Diblock copolymer-mediated lipid nanoparticles

Abstract

Block copolymer-stabilized nanoparticles (50 nm - 1000 nm in size) of oligonucleotides with a polyethylene glycol (PEG) steric stabilizing layer are provided. Such nanoparticles may be formed using stabilizing blocks of PEG with molecular weights of 1000-5000 Dalton molecular weight, and hydrophobic anchoring blocks of a degradable polylactide and / or degradable lactide-co-glycolide polymer with molecular weights of 1000-20,000 Dalton molecular weight. The anchoring blocks may be, e.g., a polycaprolactone polymer, a polylactic acid polymer, and / or a copolymer of a lactide-co-glycolide polymer. Such nanoparticles are preferably configured to be internalized into cells and transfect cells at least as effectively as a lipid nanoparticle formulated with 1500 Dalton molecular weight PEG and Dimyristoyl glycerol (DMG) anchoring lipids.

Description

[0001] DIBLOCK COPOLYMER-MEDIATED LIPID NANOPARTICLES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to U.S. Provisional Patent Application No. 63 / 528,964, filed July 26, 2023, the contents of which are herein incorporated by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to the synthesis and process optimization of diblock copolymer lipid nanoparticles for delivery of nucleic acids.

[0006] BACKGROUND

[0007] Delivery of nucleic acids through synthetic lipid nanoparticles (LNPs) has revolutionized strategies to fight against infectious diseases and treat diseases. Liver targeting of LNPs is a result of the shedding of poly(ethylene glycol) (PEG)-lipids and adsorption of apolipoprotein E (ApoE) on LNPs. leading to low-density lipoprotein receptor. Permanent PEG comonas on LNPs would prevent shedding, ApoE attachment, and targeting only to the liver. However, an additional requirement of successful nucleic acid delivery is that the LNP needs to escape from the endosome to deliver the nucleic acid to the cytosol, in the case of RNA delivery, and to the nucleus, in the case of DNA delivery. It is known that permanently attached PEG coronas on nanoparticle (NP) surfaces minimize or prevent NP recognition by cells. These are frequently referred to a “stealth” NPs. Therefore, while a permanent PEG layer may minimize recognition by cells and delivery to the liver, it may diminish transfection efficiency. Disclosed herein is the unexpected result that a permanently anchored PEG corona does not disrupt cell attachment nor transfection efficiency, when properly formulated.

[0008] BRIEF SUMMARY

[0009] In various aspects, a block copolymer-stabilized nanoparticle of oligonucleotides with a polyethylene glycol (PEG) steric stabilizing layer may be provided. The block copolymer- stabilized nanoparticle may be formed using a) stabilizing blocks of PEG with molecular weights of 1000-5000 Dalton molecular weight, and b) hydrophobic anchoring blocks of a degradable polylactide and / or degradable lactide-co-glycolide polymer with molecular weights of 1000-20,000 Dalton molecular weight. Such nanoparticles may be configured to be internalized into cells and transfect cells at least as effectively as a lipid nanoparticle formulated with 1500 Dalton molecular weight PEG and Dimyristoyl glycerol (DMG) anchoring lipids.

[0010] The hydrophobic anchoring blocks may be one or more polycaprolactone polymers. The hydrophobic anchoring blocks may be one or more polylactic acid polymers. The hydrophobic anchoring blocks may be one or more copolymers of lactide-co-glycolide polymers.

[0011] The block copolymer-stabilized nanoparticle may be 50 nm to 1000 nm in size (e.g.. effective diameter) as determined by dynamic light scattering.

[0012] The block copolymer-stabilized nanoparticle may include oligonucleotides of siRNA, RNA, mRNA, or DNA, or combinations thereof. The block copolymer-stabilized nanoparticle may include at least one additional component. The additional component(s) may include an ionizable lipid, a zwitterionic lipid, cholesterol, or a combination thereof.

[0013] In various aspects, a composite particle is provided. Such a particle may include a core nanoparticle, and at least one steric stabilizing layer around the core nanoparticle. The stabilizing layer may include a block copolymer. The block copolymer may include stabilizing blocks of PEG with molecular weights of 1000-5000 Dalton molecular weight, and hydrophobic anchoring blocks of a degradable polylactide and / or degradable lactide-co- glycolide polymer with molecular weights of 1000-20,000 Dalton molecular weight.

[0014] BRIEF DESCRIPTION OF DRAWINGS

[0015] Figures 1A and IB are graphs depicting the sizes and zeta potential of LNPs, respectively, throughout the processing conditions. “Initial'’ indicates the freshly prepared LNPs in 10% ethanol. “Dialyzed” indicates buffer exchanged LNPs through dialysis. “2d_4°C” indicates LNPs stored at 4°C after buffer exchange.

[0016] Figure 1C is a graph depicting the encapsulation efficiency of plasmid DNA (6.6 kbps) in LNPs.

[0017] Figure ID and Figure IE are graphs illustrating the transfection and cell l iability in Hela cells after LNP treatment.

[0018] Figures 2A, 2B, and 2C are graphs depicting contour plots of the transfection, size, and encapsulation efficiency (EE), respectively, at different LNP formulations; plots are described by the ratio of cholesterol to DOPE and mole % of ionizable lipid.

[0019] Figure 2D is a graph illustrating the relation between the transfection and the size of PCL block in PCL-b-PEG at different range of mole % of PCL-b-PEG. Figures 3A, Figure 3B, and Figure 3C are graphs illustrating the transfection of pDNA and cell viability in Hela cell (3 A), HepG2 cells (3B), and Jurkat cells (3C), at 24h post-LNP treatment.

[0020] Figure 3D is a graph depicting the transfection of pDNA in Hela and HepG2 cells at 4h post-LNP treatment.

[0021] Figures 3E and 3F are graphs illustrating the uptake of LNPs by Hela cells (3E) and HepG2 cells (3F).

[0022] Figure 4 is a graph illustrating the size change of LNPs throughout the processes. “Initial” indicates the freshly prepared LNPs in 10% ethanol, where the measurement time is within 1 hour of the time of initial precipitation. “Dialyzed” indicates buffer exchanged LNPs through dialysis. “Al OOF” indicates LNPs washed with HEPES buffer and concentrated via an Amicon centrifugal filter (100 kDa MWCO).

[0023] Figure 5 is a graph illustrating the size of LNPs before and after concentration using Amicon filters.

[0024] Figure 6 is a graph depicting the EE of pDNA with DMG-PEG or Vitamin E TPGS (Vit E TPGS) at different concentrations. “Eq” indicates the mole equivalent of the PEG- compound relative to the number of moles of PEG-compound used for the initial LNP synthesis via FNP.

[0025] Figure 7 is a graph depicting the size of standard LNPs (sLNPs) and next-generation LNPs (ngLNPs) after freeze-thaw with the cryoprotectants described in Table 5.

[0026] Figure 8 is a graph illustrating the size of ngLNPs combined with different processing conditions. “pre-FT” and “post-FT” indicates LNPs before and after freeze-thaw, respectively.

[0027] Figure 9 is a graph illustrating the size distribution of LNPs (FF18) throughout the processes.

[0028] Figure 10A is a graph illustrating the effect of quench buffer on initial LNP size.

[0029] Figure 1 OB is a graph depicting the size of ngLNPs throughout the processes. Figure 10C shows the EE of ngLNPs after concentration step.

[0030] DETAILED DESCRIPTION

[0031] The current disclosure presents methods for synthesis and process optimization of block copolymer (BCP) protected lipid nanoparticles (also referred to as block copolymer-stabilized nanoparticles). Further it is shown that Flash NanoPrecipitation (FNP) is an effective process to make these BCP protected LNPs. It is know n, for example, that a composite particle composed of a conventional PEG shell around a core nanoparticle can prevent interaction of the nanoparticle with cells, but such conventional PEG shells are prone to “shedding off’ in a patient’s blood stream, degrading the protection of the PEG shell. Not only do the disclosed BCPs form a steric stabilizing layer around the base nanoparticle, the disclosed PEG BCPs have larger hydrophobic blocks compared to lipid tails in PEG-lipids and therefore, “shedding off’ is minimized. Unexpectedly, the PEG to poly(e-caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG) does not compromise the transfection, when compared to the conventional PEG-lipid LNPs. In fact, with formulation of the LNP composition it is possible to achieve enhanced delivery of nucleic acids with PEG-BCP coated LNPs over the traditional PEG-lipid LNP formulations. Indeed, the block copolymer-stabilized nanoparticle may be configured to be internalized into cells and transfect cells at least as effectively as a lipid nanoparticle formulated with 1500 Dalton molecular weight PEG and Dimyristoyl glycerol (DMG) anchoring lipids.

[0032] Incorporation of BCPs in LNPs has been previously been described in US8754062B2. However, the polymers are only presented as a generic laundry list. There is no reduction to practice of using BCPs. Nor is there indication the specific BCPs disclosed herein, providing the unexpected increased transfection results as described below.

[0033] In one embodiment, BCP-mediated LNPs are composed of: 1) one or more lipids selected from cationic lipids, cholesterol, and helper lipids, 2) BCPs, 3) PEG-lipids, and 4) nucleic acids.

[0034] In some embodiments, the nanoparticles may include oligonucleotides of siRNA, RNA, mRNA, DNA, or a combination thereof.

[0035] In a general embodiment, cationic lipids include one or more alky l tails and one or more amine groups. The length of lipid tails can be varied. The lipid tails can be either saturated and / or unsaturated. Amine groups can be permanently charged quaternary' amines as well as primary, secondary , and tertiary amines with pKaS that facilitate electrostatic assembly. Lipids of this sort have been described in WO1998051278A2, CN114191561B, US8058069B2, and US11414393B2. These are included in their entirety’ in this submission.

[0036] In one embodiment. LNPs may include more than one cationic lipid, such as those described in US20220387337A1 and US11229609B2. In general, apparent pIGs of the ionizable lipids in the LNPs below’ 7 is preferred. This facilitates the electrostatic interaction mediated assembly of LNPs at low pH, while neutralizing cationic charges on LNPs in biological fluid atpH values around 7.4 (Albertsen et al.. Adv Drug Deliv Rev, 188: 114416 (2022)) More specifically, optimal apparent pKa of ionizable lipids in LNPs is between 6.2 and 6.5 for the delivery of siRNA (Jayaraman et al., Angew Chem Int Ed Engl. 51(34): 8529- 8533 (2012)).

[0037] In a general embodiment, helper lipids include zwitterionic lipids or anionic lipids that have one or more alkyl tails. The length of lipid tails can be varied. The lipid tails can be either saturated and / or unsaturated. The number of charged head group can be one or more. Commonly employed helper lipids include 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1.2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), and l,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA).

[0038] In certain embodiments, cholesterol can be substituted with various types of sterol or tocopherol molecules. The term “sterol” refers to a subgroup of steroids also known as steroid alcohols. Sterols are usually divided into two classes: (1) plant sterols also known as “phytosterols”, and (2) animal sterols also known as “zoosterols.” Non-limiting examples of phy tosterols include P-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol. Nonlimiting examples of tocopherols include a-tocopherol, p-tocopherol, y-tocopherol, 6- tocopheroL or a salt or ester thereof, such as a-tocopherol hemisuccinate.

[0039] In one embodiment, BCP indicates the linear chain polymer consisting of two types of homopolymers covalently joined together. BCP typically used in biomedical applications include PCL-b-PEG, poly(D,L-lactic acid)-block-poly(ethylene glycol) (PEG-b-PLA), and poly(L-lactide-co-glycolide)-block-poly (ethylene glycol) (PLGA-b-PEG). The usage of block copolymer as a stabilizing agent and / or a ligand anchoring site is not limited to BCPs. Other types of block copolymers are well understood. See, e.g, US20120121718, US20100003337, US8754062B2 (each of which are incorporated by reference herein in its entirety).

[0040] In some embodiments, the hydrophobic anchoring blocks may be a polycaprolactone polymer, a polylactic acid polymer, a copolymer of a lactide-co-glycohde polymer, or a combination thereof.

[0041] In a preferred embodiment, the type of BCPs in LNPs is PCL-b-PEG. The molecular weight of PCL block may be about 500 Da or 1000 Da to 15,000 Da, or 20,000 Da. The preferred molecular weight of PEG block is at about 1000 Da to 7000 Da. In some embodiments, the nanoparticle may utilize stabilizing blocks of PEG (e.g., with molecular weights of 1000-5000 Dalton molecular weight) and hydrophobic anchoring blocks of a degradable polylactide and / or lactide-co-glycohde polymer (e.g., with molecular weights of 1000-20.000 Dalton molecular weight). As used herein, the term “polylactide” generally refers to polymers of L-lactide, D- lactide. meso-lactide, blends thereof, and lactic acid polymers and copolymers in which other moieties are present in amounts less than 50 mole percent.

[0042] PEG-lipids indicates PEG conjugated to lipids. Commonly used PEG-lipids include l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), 1,2- Distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DSG-PEG), N-octanoyl- sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (Ceramide-PEG). and cholesterol-PEG2000.

[0043] PEG-lipids can be substituted by other PEG-containing compounds which includes, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, vitamin E TPGS, Pluronic L35, Pluronic L61, Pluronic L64. Pluronic F-68, or Pluronic F-127.

[0044] The molecular weight of PEG in PEG-lipids or PEG-containing compounds can vary. In preferred embodiments, molecular weight of PEG in PEG-lipids or PEG-containing compounds is about 1000 Da to 5000 Da.

[0045] In one embodiment, the LNPs include cationic lipids at a concentration of about 25 mol % to 60 mol %, a mixture of cholesterol and zwitterionic lipids at a concentration of about 30 mol % to 74 mol %, and a mixture of BCP and DMG-PEG at a concentration of about 1 mol % to 10 mol %. The mole ratios of amines in cationic lipids to phosphate groups in nucleic acids (N to P ratios) range from 3 to 12.

[0046] In a preferred embodiment, the LNPs are composed of cationic lipids at a concentration of about 35 mol % to 50 mol %, a mixture of cholesterol and zwitterionic lipids at a concentration of about 47 mol % to 64 mol %, a mixture of BCP and DMG-PEG at a concentration of about 1 mol % to 3 mol %. The preferred N to P ratio is 3 or 6.

[0047] Nucleic acids refer to a substance consisting of two or more deoxyribonucleotides or ribonucleotides, which can exist as a single strand or a double helix. The definition encompasses any form of DNA and RNA. Lipid formulations for delivery nucleic acids are known; see US8058069B2, incorporated by reference herein in its entirety.

[0048] LNPs may be synthesized via microfluidics, confined impinging jet (CIJ) mixer, or multi-inlet vortex mixer (MIVM). Synthesis of nanoparticles via CIJ or MIVM is extensively investigated by Prud’homme (US1 1376225B2, Johnson et al., Australian Journal of Chemistry, 56(10): 1021 - 1024, (2003), Markwaiter et al., J Pharm Sci, 107(9):2465-2471, (2018), Feng et al., J Transl Med, 17(l):200 (2019)c, Pagels et al., Nano Lett, 18(2): 1139-1144 (2018), Kim et al., ACS Appl. Nano Mater, 5(12): 18770-18778 (2022), each of which are incorporated herein in its entirety). In one embodiment, the solvent stream contains lipid components and BCPs dissolved in organic solvent. In a certain embodiment, the solvent stream contains PEG-lipid as well as lipids and BCP. Organic solvent is composed of one or a mixture of any of following, but not limited to: ethanol, isopropanol, methanol, acetonitrile, acetone, tetrahydrofuran, chloroform, dichloromethane, benzyl alcohol, anisole, dimethyl sulfoxide, and dimethyl sulfide. Other water miscible solvents may include, e.g., N-methyl pyrrolidone, propylene glycol, ethyl acetate, dimethyl sulfoxide, dimethyl acetamide, benzyl alcohol. 2-pyrrolidone. or benzyl benzoate. In one embodiment, the solvent stream can be one stream that contains all the components. In another embodiment, the solvent stream can be two separate streams. When more than one solvent stream is used, the location of individual components in the solvent stream can be changed depending on the formulation strategy. For instance, lipids dissolved in ethanol can be loaded in solvent stream 1 and BCP dissolved in acetonitrile is in solvent stream 2. The anti-solvent stream contains water soluble molecules, including small molecules, large molecules, nucleic acids, peptides, and proteins, dissolved in aqueous solution. In general embodiment, aqueous solution indicates salts and / or buffering reagents dissolved in water. In one embodiment, the aqueous solution is acidic buffer where cationic lipids can be fully charged. In a certain embodiment, pH of acidic buffer in antisolvent stream is between 3 and 5. Like the solvent stream, the antisolvent stream can be one stream or two separate streams.

[0049] In general embodiments, the percentage of aqueous solution in exit stream from CIJ or MIVM is 50 vol % and 24 vol %, respectively. In a certain embodiment, the percentage of aqueous solution in exit stream ranges from 10 vol % to 50 vol %.

[0050] In certain embodiments, LNPs exiting from the mixers are diluted with additional buffers such as PBS, Tris, HEPES. citrate, or acetate buffer. The concentration of organic solvent upon dilution ranges from 1 vol % to 20 vol %.

[0051] In a certain embodiment, the dilution buffer contains PEG-containing species. The amount of PEG-species dissolved in the dilution buffer does not exceed 10 mole equivalent of BCP in antisolvent stream. In another embodiment, the PEG-compound can be added in postprocessing steps such as tangential flow filtration or concentration through centrifugal filter units.

[0052] In certain embodiments, LNPs include additional imaging agents that require specific instruments to derive the qualitative or quantitative amount of one or more components of LNPs in cells or tissues. The imaging agents include, but are not limited to, fluorescent dyes, radioactive materials, lanthanide elements, or contrast agents. In various embodiments, the diameter (or effective diameter, for non-spherical particles) of LNPs ranges from about 50 nm to 1000 nm, as determined by dynamic light scattering. In various embodiments, the diameter of LNPs ranges from about 50 nm to 400 nm.

[0053] For global distribution of LNPs therapeutics, cold-chain is necessary. In certain embodiments BCP-embedded LNPs are frozen with additional stabilizers and cryoprotectants. LNPs are frozen between -20 °C and -196 °C. Stabilizers are PEG-compounds and added at a concentration of about 1 mol % to 10 mol % of final lipid nanoparticle composition as described above. Cryoprotectants are sugar molecules including, but not limited to, sucrose, trehalose, or mannitol. In a preferred embodiment, BCP-embedded LNPs are frozen with 500 mM of sucrose or 500 mM trehalose. The extended list of cryopreservation of LNPs is described in WO 2018089540 Al. The cryoprotectants of these sorts have been described in their entirety in this submission.

[0054] One application of the present disclosure is to deliver the LNP cargo wherein the BCP remains attached to NPs and does not significantly partition off.

[0055] The present disclosed LNPs can be administered through either parenteral or non- parenteral routes of administration, which includes, but not limited to, intravenous, subcutaneous, intramuscular, intradermal, intranasal, ophthalmic, oral, rectal, vaginal, buccal, or pulmonary administration.

[0056] Examples

[0057] Example 1. Formulation and screening of diblock copolymer embedded lipid nanoparticles

[0058] A dense PEG corona prevents the interaction of NPs with cells. Therefore, the Onpattro formulation, commercially available siRNA encapsulating LNP drug, utilize DMG-PEG, which rapidly shed off in the blood stream. It was expected that, since the PCL block of PCL- b-PEG on LNPs is strongly anchored, the uptake would be minimal in vitro. Surprisingly, we find, that the PCL-b-PEG. which forms a dense, stabilizing PEG layer on the surface of the nanoparticles, does not impede transfection. Similar results are found with other degradable polylactides and / or degradable lactide-co-glycolide polymers.

[0059] Flash NanoPrecipitation (FNP) is used to synthesize LNPs. The solvent stream includes DLin-KC2-DMA, cholesterol, DOPE, and PCL-b-PEG or DMG-PEG in a mixture of ethanol and acetonitrile. The total mole concentration of LNP components in the solvent stream is 9.3 mM, and the mole percentage of each component is described in Table 1. Table 1

[0060] Importantly, due to the limited solubility of the PCL block in ethanol. PCL-b-PEG is pre-dissolved in acetonitrile using a heated water bath (42 °C). The solubilized PCL-b-PEG in acetonitrile is then combined with DLin-KC2-DMA, cholesterol, and DOPE dissolved in ethanol. The volume fraction of acetonitrile in the solvent stream is 10%. The antisolvent stream includes plasmid DNA encoding luciferase dissolved in potassium acetate buffer (25 mM, pH 4). The size of the plasmid is about 6600 bps. The ratio of the amine group in DLin- KC2-DMA to the phosphate group in plasmid DNA is kept at 6, as described in Table 1. One volume of the solvent stream is impinged against one volume of the antisolvent stream using a Confined Impinging Jet (CIJ) mixer, which produces an exit stream of 50% organic solvent. The NPs eluted through the exit stream are further diluted with PBS by 5-fold in a collection vial. Therefore, the final concentration of organic solvent in the collection vial is 10 vol%. The final samples are obtained by dialyzing the remaining organic solvent out against HEPES buffer (10 mM, pH 7.5) for 5 hours against an excess of HEPES buffer. The substitution of DMG-PEG with PCL-b-PEG does not induce dramatic changes in the physicochemical properties of LNPs (FIGS. 1A and IB). However, the encapsulation efficiency (EE) is 10%- 15% lower than standard LNPs formulated with DMG-PEG (FIG. 1C).

[0061] Surprisingly, all the BCP-embedded LNPs (E29-E33) exhibited superior transfection in Hela cells compared to standard LNPs (E34) (FIG. ID). Despite the good transfection, a decrease in cell viability’ is observed as the ratio of cholesterol to DOPE decreases in LNP formulations (FIG. IE). To find the optimal design space, we screened an additional 26 formulations by individually varying the mole ratios of each LNP component as well as the block size of PCL in PCL-b-PEG (FIGS. 2A. 2B, and 2C). LNPs exhibit high transfection when the ionizable lipid content is between 45-50 mole% at different ratios of cholesterol and DOPE. At the lower amount of ionizable lipids (25 mole%-40 mole%), high transfection is observed at relatively low ratios of cholesterol and DOPE (0.5-1.5). The size of LNPs becomes smaller, and the encapsulation efficiency tends to be higher as the ratios of cholesterol and EE decrease, which is consistent with our previous observation (FIGS. 1 A and 1C). Interestingly, we do not observe a direct relationship between the size of the PCL block in PCL-b-PEG and the transfection, as well as the mole% of PCL-b-PEG and the transfection in Hela cells (FIG. 2D).

[0062] Example 2. Transfection of optimized LNPs in different model cell lines

[0063] Based on previous screening results, three formulations that exhibit high transfection are selected for further investigation (see Tables 2A and 2B below).

[0064] Table 2A, (Composition of certain LNPs).

[0065] Table 2B (phvsiochemical properties of certain LNPs and encapsulation efficiency (EE) of pDNA),

[0066] Although all three formulations show comparable or superior transfection compared to standard LNPs (sLNPs), cell viability of Hela and HepG2 cells are lower than 50 % after 24 h incubation with F10 or F16 (FIGS. 3A and 3B). On the other hand. F10 and F16 are not cytotoxic to Jurkat cells (-80% cell viability). (FIG. 3C). Surprisingly, OptLNPs that include higher mole % of ionizable lipids and lower mole % of PCL-b-PEG do not exhibit significant cy totoxicity' in any of the cell lines; viability' of tested cell lines are above 80%. Interestingly, with a short LNP exposure time (4-hour incubation time before assay), optLNPs show 2 orders of magnitude higher transfection in Hela and HepG2 cells compared to sLNPs. (see FIG. 3D) This is partially due to the higher uptake of optLNPs by Hela cells and HepG2 cells, respectively, compared to sLNPs (see FIGS. 3E and 3F).

[0067] Example 3. Optimization of concentration of BCP-embedded LNPs. LNPs are formulated with different sizes of PCL-b-PEG as well as DMG-PEG (see

[0068] Table 3, below). These samples are then diluted 4 times in HEPES buffer (10 mM, pH 7.5) and washed and concentrated 20 times via Amicon Ultra- 15 centrifugal filter (A100F). As can be seen, BCP-embedded LNPs are susceptible to aggregation upon shear force (Figure 4). To minimize the aggregation during the concentration step, different stabilizers are screened using formulation FF7 (see Table 4, below, and Figure 5). The addition of PEG compounds, either DMG-PEG or Vitamin E TPGS. abrogates the aggregation. Moreover, the encapsulation efficiency of pDNA increases as the stabilizer concentration increases (Figure 6).

[0069] Table 3, (formulations of LNPs synthesized with different molecular weights of PCL-b-PEG or DMG-PEG)

[0070] Table 4. (formulation of a certain LNP) Example 4. Optimization of freeze-thaw of BCP -embedded LNPs

[0071] Sugar compounds are screened for optimizing the freeze-thaw of BCP-embedded LNPs (see Table 5). BCP-embedded LNPs frozen with sucrose at 500 mM and trehalose at 250 mM exhibited the least size changes among the other excipients tested for freeze-thaw. However, they still lead to approximately 2-fold size change after a freeze-thaw cycle. See FIG. 7. When 1 mole equivalent of DMG-PEG (compared to the number of PCL-b-PEG in the initial precipitation of the LNPs) is added during the concentration step, BCP-embedded LNPs exhibit 2.4 % and 6.7 % of size increase after freeze-thaw cycles with 500 mM sucrose and 500 mM trehalose, respectively (Figure 8).

[0072] Table 5, (types of cryoprotectants and their concentrations)

[0073] Example 5. Formulation of BCP-embedded LNPs using pMIVM

[0074] BCP-embedded LNPs are formulated via either a confined impingingjet (CIJ) mixer or a micro multi-inlet vortex mixer (pMIVM) (see Tables 6A, 6B, and 6C, below). While CIJ has two streams, pMIVM accommodates four streams and offers flexibility in formulation. FF17 and F21 have an identical solvent stream (SS) composition, but the ratio of SS to antisolvent stream (AS) differs. This leads to the production of LNPs with a size 30 nm smaller when pMIVM is used. FF18, FF19, and FF20 have a volume ratio of SS to AS that is kept at 1 : 1, like FF17. However, LNPs synthesized with pMIVM are 4 nm to 31 nm smaller than those produced with CIJ. Note that the concentration of LNPs in the exit stream is kept constant at 4.7 mM, although the total mole concentration in the solvent stream differs between CIJ and pMIVM. We also verify that LNPs produced with pMIVM (FF18) exhibit only 5.8% size change throughout the process if the optimized process conditions above are applied (FIG. 9).

[0075] Table 6A (certain LNP formulations produced with two different types of mixers, volume ratios of solvent stream and anti-solvent streams, and the composition of streams)

[0076] Table 6B (composition of each stream for certain LNPs)

[0077] Table 6C (composition and size of certain LNPs).

[0078] Example 6. Change of sink buffer in collection vials

[0079] LNPs exiting through the mixer were diluted 5 times with PBS in the previous examples. We also examine the effect of different buffers for the dilution (see Table 7, below). Noticeably, diluting LNPs in sodium phosphate buffer results in an LNP size of 160 nm, while potassium acetate gives rise to an LNP size of 110 nm (FIG. 10A). The change in LNP size is further investigated throughout the dialysis, concentration, and freeze-thaw (FT) steps. Acetate buffer in F14 is replaced with HEPES buffer or TRIS buffer, and named F14 HEPES and F14_TRIS, respectively (FIG. 10B). Although there are slight differences in the magnitude of size changes throughout the process, the size of LNPs converges to 130 nm - 135 nm after freeze-thaw. When TRIS buffer is used as the quench buffer or LNPs are washed with TRIS buffer, the final encapsulation efficiency after concentration is 5%-10% higher than that processed with HEPES buffer (FIG. 10C).

[0080] Table 7, (Certain LNP compositions and the types of quench buffer to dilute with).

[0081] Example 7. Eormulation of LNPs that incorporate DTAP-Gd-lipid.

[0082] LNPs formulated under optimal conditions are conjugated with an antibody through the reaction between the sulfhydryl group on the antibody and the maleimide group on LPNs. LNPs are formulated with two additional components: DTPA-Gd-lipid and PCL-b-PEG-Mal (see Table 8A, below).

[0083] Gadolinium (Gd) in DTPA-Gd-lipid allows tracking the biodistribution of LNPs in vivo vialCP-OES or ICP-MS. The maleimide group on PCL-b-PEG serves as a conjugation site for Cetuximab. Due to limited solubility in ethanol, DTPA-Gd-lipid is dissolved in a mixture of chloroform and methanol at a 2: 1 volume ratio. PCL-b-PEG and PCL-b-PEG-Mal are dissolved in acetonitrile (MeCN) as previously described. This stock solution is then added to the solvent stream containing the other lipid components. The final composition of the solvent stream is described in Table 8B, below. As can be seen, chloroform amount in the formulation should be minimized to form small and homogeneous LNPs and to maintain the stability under shear forces in the concentration steps. Concentrated LNPs are stored at 4 °C until conjugation. Table 8 A (composition of certain LNPs encapsulating DTPA-Gd-lipid)

[0084] Table 8B (composition of organic solvent streams and size of LNPs for two different formulations)

[0085] Various modifications may be made to the systems, methods, apparatus, mechanisms, techniques, and portions thereof described herein with respect to the various figures, such modifications being contemplated as being within the scope of the invention. For example, while a specific order of steps or arrangement of functional elements is presented in the various embodiments described herein, various other orders / arrangements of steps or functional elements may be utilized within the context of the various embodiments. Further, while modifications to embodiments may be discussed individually, various embodiments may use multiple modifications contemporaneously or in sequence, compound modifications and the like.

Claims

What is claimed is:

1. A block copolymer-stabilized nanoparticle of oligonucleotides with a polyethylene glycol (PEG) steric stabilizing layer formed using: a) stabilizing blocks of PEG with molecular w eights of 1000-5000 Dalton molecular weight; and b) hydrophobic anchoring blocks of a degradable polylactide and / or degradable lactide-co-glycolide polymer with molecular weights of 1000-20,000 Dalton molecular weight; wherein the block copolymer-stabilized nanoparticle is configured to be internalized into cells and transfect cells at least as effectively as a lipid nanoparticle formulated with 1500 Dalton molecular weight PEG and Dimyristoyl glycerol (DMG) anchoring lipids.

2. The block copolymer-stabilized nanoparticle of claim 1, wherein the hydrophobic anchoring blocks are one or more poly caprolactone polymers.

3. The block copolymer-stabilized nanoparticle of claim 1, wherein the hydrophobic anchoring blocks are one or more polylactic acid polymers.

4. The block copolymer-stabilized nanoparticle of claim 1, wherein the hydrophobic anchoring blocks are one or more copolymers of lactide-co-glycolide polymers.

5. The block copolymer-stabilized nanoparticle of claim 1, wherein the block copolymer- stabilized nanoparticle has an effective diameter of 50 nm to 1000 nm, as determined by dynamic light scattering.

6. The block copolymer-stabilized nanoparticle of claim 1, further comprising oligonucleotides of siRNA, RNA, mRNA, or DNA, or combinations thereof.

7. The block copolymer-stabilized nanoparticle of claim 1, further comprising at least one additional component.

8. The block copolymer-stabilized nanoparticle of claim 7, wherein the at least one additional component is an ionizable lipid, a zwitterionic lipid, cholesterol, or a combination thereof.

9. A composite particle, comprising: a core nanoparticle; and at least one steric stabilizing layer around the core nanoparticle comprising a block copolymer, the block copoly mer including: stabilizing blocks of PEG with molecular weights of 1000-5000 Dalton molecular weight; and hydrophobic anchoring blocks of a degradable polylactide and / or degradable lactide-co-glycolide polymer with molecular weights of 1000-20,000 Dalton molecular weight; wherein the composite particle is configured to be internalized into cells and transfect cells at least as effectively as a lipid nanoparticle formulated with 1500 Dalton molecular weight PEG and Dimyristoyl glycerol (DMG) anchoring lipids.

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