 molar %, from about 1 molar % to about 5.5.
  • molar % from about 1.5 molar % to about 5 molar %, from about 2 molar % to about 4.5 molar %, from about 2.5 molar % to about 4 molar %, from about 3 molar % to about 3.5 molar %, from about 5.5. molar % to about 10 molar %, from about 6 molar % to about 9.5 molar %, from about 6.5 molar % to about 9 molar %, from about 7 molar % to about 8.5 molar %, from about 7.5 molar % to about 8 molar %) of the PEGylated lipid.
  • the leukocyte membrane protein can be derived from a leukocyte plasma membrane.
  • the leukocyte plasma membrane can be a human leukocyte plasma membrane.
  • the leukocyte plasma membrane can be derived from a murine leukocyte plasma membrane.
  • the leukocyte membrane protein can be a synthetic recombinant protein.
  • the leukocyte membrane protein can be lymphocyte function-associated antigen 1 (LFA-1), CD11, CD45, CD47, or any combination thereof.
  • the leukocyte membrane protein can include some or all of the peptides present in a leukocyte plasma membrane (e.g., a human leukocyte plasma membrane or a murine leukocyte plasma membrane).
  • N/P ratio refers to the nitrogen-to-phosphate ratio, where “N” represents the nitrogen atoms from the ionizable or cationic lipid, which typically contains amine groups, and “P” refers to the phosphate groups in the nucleic acid molecules, such as the phosphate backbone of siRNA or mRNA. This ratio can ensure the stability, charge balance, and transfection efficiency of the lipid nanoparticles. Its optimization can help achieve optimal encapsulation efficiency in the context of different nucleic acid payloads.
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of at least about 1 (e.g., at least about 1.25, at least about 1.5, at least about 1.75, at least about 2, at least about 2.25, at least about 2.5, at least about 2.75, at least about 3, at least about 3.25, at least about 3.5, at least about 3.75, at least about 4, at least about 4.25, at least about 4.5, at least about 4.75, at least about 5, at least about 5.25, at least about 5.5, at least about 5.75, at least about 6).
  • at least about 1 e.g., at least about 1.25, at least about 1.5, at least about 1.75, at least about 2, at least about 2.25, at least about 2.5, at least about 2.75, at least about 3, at least about 3.25, at least about 3.5, at least about 3.75, at least about 4, at least about 4.25, at least about 4.5, at least about 4.75, at least about 5, at least about 5.25, at least about 5.5, at least
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of up to about 6 (e.g., up to about 5.75, up to about 5.5, up to about 5.25, up to about 5, up to about 4.75, up to about 4.5, up to about 4.25, up to about 4, up to about 3.75, up to about 3.5, up to about 3.25, up to about 3, up to about 2.75, up to about 2.5, up to about 2.25, up to about 2, up to about 1.75, up to about 1.5, up to about 1.25, up to about 1).
  • an N/P ratio of up to about 6 e.g., up to about 5.75, up to about 5.5, up to about 5.25, up to about 5, up to about 4.75, up to about 4.5, up to about 4.25, up to about 4, up to about 3.75, up to about 3.5, up to about 3.25, up to about 3, up to about 2.75, up to about 2.5, up to about 2.25, up to about 2, up to about 1.75, up to
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.25, about 2.5, about 2.75, about 3, about 3.25, about 3.5, about 3.75, about 4, about 4.25, about 4.5, about 4.75, about 5, about 5.25, about 5.5, about 5.75, or about 6. It is considered that the biomimetic proteolipid nanovesicle can further include an N/P ratio ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of from about 1 to about 6 (e.g., from about 1.25 to about 5.75, from about 1.5 to about 5.5, from about 1.75 to about 5.25, from about 2 to about 5, from about 2.25 to about 4.75, from about 2.5 to about 4.5, from about 2.75 to about 4.25, from about 3 to about 4, from about 3.25 to about 3.75, from about 1 to about 3.5, from about 1.25 to about 3.25, from about 1.5 to about 3, from about 1.75 to about 2.75, from about 2 to about 2.5, from about 3.5 to about 6, from about 3.75 to about 5.75, from about 4 to about 5.5, from about 4.25 to about 5.25, from about 4.5 to about 5).
  • N/P ratio of from about 1 to about 6 (e.g., from about 1.25 to about 5.75, from about 1.5 to about 5.5, from about 1.75 to about 5.25, from about 2 to about 5, from about 2.25 to about 4.75, from about 2.5 to about 4.5
  • the biomimetic proteolipid nanovesicle can have a diameter of at least about 50 nm (e.g., at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at least about 170 nm, at least Docket No.10063-098WO1 about 180 nm, at least about 190 nm, at least about 200 nm).
  • nm e.g., at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 n
  • the biomimetic proteolipid nanovesicle can have a diameter of up to about 200 nm (e.g., up to about 190 nm, up to about 180 nm, up to about 170 nm, up to about 160 nm, up to about 150 nm, up to about 140 nm, up to about 130 nm, up to about 120 nm, up to about 110 nm, up to about 100 nm, up to about 90 nm, up to about 80 nm, up to about 70 nm, up to about 60 nm, up to about 50 nm).
  • a diameter of up to about 200 nm e.g., up to about 190 nm, up to about 180 nm, up to about 170 nm, up to about 160 nm, up to about 150 nm, up to about 140 nm, up to about 130 nm, up to about 120 nm, up to about 110 nm, up to about 100 nm
  • the biomimetic proteolipid nanovesicle can have a diameter of about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. It is considered that the biomimetic proteolipid nanovesicle can have a diameter ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can have a diameter of from about 50 nm to about 200 nm (e.g., from about 60 nm to about 190 nm, from about 70 nm to about 180 nm, from about 80 nm to about 170 nm, from about 90 nm to about 160 nm, from about 100 nm to about 150 nm, from about 110 nm to about 140 nm, from about 120 nm to about 130 nm, from about 50 nm to about 130 nm, from about 60 nm to about 120 nm, from about 70 nm to about 110 nm, from about 80 nm to about 100 nm, from about 120 nm to about 200 nm, from about 130 nm to about 190 nm, from about 140 nm to about 180 nm, from about 150 nm to about 170 nm).
  • a diameter of from about 50 nm to about 200 nm
  • the siRNA can be targeted to treat a cancer, inflammation, an infectious disease, or a genetic disease or disorder.
  • mRNA-CONTAINING BIOMIMETIC PROTEOLIPID NANOVESICLES in an aspect, provided is a biomimetic proteolipid nanovesicle, including: an ionizable or cationic lipid; a phosphocholine-based phospholipid; a cholesterol; a leukocyte membrane protein; and an mRNA encapsulated by the biomimetic proteolipid nanovesicle; wherein the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of from about 1:80 to about 1:100 by weight.
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of at least about 1:80 (e.g., at least about at least about 1:81, at least about 1:82, at least about 1:83, at least about 1:84, at least about 1:85, at least about 1:86, at least about 1:87, at least about 1:88, at least about 1:89, at least about 1:90, at least about 1:91, at least about 1:92, at least about 1:93, at least about 1:94, at least about 1:95, at least about 1:96, at least about 1:97, at least about 1:98, at least about 1:99, at least about 1:100) by weight.
  • lipid-to-protein ratio of at least about 1:80 (e.g., at least about at least about 1:81, at least about 1:82, at least about 1:83, at least about 1:84, at least about 1:85, at least about 1:86, at least about 1:
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of up to about 1:100 (e.g., up to about 1:99, up to Docket No.10063-098WO1 about 1:98, up to about 1:97, up to about 1:96, up to about 1:95, up to about 1:94, up to about 1:93, up to about 1:92, up to about 1:91, up to about 1:90, up to about 1:89, up to about 1:88, up to about 1:87, up to about 1:86, up to about 1:85, up to about 1:84, up to about 1:83, up to about 1:82, up to about 1:81, up to about 1:80) by weight.
  • lipid-to-protein ratio of up to about 1:100 (e.g., up to about 1:99, up to Docket No.10063-098WO1 about 1:98, up to about 1:97, up to about 1:96, up to about 1
  • the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio of about 1:80, about 1:81, about 1:82, about 1:83, about 1:84, about 1:85, about 1:86, about 1:87, about 1:88, about 1:89, about 1:90, about 1:91, about 1:92, about 1:93, about 1:94, about 1:95, about 1:96, about 1:97, about 1:98, about 1:99, or about 1:100 by weight. It is considered that the biomimetic proteolipid nanovesicle can have a lipid-to-protein ratio ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can have a lipid-to- protein ratio of from about 1:80 to about 1:100 (e.g., from about 1:81 to about 1:99, from about 1:82 to about 1:98, from about 1:83 to about 1:97, from about 1:84 to about 1:96, from about 1:85 to about 1:95, from about 1:86 to about 1:94, from about 1:87 to about 1:93, from about 1:88 to about 1:92, from about 1:89 to about 1:91, from about 1:80 to about 1:90, from about 1:81 to about 1:89, from about 1:82 to about 1:88, from about 1:83 to about 1:87, from about 1:84 to about 1:86, from about 1:90 to about 1:100, from about 1:91 to about 1:99, from about 1:92 to about 1:98, from about 1:93 to about 1:97, from about 1:94 to about 1:96)
  • At least one ionizable or cationic lipid can be selected from the group consisting of DLin-MC3-DMA, SM-102, ALC-0315, and any combination thereof.
  • the biomimetic proteolipid nanovesicle can further include at least about 40 molar % (e.g., at least about 42 molar %, at least about 44 molar %, at least about 46 molar %, at least about 48 molar %, at least about 50 molar %, at least about 52 molar %, at least about 54 molar %, at least about 56 molar %, at least about 58 molar %, at least about 60 molar % ) of the ionizable or cationic lipid.
  • the biomimetic proteolipid nanovesicle can further include up to about 60 molar % (e.g., up to about 58 molar %, up to about 56 molar %, up to about 54 molar %, up to about 52 molar %, up to about 50 molar %, up to about 48 molar %, up to about 46 molar %, up to about 44 molar %, up to about 42 molar %, up to about 40 molar %) of the ionizable or cationic lipid.
  • up to about 60 molar % e.g., up to about 58 molar %, up to about 56 molar %, up to about 54 molar %, up to about 52 molar %, up to about 50 molar %, up to about 48 molar %, up to about 46 molar %, up to about 44 molar %, up to about 42 molar %, up
  • the biomimetic proteolipid nanovesicle can further include about 40 molar %, about 42 molar %, about 44 molar %, about 46 molar %, about 48 molar %, about 50 molar %, about 52 molar %, about 54 molar %, about 56 molar %, about 58 molar %, or about 60 molar % of the ionizable or cationic lipid. It is considered that the biomimetic proteolipid nanovesicle can further include an amount of the ionizable or cationic lipid ranging from any of the minimum values described above to any Docket No.10063-098WO1 of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can include from about 40 molar % to about 60 molar % (e.g., from about 42 molar % to about 58 molar %, from about 44 molar % to about 56 molar %, from about 46 molar % to about 54 molar %, from about 48 molar % to about 52 molar %, from about 40 molar % to about 50 molar %, from about 42 molar % to about 48 molar %, from about 44 molar % to about 46 molar %, from about 50 molar % to about 60 molar %, from about 52 molar % to about 58 molar %, from about 54 molar % to about 56 molar %) of the ionizable or cationic lipid.
  • the phosphocholine-based phospholipid can be selected from the group consisting of phosphatidylcholine, egg phosphatidic acid, 1,2-dioleoyl-sn-glycerophosphocholine (DOPC), 1,2-diolyl-sn-lycerophosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn- glycerophosphocholine (DPPC), 1,2-distearoyl-sn-glycerophosphocholine (DSPC), L- ⁇ - phosphatidylserine (PS), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), and any combination thereof.
  • DOPC 1,2-dioleoyl-sn-glycerophosphocholine
  • DOPE 1,2-diolyl-sn-lycerophosphoethanolamine
  • DOPE 1,2-dipalmitoyl-sn- glycerophosphocholine
  • the biomimetic proteolipid nanovesicle can further include at least about 7 molar % (e.g., at least about 7.5 molar %, at least about 8 molar %, at least about 8.5 molar %, at least about 9 molar %, at least about 9.5 molar %, at least about 10 molar %, at least about 10.5 molar %, at least about 11 molar %, at least about 11.5 molar %, at least about 12 molar %, at least about 12.5 molar %, at least about 13 molar %, at least about 13.5 molar %, at least about 14 molar %, at least about 14.5 molar %, at least about 15 molar %) of the phosphocholine-based phospholipid.
  • at least about 7 molar % e.g., at least about 7.5 molar %, at least about 8 molar %, at least about 8.5
  • the biomimetic proteolipid nanovesicle can further include up to about 15 molar % (e.g., up to about 14.5 molar %, up to about 14 molar %, up to about 13.5 molar %, up to about 13 molar %, up to about 12.5 molar %, up to about 12 molar %, up to about 11.5 molar %, up to about 11 molar %, up to about 10.5 molar %, up to about 10 molar %, up to about 9.5 molar %, up to about 9 molar %, up to about 8.5 molar %, up to about 8 molar %, up to about 7.5 molar %, up to about 7 molar %) of the phosphocholine-based phospholipid.
  • up to about 15 molar % e.g., up to about 14.5 molar %, up to about 14 molar %, up to about 13.5
  • the biomimetic proteolipid nanovesicle can further include about 7 molar %, about 7.5 molar %, about 8 molar %, about 8.5 molar %, about 9 molar %, about 9.5 molar %, about 10 molar %, about 10.5 molar %, about 11 molar %, about 11.5 molar %, about 12 molar %, about 12.5 molar %, about 13 molar %, about 13.5 molar %, about 14 molar %, about 14.5 molar %, about 15 molar % of the phosphocholine-based phospholipid.
  • the biomimetic proteolipid nanovesicle can further include an amount of the phosphocholine-based phospholipid ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can further include from about 7 molar % to about 15 molar Docket No.10063-098WO1 % (e.g., from about 7.5 molar % to about 14.5 molar %, from about 8 molar % to about 14 molar %, from about 8.5 molar % to about 13.5 molar %, from about 9 molar % to about 13 molar %, from about 9.5 molar % to about 12.5 molar %, from about 10 molar % to about 11 molar %, from about 7 molar % to about 10.5 molar %, from about 7.5 molar % to about 10 molar %, from about 8
  • the biomimetic proteolipid nanovesicle can further include at least about 35 molar % (e.g., at least about 35.5 molar %, at least about 36 molar %, at least about 36.5 molar %, at least about 37 molar %, at least about 37.5 molar %, at least about 38 molar %, at least about 38.5 molar %, at least about 39 molar %, at least about 39.5 molar %, at least about 40 molar %, at least about 40.5 molar %, at least about 41 molar %, at least about 41.5 molar %, at least about 42 molar %, at least about 42.5 molar %, at least about 43 molar %, at least about 43.5 molar %, at least about 44 molar %, at least about 44.5 molar %, at least about 45 molar %) of the cholesterol.
  • the biomimetic proteolipid nanovesicle can further include up to about 45 molar % (e.g., up to about 44.5 molar %, up to about 44 molar %, up to about 43.5 molar %, up to about 43 molar %, up to about 42.5 molar %, up to about 42 molar %, up to about 41.5 molar %, up to about 41 molar %, up to about 40.5 molar %, up to about 40 molar %, up to about 39.5 molar %, up to about 39 molar %, up to about 38.5 molar %, up to about 38 molar %, up to about 37.5 molar %, up to about 37 molar %, up to about 36.5 molar %, up to about 36 molar %, up to about 35.5 molar %, up to about 35 molar %) of the cholesterol.
  • the biomimetic proteolipid nanovesicle can further include about 35 molar %, about 35.5 molar %, about 36 molar %, about 36.5 molar %, about 37 molar %, about 37.5 molar %, about 38 molar %, about 38.5 molar %, about 39 molar %, about 39.5 molar %, about 40 molar %, about 40.5 molar %, about 41 molar %, about 41.5 molar %, about 42 molar %, about 42.5 molar %, about 43 molar %, about 43.5 molar %, about 44 molar %, about 44.5 molar %, or about 45 molar % of the cholesterol.
  • the biomimetic proteolipid nanovesicle can further include an amount of the cholesterol ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can further include from about 35 molar % to about 45 molar % (e.g., from about 35.5 molar % to about 44.5 molar %, from about 36 molar % to about 44 molar %, from about 36.5 molar % to about 43.5 molar %, from about 37 molar % to about 43 molar %, from about 37.5 molar % to about 42.5 molar %, from about 38 molar % to about 42 molar %, from about 38.5 molar % to Docket No.10063-098WO1 about 41.5 molar %, from about 39 molar % to about 41 molar %, from about 39.5 molar % to about
  • the biomimetic proteolipid nanovesicle can further include a PEGylated lipid.
  • the PEGylated lipid can be selected from the group consisting of DMG- PEG2000, ALC-0159, and any combination thereof.
  • the biomimetic proteolipid nanovesicle can further include at least about 1 molar % (e.g., at least about 1.5 molar %, at least about 2 molar %, at least about 2.5 molar %, at least about 3 molar %, at least about 3.5 molar %, at least about 4 molar %, at least about 4.5 molar %, at least about 5 molar %, at least about 5.5 molar %, at least about 6 molar %, at least about 6.5 molar %, at least about 7 molar %, at least about 7.5 molar %, at least about 8 molar %, at least about 8.5 molar %, at least about 9 molar %, at least about 9.5 molar %, at least about 10 molar %) of the PEGylated lipid.
  • at least about 1 molar % e.g., at least about 1.5 molar %,
  • the biomimetic proteolipid nanovesicle can further include up to about 10 molar % (e.g., up to about 9.5 molar %, up to about 9 molar %, up to about 8.5 molar %, up to about 8 molar %, up to about 7.5 molar %, up to about 7 molar %, up to about 6.5 molar %, up to about 6 molar %, up to about 5.5 molar %, up to about 5 molar %, up to about 4.5 molar %, up to about 4 molar %, up to about 3.5 molar %, up to about 3 molar %, up to about 2.5 molar %, up to about 2 molar %, up to about 1.5 molar %, up to about 1 molar %) of the PEGylated lipid.
  • up to about 10 molar % e.g., up to about 9.5 molar %
  • the biomimetic proteolipid nanovesicle can include about 1 molar %, about 1.5 molar %, about 2 molar %, about 2.5 molar %, about 3 molar %, about 3.5 molar %, about 4 molar %, about 4.5 molar %, about 5 molar %, about 5.5 molar %, about 6 molar %, about 6.5 molar %, about 7 molar %, about 7.5 molar %, about 8 molar %, about 8.5 molar %, about 9 molar %, about 9.5 molar %, or about 10 molar % of the PEGylated lipid.
  • the biomimetic proteolipid nanovesicle can further include an amount of the PEGylated lipid ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can include from about 1 molar % to about 10 molar % (e.g., from about 1.5 molar % to about 9.5 molar %, from about 2 molar % to about 9 molar %, from about 2.5 molar % to about 8.5 molar %, from about 3 molar % to about 8 molar %, from about 3.5 molar % to about 7.5 molar %, from about 4 molar % to about 7 molar %, from about 4.5 molar % to about 6.5 molar %, from Docket No.10063-098WO1 about 5 molar % to about 6 molar %, from about 1 molar %
  • molar % from about 1.5 molar % to about 5 molar %, from about 2 molar % to about 4.5 molar %, from about 2.5 molar % to about 4 molar %, from about 3 molar % to about 3.5 molar %, from about 5.5. molar % to about 10 molar %, from about 6 molar % to about 9.5 molar %, from about 6.5 molar % to about 9 molar %, from about 7 molar % to about 8.5 molar %, from about 7.5 molar % to about 8 molar %) of the PEGylated lipid.
  • the leukocyte membrane protein can be derived from a leukocyte plasma membrane.
  • the leukocyte plasma membrane can be derived from a human leukocyte plasma membrane.
  • the leukocyte plasma membrane can be derived from a murine leukocyte plasma membrane.
  • the leukocyte membrane protein can be a synthetic recombinant protein.
  • the leukocyte membrane protein can be lymphocyte function-associated antigen 1 (LFA-1), CD11, CD45, CD47, or any combination thereof.
  • the leukocyte membrane protein can include some or all of the peptides present in a leukocyte plasma membrane (e.g., a human leukocyte plasma membrane or a murine leukocyte plasma membrane).
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of at least about 3 (e.g., at least about 3.25, at least about 3.5, at least about 3.75, at least about 4, at least about 4.25, at least about 4.5, at least about 4.75, at least about 5, at least about 5.25, at least about 5.5, at least about 5.75, at least about 6, at least about 6.25, at least about 6.5, at least about 6.75, at least about 7, at least about 7.25, at least about 7.5, at least about 7.75, at least about 8, at least about 8.25, at least about 8.5, at least about 8.75, at least about 9, at least about 9.25, at least about 9.5, at least about 9.75, at least about 10).
  • N/P ratio of at least about 3 (e.g., at least about 3.25, at least about 3.5, at least about 3.75, at least about 4, at least about 4.25, at least about 4.5, at least about 4.75, at least about 5, at least about 5.25,
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of up to about 10 (e.g., up to about 9.75, up to about 9.5, up to about 9.25, up to about 9, up to about 8.75, up to about 8.5, up to about 8.25, up to about 8, up to about 7.75, up to about 7.5, up to about 7.25, up to about 7, up to about 6.75, up to about 6.5, up to about 6.25, up to about 6, up to about 5.75, up to about 5.5, up to about 5.25, up to about 5, up to about 4.75, up to about 4.5, up to about 4.25, up to about 4, up to about 3.75, up to about 3.5, up to about 3.25, up to about 3).
  • an N/P ratio of up to about 10 e.g., up to about 9.75, up to about 9.5, up to about 9.25, up to about 9, up to about 8.75, up to about 8.5, up to about 8.25, up to about 8, up to about 7.75
  • the biomimetic proteolipid nanovesicle can further include an N/P ratio of about 3, about 3.25, about 3.5, about 3.75, about 4, about 4.25, about 4.5, about 4.75, about 5, about 5.25, about 5.5, about 5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, about 7.75, about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, about 9.75, or about 10. It is considered that the biomimetic proteolipid nanovesicle can have an N/P ratio ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can have an N/P ratio of from about 3 to about 10 (e.g., from about 3.25 to about 9.75, from about 3.5 to about 9.5, from about 3.75 to about 9.25, from about 4 to about 9, from about 4.25 to about 8.75, from about 4.5 to about 8.5, from about 4.75 to about 8.25, from about 5 to about 8, from about 5.25 to about 7.75, from about 5.5 to about 7.5, form about 5.75 to about 7.25, from about 6 to about 7, from about 6.25 to about 6.75, from about 3 to about 6.5, from about 3.25 to about 6.25, from about 3.5 to about 6, from about 3.75 to about 5.75, from about 4 to about 5.5, from about 4.25 to about 5.25, from about 4.5 to about 5, from about 6.5 to about 10, from about 6.75 to about 9.75, from about 7 to about 9.5, from about 7.25 to about 9.25, from about 7.5 to about
  • the biomimetic proteolipid nanovesicle can have a diameter of at least about 50 nm (e.g., at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at least about 170 nm, at least about 180 nm, at least about 190 nm, at least about 200 nm).
  • nm e.g., at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at
  • the biomimetic proteolipid nanovesicle can have a diameter of up to about 200 nm (e.g., up to about 190 nm, up to about 180 nm, up to about 170 nm, up to about 160 nm, up to about 150 nm, up to about 140 nm, up to about 130 nm, up to about 120 nm, up to about 110 nm, up to about 100 nm, up to about 90 nm, up to about 80 nm, up to about 70 nm, up to about 60 nm, up to about 50 nm).
  • a diameter of up to about 200 nm e.g., up to about 190 nm, up to about 180 nm, up to about 170 nm, up to about 160 nm, up to about 150 nm, up to about 140 nm, up to about 130 nm, up to about 120 nm, up to about 110 nm, up to about 100 nm
  • the biomimetic proteolipid nanovesicle can have a diameter of about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. It is considered that the biomimetic proteolipid nanovesicle can have a diameter ranging from any of the minimum values described above to any of the maximum values described above.
  • the biomimetic proteolipid nanovesicle can have a diameter of from about 50 nm to about 200 nm (e.g., from about 60 nm to about 190 nm, from about 70 nm to about 180 nm, from about 80 nm to about 170 nm, from about 90 nm to about 160 nm, from about 100 nm to about 150 nm, from about 110 nm to about 140 nm, from about 120 nm to about 130 nm, from about 50 nm to about 130 nm, from about 60 nm to about 120 nm, from about 70 nm to about 110 nm, from about 80 nm to about 100 nm, from about 120 nm to about 200 nm, from about 130 nm to about 190 nm, from about 140 nm to about 180 nm, from about 150 nm to about 170 nm).
  • a diameter of from about 50 nm to about 200 nm
  • the mRNA can be targeted to treat a cancer, inflammation, an infectious disease, or a genetic disease or disorder.
  • Docket No.10063-098WO1 METHODS in an aspect, provided is a method of making any of the disclosed biomimetic proteolipid nanovesicles, the method including: a) dissolving a phosphocholine-based phospholipid, an ionizable or cationic lipid, and a cholesterol in an organic solvent to produce an organic lipid solution; b) dissolving a leukocyte membrane protein and an agent in water to produce an aqueous protein solution; and c) loading the organic lipid solution into an organic phase inlet of a microfluidic mixer, and loading the aqueous protein solution into an aqueous phase inlet of said microfluidic mixer; and d) adjusting flow rates of each inlet stream and a flow ratio between each inlet stream to produce the biomimetic proteolipid nanovesicles having a specified lipid-to-protein ratio by weight
  • the agent can include a nucleic acid.
  • the agent can include an siRNA and/or an mRNA.
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of at least about 1:50 (e.g., at least about 1:55, at least about 1:60, at least about 1:65, at least about 1:66, at least about 1:67, at least about 1:68, at least about 1:69, at least about 1:70, at least about 1:71, at least about 1:72, at least about 1:73, at least about 1:74, at least about 1:75, at least about 1:76, at least about 1:77, at least about 1:78, at least about 1:79, at least about 1:80, at least about 1:81, at least about 1:82, at least about 1:83, at least about 1:84, at least about 1:85, at least about
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of up to about 1:100 (e.g., up to about 1:95, up to about 1:90, up to about 1:85, up to about 1:84, up to about 1:83, up to about 1:82, up to about 1:81, up to about 1:80, up to about 1:79, up to about 1:78, up to about 1:77, up to about 1:76, up to about 1:75, up to about 1:74, up to about 1:73, up to about 1:72, up to about 1:71, up to about 1:70, up to about 1:69, up to about 1:68, up to about 1:67, up to about 1:66, up to about 1:65) by weight.
  • a lipid-to-protein ratio of up to about 1:100 e.g., up to about 1:95, up to about 1:90, up to about 1:85, up to about 1:84, up
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of about 1:50, 1:55, 1:60, 1:65, about 1:66, about 1:67, about 1:68, about 1:69, about 1:70, about 1:71, about 1:72, about 1:73, about 1:74, about 1:75, about 1:76, about 1:77, about 1:78, about 1:79, about 1:80, about 1:81, about 1:82, about 1:83, about 1:84, about 1:85, about 1:90, about 1:95, or about 1:100 by weight.
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio ranging from any of the minimum values described above to any of the maximum values described above.
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of from about 1:65 to about 1:85 (e.g., from about 1:66 to about 1:84, from about 1:67 to about 1:83, from about 1:68 to about 1:82, from about 1:69 to about 1:81, from about 1:70 to about 1:80, from about 1:71 to about 1:79, from about 1:72 to about 1:78, from about 1:73 to about 1:77, from about 1:74 to about 1:76, from about 1:65 to about 1:75, from about 1:66 to about 1:74, from about 1:67 to about 1:73,
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of from about 1:50 to about 1:100 by weight.
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of at least about 1:80 (e.g., at least about at least about 1:81, at least about 1:82, at least about 1:83, at least about 1:84, at least about 1:85, at least about 1:86, at least about 1:87, at least about 1:88, at least about 1:89, at least about 1:90, at least about 1:91, at least about 1:92, at least about 1:93, at least about 1:94, at least about 1:95, at least about 1:96, at least about 1:97, at least about 1:98, at least about 1:99, at least about 1:100
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of up to about 1:100 (e.g., up to about 1:99, up to about 1:98, up to about 1:97, up to about 1:96, up to about 1:95, up to about 1:94, up to about 1:93, up to about 1:92, up to about 1:91, up to about 1:90, up to about 1:89, up to about 1:88, up to about 1:87, up to about 1:86, up to about 1:85, up to about 1:84, up to about 1:83, up to about 1:82, up to about 1:81, up to about 1:80) by weight.
  • up to about 1:100 e.g., up to about 1:99, up to about 1:98, up to about 1:97, up to about 1:96, up to about 1:95, up to about 1:94, up to about 1:93, up to about 1:92, up to about 1
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of about 1:80, about 1:81, about 1:82, about 1:83, about 1:84, about 1:85, about 1:86, about 1:87, about 1:88, about 1:89, about 1:90, about 1:91, about 1:92, about 1:93, about 1:94, about 1:95, about 1:96, about 1:97, about 1:98, about 1:99, or about 1:100 by weight. It is considered that the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio ranging from any of the minimum values described above to any of the maximum values described above.
  • the method can produce biomimetic proteolipid nanovesicles having a lipid-to-protein ratio of from about 1:80 to about 1:100 (e.g., from about 1:81 to about 1:99, from about 1:82 to about 1:98, from about 1:83 to about 1:97, from about 1:84 to about 1:96, from about 1:85 to about 1:95, from about 1:86 to about 1:94, from about 1:87 to about 1:93, from about 1:88 to about 1:92, from about 1:89 to about 1:91, from about 1:80 to about 1:90, from about 1:81 to about 1:89, from Docket No.10063-098WO1 about 1:82 to about 1:88, from about 1:83 to about 1:87, from about 1:84 to about 1:86, from about 1:90 to about 1:100, from about 1:91 to about 1:99, from about 1:92 to
  • the total combined flow rate of both inlet streams can be at least about 2 mL/min (e.g., at least about 3 mL/min, at least about 4 mL/min, at least about 5 mL/min, at least about 6 mL/min, at least about 7 mL/min, at least about 8 mL/min, at least about 9 mL/min, at least about 10 mL/min, at least about 11 mL/min, at least about 12 mL/min, at least about 13 mL/min, at least about 14 mL/min, at least about 15 mL/min).
  • mL/min e.g., at least about 3 mL/min, at least about 4 mL/min, at least about 5 mL/min, at least about 6 mL/min, at least about 7 mL/min, at least about 8 mL/min, at least about 9 mL/min, at least about 10 mL/min, at least about 11
  • the total combined flow rate of both inlet streams can be up to about 15 mL/min (e.g., up to about 14 mL/min, up to about 13 mL/min, up to about 12 mL/min, up to about 11 mL/min, up to about 10 mL/min, up to about 9 mL/min, up to about 8 mL/min, up to about 7 mL/min, up to about 6 mL/min, up to about 5 mL/min, up to about 4 mL/min, up to about 3 mL/min, up to about 2 mL/min).
  • mL/min e.g., up to about 14 mL/min, up to about 13 mL/min, up to about 12 mL/min, up to about 11 mL/min, up to about 10 mL/min, up to about 9 mL/min, up to about 8 mL/min, up to about 7 mL/min, up to about 6
  • the total combined flow rate of both inlet streams can be about 2 mL/min, about 3 mL/min, about 4 mL/min, about 5 mL/min, about 6 mL/min, about 7 mL/min, about 8 mL/min, about 9 mL/min, about 10 mL/min, about 11 mL/min, about 12 mL/min, about 13 mL/min, about 14 mL/min, or about 15 mL/min. It is considered that the total combined flow rate of both inlet streams can range from any of the minimum values described above to any of the maximum values described above.
  • the total combined flow rate of both inlet streams can be from about 2 mL/min to about 15 mL/min (e.g., from about 3 mL/min to about 14 mL/min, from about 4 mL/min to about 13 mL/min, from about 5 mL/min to about 12 mL/min, from about 6 mL/min to about 11 mL/min, from about 7 mL/min to about 10 mL/min, from about 8 mL/min to about 9 mL/min, from about 2 mL/min to about 9 mL/min, from about 3 mL/min to about 8 mL/min, from about 4 mL/min to about 7 mL/min, from about 5 mL/min to about 6 mL/min, from about 8 mL/min to about 15 mL/min, from about 9 mL/min to about 14 mL/min, from about 10 mL/min to about 13 mL/min
  • the flow ratio between the organic phase inlet and the aqueous phase inlet can be at least about 1:2 (e.g., at least about 1:2.5, at least about 1:3, at least about 1:3.5, at least about 1:4, at least about 1:4.5, at least about 1:5). In some aspects, the flow ratio between the organic phase inlet and the aqueous phase inlet can be up to about 1:5 (e.g., up to about 1:4.5, up to about 1:4, up to about 1:3.5, up to about 1:3, up to about 1:2.5, up to about 1:2).
  • the flow ratio between the organic phase inlet and the aqueous phase inlet can be about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, or about 1:5. Docket No.10063-098WO1 It is considered that the flow ratio between the organic phase inlet and the aqueous phase inlet can range from any of the minimum values described above to any of the maximum values described above.
  • the flow ratio between the organic phase inlet and the aqueous phase inlet can be from about 1:2 to about 1:5 (e.g., from about 1:2.5 to about 1:4.5, from about 1:3 to about 1:4, from about 1:2 to about 1:3.5, from about 1:2.5 to about 1:3, from about 1:3.5 to about 1:5, from about 1:4 to about 1:4.5)
  • the aqueous protein solution can have a protein concentration of at least about 2 mg/mL (e.g., at least about 2.5 mg/mL, at least about 3 mg/mL, at least about 3.5 mg/mL, at least about 4 mg/mL, at least about 4.5 mg/mL, at least about 5 mg/mL, at least about 5.5 mg/mL, at least about 6 mg/mL, at least about 6.5 mg/mL, at least about 7 mg/mL, at least about 7.5 mg/mL, at least about 8 mg/mL).
  • the aqueous protein solution can have a protein concentration of up to about 8 mg/mL (e.g., up to about 7.5 mg/mL, up to about 7 mg/mL, up to about 6.5 mg/mL, up to about 6 mg/mL, up to about 5.5 mg/mL, up to about 5 mg/mL, up to about 4.5 mg/mL, up to about 4 mg/mL, up to about 3.5 mg/mL, up to about 3 mg/mL, up to about 2.5 mg/mL, up to about 2 mg/mL).
  • a protein concentration of up to about 8 mg/mL e.g., up to about 7.5 mg/mL, up to about 7 mg/mL, up to about 6.5 mg/mL, up to about 6 mg/mL, up to about 5.5 mg/mL, up to about 5 mg/mL, up to about 4.5 mg/mL, up to about 4 mg/mL, up to about 3.5 mg/mL, up to about 3 mg/
  • the aqueous protein solution can have a protein concentration of about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, or about 8 mg/mL. It is considered that the aqueous protein solution can have a protein concentration ranging from any of the minimum values described above to any of the maximum values described above.
  • the aqueous protein solution can have a protein concentration of from about 2 mg/mL to about 8 mg/mL (e.g., from about 2.5 mg/mL, from about 7.5 mg/mL, from about 3 mg/mL to about 7 mg/mL, from about 3.5 mg/mL to about 6.5 mg/mL, from about 4 mg/mL to about 6 mg/mL, from about 4.5 mg/mL to about 5.5 mg/mL, from about 2 mg/mL to about 5 mg/mL, from about 2.5 mg/mL to about 4.5 mg/mL, from about 3 mg/mL to about 4 mg/mL, from about 5 mg/mL to about 8 mg/mL, from about 5.5 mg/mL to about 7.5 mg/mL, from about 6 mg/mL to about 7 mg/mL).
  • a protein concentration of from about 2 mg/mL to about 8 mg/mL (e.g., from about 2.5 mg/mL, from about 7.5 mg/mL, from about
  • the method can yield at least about 60% (e.g., at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least Docket No.10063-098WO1 about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%,
  • step c) can be performed at a temperature of at least about 40°C (e.g., at least about 41°C, at least about 42°C, at least about 43°C, at least about 44°C, at least about 45°C, at least about 46°C, at least about 47°C, at least about 48°C, at least about 49°C, at least about 50°C).
  • a temperature of at least about 40°C e.g., at least about 41°C, at least about 42°C, at least about 43°C, at least about 44°C, at least about 45°C, at least about 46°C, at least about 47°C, at least about 48°C, at least about 49°C, at least about 50°C.
  • step c) can be performed at a temperature of up to about 50°C (e.g., up to about 49°C, up to about 48°C, up to about 47°C, up to about 46°C, up to about 45°C, up to about 44°C, up to about 43°C, up to about 42°C, up to about 41°C, up to about 40°C).
  • step c) can be performed at a temperature of about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C.
  • step c) can be performed at a temperature ranging from any of the minimum values described above to any of the maximum values described above.
  • step c) can be performed at a temperature of from about 40°C to about 50°C (e.g., from about 41°C to about 49°C, from about 42°C to about 48°C, from about 43°C to about 47°C, from about 44°C to about 46°C, from about 40°C to about 45°C, from about 41°C to about 44°C, from about 42°C to about 43°C, from about 45°C to about 50°C, from about 46°C to about 49°C, from about 47°C to about 48°C).
  • a method for the delivery of an agent into a cell including introducing into the cell any of the disclosed biomimetic proteolipid nanovesicles.
  • the agent can include a nucleic acid.
  • the agent can include an siRNA and/or an mRNA.
  • the method can be used to target delivery of the agent to inflamed tissues.
  • the method can be performed in vivo, in vitro, or ex vivo.
  • the cell can be human.
  • a method of treating a disease or disorder in a subject in need thereof the method including administering to the subject any of the disclosed biomimetic proteolipid nanovesicles.
  • the agent can include a nucleic acid. In some such aspects, the agent can include an siRNA and/or an mRNA. In some aspects, the disease or disorder can be a cancer. In some such aspects, the disease or disorder can be triple negative breast cancer. Additionally or alternatively, in other aspects, the disease or disorder can be inflammation. In some such aspects, the disease or disorder can be a tumor, sepsis, a traumatic brain injury, inflamed epithelia, atherosclerosis, or post traumatic osteoarthritis. Docket No.10063-098WO1 EXAMPLES Example 1: siRNA Loading into Leukosomes In addition to the standard-of-care utilized in the treatment of TNBC, targeted delivery of RNA represents a promising alternative approach.
  • siRNA can serve as molecular brakes that reduce the expression of proteins activating signaling within these pathways [1, 2] .
  • administration of these molecules requires the use of a protective carrier that takes these molecules to the target site [3] .
  • the harsh in vivo environment exposes the siRNA to enzymes that quickly degrade the molecules upon systemic administration [4] .
  • NPs have been utilized as protective drug delivery carriers of siRNA [5] .
  • NP-based siRNA delivery has been tested using polymeric, lipid and inorganic NPs [4, 6] .
  • lipid NPs have demonstrated the most promising potential as carriers of siRNA [7] .
  • Recent FDA approval of two lipid-based NP-siRNA formulations has validated the use of these NPs for the treatment of liver diseases [8] . This has been primarily due to the natural homing properties of these lipidic formulations to the liver [9-11] .
  • researchers have explored ways to further the utility of these NPs for other organs, which has required iterative testing of formulations for organ-specific targeting [12] . Attempts to address this current gap in the application of this technology to other diseases have included synthesis of novel lipids and tuning the lipid contents of the NP formulation [13, 14] .
  • Protein extraction for NPs synthesis Protein extraction of the membrane proteins from J774 cells was performed using a Calbiochem ProteoExtract Kit. Approximately 50 million cells were split evenly into 10 tubes (5 million cells/tube), washed twice with provided Wash Buffer and centrifuged at 200g for 10 min at 4°C following each wash. The supernatant was removed, pellet resuspended in 2mL of extraction buffer 1 and incubated on ice for 10 min under gentle agitation. Samples were then centrifuged at 16000g for 15 min at 4°C, followed by removal of the supernatant. Next, the pellet was resuspended in 1mL of extraction buffer 2 (EB2) and incubated on ice for 30 min under gentle agitation.
  • EB2 extraction buffer 2
  • siRNA Scramble siRNA, also known as Negative Control siRNA #1, was purchased from Ambion.
  • STAT3 siRNA for murine cells was purchased from Qiagen, while GFP siRNA purchased from Ambion.
  • Synthesis of liposomes and leukosomes with siRNA involves synthesis of 3 different lipid formulations. 3 lipid backbones were tested with the cationic or ionizable lipid being different in each one.
  • DOTAP was tested a cationic lipid for siRNA loading, while 16:0 DAP and DLin-MC3-DMA were tested as ionizable lipids of choice.
  • Two phases i.e. aqueous and organic
  • Lipids and ethanol made up the organic phase, while acidic sodium acetate buffer, siRNA and protein or protein buffer comprised the aqueous phase.
  • NPs were in dialyzed overnight in a two-step process utilizing acidic sodium acetate buffer to remove residual ethanol followed by 1X PBS.
  • NPs were filtered using a 0.22 ⁇ M PVDF filters prior to characterization and treatment of cells or mice.
  • Fluorescently labeled siRNA-NPs were synthesized using the addition of a rhodamine-lipid during the synthesis process, with no changes to the parameters on the NanoAssemblrTM or post-synthesis purification methods.
  • Dynamic light scattering, zeta potential and concentration of NPs Malvern Zetasizer was utilized to measure the size, polydispersity index (PDI) and zeta potential of the NPs. For size and PDI measurements, samples were diluted 1:1000 in MilliQ water and characterized with 3 Docket No.10063-098WO1 measurements of 15 runs for each.
  • RNA loading quantification A modified Ribogreen ⁇ assay was utilized for the quantification of siRNA loaded within the synthesized NPs, as previously described [15] .
  • NP samples were first diluted 1:100 in 1X TE buffer, followed by 50uL of each diluted sample added to either 50uL of 1X TE buffer or 2% Triton-X 100 in 1X TE buffer.
  • RNA standard curve ranging from 0.1-2.5 ⁇ g/mL, was prepared with using a combination of 2% Triton-X 100 in 1X TE buffer and 1X TE buffer as the diluent (TABLE 1). All samples were run in duplicate and incubated at 37C for 10 minutes. Ribogreen reagent was diluted 1:100 in 1X TE buffer, with 100uL added to each well. Following a 4 min incubation at room temperature, plate was read at Ex/Em of 480nm/520nm. TABLE 1. Sample preparation of RNA standards for Ribogreen TM kit RNA concentration RNA stock TE buffer Triton buffer Total volume per e at a seeding density of 8,000 cells/well.
  • NP were resuspended in Optimem media and added to the cells at the following concentrations: 10, 25, 50, 100, 250, 500 and 1000 ⁇ M. These concentrations were based on the lipid concentration of the NP after synthesis.
  • Optimem media was aspirated and replaced with complete DMEM HG media.
  • Toxicity of NP treatment was evaluated using an MTT assay at 3 timepoints – 24, 48 and 72h. At each timepoint, media was aspirated and replaced with MTT resuspended in completed media at a concentration of 0.5 mg/ml. After 2 h, MTT reagent was aspirated and replaced with equal volume DMSO.
  • TNBC model was established by injecting a total of 3x10 5 4T1-Red-FLuc (PerkinElmer, Waltham MA) cells, suspended in 50 ⁇ l of 1X PBS, subcutaneously into the mammary fat pad of 10-week-old BALB/c female mice (Charles River Laboratories, Wilmington, MA). 10-12 days after tumor inoculation, mice were divided into 3 groups – untreated control, siRNA-liposomes and siRNA-leukosomes.
  • lipid backbones were screened for their ability to effectively load siRNA, maintain key NP physiochemical properties and exhibit in vitro knockdown efficiencies with minimal cytotoxicity to TNBC cells.
  • Liposomes comprised of 3 discrete lipid formulations and loaded with scramble siRNA were synthesized on the NanoAssemblr and characterized for their physiochemical properties. All 3 lipid formulations maintained a size below 200nm and a PDI less than 0.2 (FIGS. 1A-1B). However, significant differences could be observed in the zeta potential of the 3 formulations, with DOTAP lipid nanoparticles having a positive charge following synthesis and dialysis processing (FIG. 1C).
  • DOTAP exhibited the lowest siRNA encapsulation efficiency among the 3 lipid backbones screened, with only 50% of siRNA encapsulated within the NPs (FIG.1D).
  • cytotoxic profile of each liposome formulation was evaluated on 4T1 cells (FIGS.2A-2B). In both formulations, analysis indicated minimal decreases in viability for up to 72h at concentrations less than 500 ⁇ M. Following establishment of the max NP concentration threshold, siRNA knockdown efficiency of both formulations was determined.
  • Treatment of 4T1 cells with DAP NPs loaded with STAT3- siRNA resulted in only a 40% knockdown of STAT3 mRNA levels after 24h (FIG.3A).
  • NPs made utilizing the DLin-MC3 formulation were determined to be the superior candidate for moving forward with the leukosome formulation.
  • siRNA encapsulation was quantified both before and after the filtration process. Indeed, filtration of the siRNA-leukosomes resulted in almost 70% loss of siRNA, while liposomes only had about a 30% in loss (FIG.4F).
  • filtration of the siRNA-leukosomes resulted in almost 70% loss of siRNA, while liposomes only had about a 30% in loss (FIG.4F).
  • PEG lipid Upon understanding the role of the PEG lipid in stabilizing the siRNA-leukosomes, efforts were shifted towards understanding how the PEG concentration can be tuned to further stabilize the leukosomes. To this end, NPs were synthesized using 2-fold and 4-fold more PEG lipid than the original lipid backbone with the lowest PEG content.
  • NPs with increasing PEG content demonstrated Docket No.10063-098WO1 an incremental increase in the PDI, with both Lipo- and Leuko-PEG 6% having a PDI between 0.2-0.25 (FIG. 5B).
  • siRNA encapsulation efficiency in both siRNA-liposomes and leukosomes was found to increase by 20% when increasing the PEG content (FIG. 5C).
  • evaluation of siRNA loss during the filtration process was found to significantly decrease with increasing PEG formulations of both liposomes and leukosomes, with less than 20% loss in the highest PEG NPs (FIG.5D).
  • the flow rate (FR) utilized during the synthesis on the NanoAssemblrTM was hypothesized to have an effect on the resulting NPs [17] .
  • synthesis of siRNA-leukosomes with varying concentrations of PEG at a low, mid and high flow rates was found to have minimal effects on the size and PDI of the NPs (FIGS.6A-6D).
  • the synthesis of the leukosomes at the highest flow rate Given that both the mid and high PEG percentage NPs demonstrated stabilization of the NPs, especially in terms of size and siRNA encapsulation, both of these of PEG ratios were selected to continue to in vitro and in vivo evaluation. Both of these formulations were synthesized using the mid flow rate.
  • Cytotoxicity profile of 2 candidate siRNA-leukosomes formulations Prior to validating the knockdown efficiency of siRNA-loaded NPs, the 2 selected siRNA-leukosome formulations were evaluated for their cytotoxic profile on 4T1 cells. siRNA leukosomes with the mid PEG percentage exhibited minimal decreases in cell viability, with cells demonstrating a recovery in viability after the first 24h (FIG.7A). Treatment with leukosomes of the highest concentration of PEG resulted in minimum decrease in cell viability for 72h at concentrations below 250 ⁇ M (FIG. 7B). Both concentrations above this threshold resulted in a 40% decrease in cell viability 72h after NP treatment (FIG. 7B).
  • siRNA-NPs with mid PEG percentage exhibited up to a 75% knockdown in STAT3 mRNA expression within 48h, with both liposomes and leukosomes outperforming the Lipofectamine control (FIG.8A).
  • TNBC tumor targeting and biodistribution of 2 candidate siRNA-leukosome formulations A 4T1 tumor model of TNBC was utilized for the evaluation of the biodistribution and tumor targeting properties of the selected siRNA-leukosome formulations.
  • NPs of both PEG ratios demonstrated similar levels of accumulation within the tumor, with little differences observed between the liposomes and leukosomes (FIGS. 10A).
  • the decrease in NP signal in the liver over time was indicative of NP clearance, while maintaining signal within the tumor (FIGS.10B).
  • the latter observation required further analysis of the kinetics of NP behavior within the tumor environment. Discussion This study demonstrated the development of a leukosome formulation capable of being loaded with RNA molecules.
  • the loss of siRNA during this purification step indicated the loss of material, specifically NPs and the associated protein components on their surface hypothesized to mediate their targeting behavior in vivo. Nonetheless, this initial set of analysis proved vital for recognizing not only the need to analyze these NPs further, but also enabling an understanding of what key factors can be tuned to stabilize these particles during the synthesis process.
  • the tuning of the siRNA-NP formulation provided valuable insights on the effect of 2 key variables – PEG content and flow rate for mixing – on the final siRNA-leukosome formulation. In contrast to the previously known behavior of PEG in minimizing NP and cellular protein interactions, the results from this study indicated that the PEG presence was indeed necessary for the stabilization of the NPs in terms of size and PDI.
  • both formulations of NPs demonstrated the maximum accumulation within the liver, which aligns with previous studies utilizing this lipid backbone for gene delivery [11] .
  • the difference observed in the levels of accumulation within the liver when increasing the PEG ratio highlighting the underlying role of the PEG content in mediating the overall biodistribution of the NPs.
  • the increased PEG content appears to support longer retention of the NPs within the tumor environment.
  • the presence of the proteins in the siRNA-leukosomes did not demonstrate an improved targeting to the tumor site.
  • the combined findings from the in vitro and in vivo analysis have emphasized the subsequent effects on the biological behavior of these NPs when tuning the PEG content.
  • FIG. 14 shows day 0 post synthesis.
  • FIGS. 14A-14D show that the size (FIG. 15A) and PDI (FIG. 15B) of Leukosome-LNPs improved.
  • the N/P ratio was 3
  • the flow rate ratio was 1:3
  • the total flow rate was 5 mL/min
  • the ionizable lipid was SM-102.
  • the protein stock concentration was 4 mg/mL. TABLE 5. Iteration 4, no protein.
  • FIGS.16A-16C show in vivo downregulation tests for siRPL39 Leukosomes-LNPs.5x10 6 MDA-MB-321 cells were injected in the mammary fat pad of NSG mice (xenografts).
  • FIGS. 17A-17C show in vivo targeting tests for siRPL39 Leukosomes-LNPs.
  • a metaplastic breast cancer model was developed with patient derived xenograft (PDX4664, RPL39 A14V).
  • Example 3 mRNA Payload
  • Leukosome-LNP formulations were designed to improve quality attributes of the final product (physio-chemical properties and encapsulation efficiency).
  • Leukosome-LNPs were designed to improve quality attributes of the final product (physio-chemical properties and encapsulation efficiency).
  • the ionizable lipid was SM-102, the N/P ratio was 6, the flow rate ratio was 1:3, the total flow rate was 12 mL/min, and the protein source was membrane proteins from J774 murine macrophages. TABLE 10. Iteration 2. Leukosome-LNPs (protein:lipid ratio 1:100) Size (nm) PDI (a.u.) ZP (mV) RNA Encapsulation efficiency (EE%) 113.67 0.17 -13.9 80 to evaluate in vivo biodistribution and targeting in a Triple Negative Breast Cancer (TNBC) murine model.
  • TNBC Triple Negative Breast Cancer
  • IV intravenous
  • SC subcutaneous
  • SC injection of DiD-Labeled LNPs No membrane proteins, n
  • FIGS. 20A- 20B and FIGS.21A-21B The results of in vivo biodistribution and targeting of iteration 2 is shown in FIGS. 20A- 20B and FIGS.21A-21B.
  • IV administered LNPs had predominantly hepatic accumulation (FIG. 21A), consistent with previous results.
  • a trend of increased tumor accumulation was identified for Leukosome-LNPs when compared with regular LNPs (FIG. 21B), despite the absence of significance.
  • SC administered LNPs did not move from the injection area, as demonstrated by the absence of signal in organs (FIG.22A) and tumors (FIG.22B). This suggests that targeting cannot be achieved via SC administration of Leukosome-LNPs.
  • the Leukosome-LNP formulation was tested to evaluate feasibility of using a different lipidic composition while maintaining acceptable “quality attributes” of the formulation.
  • changes involved the following items: (i) the use of a different ionizable lipid (ALC-0315 instead of SM-102) and a difference in its molar %, (ii) the use of two different helper lipids (DOPG and PS instead of DSPC), (iii) the use of a different pegylated lipid (ALC-0159 instead of DMG-PEG2000) and an increase in its molar % (from 3.5% to 4%).
  • the ionizable lipid was ALC- 0315, the helper lipids were DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)) and Brain PS (L- ⁇ -phosphatidylserine (Brain, Porcine) (sodium salt)), the PEGylated lipid was ALC-0159, the N/P ratio was 6, the flow rate ratio was 1:3, the total flow rate was 12 mL/min, the protein:lipid ratio was 1:90, and the protein source was recombinant protein LFA1. TABLE 12. Iteration 3.
  • DOPG 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)
  • Brain PS L- ⁇ -phosphatidylserine (Brain, Porcine) (sodium salt)
  • the PEGylated lipid was ALC-0159,

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Nanotechnology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne une nanovésicule protéolipidique biomimétique, comprenant : un lipide ionisable ou cationique ; un phospholipide à base de phosphocholine ; un cholestérol ; une protéine membranaire leucocytaire ; et un ARNsi encapsulé par la nanovésicule protéolipidique biomimétique ; la nanovésicule protéolipidique biomimétique ayant un rapport lipide-protéine d'environ 1:65 à environ 1:85 en poids. L'invention concerne en outre une nanovésicule protéolipidique biomimétique, comprenant : un lipide ionisable ou cationique ; un phospholipide à base de phosphocholine ; un cholestérol ; une protéine membranaire leucocytaire ; et un ARNm encapsulé par la nanovésicule protéolipidique biomimétique ; la nanovésicule protéolipidique biomimétique ayant un rapport lipide-protéine d'environ 1:80 à environ 1:100 en poids.
PCT/US2024/031899 2023-06-02 2024-05-31 Nanovésicules protéolipidiques biomimétiques pour l'administration d'acides nucléiques Ceased WO2024249782A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363505913P 2023-06-02 2023-06-02
US63/505,913 2023-06-02

Publications (2)

Publication Number Publication Date
WO2024249782A2 true WO2024249782A2 (fr) 2024-12-05
WO2024249782A3 WO2024249782A3 (fr) 2025-01-16

Family

ID=93658448

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/031899 Ceased WO2024249782A2 (fr) 2023-06-02 2024-05-31 Nanovésicules protéolipidiques biomimétiques pour l'administration d'acides nucléiques

Country Status (1)

Country Link
WO (1) WO2024249782A2 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2779099C (fr) * 2009-10-30 2021-08-10 Northwestern University Nanoconjugues formes sur matrice
EP2714017B1 (fr) * 2011-06-02 2018-08-01 The Regents of The University of California Nanoparticules encapsulées dans une membrane et leur procédé d'utilisation
KR102396026B1 (ko) * 2014-01-21 2022-05-09 안자리움 바이오사이언시스 아게 하이브리도좀, 이를 포함하는 조성물, 이의 제조 방법 및 이의 용도
WO2019141267A1 (fr) * 2018-01-22 2019-07-25 北京茵诺医药科技有限公司 Système de distribution de nanovecteur de type recombinant bionique destiné à cibler une molécule cd44 active, son procédé de préparation et ses applications
CA3094859A1 (fr) * 2020-10-01 2022-04-01 Entos Pharmaceuticals Inc. Vesicules de proteolipide formulees avec petites proteines transmembranaires associees a la fusion
US20220280429A1 (en) * 2021-03-03 2022-09-08 The Methodist Hospital Tunable leukocyte-based biomimetic nanoparticles and methods of use

Also Published As

Publication number Publication date
WO2024249782A3 (fr) 2025-01-16

Similar Documents

Publication Publication Date Title
US12569438B2 (en) Nanomaterials containing constrained lipids and uses thereof
US12065664B2 (en) Method for treatment of a leukocyte related disease by delivery of nucleic acid molecules to leukocytes using targeted lipid particles
Zhang et al. Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy
KR102541164B1 (ko) 하이브리도좀, 이를 포함하는 조성물, 이의 제조 방법 및 이의 용도
US20220280429A1 (en) Tunable leukocyte-based biomimetic nanoparticles and methods of use
JP7017018B2 (ja) エキノマイシン製剤、その製造法および使用法
US12599574B2 (en) Process of making membrane lipid coated nanoparticles
TWI221421B (en) Artificial kidney having function of metabolizing protein and method of constructing the same
Dreaden et al. RNA‐peptide nanoplexes drug DNA damage pathways in high‐grade serous ovarian tumors
Zhang et al. Advances in novel biomaterial-based strategies for spinal cord injury treatment
Zhao et al. Nanomaterials for acute myeloid leukemia therapy: current progress and future perspectives
WO2024249782A2 (fr) Nanovésicules protéolipidiques biomimétiques pour l'administration d'acides nucléiques
JP7585166B2 (ja) 物質送達キャリア及び組成物
Alsaidan Current trends in exosomes as therapeutic drug delivery systems
Yin et al. Extracellular Vesicle‐Based Nanodrug Delivery
She et al. Breaking through the blood-brain barrier for different kinds of drugs: Challenges, solutions, and perspectives
CN114369575B (zh) 无促瘤功能的脑胶质瘤细胞来源外泌体及其制备方法与应用
Yan New lipids and polymers based nanotherapeutics and their applications in disease treatment
US20260102468A1 (en) Compositions and methods for marco inhibition and improved drug delivery
CN121774885A (zh) 一种榄香烯卡巴他赛工程化仿生脂质体、制备方法及应用
Sushnitha Engineering Leukocyte Mimicking Nanoparticles for Targeted Delivery in Triple-Negative Breast Cancer: Biological versus Cargo-Based Approach
WO2024210160A1 (fr) Complexe de conjugaison
Ren et al. Mannosylated siRNA/PERKi nanoassemblies reprogram macrophages to block tumor metastasis
WO2026046110A1 (fr) Nouveau système d'administration de médicament basé sur des globules rouges modifiés
CN115919797A (zh) 一种类外泌体负载阿霉素在治疗神经母细胞瘤的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24816518

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE