Lipid-polymer hybrid nanoplexs and method of synthesis

Abstract

The present invention provides lipid-polymer hybrid nanoplexes for delivery of negatively charged biomolecular payloads, and to method of preparing these nanoplexes. The disclosed hybrid delivery system exhibits enhanced cellular uptake, stability, and transfection efficiency.

Description

DescriptionTitle of Invention: LIPID-POLYMER HYBRID NANOPLEXS AND METHOD OF SYNTHESISFIELD OF THE INVENTION

[0001] The present invention relates to lipid-polymer hybrid nanoplexes for non-viral delivery of negatively charged biomolecular payloads, including nucleic acids and / or ribonucleoproteins and to method for synthesizing and formulating the lipid-polymer hybrid nanoplexes. The disclosed hybrid delivery system exhibits enhanced cellular uptake, stability, and transfection efficiency.BACKGROUND OF THE INVENTION

[0002] The CRISPR / Cas system has emerged as a transformative genome-editing tool, offering precision and efficiency in modifying genetic sequences. Comprising Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the Cas9 endonuclease, derived from Streptococcus pyogenes (SpCas9 this system enables targeted double-strand breaks (DSBs), which are repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). The system has been widely explored for gene therapy, disease modeling, and functional genomics, with several ongoing clinical trials evaluating its therapeutic potential.

[0003] The emergence of genetically evolved nuclease-null deactivated or dead Cas protein (dCas) shows outstanding epigenome editing efficiency. Its DNA binding ability, sitespecific at primer (1 nt) level and chromatin rearrangement properties have made it an effective epi-effector (EE). EEs are epigenetic modifying proteins or enzymes conjugated with dCas9 protein to form a dCas9-EE complex. This complex can be activated by coupling with a functional, single guide RNA (sgRNA). This entire functional conjugate can be examined and translated for epigenetic modification. Depending on the mechanism of action and nature, EEs are categorized as epigenetic modifiers (EM) and transcription modifiers (TM). EMs such as p300, TET1, LSD1 and DNMT3 are enzymes that promote the alteration of epigenetic markers. Whereas TMs are transcriptional factors that can act as either activator (Herpes simplex viral protein VP 16, VP48, VP64, VP 120 and transactivator domain of NF-KB- p65 (nuclear factor kappa B) or repressor (Kriippel associated box, KRAB), SID4x, WRPW,chromo shadow (CS), etc., Eventually, using these insights, a few research groups explored the epigenetic efficacy of dCas9-EE complexes such as dCas9-DNMT3 A for DNA methylation, dCas9-TETl for DNA demethylation, dCas9-p300, dCas9-LSDl and combination of three effectors likes dCas9-VPR (VP64, p65 and Rta) and DNMT3ACD-DNMT3L-KRAB etc.

[0004] Several transcriptional activators have been utilized in CRISPR / dCas9 based epigenome editing to achieve robust and controllable gene activation. Chavez et al. demonstrated that dCas9-VP64 could moderately activate endogenous genes in human cells, while the tripartite activator dCas9-VPR (VP64-p65-Rta) produced substantially stronger upregulation of silent genes. As reported by Tanenbaum et al. the SunTag- VP64 system, which recruits multiple copies of VP64 via a peptide array, has also been shown to amplify transcription at target loci. In addition, Thakore PI et al., demonstrated that epigenetic activators such as dCas9-p300, a histone acetyltransferase fusion, can increase transcription by locally acetylating histones at promoters or enhancers, providing a more physiologically relevant activation without altering DNA sequences. Further, the CRISPR-SAM system combines dCas9-VP64 with MS2-tagged gRNAs and MS2-p65-HSF 1 to achieve synergistic, robust activation of even silent genes, as shown by Konermann et al.

[0005] So far, three types of adaptive delivery modes of CRISPR such as plasmid, mRNAthat encodes gRNA and Cas protein, and ribonucleoprotein complex (gRNA and Cas protein complex) have been explored as effective genome / epigenome editing approaches. These delivery modes provide their own advantages and disadvantages in their efficacy, specificity, cost, and off-target effects. Plasmid delivery in CRISPR which encodes both Cas protein and sgRNA in the same vector is one of the simple and commonly used methods. Thus, multiple transfections can be omitted. Still, plasmid delivery exhibits few shortcomings as it is time consuming and needs to be delivered specifically into the nucleus of the cells. Another approach is to deliver sgRNA and Cas9 mRNA separately, where Cas9 mRNA translates to the protein known as Cas9. This Cas9 protein conjugates / complexes with sgRNA to form the functional RNPs. This mode of delivery reduces off-target / non-specific binding and needs to be delivered only to the cytoplasm.

[0006] The other challenges hindering CRISPR / Cas9 -based genome editing is the effective and safe delivery of CRISPR components into target cells. The large molecular size, negative charge, hydrophilicity, and susceptibility to extracellular degradation pose significant barriers to efficient delivery. Conventional viral vector-based delivery systems, such as adeno-associated viruses (AAVs), have been explored to address these challenges. However, these systems and methods suffer from limitations including limited payload capacity (~4.7 kb), immunogenicity, risk of insertional mutagenesis, and reduced targeting flexibility, making them less ideal for clinical applications.

[0007] Several studies have demonstrated the feasibility of non-viral CRISPR / Cas based RNP delivery using biodegradable polymeric, lipid-based nanoparticles achieving efficient gene editing across ocular, muscular, hepatic, and neural tissues in vivo. Chen et al. reported the development of a biodegradable nanocapsule system encapsulating Cas9 ribonucleoproteins, with an average hydrodynamic diameter of approximately 25 nm. Upon local administration, this nanocarrier demonstrated efficient in vivo gene-editing activity in murine retinal pigment epithelial tissue as well as skeletal muscle. Polymeric nanocarriers offer significant versatility due to their capacity for structural and chemical modifications, allowing precise tuning for efficient gene delivery. Lu et al. demonstrated that micelles synthesized from polymers containing imidazole rings could promote endosomal escape, thereby enhancing intracellular cargo release. In a separate study, Kim et al. investigated the delivery of CRISPR / Cas components using polyethylene glycol monomethyl ether (mPEG) conjugated with chitosan, reporting that low molecular weight PEGylated chitosan achieved optimal transfection at an N / P ratio of 20, whereas medium molecular weight PEGylated chitosan showed maximal efficiency at an N / P ratio of 5. Additionally, Liu et al. developed cationic lipid-assisted polymeric nanoparticles (CLANs) based on PEG-b-PLGA, which successfully delivered CRISPR / Cas9 plasmids to target cells, effectively disrupting the BCR-ABL fusion gene in a chronic myeloid leukemia mouse model and improving survival outcomes.

[0008] Despite these advancements, current non-viral delivery strategies still face challenges such as limited endosomal escape, inefficient nuclear transport, and batch-to-batch variability in synthesis. This invention aims to overcome these challenges bydeveloping an advanced delivery system that enhances stability, biocompatibility, and cellular uptake of CRISPR components, ensuring high transfection efficiency with minimal cytotoxicity. Furthermore, the system can be adapted for the delivery of additional genetic cargo, including mRNA, siRNA, therapeutic peptides, and ribonucleoproteins (RNPs), expanding its potential applications in gene therapy, regenerative medicine, and infectious disease treatments.

[0009] By improving the efficiency, safety, and specificity of gene delivery, this invention provides a scalable and clinically viable approach to overcome the current limitations in genome and epigenome editing and therapeutic interventions.SUMMARY OF THE INVENTION

[0010] The present invention provides nanoscale lipid-polymer hybrid nanoplexes that utilize:• Electrostatic complexation between a biodegradable cationic polymer and a negatively charged payload including nucleic-acid and / or riboucleproteins • A lipid component that improves nanoplex formation, structural stability, and end use performance such as cellular uptake.

[0011] A lipid-polymer hybrid nanoplex comprising: (i) a biodegradable cationic polymer, preferably a cationic PEG-polycarbonate-based copolymer; (ii) a lipid, preferably selected from cholesterol, P-sitosterol, Precirol® ATO 5, GMS, Capmul GMO-50 EP, and Compritol® 888 ATO; and (iii) a nucleic-acid and / or ribonucleoprotein containing payload electrostatically complexed to the lipid polymer hybrid nanoplexes through cationic polymer.

[0012] In one aspect, the invention provides a lipid- polymer hybrid nanoplexes particularly suitable for delivering a nucleic-acid containing payload including but not limited to nucleic acid (DNA / RNA, including plasmid DNA, mRNA, miRNA / siRNA, RNP), a gene-editing component (including CRISPR / Cas tools in forms including pDNA encoding Cas or dCas and gRNA, mRNA encoding for Cas or dCas protein and sgRNA and / or RNPs (Cas or dCas protein complexed with gRNA) and a epigenome editing component (including CRISPR / dCas modified with epi-effectors)

[0013] In the present invention, lipid-polymer hybrid nanoplexes are prepared by first synthesizing a cyclic carbonate monomer, followed by preparing an amphiphilic block copolymer backbone, thereafter, converting the backbone into a cationic copolymer, and formulating the cationic polymer into lipid-polymer hybrid nanoplexes using a double emulsion solvent evaporation process followed by electrostatic complexation with nucleic-acid containing payload. In this embodiment a method of preparing a lipid-polymer hybrid nanoplex, the method comprising:a) forming an organic phase comprising a biodegradable cationic polymer and lipids in an organic solvent;b) adding a first aqueous phase to the organic phase and emulsifying to form a primary emulsion;c) adding the primary emulsion to a second aqueous phase and emulsifying to form a secondary emulsion;d) removing the organic solvent to obtain blank nanoplexes;e) contacting the blank lipid-polymer hybrid nanoplexes with a negatively charged biomolecular payloads to form the lipid-polymer hybrid nanoplex by electrostatic complexation.

[0014] In embodiments, the nanoplexes are prepared by forming lipid-polymer hybrid particles using an emulsion-based solvent evaporation method and thereafter complexing the nucleic-acid containing payload via electrostatic interaction with the lipid-polymer hybrid nanoplexes through cationic polymer. This approach reduces the need to expose fragile nucleic-acid containing payloads to harsh chemical conditions, thereby preserving integrity, and improving functional delivery.OBJECT OF THE INVENTION

[0015] The primary object of the present invention is to provide an efficient, biocompatible non-viral delivery system for delivering genome editing and epigenome editing tools, including CRISPR / Cas components, and the other nucleic acid component to overcome limitations associated with conventional viral, and non-viral delivery approaches.

[0016] Another object of the invention is to provide a lipid-polymer hybrid nanoplexes to complex, protect, and deliver nucleic-acid containing payloads through electrostatic interactions, thereby avoiding harsh chemical reactions and maintaining the stability and enhanced bioactivity of the payload.

[0017] A further object of the invention is to provide a lipid-polymer hybrid nanoplexes capable for delivering CRISPR / Cas payloads such as CRISPR / Cas expressing plasmids, ribonucleoproteins (RNPs) complexes, and mRNA encoding for Cas or dCas protein or dCas modified with epi-effectorsand sgRNA, with enhanced transfection efficiency and minimal cytotoxicity.

[0018] Yet another object of the present invention is to provide a scalable, cost-effective, and clinically adaptable nanoplexes that can be customized for various therapeutic applications, including gene therapy, targeted drug delivery, and regenerative medicine.

[0019] It is also an object of the present invention to enable effective gene-editing and epigenome editing applications ensuring precise localization and effective genome modifications in specific tissues or cells.

[0020] A further obj ect of the invention is to provide a process for synthesis including a double emulsion solvent evaporation approach for preparing blank nanoplexes and lipid- polymer hybrid nanoplexes, followed by electrostatic complexation with a negatively charged biomolecular payloads.

[0021] It is also an object of the invention to provide a process for syntheses of lipid-polymer hybrid nanoplexes with improved transfection efficiency without compromising efficacy and toxicity, thereby reducing dosing frequency.DETAILED DESCRIPTION OF THE ACCOMPANIED DIAGRAMS

[0022] The above and other aspects of the present invention will be more apparent and better understood when considered in conjunction with the following detailed descriptionand accompanying drawings in which like characters represent like parts throughout the drawings, wherein:Figure 1. Schematic representation of the synthesis of mPEG-Z>-p(CB-{g-cationic chain})Figure 2. Characterization of cationic polymer (PLM-35) using NMR and GPC. 1H NMR of a) 2-methyl-2-benzyloxycarbonyl propylene carbonate (MBC) b) mPEG-b-p(CB), c) mPEG-b-p(CB-{g-COOH}) and d) mPEG-b-p(CB-{g-Cationic chain}), e) Gel permeation chromatograph of mPEG-b-p(CB-{g-cationic chain}).Figure 3. Optimization of particles size and zeta potential of blank cationic nanoplexes and blank cationic lipid-polymer hybrid nanoplexes. a and b) Particles size distribution curves and zeta potential curve of cationic nanoplexes fabricated using four different cationic polymers (PLM-84, PLM-65, PLM-53 and PLM-35). c and d) Particles size distribution curve and zeta potential curve of cationic lipid-polymer hybrid nanoplexes (BNPX-1, BNPX-2, BNPX-3, BNPX-4) prepared using only cationic polymer (PLM-35) and varying amounts of cholesterol (viz. 0, 1, 2.5 and 5 mg), e and f) Particles size distribution curve and zeta potential curve of cationic lipid-polymer hybrid nanoplexes (BNPX-3, BNPX-CA, BNPX-GM, BNPX-SI, BNPX-PR, BNPX-CO) prepared using only cationic polymer (PLM-35) and different lipids (2.5 mg).Figure 4. Gel retardation assay of (a) pcDNA3-EGFP plasmid complexed with BNPX-1, BNPX-2, BNPX-3, and BNPX-4 lipid-polymer hybrid nanoplexes where the lipid-polymer hybrid nanoplexes were fabricated using a constant amount of the cationic polymer PLM-35 (15 mg) with varying cholesterol contents (0 mg, 1 mg, 2.5 mg and 5 mg), (b) CRISPRi plasmid complexed with BNPX-3 lipid-polymer hybrid nanoplexes. The N / P ratio signifies the ratio of blank LPH nanoplexes to plasmid. Figure 5. Agarose gel electrophoresis assay showing the complexation efficiency of dCas9-VP64 RNP formed by complexation of PEDF -targeting SERPINF1 specific sgRNA (PEDF sgRNA) with dCas9-VP64 proteins. dCas9-VP64 RNP complexes formed at varying sgRNA-to-dcas9-VP64 protein molar ratios. The extent of sgRNA band disappearance in the gel was used to evaluate RNP complex formation and complexation efficiency.Figure 6. a and b) Particle size distribution and zeta potential curves of BNPX-3 before and after complexation with the pcDNA3-EGFP plasmid, c and d) Particle size distribution and zeta potential curves of BNPX-3 before and after complexation withthe CRISPRi plasmid. SEM morphology of BNPX-3 e) before and f) after complexation with the CRISPRi plasmid. BNPX-3, prepared using the cationic polymer PLM-35 (15 mg) and cholesterol (2.5 mg).Figure 7. a and b) Particle size distribution and zeta potential curves of BNPX-3 before and after complexation with the dcas9-VP64 RNP. SEM morphology of BNPX-3 c) before and d) after complexation with the dcas9-VP64 RNP. BNPX-3, prepared using the cationic polymer PLM-35 (15 mg) and cholesterol (2.5 mg).Figure 8. Heparin competition assay of a) pcDNA3-EGFP and b) CRISPRi plasmids from BNPX-3, prepared using the cationic polymer PLM-35 (15 mg) and cholesterol (2.5 mg).Figure 9. Cytocompatibility study performed on a) Human Embryonic Kidney (HEK 293) cells and b) Statens Seruminstitut Rabbit Cornea (SIRC) cells c) Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells using blank cationic lipid-polymer hybrid nanoplexes (BNPX-3).Figure 10. In vitro transfection of pcDNA3-EGFP and CRISPRi plasmid in HEK 293 cells, a and c) Qualitative and b and d) Quantitative uptake, respectively, using lipofectamine 3000 as control, pcDNA3-EGFP BNPX-1 and CRISPRi BNPX-1 nanoplexes were prepared using the cationic polymer PLM-35 alone (BNPX-1) and subsequently complexed with the respective plasmids. pcDNA3-EGFP BNPX-3 and CRISPRi BNPX-3 nanoplexes were formulated as lipid-polymer hybrid systems (BNPX-3) using PLM-35 (15 mg) and cholesterol (2.5 mg) and subsequently complexed with the respective plasmids.Figure 11. In vitro transfection of pcDNA3-EGFP and CRISPRi plasmid in SIRC cells, a and c) Qualitative and b and d) Quantitative uptake, respectively, using lipofectamine 3000 as control, pcDNA3-EGFP BNPX-1 and CRISPRi BNPX-1 nanoplexes were prepared using the cationic polymer PLM-35 alone (BNPX-1) and subsequently complexed with the respective plasmids. pcDNA3-EGFP BNPX-3 and CRISPRi BNPX-3 nanoplexes were formulated as lipid-polymer hybrid systems (BNPX-3) using PLM-35 (15 mg) and cholesterol (2.5 mg) and subsequently complexed with the respective plasmids.Figure 12. Endocytic uptake pathway mechanism of CRISPRi BNPX-3 nanoplexes, formulated using PLM-35 (15 mg) and cholesterol (2.5 mg) and subsequentlycomplexed with CRISPRi plasmids, (a) Qualitative and (b) quantitative uptake in HEK-293 cells, and (c) qualitative and (d) quantitative uptake in SIRC cells.Figure 13. In vitro transfection of CRISPR / dCas9 (PEDF sgRNA / dcas9-VP64 RNP) based RNPs on both HEK 293 cells and ARPE-19 cells, a) Qualitative and b) and c) Quantitative uptake on HEK 293 cells; and d) Qualitative and e) and f) Quantitative uptake on ARPE-19 cells; Lipofectamine™ CRISPRMAX™ using as control, BNPX- 3 nanoplexes prepared by 15 mg of cationic polymer (PLM-35) and 2.5 mg of Cholesterol; lipid-polymer hybrid nanoplexes; ns: not significant; ****p < 0.0001.Figure 14. Hematoxylin and eosin (H&E)-stained paraffin-embedded retinal crosssections of Wistar rats following treatment with cationic blank lipid-polymer hybrid nanoplexes (BNPX-3) at different concentrations after 7 days. Representative micrographs show preserved retinal architecture with a normal appearance of the retinal pigment epithelium (RPE) and no observable histopathological alterations compared to control.Figure 15. (a) Transverse and (b) circumferential sections of retinal tissue showing the time-dependent distribution of BNPX-3 -dCas9-VP64 RNPs. Naked FITC-tagged dCas9-VP64 RNPs were used as a control. BNPX-3 nanoplexes were formulated using 15 mg of cationic polymer (PLM-35) and 2.5 mg of cholesterol to form lipid-polymer hybrid nanoplexes.DESCRIPTION OF THE INVENTION

[0023] The following description is provided for the purpose of illustration and explanation of the invention and is not intended to limit the scope of the invention. The invention may be practiced in various embodiments, and functionalization and variations may be made without departing from the scope of the invention as defined by the claims.

[0024] The use of the terms “comprising,” “including,” “having,” or “containing” in this specification denotes the presence of stated features and does not preclude the presence of additional features. The embodiments described herein are illustrative in nature and are not intended to limit the invention to the specific forms disclosed.

[0025] Features described in relation to one embodiment may be combined with features of other embodiments, and the omission of a particular feature in any embodiment should not be construed as limiting or excluding such features unless expressly stated.

[0026] As used herein, the term “Nanoplex” refers to a nanoscale complex comprising at least one carrier component and at least one cargo component, wherein association is attained by electrostatic interaction, encapsulation, adsorption, covalent linkage, or combinations thereof. Carrier component comprises a polymeric component and a lipid component arranged as a hybrid nanoscale structure configured to associate with the cargo component.

[0027] As used herein, the term “Hybrid” refers to the presence of both polymer and lipid elements forming a single delivery structure.

[0028] As used herein, the term Payload (or “cargo”) means a negatively charged bioactive nucleic acid containing component including (i) a nucleic acid (ii) a gene-editing component (iii) a epigenome editing component.

[0029] The present invention provides to a non-viral delivery platform, particularly a lipidpolymer hybrid nanoplexes for the efficient delivery of negatively charged biomolecular payloads. The present invention also discusses the process of synthesizing these nanoplexes, which exhibit enhanced cellular uptake, stability, and transfection efficiency.

[0030] In the main embodiment, the present invention provides a lipid-polymer hybrid nanoplexes for non-viral delivery of anionic biomolecular components. Each nanoplex comprises (i) a biodegradable cationic polymer, (ii) a lipid, wherein the polymer and lipid cooperatively form a hybrid nanoscale complex that improves cellular uptake, colloidal stability, and functional delivery performance including transfection efficiency and (iii) a negatively charged biomolecular payload electrostatically complexed to the cationic polymer of the lipid-polymer hybrid nanoplex.

[0031] In this embodiment, the biodegradable cationic polymer comprises a cationic polymer selected from cationic polymers preferably cationic PEG-polycarbonate-based copolymers.

[0032] In preferred embodiments, the polymer is an amphiphilic copolymer having a poly(ethylene glycol) (PEG) segment, and a polycarbonate segment bearing functional groups coupled with cationic chains. In an exemplary and preferred embodiment, the polymer comprises mPEG-b-P(CB-{g-cationic chain}), where CB denotes polycarbonate repeating units derived from a cyclic carbonate monomer, and the cationic chain is covalently attached to pendant carboxyl groups along the backbone. The mPEG-b-P(CB-{g-cationic chain}) comprises a cationic chain having primary, secondary and / or tertiary amine groups. In one embodiment, the cationic chain comprises N,N-dimethyldipropylenetriamine coupled via carbodiimide chemistry (for example, EDC / HOBt coupling).

[0033] The polymer component is configured to carry a net positive charge under formulation conditions, thereby enabling formation of nanoplexes via electrostatic association with one or more anionic payloads. In certain embodiments, the polymer component is provided as a set of polymer variants differing in molecular weight, including by way of example polymer variants designated PLM-84, PLM-65, PLM-53, and PLM-35 (or equivalents), such that molecular-weight enables control of nanoplex properties including particle size and surface charge. In particular embodiments, a lower molecular-weight polymer variant yields a reduced hydrodynamic particle size while substantially maintaining a positive surface charge, thereby supporting improved delivery performance for one or more payload classes.

[0034] The nanoplexes further comprise at least one lipid component preferably natural and / or pharmaceutically acceptable solid or semi-solid lipids incorporated into the nanoplex to form a lipid-polymer hybrid assembly. In various embodiments, the lipid component may be selected from one or more lipid classes including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, and combinations thereof, including derivatives, analogs, salts, esters, and prodrug forms thereof. In preferred embodiments, the lipid preferably selected from cholesterol, P-sitosterol, Precirol® ATO 5, glyceryl monostearate (GMS), Capmul GMO-50 EP, and Compritol® 888 ATO, or combinations thereof. The lipid component contributes to at least one of enhanced cellular interaction, improved nanoplexstability, improved payload retention, and improved end-use performance during delivery.

[0035] In one aspect of this embodiment, the nanoplexes further comprise an anionic payload component including a gene-editing component and other nucleic acid components. The gene-editing component, particularly the CRISPR / Cas component are electrostatically complexed to the cationic polymer, wherein the payload is associated with the polymer through charge interactions and is at least partially protected within and / or upon the hybrid nanoplexes.

[0036] In embodiments, the payload is particularly a negatively charged biomolecular payloads including but not limited to nucleic acid (DNA / RNA, including plasmid DNA, mRNA, miRNA / siRNA, RNP), a gene-editing component (including CRISPR / Cas tools in forms including pDNA encoding Cas or dCas and gRNA, mRNA encoding for Cas or dCas protein and sgRNA and / or RNPs (Cas or dCas protein complexed with gRNA) and a epigenome editing component (including CRISPR / sCas or CRIPSR / dCas modified with epi -effectors) or combination thereof.

[0037] In certain preferred embodiments the nanoplexes is particularly suitable where pDNA or ribonucleoproteins is a principal payload due to its relatively large size; the nanoplexes facilitate payload association and protect payload integrity during handling and delivery.

[0038] In certain embodiments, the nanoplexes, by way of non-limiting example, exhibit an average size of about 50-250 nm, including about 70-200 nm, while maintaining a neutral to positive zeta potential of +35 mV effective for electrostatic association with anionic payloads and for interaction with cellular membranes. In particular embodiments, the combination of particle size within the foregoing ranges and positive surface potential provides an advantageous balance of payload association, colloidal stability, and cellular uptake.

[0039] In one main embodiment, lipid-polymer hybrid nanoplexes are prepared by first synthesizing a cyclic carbonate monomer, followed by preparing an amphiphilic blockcopolymer backbone, thereafter, converting the backbone into a cationic copolymer, and formulating the cationic polymer into lipid-polymer hybrid nanoplexes using a double emulsion solvent evaporation process followed by electrostatic complexation with nucleic acid payload. In this embodiment a method of preparing a lipid-polymer hybrid nanoplex, the method comprising:a) forming an organic phase comprising a biodegradable cationic polymer and lipid in an organic solvent;b) adding a first aqueous phase to the organic phase and emulsifying to form a primary emulsion;c) adding the primary emulsion to a second aqueous phase and emulsifying to form a secondary emulsion;d) removing the organic solvent to obtain blank nanoplexes; ande) contacting the blank lipid-polymer hybrid nanoplexes with negatively charged biomolecular payloads payload to form the lipid-polymer hybrid nanoplex by electrostatic complexation.

[0040] In embodiments, the nanoplexes are prepared by forming lipid-polymer hybrid particles using an emulsion-based solvent evaporation method and thereafter complexing the nucleic-acid payload via electrostatic interaction with the cationic polymer. This approach reduces the need to expose fragile nucleic-acid payloads to harsh chemical conditions, thereby preserving integrity and improving functional delivery.

[0041] In one embodiment the process comprises preparing an organic phase by dissolving a defined amount of the cationic polymer, in a volatile, water-immiscible organic solvent to obtain a clear polymer solution, optionally together with the lipid component dissolved or dispersed in the same organic phase. In one implementation, about 15 mg of the cationic polymer is dissolved in about 600 pL of di chloromethane to form the organic phase.

[0042] A primary aqueous phase comprising a buffer is then introduced into the organic phase to form a first (water-in-oil) emulsion, wherein the primary aqueous phase is added in a controlled volume and the mixture is subjected to energy input to generate fine aqueous droplets within the organic phase; in one implementation, about 150 pL of aHEPES buffer at about pH 6.0 is added and the mixture is probe-sonicated for about 30 seconds to form the primary emulsion.

[0043] The primary emulsion is thereafter introduced into a secondary aqueous phase comprising the same or a compatible buffer to form a second (water-in-oil-in-water) emulsion, wherein the secondary aqueous phase volume is sufficient to stabilize the dispersed droplets and the mixture is further emulsified by probe sonication under controlled amplitude and duration; in one implementation, the primary emulsion is added to about 3 mL of HEPES buffer at about pH 6.0 and emulsified by probe sonication at about 20% amplitude for about 3.5 minutes to form a stable double emulsion.

[0044] Following formation of the double emulsion, the volatile organic solvent is removed under reduced pressure, such as by rotary evaporation or equivalent vacuum-assisted evaporation, thereby driving self-assembly and / or solidification of the polymer-lipid components into nanoscale hybrid structures and yielding an aqueous dispersion of lipid-polymer hybrid nanoplexes. In this manner, lipid-polymer hybrid nanoplexes are obtained as a clear or colloidally stable aqueous dispersion, wherein the nanoplexes are cationic due to the cationic functionality of the polymer and wherein the lipid component is associated with the polymeric nanostructure to form a hybrid architecture.

[0045] In various embodiments, the lipid component is selected from one or more lipid classes including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, and combinations thereof, including derivatives, analogs, salts, esters, and combinations thereof. In preferred embodiments, the lipid component is selected from one or more lipids, including P-sitosterol, Precirol® ATO 5, glyceryl monostearate (GMS), Capmul GMO-50 EP, and Compritol® 888 ATO and is incorporated in varying amounts relative to the polymer to tune the hybrid character of the nanoplexes, while retaining the same emulsification and solvent-evaporation sequence. The lipid is introduced with cationic polymer, such that the lipid associates with the polymeric component to yield the lipid-polymer hybrid assembly.

[0046] In additional embodiments, the cationic polymer used in the main embodiment is selected from a set of cationic copolymers having different molecular weights and / or different backbone lengths and / or different degrees of cationic substitution and cholesterol is included at about 0 , about Img, about 2 mg and 5 mg per formulation batch corresponding to the polymer amount used, thereby yielding distinct lipidpolymer hybrid nanoplex formulations that differ in lipid content while remaining synthesized by the same double emulsion solvent evaporation method. The cationic polymer comprises a selected from mPEG-b-P(CB-{g-cationic chain}).

[0047] In additional embodiments, the cationic polymer in the main embodiment is prepared by synthesizing a cyclic carbonate monomer, polymerizing the monomer to form an mPEG-based an amphiphilic copolymer containing protected pendant groups, converting the pendant groups to free carboxyl groups, and coupling a cationic amine- containing chain to at least a portion of the carboxyl groups via carbodiimide-mediated coupling to yield mPEG-b-P(CB-{g-cationic chain}). In one implementation, a cyclic monomer 2-methyl-2-(benzyloxycarbonyl)propylene carbonate (MBC) is prepared via benzyl-protection and cyclization chemistry, the monomer is polymerized by ringopening polymerization using mPEG as a macroinitiator and a tin(II) 2-ethylhexanoate catalyst under inert atmosphere to yield mPEG-b-p(CB) bearing benzyl -protected groups, the benzyl groups are removed by catalytic hydrogenation using palladium on carbon under hydrogen pressure to generate mPEG-b-P(CB-{g-COOH}), and the free carboxyl groups are coupled with N,N-dimethyldipropylenetriamine using EDC and HOBt to form the cationic graft copolymer, which is purified by precipitation and vacuum drying.

[0048] In further embodiments, the ring-opening polymerization is conducted either under conventional thermal heating or under microwave-assisted heating to control reaction time while achieving comparable polymer formation, and the degree of polymerization and density is adjusted to obtain polymers of different molecular weights, such as those corresponding to PLM-84, PLM-65, PLM-53, and PLM-35.

[0049] In embodiments, the efficacy and multifunctionality of these nanoplexes may be further enhanced by surface modification with targeting ligands, such as peptides, antibodies, aptamers, sugars, small molecules that bind cell-surface markers associatedwith specific tissues or disease states to enhance uptake and / or functional delivery in selected cell populations.

[0050] In embodiments, the nanoplexes demonstrate cytocompatibility in vitro across relevant cell types, supporting suitability for biomedical applications.

[0051] The detailed operational parameters, reagent quantities, processing times, and instrument settings for representative preparations are provided in the Working of the Invention / Working Examples to avoid repetition herein, while this embodiment defines the core synthesis architecture and formation pathway.Working examples and Experimental Data1. Synthesis and characterization of monomers and cationic polymer mPEG-b- P(CB-{g-cationic chain})

[0052] This included the creation of a cyclic monomer, 2-methyl-2- benzyloxycarbonylpropylene carbonate (MBC), followed by the synthesis of an amphiphilic copolymer, mPEG-b-p(CB). After copolymer synthesis, a reduction step using palladium on carbon (Pd / C) in the presence of hydrogen gas at 45 psi pressure for 6 h and cationic chain (N, N-dimethyldipropylenetriamine) coupling using 1-ethyl- 3 -(3 -dimethylaminopropyl) carbodiimide (EDC) / HOBt chemistry were performed (Figure 1).

[0053] The cyclic monomer MBC was prepared through a two-step reaction process. Initially, a mixture of 2,2-bis(hydroxymethyl) propionic acid and potassium hydroxide were reacted with benzyl bromide in dimethylformamide (DMF) at 100°C for 15 h. The crude product was then processed and recrystallized in toluene, yielding pure benzyl- 2,2-bis(methylol)propionate as an intermediate. In the subsequent step, benzyl 2,2- bis(methylol)propionate was reacted with triphosgene in a dichloromethane-pyridine solvent mixture to produce MBC, which was then recrystallized from ethyl acetate to yield white crystalline MBC.

[0054] The polymer, mPEG-b-p(CB), was synthesized via ring-opening polymerization, where mPEG was reacted with MBC in the presence of tin(II) 2-ethylhexanoate as acatalyst (10 mol% of mPEG) at 130°C under nitrogen for 20 h. The crude product was then dissolved in chloroform and purified by precipitating with diethyl ether and isopropyl alcohol, followed by vacuum drying. Alternatively, the polymer mPEG-b- p(CB) was synthesized via ring-opening polymerization. Briefly, mPEG was reacted with MBC in the presence of tin(II) 2-ethylhexanoate [Sn(Oct)2] as a catalyst (10 mol% with respect to mPEG) at 130 °C under a nitrogen atmosphere for 1 h using a microwave synthesis reactor (Monowave 400, Anton Paar). The purification procedure was kept the same as described previously. To obtain mPEG-b-p(CB-{g-COOH}) with free carboxyl groups on its backbone, the protective benzyl groups were removed through catalytic hydrogenation using Pd / C under 40 psi.

[0055] The carboxyl-functionalized polymer, mPEG-b-P(CB-{g-COOH}), was then modified through l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxybenzotriazole (HOBt) coupling to attach N,N-dimethyldipropylenetriamine on the free carboxyl groups, producing four cationic polymers (PLM-84, PLM-65, PLM-53, and PLM-35) with varying molecular weights based on the attached of different units of monomer (MBC) as given in Table 1. The polymers were further purified by precipitation in isopropanol and diethyl ether, followed by drying under vacuum.

[0056] To characterize these polymers,NMR spectroscopy was employed to confirm structural details, composition, and molecular weight. For this analysis, polymers were dissolved in DMSO-d6 and examined with a Bruker 400 MHz NMR spectrometer. The molecular weight of the mPEG-b-P(CB-{g-cationic chain}) polymer was also determined through gel permeation chromatography (GPC) using a Waters 515 HPLC system equipped with a Styragel® HR4 column (300 mm x 7.8 mm) and a differential refractive index detector (Waters 2414). The elemental composition of the synthesized polymers was analyzed using an Elementar Vario Micro Cube C-H-N analyzer, employing combustion analysis. The weighed amount of sample was combusted in excess oxygen, and the resulting combustion products, including carbon dioxide, water, and nitrogen oxides, were captured in separate traps. The quantities of these combustion products were then used to determine the percentages of carbon, hydrogen, and nitrogen in the polymers.

[0057] The 'H NMR spectrum ofMBC, displayed characteristic signals at 5 1.2 (CH3, singlet, 3H), 54.3 (CH2, doublet, 2H), 54.7 (CH2, doublet, 2H), 55.2 (CH2, singlet, 2H), and 5 7.3 (C6H5, multiplet, 5H) (Figure 2a). Subsequently, the ring-opening polymerization of MBC with mPEG, using tin(II) 2-ethylhexanoate as a catalyst, resulted in the formation of the polymer mPEG-b-P(CB), achieving a 78% practical yield. TheTH NMR (400 MHz, CDC13) of the synthesized polymer showed mPEG- related peaks at 53.56 (CH2, multiplet, 4H) and signals corresponding to CB units at 5 1.24 (CH3, singlet, 3H), 54.3 (CH2, multiplet, 4H), 55.2 (CH2, singlet, 2H), and 5 7.3 (C6H5, multiplet, 5H) (Figure 2b). The absence of two peaks from the MBC monomer at 54.3 and 54.7, and the presence of a single proton peak at 54.3 (CH2, multiplet, 4H), confirmed the completion of the ring-opening polymerization. Further, to generate free carboxylic acid groups in the polymer backbone, the mPEG-b-P(CB) polymer underwent catalytic hydrogenation, yielding mPEG-b-P(CB-{g-COOH}) with a practical yield exceeding 82% (Figure 2c).

[0058] The 'H NMR spectrum of cationic polymers displayed peaks characteristic of mPEG and CB units, along with additional signals associated with N, N- dimethyldipropylenetriamine, including 52.4 (NH, singlet, 1H) and 57.4 (amidicNH, singlet, 1H) (Figure 2d). These findings confirmed the effective grafting of the ligands onto the polymer backbone. Additionally, the molecular weight of the polymer (PLM- 35) was analyzed using 'HNMR and GPC, which determined values of 14893 Da and 15160 Da, respectively (Figure 2e). Elemental analysis of the cationic copolymer (PLM-35) revealed its composition as 48.49% carbon (C), 5.57% hydrogen (H), and 6.90% nitrogen (N).2. Preparation and characterization of blank lipid-polymeric hybrid nanoplexes

[0059] Nanoplexes were formulated using four copolymers with varying molecular weights (PLM-84, PLM-65, PLM-53 and PLM-35) utilizing double emulsion solvent evaporation (DE) method. Specifically, 15 mg of mPEG-b-(CB-g-cationic chain) polymer was accurately weighed and dissolved in 600 pL of di chloromethane (DCM). To prepare the primary emulsion (w / o), 150 pL of HEPES buffer, pH 6.0 was added to the polymer solution, followed by probe sonication for 30 sec. The primary emulsion was then introduced into a secondary aqueous phase (3 mL HEPES buffer, pH 6.0) and emulsified by probe sonication (20% amplitude, 3.5 minutes) to form astable secondary emulsion (w / o / w). The resulting emulsion was subjected to vacuum using a rotary evaporator to remove DCM, yielding a clear solution of blank cationic nanoplexes. Similarly, lipid-polymer hybrid nanoplexes (BNPX-1, BNPX-2, BNPX- 3, and BNPX-4) were formulated using the double emulsion solvent evaporation method by incorporating the cationic polymer PLM-35 with varying cholesterol amounts of 0 mg, 1 mg, 2.5 mg, and 5 mg.

[0060] Following the initial formulation optimization, different solid and semi-solid lipids, namely P-sitosterol Precirol® ATO 5, glyceryl monostearate (GMS), and Capmul GMO-50 EP, Compritol® 888 ATO were investigated for their suitability in the formation of the lipid-polymer hybrid nanoplexes system. All formulations were prepared using the same procedure and identical composition, as described previously, with the lipid component being the only variable. The prepared lipid-polymer hybrid nanoplexes were evaluated for particle size and zeta potential in order to assess the successful formation and stability of the lipid-polymer hybrid system incorporating the cationic polymer (PLM-35). Particle size analysis was performed to determine the influence of lipid type on nanocarrier size distribution, while zeta potential measurements were used to evaluate surface charge characteristics and confirm the interaction between the different lipid matrix and the cationic polymer (PLM-35).

[0061] Cationic polymers (PLM-84, PLM-65, PLM-53, and PLM-35) and blank cationic nanoplexes characterized for particle size, zeta potential, and PDI, (Table 1, Figure 3 a and b). Among these, nanoplexes fabricated using PLM-35 were selected based on their smallest particle size compared to the other polymers, with no significant compromise in zeta potential. The reduced particle size can be attributed to the lower molecular weight of PLM-35, as it contains lesser monomer (MBC) units. Additionally, it was expected that incorporating cholesterol during the formulation of lipid-polymer hybrid nanoplexes, along with plasmid complexation, would result in an increase in particle size.

[0062] The blank nanoplexes were prepared using double emulsion solvent evaporation method, and their particle size, poly dispersity index (PDI), and zeta potential were measured using a Malvern Zetasizer, as summarized in Table 2 (Figure 3 c and d). These blank nanoplexes were subsequently utilized to evaluate their complexationefficiency. Subsequently, blank nanoplexes prepared with different lipids were characterized and are summarized in Table 3 (Figure 3e and f).

[0063] Table 1. Characterization of blank cationic nanoplexes prepared using four different cationic polymers.Cationic Polymers Blank Nanoplexes PS (nm)±SD PDI+SD ZP (mV)±SD (Cationic polymerscode)mPEG-b-P(CB84-{g- PLM-84 216.56 + 3.45 0.166+0.07 19.8+1.16 Cation chain24})mPEG-b-P(CB65-{g- PLM-65 165.23+4.20 0.176+0.02 20.1+1.34 Cation chain26})mPEG-b-P(CB53-{g- PLM-53 136.63+6.49 0.151+0.04 22.0+1.22 Cation chain31})mPEG-b-P(CB35-{g- PLM-35 73.20+3.86 0.123+0.09 23.7+0.67 Cation chain29})[n=3, Mean +SD], PS: Particle Size, PDL Poly dispersity Index, ZP: Zeta potential, SD: Standard Deviation

[0064] Table 2. Characterization of blank cationic lipid-polymer hybrid nanoplexes prepared using cationic polymer (PLM-35) and varying amount of cholesterol.Blank Nanoplexes PS (nm)±SD PDI+SD ZP (mV)±SD0 mg Cholesterol + 15 mg 73.20+3.86 0.142+0.03 23.7+0.67 Cationic Polymer, PLM-35(BNPX-1)1 mg Cholesterol + 15 mg 86.99±6.22 0.111+0.04 22.0+1.46 Cationic Polymer, PLM-35(BNPX-2)2.5 mg Cholesterol + 15 mg 99.80+8.67 0.134+0.02 19.7+1.68 CationicPolymer, PLM-35(BNPX-3)5 mg Cholesterol + 15 mg 109.2+7.48 0.161+0.06 20.5+1.10 Cationic Polymer, PLM-35(BNPX-4)[n=3, Mean +SD], PS: Particle Size, PDI: Poly dispersity Index, ZP: Zeta potential, SD: Standard Deviation

[0065] Table 3 Characterization of blank cationic lipid-polymer hybrid nanoplexes prepared using cationic polymer (PLM-35) and different lipids.Code Blank Nanoplexes PS (nm)± SD PDI+ SD ZP(mV)± SDBNPX- Cholesterol (2.5 mg) + 99.80+8.67 0.134+0.02 19.7+1.68 3 Cationic Polymer, PLM-35(15 mg)BNPX- P-sitosterol (2.5 mg) + 115.4+5.08 0.149+0.04 19.1+2.92 SI Cationic Polymer, PLM-35(15 mg)BNPX- Precirol® ATO 5 (2.5 mg) + 131.5+6.77 0.190+0.12 18.4+1.64 PR Cationic Polymer, PLM-35(15 mg)BNPX- Glyceryl monostearate 150.8+4.32 0.257+0.08 18.7+2.39 GM (GMS) (2.5 mg) + CationicPolymer, PLM-35 (15 mg)BNPX- Capmul GMO-50 EP, 181.0+5.11 0.278+0.16 17.5+2.17 CA NF(GMO) (2.5 mg) +Cationic Polymer, PLM-35(15 mg)BNPX- Compritol® 888 ATO (2.5 167.2±7.49 0.147±0.07 18.3±1.25 CO mg) + Cationic Polymer,PLM-35 (15 mg)3. Evaluation of Complexation Efficiency

[0066] The complexation efficiency of lipid-polymer hybrid nanoplexes with anionic plasmids, specifically pcDNA3-EGFP and pX458-Efla-dCas9-KRAB-MECP2-H2B- GFP (CRISPRi), was assessed through an agarose gel electrophoresis-based mobility shift assay. In this assay, a constant plasmid amount of 200 ng was incubated with varied amounts of four different blank lipid-polymer hybrid nanoplexes at differing N / P ratios (where N / P is defined as blank lipid-polymer nanoplexes to plasmid in ratio of moles). Therefore, agarose gel retardation assay was conducted to assess the complexation efficiency of pcDNA3-EGFP with different blank lipid-polymer hybrid nanoplexes at varying N / P ratios of 2.5, 5, 10, 15, and 20. Each sample was incubated at room temperature for 30 minutes, followed by electrophoresis on a 1% agarose gel at 100 V for 45 minutes. The gel was then visualized with the Gel Doc XR+ system to observe the retardation of plasmid mobility, serving as an indicator of complexation. Similarly, for the pX458-Efla-dCas9-KRAB-MECP2-H2B-GFP (CRISPRi) plasmid, the complexation efficiency was evaluated using the same procedure with a fixed N / P ratio. The naked CRISPRi plasmid and blank lipid-polymer hybrid nanoplexes (BNPX-3) were used as controls to assess comparative mobility shifts.

[0067] The results showed complete inhibition of the electrophoretic mobility of the pcDNA3-EGFP plasmid at an N / P ratio of 15 for both nanoplexes prepared with PLM- 35 cationic polymer alone (BNPX-1) and PLM-35 cationic polymer combined with 1 mg of cholesterol (BNPX-2), indicating complete complex formation (Figure 4a). In contrast, nanoplexes containing PLM-35 and 2.5 mg cholesterol (BNPX-3) and 5 mg cholesterol (BNPX-4) achieved complete complexation at a lower N / P ratio of 10 (Figure 4a). Among the formulations, BNPX-3 was selected for further studies based on its particle size, poly dispersity index (PDI), zeta potential and complexation efficiency as determined by the agarose gel assay. This selection was critical to ensure that the final particle size post-complexation remained within an acceptable range i.e. < 150-200 nm, as excessively large particle sizes could hinder their application.Although all formulations displayed nanometer-scale particle sizes (Table 2), BNPX- 3, comprising 15 mg of PLM-35 and 2.5 mg of cholesterol, exhibited the most suitable characteristics for subsequent investigation. The particle size, PDI, and zeta potential of BNPX-3 nanoplexes were evaluated before and after complexation with the pcDNA3-EGFP plasmid at a fixed N / P ratio of 10. Prior to complexation, the nanoplexes exhibited particle size of 93.60 nm, PDI of 0.116, and zeta potential of 21.9 mV. Following complexation with the pcDNA3-EGFP plasmid, the particle size increased to 123.4 nm, the PDI slightly increased to 0.128, and the zeta potential decreased to 12.2 mV (Figure 6a and b). Further, these changes reflect the successful electrostatic interaction between the nanoplexes and the plasmid.

[0068] Additionally, the complexation efficiency of the CRISPRi plasmid with the blank lipid-polymer hybrid nanoplexes was also evaluated using the agarose gel electrophoresis assay, focusing specifically on the BNPX-3 formulation. Complete complexation of the CRISPRi plasmid with BNPX-3 was observed at an N / P ratio of 20 (Figure 4b). This higher N / P ratio requirement compared to the pcDNA3-EGFP plasmid can be attributed to the difference in molecular weight between the two plasmids. The pcDNA3-EGFP plasmid has a molecular weight of approximately 3880 kDa (6159 bp), whereas the CRISPRi plasmid has a molecular weight of about 7098 kDa (11,266 bp), nearly double that of the pcDNA3-EGFP plasmid. The increased molecular weight and size of the CRISPRi plasmid likely necessitated a higher N / P ratio to achieve efficient complexation. Subsequently, the particle size, poly dispersity index (PDI), and zeta potential of BNPX-3 nanoplexes were evaluated before and after their complexation with the CRISPRi plasmid at a fixed N / P ratio of 20. The nanoplexes showed a particle size of 93.60 nm, a PDI of 0.116, and a zeta potential of 21.9 mV prior to complexation. Following complexation with the CRISPRi plasmid, the particle size increased to 131.6 nm, the PDI slightly rose to 0.142, and the zeta potential decreased to 10.5 mV (Figure 6 c and d). These changes confirm the successful electrostatic complexation between the nanoplexes and the CRISPRi plasmid.

[0069] Single-guide RNA (sgRNA) required for ribonucleoprotein (RNP) complex formation was generated via an in vitro transcription (IVT) approach using a double-stranded DNA (dsDNA) template. Briefly, target genomic regions were analyzed for thepresence of appropriate protospacer adjacent motif (PAM) sequences, and sgRNAs were designed using CRISPOR bioinformatics tools to achieve high targeting efficiency with minimal off-target effects. Corresponding forward and universal reverse primers were designed and evaluated for specificity, followed by amplification of the sgRNA template by PCR to obtain dsDNA. The purified dsDNA templates were subsequently transcribed in vitro using the MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s guidelines to produce sgRNA. All transcription and handling steps were performed under RNase- free conditions, and the synthesized sgRNA was purified and stored appropriately until further use.

[0070] For epigenome editing studies, dCas9-based transcriptional modulators (dCas9-VP64) were complexed with corresponding sgRNA using the same protocol. CRISPR / dCas9- VP64 ribonucleoprotein (RNP) complexes were prepared by complexing purified dCas9-VP64 protein with sequence-specific guide RNA (sgRNA) under RNase-free conditions. Briefly, the sgRNA was diluted in nuclease-free water (RNase free) and incubated with dCas9-VP64 protein at a predefined different molar ratio such as 5:1, 2.5: 1,1.5: 1, 1.25:1 and 1:1 sgRNA: dCas9-VP64 to ensure complete complexation. The mixture was gently mixed and incubated at room temperature for 30 min to allow efficient formation of the RNP complex through electrostatic and structural interactions. Gel retardation, an electrophoretic mobility shift assay was performed to confirm RNP complex formation. Briefly, electrophoresis was carried out on a 1% agarose gel at 100 V for 45 min. The gel was subsequently visualized using a Gel Doc XR+ imaging system (Bio-Rad) to assess the retardation in nucleic acid mobility, which served as an indicator of successful RNP complexation. Furthermore, the surface charge (zeta potential) of the formed complexes was evaluated using a Malvern Zetasizer to further confirm electrostatic interaction and complex stability. The freshly prepared RNP complexes were immediately used for subsequent complexation with lipid-polymer hybrid nanoplexes (BNPX-3) to minimize degradation and preserve biological activity. All procedures were carried out using RNase-free consumables to prevent RNA degradation.

[0071] Ribonucleoprotein (RNP) complexes were formed using Single guide RNA (sgRNA) targeting the SERPINFl gene encoding pigment epithelium-derived factor (PEDF)and dCas9-VP64 protein. The sgRNA was synthesized by in vitro transcription (IVT), followed by purification. The purity of the synthesized sgRNA was confirmed by UV spectrophotometric analysis, yielding an A260 / A 280 ratio of 2.0, indicating high purity suitable for complex formation. To optimize RNP complex, sgRNA was incubated with dCas9-VP64 protein at room temperature using different molar ratios of sgRNA to protein (5:1, 2.5:1, 1.5:1, 1.25:land 1:1). The extent of complex formation was evaluated by agarose gel electrophoresis. As the molar ratio of sgRNA to dCas9-VP64 was decreased from 5:1 to 1:1, a progressive decrease in intensity of the sgRNA band was observed, indicating increasing complexation with the protein (Figure 5). Complete complex formation was achieved at a 1:1 molar ratio of sgRNA to dCas9-VP64 (corresponding to a 1:5 w / w ratio), as evidenced by the complete disappearance of the sgRNA band, with the RNP band retained in the loading well (Fig. 5). This result confirms efficient complexation between sgRNA and dCas9-VP64 protein at the optimized ratio. Following confirmation of RNP formation, zeta potential analysis was performed to evaluate the surface charge characteristics of the complexes. Free sgRNA exhibited a negative surface charge, whereas complexation with the positively charged dCas9-VP64 protein resulted in a marked shift toward neutral zeta potential values. The final surface charge of the optimized RNP complex was determined to assess its suitability for subsequent formulation with positively charged lipid-polymer hybrid nanoplexes (BNPX-3). The zeta potential of naked sgRNA was measured to assess its surface charge characteristics and was found to be -13.9 ± 1.29 mV. The zeta potential values of ribonucleoprotein (RNP) complexes formed at different sgRNA to dCas9-VP64 protein ratios are summarized in Table 4. Overall, these findings confirm the successful formation of stable PEDF sgRNA / dCas9-VP64 RNP complexes with defined stoichiometry and favourable surface charge properties, supporting their incorporation into lipid-polymer hybrid (BNPX-3) nanoplexes delivery systems.

[0072] Table 4. Characterization CRISPR / dcas9 based RNPs Complexation Efficiency RNP Code sgRNA: dcas9 based ZP (mV) ± SD protein= RNP(Molar ratio)PEDF sgRNA / dcas9-VP64 2.5:1 -6.8±1.16PEDF sgRNA / dcas9-VP64 1.5:1 -5.7±2.23PEDF sgRNA / dcas9-VP64 1.25:1 -5.2±1.51PEDF sgRNA / dcas9-VP64 1:1 -4.8±1.43[n=3, Mean ±SD], RNP: Ribonucleoprotein, ZP: Zeta potential, SD: Standard Deviation

[0073] The complexation efficiency of anionic ribonucleoprotein (RNP) complexes [PEDF sgRNA: dCas9-VP64 prepared at a 1 : 1 molar ratio subsequently complexed with lipidpolymer hybrid nanoplexes (BNPX-3). RNP complexed nanoplexes were prepared at varying RNP: BNPX-3 molar ratios of 1 : 1 and 1 :2 to optimize electrostatic interactions between the negatively charged RNPs and the cationic carrier system. The resulting complexes were characterized for particle size, zeta potential, and poly dispersity index (PDI) using dynamic light scattering (DLS) and electrophoretic light scattering on a Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Measurements were conducted at 25 °C after appropriate dilution of the samples with RNase-free, nuclease-free water to minimize multiple scattering effects.

[0074] The formulation comprising the cationic polymer PLM-35 and cholesterol was selected for complexation studies. Complex formation between ribonucleoproteins (RNPs) and lipid-polymer hybrid nanoplexes (BNPX-3) was evaluated at different ratios of RNP-to-BNPX-3 ratios to assess their effect on particle size and zeta potential. Complexation studies were performed at RNP: BNPX-3 nanoplex ratios of 1:1 and 1:2. The resulting complexes were characterized in terms of particle size and zeta potential, and the obtained values are summarized in Table 5. Among the evaluated ratios, the 1:2 RNP: BNPX-3 nanoplexes ratio exhibited a desirable particle size and zeta potential. Compared with blank BNPX-3 nanoplexes, BNPX-3 -dCas9- VP64 RNP nanoplexes showed a reduction in positive zeta potential from 19.1 mV to 8.7 mV, accompanied by an increase in particle size from 106.23 nm to 149.11 nm. These changes indicate effective complexation between the RNPs and BNPX-3nanoplexes while maintaining suitable surface charge characteristics. Based on these findings, the 1:2 RNP: BNPX-3 nanoplexes ratio was selected for subsequent in vitro and in vivo transfection efficiency studies (Figure 7 a, and b). As the other BNPX-3 nanoplexes formulations also exhibited particle sizes and zeta potential values within a similar range, it is anticipated that these formulations would demonstrate comparable complexation behaviour with RNPs, exhibiting similar interaction patterns and complexation efficiency.

[0075] Table 5: Evaluation of complexation efficiency of CRISPR / dCas9-VP64 ribonucleoproteins (RNP) and lipid-polymer hybrid nanoplexes (BNPX-3)Code RNP: LPH PS (nm)±SD PDI±SD ZP (mV)±SD (molar ratio)BNPX-3 -dcas9-VP64 1:1 128.53±8.23 0.146±0.08 5.8±1.46 RNP BNPX-3 -dcas9-VP64 1:2 149.87±7.22 0.113±0.02 8.9±1.64 RNP[n=3, Mean ±SD], PS: Particle Size, PDI: Poly dispersity Index, ZP: Zeta potential, SD: Standard Deviation; dcas9-VP64 RNP: PEDF targeting SERPINFl sgRNA(PEDF sgRNA) : dcas9-VP64 protein.sgRNA Primers for SERPINFl gene for targeting PEDF4. Heparin competition Assay

[0076] A heparin competition assay was performed to investigate the release profile of plasmids from lipid-polymer hybrid nanoplexes (BNPX-3). In this assay, heparin, acommonly used anionic competitor, facilitates the release of complexed DNA / RNA from the cationic components of polymers and lipids. Complexed pcDNA3-EGFP plasmid-loaded lipid-polymer hybrid nanoplexes were incubated with varying concentrations of heparin (5, 10, 15, 20, and 25 IU) at 37°C for 30 minutes. After incubation, a loading dye (5 pL) was added, and samples were analyzed by agarose gel electrophoresis as previously described. Naked plasmid and lipid-polymer hybrid nanoplexes (BNPX-3) served as controls. Similarly, heparin competition assay was performed on CRISPRi plasmid-loaded lipid-polymer hybrid nanoplexes (BNPX-3), altering the heparin concentration to 10, 20, 30, 40, and 50 IU, while maintaining all other parameters constant.

[0077] The results demonstrated that at 15 IU of heparin, the pcDNA3-EGFP plasmid was successfully released, whereas 30 IU was required to release the CRISPRi plasmid (Figure 8 a and b). This variation is likely due to the higher molecular weight of the CRISPRi plasmid, which requires a greater N / P ratio for efficient incorporation into the nanoplexes, resulting in the need for a higher concentration of heparin to facilitate its complete dissociation.5. Scanning electron microscopy (SEM)

[0078] The morphological analysis of lipid-polymer hybrid nanoplexes was conducted using a Field Emission Scanning Electron Microscope (FESEM) (FEI, Apreo LoVac) to examine their shape and size both before and after complexation with the CRISPRi plasmid. For sample preparation, a 5 mg / ml solution of blank as well as plasmid- loaded nanoplexes was diluted ten fold, and a 20 pl aliquot was placed onto a coverslip, then allowed to dry at 37°C. The dried samples were mounted on stubs and coated with a thin layer of gold using a sputter coater (LEICA EM ACE-200) prior to imaging.

[0079] The morphological evaluation was conducted before and after the complexation of CRISPRi plasmid with BNPX-3 nanoparticles, revealing spherical particles in both cases (Figure 6 e and f). The particle diameters were measured from the SEM images using Imaged software. The average size of the blank lipid-polymer hybrid nanoplexes was found to be 106.1 nm, while the size increased to 142.8 nm after complexation with the CRISPRi plasmid. These results are consistent with the sizes obtained through Malvern Zetasizer analysis.

[0080] Morphological characterization of blank lipid-polymer hybrid nanoplexes (BNPX-3), RNP-complexed lipid-polymer hybrid nanoplexes (BNPX-3 -dca9-VP64 RNP), was performed using a field-emission scanning electron microscope (FE-SEM) (FEI Apreo LoVac, Hillsboro, Washington, USA). The analysis was carried out to examine particle morphology and size prior to complexation, following RNP complexation. For sample preparation, blank BNPX-3, RNP-complexed BNPX-3 nanoplexes (BNPX-3 -dca9- VP64 RNP) were prepared at a concentration of 5 mg / mL. A 20 pL aliquot of each sample was deposited onto a clean coverslip and allowed to dry at 37 °C. The dried samples were mounted on aluminium stubs and sputter-coated with a thin layer of gold using a sputter coater (LEICA EM ACE-200) prior to FE-SEM imaging.

[0081] The surface morphology and size characteristics of the lipid-polymer hybrid nanoplexes (BNPX-3) were examined using scanning electron microscopy (SEM). Morphological assessment was carried out for both blank lipid-polymer hybrid nanoplexes (BNPX-3) and RNP-complexed LPH (BNPX-3 -dca9-VP64 RNP) nanoplexes. SEM images revealed that the nanoplexes retained a predominantly spherical morphology before and after RNP complexation (Figure 7 c and d). Particle size analysis based on SEM micrographs indicated that the mean diameter of blank BNPX-3 was 98.95 nm, which increased to 143.25 nm following complexation with RNPs (BNPX-3 -dca9-VP64 RNP). These observations correlate with the particle size measurements.6. In Vitro Cell Culture Studies

[0082] Human Embryonic Kidney (HEK 293) cells and Statens Seruminstitut Rabbit Cornea (SIRC) cells were procured from the National Centre for Cell Science (NCCS), Pune. ARPE-19 cells were received as a kind gift from Dr. Vivek Singh, Senior Scientist, L.V. Prasad Eye Institute (LVPEI), Hyderabad, India. These cells were cultured in Minimum Essential Medium Eagle (MEM), supplemented with 10% FBS, ImM Sodium pyruvate 50 U / mL Penicillin and 50 U / mL streptomycin. The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.7. Cytotoxicity assay of blank lipid-polymeric hybrid nanoplexes

[0083] The cytocompatibility of the formulated lipid-polymer hybrid nanoplexes (BNPX-3) on HEK 293 and SIRC cells was assessed using the MTT (3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) assay. HEK 293 and SIRC cells were seeded into a 96-well plate at a density of 5 x io3cells per well and incubated in MEM growth medium at 37°C with 5% CO2 for 24 h. Similarly, ARPE-19 cells were seeded into a 96- well plate at a density of 5 x 103cells per well and incubated in DMEM growth medium at 37°C with 5% CO2 for 24 h. After incubation, the cells were exposed to varying concentrations of lipid-polymer hybrid nanoplexes (1, 10, 20, 40, 80, 100, 200, and 500 pg / mL). The cell viability was determined after 72 h of treatment using MTT assay as reported earlier. Briefly, after 72 h of treatment, 100 pL of fresh MEM / DMEM supplemented with 10% FBS and 0.5 mg / mL MTT was added to each well. The cells were then incubated for another 4 h at 37°C and 5% CO2. Following this, the formazan crystals formed through mitochondrial reduction of MTT were solubilized with 200 pL of DMSO per well. Absorbance readings were taken at 570 nm and 630 nm using a BioTek Epoch microplate reader, and cell viability was calculated using the following formula:Absorbance at 570 nm — Absorbance at 630 nm% Cell Viability = - - - - - - - - - x 100Absorbance of control

[0084] The results demonstrated that BNPX-3 exhibited minimal toxicity, maintaining over 80% cell viability at concentrations up to 100 pg / mL in all three cell lines (Figure 9 a, b and c). Since the polymer is cytocompatible up to 100 pg / mL, the corresponding N / P ratios of 10 and 20, required for the complexation of pcDNA3-EGFP and CRISPRi plasmids respectively, remain within acceptable cytocompatibility limits. These findings confirm that the polymer concentrations needed for effective plasmid complexation are both safe and cytocompatible, supporting their suitability for further investigations. Since the cationic polymer in the BNPX-3 nanoplexes is cytocompatible up to 100 pg / mL, the selected RNP: BNPX-3 nanoplexes complexation ratio of 1:2 remains within acceptable cytocompatibility limits. These results indicate that the cationic polymer and lipid concentrations required for effective RNP complexation are safe, supporting their suitability for further in vitro and in vivo investigations.8. Transfection Efficiency

[0085] To assess transfection efficiency, nanoplexes, including pcDNA3-EGFP and CRISPRi nanoplexes (BNPX-3), were employed and analyzed using fluorescence microscopy and flow cytometry as reported earlier. In brief, HEK 293 and SIRC cells were seeded at a density of 5*104cells per well in 24-well plates and allowed to adhere for 24 h. One hour prior to treatment, the culture medium was replaced with Opti-MEM. The cells were then exposed to all treatment groups for 6 h. As controls, naked plasmids (500 ng) and Lipofectamine 3000® / plasmid lipoplexes (plasmid equivalent to 500 ng) were used as negative and positive controls, respectively. Test samples included plasmid-loaded nanoplexes formed through electrostatic interactions with the cationic polymer (PLM-35) alone, as well as nanoplexes (BNPX-3) containing both the cationic polymer (PLM-35) and cholesterol. After 6 hours of incubation, the Opti- MEM medium was replaced with fresh MEM medium containing 10% fetal bovine serum, 50 U / mL Penicilline, 50 U / mL streptomycin and ImM sodium pyruvate, and the cells were analyzed at 24 h post-treatment. At this time point, cells were washed with PBS, stained with Hoechst dye, and observed under a fluorescence microscope (Zeiss Vert Al). For the quantitative uptake study, both cell lines were seeded at a density of 5*105cells per well in 6-well plates and allowed to adhere for 24 h. Treatments were given under identical conditions for both cell lines, with the plasmid concentration standardized to 2500 ng per well. After the treatment period, the cells were trypsinized, centrifuged at 1200 rpm for 5 minutes at 4°C, resuspended in PBS, and analysed using a Cytoflex flow cytometer (Beckman Coulter, USA). Flow cytometric data were analyzed using CytExpert 300 software (version 2.3), with the positive control acting as the reference point for assessing transfection efficiency in this study.

[0086] The results indicated successful plasmid delivery, with green fluorescence confirming the efficient transfection of the plasmids tagged with EGFP / GFP through the cationic nanoplexes (BNPX-1) and lipid-polymeric hybrid nanoplexes (BNPX-3) in both cell lines (Figure 10a and 10c and Ila and 11c). Intense green fluorescence was observed with BNPX-3 compared to nanoplexes prepared with cationic polymer alone (BNPX- 1), and the transfection efficiency was found to be comparable to that of the standard transfection reagent, Lipofectamine 3000. The blue fluorescence corresponds to Hoechst-stained nuclei, while the green fluorescence represents the EGFP from thepcDNA3-EGFP plasmid and GFP from the CRISPRi plasmid. To further validate the transfection efficiency, flow cytometry analysis was performed, providing quantitative confirmation of the results obtained with BNPX-3 and BNPX-1.

[0087] Flow cytometry analysis of HEK293 cells revealed that pcDNA3-EGFP-loaded nanoplexes (BNPX-3) achieved a transfection efficiency of 60.09%, while BNPX-1 showed a transfection efficiency of 49.93%, and Lipofectamine 3000 resulted in a transfection efficiency of 58.98% (Figure 10b). Similarly, for CRISPRi plasmid- loaded nanoplexes, BNPX-3 exhibited a transfection efficiency of 48.02%, compared to 35.22% for CRISPRi plasmid-loaded nanoplexes, BNPX-1 and 44.59% for Lipofectamine 3000 (Figure lOd). The results from both plasmids indicate a consistent trend, suggesting that the inclusion of cholesterol in the formulation of BNPX-3 enhances cellular uptake and transfection efficiency of both plasmids, as compared to BNPX-1.

[0088] In addition to this, flow cytometry analysis of using SIRC cells revealed a consistent trend in transfection efficiency across both plasmids. For pcDNA3-EGFP-loaded nanoplexes, BNPX-3 achieved a transfection efficiency of 56.04%, BNPX-1 showed 36.76%, and Lipofectamine 3000 resulted in 51.48% efficiency (Figure 11b). Similarly, for CRISPRi plasmid-loaded nanoplexes, BNPX-3 exhibited a transfection efficiency of 40.34%, compared to 30.31% for BNPX-1 and 42.53% for Lipofectamine 3000 (Figure lid). These results suggest that the incorporation of cholesterol in BNPX-3 enhances cellular uptake and improves transfection efficiency compared to BNPX-1, making it comparable to the well-established transfection reagent, Lipofectamine 3000.

[0089] Further, transfection studies were performed using lipid-polymer hybrid nanoplexes complexed with both types of ribonucleoproteins (RNPs), containing sgRNAs targeting PEDF for SERPINF1 gene (PED sgRNA), with FITC tagged dCas9-VP64. Cellular uptake and intracellular localization were analysed by confocal laser scanning microscopy (CLSM) and quantified by flow cytometry following previously reported methods. Briefly, HEK 293 and ARPE-19 cells were harvested by trypsinization and seeded at a density of 6.5 x io4cells per coverslip in 6-well plates. Cells were allowed to adhere for 24 h under standard culture conditions. Prior to treatment, the culturemedium was replaced with Opti-MEM, and cells were incubated for 1 h. Cells were then exposed to different formulations to assess time-dependent uptake at 1, 3, and 6 h. Naked FITC-labelled dCas9-VP64 RNP (PEDF sgRNA: dCas9-VP64= 1:1 molar ratio) and Lipofectamine™ CRISPRMAX™-mediated FITC-dCas9-VP64 RNP complexes (CRISPRMAX-dCas9-VP64 RNP) (equivalent amounts of PEDF sgRNA and dCas9-VP64 RNP protein) were used as negative and positive controls, respectively. Test formulations included BNPX-3-dCas9-VP64 RNP (PEDF sgRNA and FITC-labelled dCas9-VP64 RNP complexed with cationic BNPX-3). Following incubation for the designated time points, cells were washed with phosphate-buffered saline (PBS), fixed with 2% paraformaldehyde, and counterstained with DAPI to visualize cell nuclei. The intracellular internalization of CRISPR / dCas9 RNP complexes was subsequently examined using a confocal laser scanning microscope (CLSM, Carl Zeiss, Germany). The internalization of CRISPR / dCas9-VP64 RNPs was analysed based on intracellular green fluorescence using Zeiss ZEN software (version 3.7).

[0090] For quantitative analysis of cellular internalization, HEK 293 and ARPE-19 cells were seeded at a density of 5 * 105cells per well in 6-well culture plates and incubated for 24 h to ensure adequate cell attachment. All formulations were administered under identical experimental conditions for both cell lines. The RNP amount was standardized to a 1:1 molar ratio of PEDF sgRNA to FITC-tagged dCas9-VP64 per well. Following treatment, cells were enzymatically detached using trypsin, collected by centrifugation at 1200 rpm for 5 min at 4 °C, and resuspended in phosphate-buffered saline (PBS). Quantitative uptake was subsequently evaluated using a CytoFLEX flow cytometer (Beckman Coulter, USA). Flow cytometry data were acquired and analyzed using CytExpert 300 software (version 2.3), with the positive control serving as the reference standard for determining transfection efficiency.

[0091] To evaluate the transfection efficiency of BNPX-3 nanoplexes in HEK293 and ARPE- 19 cells, the cells were treated with RNP-complexed BNPX-3 nanoplexes containing FITC-dCas9-VP64 RNPs which is having sgRNAs targeting PEDF (SERPINFl gene). The amount of RNPs used corresponded to 1 : 1 molar ratio of PEDF sgRNA: FITC tagged dcas9-VP64 protein, and the cells were incubated for 1, 3, and 6 h. Following incubation, the cells were fixed and stained with DAPI to visualize the nuclei andsubsequently analyzed using confocal microscopy. Confocal imaging revealed successful intracellular delivery of RNPs in both cell lines, as evidenced by the presence of green fluorescence (Figure 13 a and d). Fluorescence intensity increased in a time-dependent manner, with higher signal intensity at 3 h and further enhancement at 6 h compared to 1 h, indicating improved cellular uptake and transfection with prolonged incubation. The transfection efficiency achieved with the BNPX-3 nanoplexes was found to be comparable to that of the standard transfection reagent, Lipofectamine™ CRISPRMAX™. In the confocal images, blue fluorescence corresponds to DAPI stained nuclei, whereas green fluorescence represents FITC from dCas9-VP64 RNPs.

[0092] To further validate transfection efficiency, flow cytometry analysis was performed to quantitatively confirm the results obtained with BNPX-3 nanoplexes. Flow cytometric analysis of HEK293 cells demonstrated that FITC-tagged dCas9-VP64 RNP loaded BNPX-3 nanoplexes (BNPX-3 -dCas9-VP64 RNP) achieved transfection efficiencies of 48.67%. In contrast, naked dCas9-VP64 RNP exhibited a markedly lower transfection efficiency of 2.34%, while Lipofectamine™ CRISPRMAX™ (CRISPRMAX-dCas9-VP64 RNP) resulted in a transfection efficiency of 49.00% after 6 h of transfection (Figure 13 b and c). Similarly, flow cytometric analysis of ARPE-19 cells revealed that BNPX-3 -dCas9-VP64 RNP achieved transfection efficiencies of 54.05%. Naked FITC tagged dCas9-VP64 RNP showed minimal transfection efficiency (3.51%), whereas Lipofectamine™ CRISPRMAX™ (CRISPRMAX-dCas9-VP64 RNP) achieved a transfection efficiency of 47.16% (Figure 13 e and f). Overall, the results obtained from both cell lines indicate a consistent trend, demonstrating that BNPX-3 nanoplexes encapsulating RNP exhibit transfection efficiencies comparable to those achieved with Lipofectamine™ CRISPRMAX™, thereby highlighting the effectiveness of the BNPX-3 nanoplex delivery system.9. Endocytosis Uptake Pathway

[0093] HEK293 and SIRC cells were seeded into 24-well plates at a density of 5*104cells per well and allowed to adhere overnight at 37°C in a humidified incubator with 5% CO2. After incubation, the cells were rinsed with PBS, and complete MEM growth media containing specific endocytic inhibitors including, nystatin (27 pM),chlorpromazine (10 pM), methyl P-cyclodextrin (3 mM), and amiloride (1 mM) were added. The cells were then incubated with the inhibitors for 1 h at 37°C under 5% CO2 . Following inhibitor treatment, the cells were washed with PBS and exposed to CRISPRi -loaded lipid-polymer hybrid nanoplexes (BNPX-3) in optiMEM media for 6 hours. The media was then replaced with fresh MEM growth media, and the cells were incubated for an additional 24 h. After washing the cells with PBS, Hoechst dye was used for nuclear staining, and the cells were visualized under a fluorescence microscope (Vert.Al, ZEISS, Oberkochen, Germany). To evaluate plasmid uptake, fluorescence microscopy was performed to measure the fluorescence intensity of GFP expression from the CRISPRi plasmid. Additionally, for the quantitative uptake study, both cell lines were seeded at a density of 5 * 105cells per well in 6-well plates and allowed to adhere for 24 hours. After incubation, the cells were washed with PBS and treated with complete MEM growth media containing specific endocytic inhibitors- ny statin (27 pM), chlorpromazine (10 pM), methyl P-cyclodextrin (3 mM), and amiloride (1 mM). The inhibitors were incubated with the cells for 1 hour at 37°C under 5% CO2. Subsequently, treatments were administered under identical conditions for both cell lines, with a standardized plasmid concentration of 2500 ng per well. This concentration was consistent across all groups. After the treatment period, the cells were trypsinized, centrifuged at 1200 rpm for 5 minutes at 4°C, resuspended in PBS, and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA). The flow cytometric data were processed using CytExpert 300 software (version 2.3), with the treatment group (CRISPRi loaded BNPX-3) serving as the reference for evaluating transfection efficiency in the study.

[0094] The results indicated that the uptake mechanism for these nanoplexes was lipid raft- mediated endocytosis, as marked reduced cellular uptake was observed upon treatment with methyl P-cyclodextrin. Additionally, chlorpromazine treatment also led to a noticeable decrease in uptake, as evidenced by a significant reduction in GFP fluorescence intensity within the cytoplasm. This suggests involvement of clathrin- mediated endocytosis and lipid raft-mediated endocytosis play significant role in the delivery of CRISPRi plasmids via these lipid-polymer hybrid nanoplexes (Figure 12a and 12c). Further, the quantitative uptake analysis revealed a consistent trend in both HEK293 and SIRC cell lines upon transfection with CRISPRi plasmid-loaded BNPX- 3 nanoplexes. In HEK293 cells, the nanoplexes demonstrated a high uptake efficiencyof 53.55%, which significantly reduced to 3.55% when chlorpromazine was used as an inhibitor, indicating a clathrin mediated uptake pathway. Similarly, a marked reduction in GFP expression (14.74%) was observed when the lipid-mediated pathway was inhibited using P-cyclodextrin, confirming its role in the uptake process. In contrast, no significant changes in uptake were observed upon inhibition of caveolae- mediated and macropinocytosis pathways using nystatin and amiloride, with uptake efficiencies remaining at 51.64% and 51.80%, respectively (Figure 12b). In SIRC cells, a similar pattern was observed, with the transfection efficiency of CRISPRi plasmid-loaded BNPX-3 nanoplexes exhibited at 41.24%. This efficiency significantly decreased to 2.87% and 8.46% upon inhibition with chlorpromazine and P- cyclodextrin, respectively, confirming the involvement of clathrin-dependent and lipid-mediated pathways. However, inhibition of caveolae-mediated and macropinocytosis pathways using nystatin and amiloride showed negligible effects, with transfection efficiencies of 40.33% and 39.15%, respectively. Collectively, these findings highlight the significant role of lipid-mediated and clathrin-dependent pathways in the cellular uptake of BNPX-3 nanoplexes (Figure 12d)10. In vivo evaluation of RNP containing lipid-polymer hybrid nanoplexes

[0095] All experimental procedures involving animals were conducted following prior approval from the Institutional Animal Ethics Committee (IAEC; Protocol No. IAEC / RES / 39 / 13). The study was carried out in strict accordance with the guidelines prescribed by the Committee for Control and Supervision of Experiments on Animals (CCSEA). Healthy adult male Wistar rats, weighing up to 250 g, were procured from the Central Animal Facility, BITS Pilani, Rajasthan. Throughout the experimental period, animals were maintained under standard laboratory conditions with unrestricted access to standard pellet diet and drinking water.11. Evaluation of Retinal Toxicity

[0096] Toxicity is a significant concern for cationic polymeric and lipid-based nanocarriers;therefore, the safety of the lipid-polymer hybrid system (BNPX-3) was evaluated in rats following intravitreal administration. Healthy rats were randomly assigned to experimental groups and anesthetized via intraperitoneal injection with ketamine (80 mg / kg) and xylazine (10 mg / kg). The animals were randomly divided into three experimental groups. Each group received a single intravitreal injection of 10 pL ofcationic blank lipid-polymer hybrid nanoplexes (BNPX-3) at doses of 50 pg, 100 pg, or 250 pg administered to the right eye. The contralateral (left) eye of each animal received an intravitreal injection of 10 pL of sterile normal saline and served as the control. Following intravitreal injection, the animals were monitored for 7 days for any signs of ocular toxicity, such as redness, bulging, or pigmentation changes. After the observation period, the animals were euthanized, and the eyes were harvested, fixed in Davidson’s fixative, and processed for histological analysis. Hematoxylin and eosin (H&E) staining was performed, and the retinal and ocular tissues were examined under a microscope (Vert Al, Zeiss) at 4* and 40* magnifications to assess cellular damage and infiltration.

[0097] The retinal safety of BNPX-3 nanoplexes was assessed in vivo at doses of 50 pg, 100 pg, and 250 pg, and compared with the negative control group, which received 10 pL of sterile normal saline. Visual inspection of the eyes revealed no signs of ocular irritation or distress, such as conjunctival redness, excessive tearing, vitreous hemorrhage, or abnormal animal behavior. Histological analysis was further performed using hematoxylin and eosin (H&E) staining. As illustrated in Figure 14, retinal tissue from nanoplex-treated eyes displayed normal morphology with no detectable signs of cellular damage or structural abnormalities, comparable to the control group. These observations indicate that lipid-polymer hybrid nanoplexes (BNPX-3) are well tolerated in the ocular environment and do not induce significant retinal toxicity at the tested doses.12. Retinal Tissue Distribution

[0098] This study aimed to develop a nanocarrier system for the efficient delivery of PEDF sgRNA / dCas9 based ribonucleoprotein (RNP) complexes to the posterior segment of the eye, specifically the retina, by overcoming the vitreous barrier. The retinal distribution of both RNPs encapsulated within BNPX-3 nanoplexes were systematically evaluated. For in vivo delivery, 8 pg of FITC-labelled dCas9-VP64 RNPs containing BNPX-3 nanoplexes (BNPX-3 -dcas9-VP64 RNP) were administered in a final volume of 10 pL via intravitreal injection into the right eye of rats using a Hamilton syringe. An equivalent dose of the corresponding naked RNPs was administered to the left eye, which was used as the control. Animals were sacrificed at designated time intervals (12, 24, and 48 h post-injection), and the eyeswere immediately excised. Enucleated eyes were fixed in Davidson’s fixative and sectioned circumferentially. Retinal tissues from the posterior segment were further processed by preparing thin sections, which were mounted onto glass slides and fixed. Nuclear staining was performed using DAPI, followed by visualization of fluorescence signals within the retinal layers.Technical applications of the invention• The lipid-polymer hybrid nanoplexes ensure efficient delivery of CRISPR-Cas components by improving cellular uptake and maintaining bioactivity, thereby increasing therapeutic outcomes.• The system can accommodate various therapeutic agents, including nucleic acids, peptides, and proteins, making it a multifunctional platform adaptable for different disease conditions.• The incorporation of tertiary amine groups enables endosomal escape through the proton sponge effect, enhancing intracellular delivery and reducing degradation of therapeutic components.• Electrostatic interactions used for complexation avoid the need for harsh chemical treatments, preserving the stability and functionality of sensitive biomolecules like CRISPR-Cas components.• Surface modification with disease-specific targeting ligands enhances precision, enabling selective delivery to diseased cells while minimizing off-target effects. • The invention is applicable to a wide range of diseases, including ocular disorders, cancer, and diabetes-related complications, highlighting its potential for clinical use in diverse areas.• The design and fabrication of the hybrid nanoplexes are scalable and suitable for large-scale production, paving the way for future commercialization.• The use of biocompatible lipids and polymers ensures minimal toxicity, making the system safe for clinical applications.

Claims

CLAIMS:

1. A lipid-polymer hybrid nanoplexes for the delivery of anionic biomolecular components, each nanoplex comprisesa) a biodegradable cationic polymer;b) a lipid, wherein the polymer and lipid cooperatively form a hybrid nanoscale complex that improves cellular uptake, colloidal stability, and functional delivery performance including transfection efficiency; andc) a negatively charged biomolecular payload is electrostatically complexed to the cationic polymer.

2. The nanoplex as claimed in claim 1, wherein the biodegradable cationic polymer is a cationic PEG-polycarbonate copolymer comprises mPEG-b-P(CB-{g-cationic chain}).

3. The nanoplex as claimed in claim 2, wherein the mPEG-b-P(CB-{g-cationic chain}) comprises a cationic chain having primary, secondary and / or tertiary amine groups.

4. The nanoplex as claimed in claim 2, wherein the cationic chain comprises N,N dimethyldipropylenetriamine.

5. The nanoplex as claimed in claim 1, wherein the lipid is selected from fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, and derivatives, analogs, salts, esters, prodrugs, and combinations thereof.

6. The nanoplex as claimed in claim 5, wherein the lipid is selected from cholesterol, P- sitosterol, Precirol® ATO 5, glyceryl monostearate (GMS), Capmul GMO-50 EP, and Compritol® 888 ATO, or combinations thereof.

7. The nanoplex as claimed in claim 1, wherein the biodegradable cationic polymer is selected from polymer variants corresponding to PLM-84, PLM-65, PLM-53, and PLM- 35.

8. The nanoplex as claimed in claim 1, wherein the payload comprises at least one selected from DNA / RNA, including plasmid DNA, mRNA, sgRNA / siRNA, ribonucleoprotein complexes9. The nanoplex as claimed in claim 1, wherein the nanoplex exhibits a neutral to positive zeta potential of +35 mV.

10. The nanoplex as claimed in claim 1, the nanoplex having an average particle size of about 50 nm to about 250 nm.

11. A method of preparing a lipid-polymer hybrid nanoplex, the method comprising: a) forming an organic phase comprising a biodegradable cationic polymer and lipid in an organic solvent;b) adding a first aqueous phase to the organic phase and emulsifying to form a primary emulsion;c) adding the primary emulsion to a second aqueous phase and emulsifying to form a secondary emulsion;d) removing the organic solvent to obtain blank nanoplexes; ande) contacting the blank lipid-polymer hybrid nanoplexes with a negatively charged biomolecular payload to form the lipid-polymer hybrid nanoplex by electrostatic complexation.

12. The method as claimed in claim 11, wherein the first aqueous phase and the second aqueous phase comprise a buffered aqueous solution having a pH from about 4.5 to about 8.0.

13. The method as claimed in claim 11, wherein the cationic polymer is an amphiphilic copolymer comprising a hydrophilic segment and a polycarbonate-containing segment bearing pendant functional groups modified to provide cationic character.

14. The method as claimed in claim 11, wherein the cationic polymer is selected from polymers corresponding to PLM-84, PLM-65, PLM-53, and PLM-35.

5. The method of claim 11, wherein step (e) comprises contacting the blank lipid-polymer hybrid nanoplexes with the negatively charged payload under conditions effective to allow electrostatic complexation between the cationic polymer and the negatively charged payload to form the lipid-polymer hybrid nanoplex.

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