Liposomal nanoparticles for crispr / cas9 delivery

Abstract

Disclosed herein are lipid nanoparticles comprising one or more neutral lipid, one or more sterol, one or more cationic lipid, one or more ionizable lipid, and one or more PEG- modified lipid; in particular, the lipid nanoparticles comprising 1,2-dioleoyl-sn-glycero- 3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), 6-((2-hexyldecanoyl)oxy)-N-(6- ((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 aminium (ALC-0315), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)- 2000] (DSPE-PEG2000); compositions comprising such lipid nanoparticles, and methods of using such lipid nanoparticles and compositions.

Description

[0001]Liposomal nanoparticles for CRISPR / Cas9 delivery FIELD OF INVENTION The present invention relates to lipid nanoparticles suitable for delivery of various agents into the cells, in particular, suitable for delivery of CRISPR / Cas9 complexes. BACKGROUND Discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR- associated protein 9 (Cas9) system added a robust tool to the genome editing toolbox (Doudna et.al., 2014, Gasiunas et.al., 2012). In order to use Cas9 in genome editing, minimum two components, the Cas9 protein and a single guide RNA (sgRNA) targeting the genomic site of interest, need to be delivered into a cell. When designing and preparing a delivery system, it is necessary to focus on maintaining the nuclease activity of Cas9 and protecting the ribonucleoprotein (RNP) that comprises the Cas9 protein in complex with a targeting sgRNA, against proteases, nucleases, antibodies, and T cell recognition in the serum and body fluids. Once entered the target cell, the delivery system should help the RNP be released from the endosome to the cytoplasm and enable its function. Efficient delivery of CRISPR / Cas9 to target tissues or cells faces considerable challenges. The current CRISPR / Cas9 delivery strategies are as follows: 1. Physical methods such as electroporation and microinjection (Huang et.al., 2022). 2. Virus-mediated delivery (adeno-associated viruses (AAVs), adenovirus (AdVs), lentivirus) (Huang et.al., 2022). 3. Delivery via non-viral vectors (lipid nanoparticles, polymer nanoparticles, DNA nanoclew, inorganic nanoparticles) (Huang et.al., 2022). Physical delivery methods are relatively straightforward to perform but are mostly applicable to in vitro systems. Electroporation uses strong currents that can cause cell death, so this method is not suitable for stress-sensitive cells. Virus-mediated delivery of the components of CRISPR / Cas9, is one of the most effective and commonly used methods. During this process, however, the viral vectors may integrate into host cells and cause problems such as mutations, carcinogenesis, and an immune response (Xu et al., 2019; Yip, et.al, 2020). Also, the physical capacity of viral vectors is often a challenge for delivery of large CRISPR / Cas protein complexes as the maximum capacity of an adeno-associated virus is approximately 4.7 kb (Glass et.al., 2018). The application of biomaterials as non-viral vectors has gained popularity in recent years due to their versatility, biocompatibility and increasing transfection efficiency. As a non-viral delivery system, liposomal nanoparticles (NP) offer many advantages including formulation simplicity, self-assembly, biocompatibility, high bioavailability, ability to carry large payloads and a range of physicochemical properties that can be controlled to modulate their biological characteristics (Mitchell et.al., 2021). Liposomal nanoparticles (LNPs) have been used for drug delivery for multiple decades (Allen et.al., 2004) and LNPs components were systematically optimized for efficient CRISPR / Cas9 delivery (Witzigmann et.al., 2020, Sercombe et.al., 2015). Capacity limitations are no longer an issue and nanoparticles can be further customized to improve tissue specificity as well as nuclear transport (Mitchell et.al., 2021). Current research is focused on improving the properties of non-viral nanoparticles with respect to cell-penetration, endosome escape, reducing toxicity, degradation, and improving long-term storage stability (Sinclair et.al., 2023). However, despite already available delivery tools, the development of safe and optimally efficient delivery systems for CRISPR / Cas9 elements capable of achieving specific targeting of gene therapy to the location of interest without off-target effects remains a primary challenge for clinical therapeutics. SUMMARY OF INVENTION Disclosed herein are lipid nanoparticles comprising one or more neutral lipid, one or more sterol, one or more cationic lipid, one or more ionizable lipid, and one or more PEG-modified lipid; in particular, the lipid nanoparticles comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-3- trimethylammoniumpropane (DOTAP), 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 aminium (ALC-0315), and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000); compositions comprising such lipid nanoparticles, and methods of using such lipid nanoparticles and compositions. Further, the present invention provides said lipid nanoparticles that are loaded with Cas9:sgRNA (CRISPR / Cas9) complex, as well as a method for encapsulation of said complexes into the lipid nanoparticles according to the present invention. The presently provided stable lipid nanoparticles are most suitable for CRISPR / Cas9 efficient encapsulation and delivery to the cells. Another aspect of the invention concerns a method for analysis of fusion mechanism between lipid nanoparticles and phospholipid membranes – such a method allows analysis in vitro utilizing nontargeted / targeted membrane fusion between liposomal NPs and an artificial phospholipid membrane. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. The illustration of liposomal NPs with different lipid compositions and loading of the ribonucleoprotein (RNP) complex. Figure 2. Stability of liposomal NPs sizes before (left) and after (Right) loading with BSA determined by DLS. Figure 3. Liposomal NPs sizes before and after BSA (Left) and sgRNA / Cas9 complex (Right) loading by DLS. Composition of liposomal NPs: DOPE / DOPC / Chol / DOTAP / ALC0315 / DSPE- PEG2000 (20 / 10 / 30 / 20 / 15 / 5). Figure 4. (Left) BSA encapsulation efficiency into liposomal NPs of different compositions. (Right). BSA and sgRNA / Cas9 encapsulation efficiency into liposomal NPs. Figure 5. The influence of Cas9-GFP concentration (9-35 nM) and incubation time (3-24 h) to liposomal NPs delivery of Cas9-GFP and sgRNA complexes to HEK293T / 17 cells. Left – the percentage of cells containing Cas9-GFP in the cytoplasm. Right – the percentage of cells containing Cas9-GFP in the nucleus. Data are represented as mean ± standard error of mean. Figure 6. The stability of liposomal NPs for the delivery of Cas9-GFP: sgRNA complexes to HEK293T / 17 cells. The percentage of cells containing Cas9-GFP inside the cytoplasm (left) or the nucleus (right) using freshly prepared liposomal NPs (0 days), 2 or 7 days after preparation. Cells were treated with 15 nM of Cas9-GFP:sgRNA complexes for 24 h. Data are represented as mean ± standard error of mean. Figure 7. The delivery of Liposomal NPs / Cas9-GFP: sgRNA complexes to brain endothelial cells. The percentage of brain endothelial cells containing Cas9-GFP inside the cytoplasm (left) or the nucleus (right). Cells were treated with liposomal NPs containing 22.7 nM of Cas9-GFP and sgRNA complexes for 24 or 48 h. Data are represented as mean ± standard error of mean. Figure 8. The fusion of liposomal NPs with artificial phospholipid membranes. Fusion / Lipid exchange: Liposomal NPs: DOPE / Chol / DOTAP / ALC0315 / DSPE-PEG2000 (30 / 30 / 20 / 15 / 5), phospholipid membrane (tBLM): DOPC. Figure 9. The lateral lipid distribution after liposomal NPs fusion with phospholipid membrane by FLIM. Donor - phospholipid membrane (tBLM, DOPC) with Cys3 and Acceptor – liposomal NPs (DOPC / Chol / DSPE-PEG2000) with Cy5. The parameter ^ allows us to quantify the fusion of liposomal NPs with lipid membranes. DETAILED DESCRIPTION As a non-viral delivery system, lipid-based nanoparticles (LNPs) offer many advantages including formulation simplicity, self-assembly, biocompatibility, high bioavailability, ability to carry large payloads and a range of physicochemical properties that can be controlled to modulate their biological characteristics (Mitchell et.al., 2021). The present invention provides a system of various composition liposomal NPs with enhanced cellular entry. It is known, that by changing the structure and the composition of liposomal NPs, it is possible to generate functionally different lipid nanoparticles delivering genes. (Sercombe et.al., 2015, Mitchell et.al., 2021). Selective organ-targeting (SORT) lipid-based NPs are a series of NPs designed recently for tissue specific delivery of mRNA and CRISPR / Cas9 genome editing tool (Cheng, et.al., 2020). Recently, surface engineering of LNPs with integrated membrane proteins from human pluripotent stem-cell-derived neurons showed a reproducible production of efficient neuron-targeting LNPs (Zinger et.al. 2021). One of the strategies for improving Cas9 delivery is the enhancing cell entry by modifying LNPs with cell penetrating peptides (CPPs) or targeting ligands (aptamers, hyaluronan, etc.) (Eoh and Gu, 2019; WO2022 / 192879A1). Moreover, the positive charge of CPPs such as the Tat peptide leads to unfavorable interaction with blood components. Whereas, efficient delivery of CRISPR / Cas9 to living cells is still a major challenge, we developed a method to study the CRISPR / Cas9 entry into the cytosol of live cells in vitro utilizing nontargeted / targeted membrane fusion between liposomal NPs and plasma membrane. In the present disclosure, liposomal nanoparticles of various lipid compositions were designed which were characterized by the dynamic light scattering method, and their stability was evaluated by the calcein release method or the release of fluorescently labeled BSA. The composition of the presently disclosed liposomal NPs provides various advantageous properties. The liposomal NPs comprise cationic lipids (such as, e.g. DOTAP) and ionizable lipids (such as, e.g. ALC-0315) that are useful for sgRNA complexation and for aid endosomal escape. Liposomal NPs further contain neutral lipids (such as, e.g., DOPC and / or DOPE) for steric stabilization, and to promote cellular uptake, membrane fusion, and intracellular processing. Liposomal NPs further contain Cholesterol and PEGylated lipids (such as, e.g., DSPE-PEG2000) which serve to improve stability, fusion and circulation of liposomal NPs. A lipid nanoparticle which comprises combinations of lipids as disclosed herein, provides additional advantages over the lipid nanoparticles available in the art. In particular, the present disclosure provides a lipid nanoparticle comprising: one or more neutral lipid, one or more sterol, one or more cationic lipid, one or more ionizable lipid, and one or more PEG-modified lipid. Preferably, in such a lipid nanoparticle, the lipids are selected from: a) the one or more neutral lipid is selected from: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-3 -phosphoethanolamine (POPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE) (DPhPC); b) the sterol is cholesterol, c) the one or more cationic lipid is selected from: 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC); d) the one or more ionizable lipid is selected from: 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 aminium (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM- 102), Di((Z)-Non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (LIPID L319); e) the one or more PEG-modified lipid is selected from: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (PEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG), PEG-modified phosphatidylethanolamine, a PEG-modified ceramide, a PEG-modified diacylglycerol. In some embodiments, the lipid nanoparticle comprises one or more neutral lipids such as, e.g., DOPC and / or DOPE, a sterol, such as, e.g., cholesterol, a cationic lipid, such as, e.g., DOTAP, an ionizable lipid, such as, e.g., ALC-0315, and a PEG-modified lipid, such as, e.g., DSPE- PEG2000. In some embodiments, in the lipid nanoparticle of the present invention, the one or more neutral lipid is DOPC and / or DOPE. In some embodiments, the one or more sterol is cholesterol. In some embodiments, the one or more cationic lipid is DOTAP. In some embodiments, the one or more ionizable lipid is ALC-0315. In some embodiments, the one or more PEG-modified lipid is DSPE-PEG2000. As disclosed herein, the lipid nanoparticle of the present invention, preferably comprises: neutral lipids DOPC and DOPE, cholesterol, cationic lipid DOTAP, ionizable lipid ALC-0315, and PEG- modified lipid DSPE-PEG2000. In some embodiments, in such a lipid nanoparticle, the lipids are provided at the following molar ratio: a) DOPC is provided at molar ratio from 15 to 30; for example, it may be present at molar ratio of 15, 17, 20, 25, 30, or any other molar ratio in between; b) DOPE at molar ratio from 10 to 20; for example, it may be present at molar ratio of 10, 12, 15, 17, 20, or any other molar ratio in between; c) cholesterol at molar ratio from 20 to 30; for example, it may be present at molar ratio of 20, 22, 25, 27, 30, or any other molar ratio in between; d) DOTAP at molar ratio from 15 to 30; for example, it may be present at molar ratio of 15, 17, 20, 25, 30, or any other molar ratio in between; e) ALC-0315 at a molar ratio from 10 to 20; for example, it may be present at molar ratio of 10, 12, 15, 17, 20, or any other molar ratio in between; f) DSPE-PEG2000 at a molar ratio from 5 to 10; for example, it may be present at molar ratio of 5, 7, 10 or any other molar ratio in between. As used herein, the molar ratio defines the molar proportion of different lipids in a lipid nanoparticle. It will be understood that, as the ratio of different lipids is selected, the amounts are chosen within the molar ratio range as indicated here above; also, when some of the lipids are added in a larger molar ratio (within their preferred molar ratio range), that, respectively, other lipids would then be present in a smaller molar ratio (within their preferred molar ratio range). That is, the molar ratios of all lipids in parts a) to f) as above, amount to a number 100 in a lipid nanoparticle as disclosed herein. In some embodiments, the lipid nanoparticle of the present invention, comprises neutral lipids DOPC and DOPE, cholesterol, cationic lipid DOTAP, ionizable lipid ALC-0315, and PEG- modified lipid DSPE-PEG2000, wherein the molar ratio of DOPC / DOPE / cholesterol / DOTAP / ALC-0315 / DSPE-PEG2000 is 20 / 10 / 30 / 20 / 15 / 5. In further embodiments, the lipid nanoparticle of the present invention, comprises neutral lipids DOPC and DOPE, cholesterol, cationic lipid DOTAP, ionizable lipid ALC-0315, and PEG-modified lipid DSPE-PEG2000, wherein the molar ratio of DOPC / DOPE / cholesterol / DOTAP / ALC- 0315 / DSPE-PEG2000 is 20 / 10 / 30 / 20 / 15 / 5. In further embodiments, the lipid nanoparticle of the present invention consists of DOPC / DOPE / cholesterol / DOTAP / ALC-0315 / DSPE-PEG2000 at a molar ratio of 20 / 10 / 30 / 20 / 15 / 5. In some embodiments, the lipid nanoparticle of the present invention is from about 50 nm to about 56 nm in diameter. For example, the lipid nanoparticle may be of 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, or 56 nm diameter. As further provided herein, the lipid nanoparticle of the present invention, may further comprise one or more nucleic acid, one or more protein, a complex of one or more nucleic acid and one or more protein, and / or one or more therapeutically active molecule; in such case it means that these substances are encapsulated into the lipid nanoparticle. The lipid composition as presently provided improves the encapsulation efficiency and enhances cellular entry. In some examples, when the lipid nanoparticle comprises the one or more nucleic acid, one or more protein, a complex of one or more nucleic acid and one or more protein, and / or one or more therapeutically active molecule, and the ratio of the one or more nucleic acid, the one or more protein, the complex of one or more nucleic acid and one or more protein, and / or the one or more therapeutically active molecule to the lipid nanoparticle is from about 1:6 to 1:20. As an example, the lipid nanoparticle is encapsulated with a protein and / or a nucleic acid, wherein the ratio is 1:12. As further disclosed herein, the lipid nanoparticle of the present invention comprises a Cas9:sgRNA (RNP) complex. In some embodiments, the ratio of Cas9:sgRNA (RNP) complex to the lipid nanoparticle is from 1:6 to 1:20. In some examples, the lipid nanoparticle comprises neutral lipids DOPC and DOPE, cholesterol, cationic lipid DOTAP, ionizable lipid ALC-0315, and PEG-modified lipid DSPE-PEG2000, wherein the molar ratio of DOPC / DOPE / cholesterol / DOTAP / ALC-0315 / DSPE-PEG2000 is 20 / 10 / 30 / 20 / 15 / 5, and is encapsulated with Cas9:sgRNA (RNP) complex. In further embodiments, in such lipid nanoparticle, the ratio of Cas9:sgRNA (RNP) complex to the lipid nanoparticle is from 1:12. Further disclosed herein is a pharmaceutical composition comprising the lipid nanoparticle of the present invention and a pharmaceutically acceptable carrier. The present disclosure further provides a method for delivering one or more nucleic acid, one or more protein, a complex of one or more nucleic acid and one or more protein, and / or one or more therapeutically active molecule to a cell comprising: a) contacting the cell with a lipid nanoparticle of the present invention, or b) contacting the cell with a pharmaceutical composition of the present invention. Further disclosed herein is a lipid nanoparticle for use in therapy and / or medical treatment. In some embodiments, a pharmaceutical composition, comprising the lipid nanoparticle of the present invention and a pharmaceutically acceptable carrier for use in therapy and / or medical treatment is provided. In a further aspect, a method for encapsulation of Cas9:sgRNA (RNP) complex into the lipid nanoparticle of the present invention, is provided, wherein the method comprises the steps: a) Sonicating of mixture of lipid nanoparticle / RNP complex and applying two cycles of freeze- thawing; b) Extruding lipid nanoparticle / RNP complex preparations through 200 nm polycarbonate membrane; c) Centrifugating lipid nanoparticle / RNP complex preparations using 300kDa centrifuge filter devices. As further disclosed herein, a method was developed to investigate the fusion mechanism between liposomal NPs and phospholipid membranes. Significant efforts have been given in the last couple of decades to understand the mechanistic role of lipid composition on the membrane fusion (Meher, 2019). Therefore, stable and biologically relevant phospholipid membrane models for liposomal NPs fusion studies as provided by the present disclosure are advantageous. Even though, vesicular and micellar lipid matrixes was extensively used, their utility is quite limited to study structural properties of the phospholipid bilayers. Therefore, the presently provided method uses solid supported tethered bilayer lipid membrane (tBLM) (McGillivray et.al., 2007, Budvytyte et.al., 2021) systems, which will mimic composition of neurons and other brain cells. tBLMs, in contrast to classical black lipid membranes (BLM), exhibit exceptional stability and may be made in the form of large (up to 3 inch diameter) flat surfaces suitable for high resolution structural measurements by neutron reflectometry (NR) (Budvytyte et.al., 2013). The plane geometry of the tBLMs allowed us to measure the efficiency of liposomal NPs fusion with lipid membranes and the structure and morphology of lipids in the membrane (by FRET-FLIM) and the structural changes of liposomal NPs (by DLS). Therefore, in yet further aspect, the present disclosure provides an advantageous method for analysis of fusion mechanism between lipid nanoparticles and phospholipid membranes comprising contacting lipid nanoparticles with artificial phospholipid membranes (tBLM), and analyzing lipid exchange between lipid nanoparticles and artificial phospholipid membranes, wherein: a) lipid nanoparticles are labeled with a first label, or b) lipid nanoparticles are labeled with a first label, and artificial phospholipid membrane (tBLM) is labeled with a second label. The labels that are used, should be compatible with use in such method setup. In some embodiments, the labels are selected from various reporters, for example, fluorescent dyes, such as Cy3 or Cy5. It is preferred that the first and second label are different, and it will be understood that as long as the first and second label signals can be distinguished (for example, are detected in different wave lengths), any such labels are suitable for use in the presently disclosed method. The presently disclosed method is advantageous in studying the CRISPR / Cas9 entry into the cytosol of live cells in vitro utilizing nontargeted / targeted membrane fusion between liposomal NPs and plasma membrane. EXAMPLES Materials: All phospholipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-Dioleoyl-3- trimethylammoniumpropane (DOTAP), cholesterol (Cho) and 6-((2-hexyldecanoyl)oxy)-N-(6- ((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 aminium (ALC-0315), were purchased from Avanti Polar Lipids (Alabaster, USA). All other chemicals were used as analytical grade (Sigma-Aldrich (St. Louis, MO). Ultrapure nuclease-free water (UPH2O) was used in all stages needed. Cas9 Protein, Cas9-GFP were purchased from Sigma-Aldrich (ST Louis, MO). Bovine Serum Albumin (BSA), Alexa Fluor™ 488 conjugate and sgRNA (TrueGuide, sgRNA Negative control, Invitrogen) were purchased from Thermo Fisher Scientific (Waltham, MA). Preparation of the liposomal NPs The liposomal NPs were prepared by the thin-film hydration method (Zhang., 2017). In detail, 10mM solutions of DOPC, DOPE, cholesterol, DSPE-PEG2000 and ACL-0315 were prepared in chloroform. A Mixture of 10mM of DOPC / DOPE / Chol / DOTAP / ALC0315 / DSPE-PEG2000 at different molar ratio in chloroform were prepared. The solvent was removed by evaporating 1 mL of the chloroform solution in a gentle stream of nitrogen followed by vacuum drying for 1h. The dried film was rehydrated with the PBS solution (PH7.4) to obtain the multilamellar liposomal formulation. The multilamellar vesicles were homogenized with a ultrasonicator for 60 min, and incubated with occasional vortexing, as needed, until the lipid film was no longer visible. The liposomes preparation was then extruded 21 times through a 100, 200 and 400 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL). The liposomal NPs sizes were determined by dynamic light scattering (DLS). Preparation of RNP complex and loading to liposomal NPs The Cas9:sgRNA (RNP) complex was prepared by mixing Cas9-GFP (350 nM) and sgRNA (1050 nM) with a molar ratio of 1:3 on ice for at least 20 min before use or to be stored at −80°C. Liposomal NPs / Cas9-GFP:sgRNA complexes were formed by electrostatic interactions between cationically charged liposomes and anionically charged RNPs. The optimal complex formation was of the cationic liposomal NPs and Cas9-GFP:sgRNA at molar ratio 12:1 (Lipids:RNP). For loading liposomal NPs experiments, an evaporated lipid films (with optimal composition) were resuspended in PBS containing RNP complex. The mixture was sonicated for 5 min followed by two cycles of freeze-thawing method. A single freeze-thaw cycle consisted of freezing for 5 min at -800C and thawing for 5 min in a water bath at 37oC. Then 5 min sonication step was repeated and liposomes were kept at room temperature for 10min. The liposomal NPs / RNP complex preparations were then extruded 21 times through a 200 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL). A loaded liposomal NPs with RNP complex was centrifuged at 3000 rpm for 10 min by using Microcon - 300kDa centrifuge filter device to isolate the unloaded RNP complex. The sizes of LNPs loaded RNPs were determined by dynamic light scattering (DLS). Encapsulation Efficiency of Liposomal NPs The encapsulation efficiency of RNP complex or BSA within liposomal NPs was measured using a centrifugal filter system. 500 μL of the liposome suspension was placed in an Microcon - 300kDa centrifuge filter device and centrifuged at 3000 rpm for 10 min. The encapsulation efficiency was calculated as the percentage of Cas9 protein incorporated into the liposome relative to the total amount of Cas9 used initially according to the following formula: Cas9-GFP sgRNA ribonucleoprotein delivery to mammalian cells via liposomal NPs HEK293T / 17 (RRID:CVCL 1926) cells (American Type Culture Collection, ATCC) were maintained in T25 flasks with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U / ml penicillin, 100 µg / ml streptomycin. The cell culture was maintained at 37 °C and 5% CO2. The cells were harvested using TrypLE™ (Thermo Fisher Scientific) solution. To evaluate the liposomal Cas9-GFP delivery, the cells were plated at a density of 250 cells / mm2onto 24-well plates (Thermo Fisher Scientific) that were pre-coated with poly-L-lysine (0.001% in PBS) for at least 20 min. at RT. The next day liposomal nanoparticles / Cas9-GFP and sgRNA complexes were delivered to HEK293T / 17 cells. First, we assessed the required Cas9 concentration (9-35 nM) in liposomes and time (3-24 h) required to reach the cell and its nucleus. After liposomal RNP addition cells were washed with PBS to eliminate the remaining liposomal NPs, fixed with 4% paraformaldehyde (PFA) in PBS for 20 min. at room temperature (RT). After rinsing with PBS, residual PFA was quenched with 30 mM glycine in PBS, and the cells were washed twice with PBS. All nuclei were stained with DAPI (1 μg / ml) for 10 min. at RT and washed three times. Then cells were investigated under the Olympus IX83 widefield microscope with 20x / 0.75NA air objective using DAPI and FITC fluorescence filter cubes. The images were analysed using the open-source CellProfiler software (RRID:SCR_007358). Cell nuclei were identified using DAPI channel and Identify primary objects function. The cell contour was reproduced by using Identify secondary objects function and identifying the cell cytoplasm compartment. The integrated intensity of GFP was measured in each cell cytoplasm and nucleus of an image (n = 4-6 images for each well, all conditions were repeated in triplicates). Then the percentage of cells with GFP fluorescence in cytoplasm or nucleus compartment was calculated. After this initial experiment three biological replicates were performed using optimal Cas9 concentration (15 nM) and incubation time (24 h). Also, the same experimental pipeline was performed in liposomal NPs stability experiment. Tethered Phospholipid Bilayer Membrane (tBLM) Formation Gold substrates for fluorescence microscopy measurements were prepared on mica. Gold layers 100 nm were deposited by the magnetron sputtering using a PVD75 (Kurt J. Lesker Co., Jefferson Hills, PA, USA) system. For the formation of the self-assembled monolayer, the Au-coated mica was immediately immersed in ethanolic solutions of mixtures of WC14 (20-tetradecyloxy- 3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol, was synthesized and described in McGillivray et.al., 2007) and ^ME (^-mercaptoethanol from Sigma-Aldrich, St. Louis, MO) (in molar ratio 30:70) and incubated at least 2.5 h for self- assembly. The details on the synthesis and properties of the self-assembled monolayers used in this work were described earlier (McGillivray et.al., 2007, Budvytyte et.al., 2013). The arrangement of tBLM were completed by multilamellar vesicle fusion method described in previous paper (Ragaliauskas et.al., 2017; Lithuanian patent: LT6424B). Fluorescence lifetime imaging microscopy (FLIM) FLIM was done with a Leica microsystems TCS SP8 confocal microscope with a 63°— / 1.4 oil immersion objective (pixel size 0.18 °— 0.18 μm). The fluorescence decay signal was measured over 505–550 nm range using 488 nm white light laser excitation line. The instrument response function (IRF) was measured by recording the laser reflection signal off a glass coverslip. The formation and characterization of various composition liposomal NPs for CRISPR / Cas9 delivery The particle size distribution of liposome formulations plays a crucial role in determining their efficacy. After applying the calcein release assay (Maherani, et.al., 2013) it was found that liposomal NPs containing additional cationic lipids (DOTAP) and ionizable lipids (ALC-0315, which ensured CRISPR / Cas9 encapsulation, and PEGylated lipids (DSPE-PEG2000), which improved the stability of liposomal NPs, stood out for their special stability, because the extent of calcein release was the lowest and did not change with time. To further increase the stability and loading efficiency, we optimized a variety of formulation parameters used for the assembly of these liposomal NPs, including the excipient phospholipid identity, the molar composition ratios of the six components of the liposomal NP formulation, and the lipid / Cas9 / sgRNA (BSA) weight ratio. BSA loading experiments were performed to optimize the liposomal formulations for increased encapsulation efficiency. The stability of lipid particle size experiments on the over time, have shown, that the size of lipid particles was different before and after the loading with BSA and was dependent on the lipid composition (Table 1). Table 1. LNPs sizes before and after BSA loading by DLS. The liposome preformulations and final formulations obtained within the scope of this study are in accordance with the general liposome characteristics specified in the literature. The optimized final formulation of liposomal NPs contains: DOPE / DOPC / Chol / DOTAP / ALC0315 / DSPE- PEG2000 at molar ratio 20 / 10 / 30 / 20 / 15 / 5 (Figure 1) average diameter was 53 ± 3nm, after BSA loading the diameter of liposomal NPs increased (Table 1). Monodispersity is an essential characteristic in the preparation of liposomes, as it affects the encapsulation efficiency and stability. The values of polydispersity index (PI) lower than 0.3 of liposomal dispersion can be considered homogeneous and monodisperse (Bahari, 2016). The PI of all liposomal NPs formulations produced in this study, was below 0.3. All of the formulations were monodispersed. The sizes of liposomal NPs were stable before and after loading with BSA over 5 days (Figure 2). The composition of liposomal NPs: DOPE / DOPC / Chol / DOTAP / ALC0315 / DSPE-PEG2000 (20 / 10 / 30 / 20 / 15 / 5) was optimal and was chosen for Cas9:sgRNA complex loading experiments. An empty liposomal NPs were in average in size 54 ± 4 nm. Whereas, after BSA loading, their size increased to 89 ± 10 nm, and after sgRNA / Cas9 complex loading even up to 99 ± 18 nm (Figure 3). Encapsulation Efficiency of Liposomal NPs Different formulations of liposomal NPs have been tested to further increase the encapsulation efficiency. The encapsulation efficiency was determined for each composition (Figure 4. Left). It was found that liposomal NPs containing cationic and ionizable lipids showed the highest encapsulation efficiency. LNPs with this optimal ratio of six components (DOPE / DOPC / Chol / DOTAP / ALC0315 / DSPE-PEG2000 (20 / 10 / 30 / 20 / 15 / 5) were selected for further studies. The efficiency of encapsulation of the sgRNA / Cas9 complex in such liposomal NPs reached about 60% (Figure 5). Delivery of liposomal NPs / Cas9-GFP:sgRNA complex to the cells Overall, the uptake efficiency of Cas9 RNPs varies depending on the cell type and the delivery method employed. Liposomal NPs containing Cas9-GFP:sgRNA was designed to allow for convenient visualization and localization of GFP by confocal microscopy The Cas9-GFP concentration (9-35 nM) and incubation time (3-24 h) influence the delivery of liposomal NPs / Cas9-GFP:sgRNA complexes to HEK293T / 17 cells (Figure 5). Hereby, a high level (more than 60%) of cellular uptake of into HEK293T / 17 was determined for the liposomal nanoparticles / Cas9-GFP:sgRNA complex (12:1) at 15 nM and 35nM (more than 70% ) after 24h of incubation according to the quantitative analysis of cellular uptake studies (Figure 5 Left). The percentage of cells containing Cas9-GFP in the nucleus was more than 50% at 15 nM and 35nM after 24h of incubation (Figure 5 Right). The stability of liposomal NPs for the delivery of Cas9-GFP:sgRNA complexes to HEK293T / 17 cells was evaluated (Figure 6). The liposomal NPs containing Cas9-GFP:sgRNA were stored for 2 and 7 days before the delivery to the cells. The percentage of cells containing Cas9-GFP inside the cytoplasm was about 80% (Figure 6 Left) and 70% in the nucleus (Figure 6 Right) and did not change over 7 days. Cells were treated with 15 nM of Cas9-GFP:sgRNA complexes for 24 h. Liposomal NPs / Cas9-GFP:sgRNA crossing blood-brain barrier The blood−brain barrier (BBB) is considered as the most challenging barrier in brain drug delivery. BBB is one of the main reasons why the delivery of therapeutic molecules inside the brain is so complex. The tight junctions of the endothelial cells make almost impossible of delivering drugs into the brain if they are not functionalized or if they are not encapsulated inside other nanostructures (Amiri et.al., 2021). The development of drug delivery approaches that overcome BBB restrictions to achieve drug efficiency is in high demand. We were able to show that our developed of liposomal NPs nanostructures can penetrate to brain endothelial cells and deliver a Cas9-GFP: sgRNA complex (Figure 7) into the cells. Cells were treated with liposomal NPs containing 22.7 nM of Cas9-GFP:sgRNA complex for 24 or 48 h. The percentage of brain endothelial cells containing Cas9-GFP inside the cytoplasm (Figure 7 Left) or in the nucleus (Figure 7 Right) was more than 40% after 48h of incubation. This means that liposomal NPs described herein are suitable for crossing the blood-brain barrier and can be used for delivery of therapeutic molecules, or other compounds or complexes, such as CRISPR / Cas complexes, into the brain. The efficiency of liposomal NPs fusion with phospholipid membranes The fusion mechanism of LNPs with tBLMs was examined by fluorescence microscopy and FLIM. To assess whether lipid composition has a synergistic or opposing effect on membrane fusion, we monitored the efficiency of liposomal NPs fusion with artificial lipid membranes by changing the lipid composition of the membranes. In order to evaluate the efficiency of membrane fusion, we followed the fusion / lipid exchange between artificial membranes (tBLM) and liposomal NPs of various compositions by fluorescence microscopy. Using labelled liposomal NPs with Cys5 (DOPE / Chol / DOTAP / ALC0315 / DSPE-PEG2000 (30 / 30 / 20 / 15 / 5), it was found that complete fusion of vesicles with the phospholipid membrane occurs after 10 min (Figure 8). By applying FLIM the lateral lipid distribution and changes in molecular conformation after fusion events were determined. In these experiments donor is phospholipid membrane (tBLM) labeled with Cys3 and Acceptor is liposomal NPs labelled with Cy5 (Figure 9). By keeping the number of donor molecules in the membrane constant, and by having the same amount of acceptor molecules in liposomal NPs in each experiment, we compared the fractions of interacting molecules after liposomal NPs fuse with phospholipid membrane. So, if more acceptor molecules are transferred from the liposomal NPs to the phospholipid membrane, the parameter ^ will be larger, this allows us to quantify the fusion of liposomal NPs with lipid membranes (Figure 9). Moreover, FLIM also gives information about the spatial distribution of molecular interactions – we can see whether acceptors are transferred evenly or heterogeneously. REFERENCES Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science, 2004;303:1818e22. Amiri M, Jafari S, Kurd M, Mohamadpour H, Khayati M, Ghobadinezhad F, Tavallaei O, Derakhshankhah H, Sadegh Malvajerd S, Izadi Z. Engineered Solid Lipid Nanoparticles and Nanostructured Lipid Carriers as New Generations of Blood-Brain Barrier Transmitters. ACS Chem Neurosci. 2021;12(24):4475-4490. Bahari ASL, Hamishehkar H. The Impact of Variables on Particle Size of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers; A Comparative Literature Review. Adv Pharm Bull. 2016;6(2):143-51. Budvytyte R., Valincius G, Niaura G., Voiciuk V, Stauffer H., Shekhar P., Heinrich F., Shenoy S., Vanderah D.J., and Lösche M. Anchor Molecules Affect Structure and Properties of Tethered Bilayer Lipid Membranes. Langmuir, 2013;29(27):8645–8656. Budvytyte, R., Milasiute, A., Vitkus, D., Strupas, K., Gulla, A., Sakinyte I., Razumiene, J. Tethered Lipid Membranes as a Nanoscale Arrangement towards Non-Invasive Analysis of Acute Pancreatitis. Biomedicines 2021;9:755. Cheng, Q., Wei, T., Farbiak, L. et al. Selective organ targeting (SORT) nanoparticles for tissue- specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol., 2020;15:313–320. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 2014;346:1258096. Huang J, Zhou Y, Li J, Lu A, Liang C. CRISPR / Cas systems: Delivery and application in gene therapy. Front Bioeng Biotechnol. 2022;10:942325. Eoh J , Gu L . Biomaterials as vectors for the delivery of CRISPR-Cas9. Biomater Sci., 2019;7(4):1240-1261. doi: 10.1039 / c8bm01310a. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. PNAS, 2012;109(39):2579- 86. Glass Z., M. Lee, Y. Li and Q. Xu, Engineering the Delivery System for CRISPR-Based Genome Editing, Trends Biotechnol., 2018, 36, 173–185. McGillivray DJ, Valincius G, Vanderah DJ, Febo-Ayala W, Woodward JT, Heinrich F, Kasianowicz JJ, Lösche M. Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes. Biointerphases, 2007;2:21-32. Maherani B, Arab-Tehrany E, Kheirolomoom A, Geny D, Linder M. Calcein release behavior from liposomal bilayer; influence of physicochemical / mechanical / structural properties of lipids. Biochimie. 2013;95(11):2018-33. Meher G, Chakraborty H. Membrane Composition Modulates Fusion by Altering Membrane Properties and Fusion Peptide Structure. J Membr Biol. 2019;252(4-5):261-272. Mitchell, M.J., Billingsley, M.M., Haley, R.M. et al. Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov., 2021;20:101–124. Sercombe, L. et al. Advances and challenges of liposome assisted drug delivery. Front. Pharmacol., 2015;6:286. Sinclair, F., Begum, AA., Dai, CC. et al. Recent advances in the delivery and applications of nonviral CRISPR / Cas9 gene editing. Drug Deliv. and Transl. Res. 2023;13:1500–1519. Ragaliauskas, T.; Mickevicius, M.; Rakovska, B.; Penkauskas, T.; Vanderah, D.J.; Heinrich, F.; Valincius, G. Fast Formation of Low-Defect-Density Tethered Bilayers by Fusion of Multilamellar Vesicles. BBA Biomembr. 2017, 1859: 669–678. Witzigmann D, Kulkarni JA, Leung J, Chen S, Cullis PR, van der Meel R. Lipid nanoparticle technology for therapeutic gene regulation in the liver. Adv Drug Deliv Rev 2020;159:344e63. Xu, C.L., Ruan, M. Z. C., Mahajan, V. B., and Tsang, S. H. (2019). Viral delivery systems for CRISPR. Viruses, 2019; 11:28. doi: 10.3390 / v11010028 Yip, B.H. Recent advances in CRISPR / Cas9 delivery strategies. Biomol. Ther.2020: 10:839. doi: 10.3390 / biom10060839 Zinger A., Caroline Cvetkovic, Manuela Sushnitha, Tomoyuki Naoi, Gherardo Baudo,Morgan Anderson, Arya Shetty, Nupur Basu, Jennifer Covello, Ennio Tasciotti,Moran Amit, Tongxin Xie, Francesca Taraballi, and Robert Krenci. Humanized Biomimetic Nanovesicles for Neuron Targeting. Advance Science., 2021;8:2101437. Zhang H. Liposomes: Methods and Protocols , G. G. M. D'Souza Springer New York, New York, NY, 2017, 17–22

Claims

CLAIMS:

1. A lipid nanoparticle comprising: a) one or more neutral lipid, b) one or more sterol, c) one or more cationic lipid, d) one or more ionizable lipid, and e) one or more PEG-modified lipid.

2. The lipid nanoparticle according to claim 1, wherein the lipids are selected from: a) the one or more neutral lipid is selected from: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-3 -phosphoethanolamine (POPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE) (DPhPC); b) the sterol is cholesterol, c) the one or more cationic lipid is selected from: 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC); d) the one or more ionizable lipid is selected from: 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1 aminium (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), Di((Z)-Non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (LIPID L319); e) the one or more PEG-modified lipid is selected from: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)- 2000] (PEG-DSPE),1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG), PEG-modified phosphatidylethanolamine, a PEG-modified ceramide, a PEG-modified diacylglycerol.

3. The lipid nanoparticle according to claim 1 or 2, wherein the one or more neutral lipid is 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) and / or 1,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).

4. The lipid nanoparticle according to any one of claims 1 to 3, wherein the one or more sterol is cholesterol.

5. The lipid nanoparticle according to any one of the preceding claims, wherein the one or more cationic lipid is 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP).

6. The lipid nanoparticle according to any one of the preceding claims, wherein the one or more ionizable lipid is 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1 aminium (ALC-0315).

7. The lipid nanoparticle according to any one of the preceding claims, wherein the one or more PEG-modified lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG2000).

8. The lipid nanoparticle according to any one of the preceding claims, comprising neutral lipids DOPC and DOPE, cholesterol, cationic lipid DOTAP, ionizable lipid ALC-0315, and PEG- modified lipid DSPE-PEG2000.

9. The lipid nanoparticle according to claim 8, comprising the lipids at the following molar ratio: a) DOPC at molar ratio from 15 to 30; b) DOPE at molar ratio from 10 to 20; c) cholesterol at molar ratio from 20 to 30; d) DOTAP at molar ratio from 15 to 30; e) ALC-0315 at a molar ratio from 10 to 20; f) DSPE-PEG2000 at a molar ratio from 5 to 10.

10. The lipid nanoparticle according to claim 9, wherein the molar ratio of DOPC / DOPE / cholesterol / DOTAP / ALC-0315 / DSPE-PEG2000 is 20 / 10 / 30 / 20 / 15 / 5.

11. The lipid nanoparticle according to claim 10, consisting of DOPC / DOPE / cholesterol / DOTAP / ALC-0315 / DSPE-PEG2000 at a molar ratio of 20 / 10 / 30 / 20 / 15 / 5.

12. The lipid nanoparticle according to any one of the preceding claims, wherein the lipid nanoparticle is from about 50 nm to about 56 nm in diameter.

13. The lipid nanoparticle according to any one of the preceding claims, further comprising one or more nucleic acid, one or more protein, a complex of one or more nucleic acid and one or more protein, and / or one or more therapeutically active molecule.

14. The lipid nanoparticle according to claim 13, wherein the ratio of the one or more nucleic acid, the one or more protein, the complex of one or more nucleic acid and one or more protein, and / or the one or more therapeutically active molecule to the lipid nanoparticle is from 1:6 to 1:

20.

15. The lipid nanoparticle according to claim 13 or 14, wherein the lipid nanoparticle comprises Cas9:sgRNA (RNP) complex.

16. The lipid nanoparticle according to claim 15, wherein the ratio of Cas9:sgRNA (RNP) complex to lipid nanoparticle is from 1:6 to 1:

20.

17. A pharmaceutical composition comprising a lipid nanoparticle according to any one of the preceding claims and a pharmaceutically acceptable carrier.

18. A method for delivering one or more nucleic acid, one or more protein, a complex of one or more nucleic acid and one or more protein, and / or one or more therapeutically active molecule to a cell comprising: c) contacting the cell with a lipid nanoparticle according to any one of claims 13 to 16, or d) contacting the cell with a pharmaceutical composition according to claim 17.

19. A lipid nanoparticle according to any one of claims 1 to 16 for use in therapy and / or medical treatment.

20. A pharmaceutical composition according to claim 17 for use in therapy and / or medical treatment.

21. A method for encapsulation of Cas9:sgRNA (RNP) complex into the lipid nanoparticle according to any one of claims 1 to 12, comprising the steps: a) Sonicating of mixture of lipid nanoparticle / RNP complex and applying two cycles of freeze-thawing; b) Extruding lipid nanoparticle / RNP complex preparations through 200 nm polycarbonate membrane; c) Centrifugating lipid nanoparticle / RNP complex preparations using 300kDa centrifuge filter devices.

22. A method for analysis of fusion mechanism between lipid nanoparticles and phospholipid membranes comprising contacting lipid nanoparticles with artificial phospholipidmembranes (tBLM), and analyzing lipid exchange between lipid nanoparticles and artificial phospholipid membranes, wherein a) lipid nanoparticles are labeled with a first label, or b) lipid nanoparticles are labeled with a first label, and artificial phospholipid membrane (tBLM) is labeled with a second label.

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