Novel lipid and lipid nanoparticle formulations

Abstract

The present invention is in the field of drug delivery and relates to relates to novel cationic lipids that can be used alone or in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the targeted intracellular delivery of therapeutic nucleic acids.

Description

[0001] NOVEL LIPID AND LIPID NANOPARTICLE FORMULATIONS

[0002] FIELD OF TH E INVENT ION

[0003]

[0001] The present disclosure is in the field of drug delivery and particularly using lipid nanoparticles (LNP) as the preferred mode of delivering drug products in vivo.

[0004]

[0002] The present disclosure generally relates to novel cationic lipids that can be used alone or in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g. oligonucleotides, messenger RNA, siRNA, shRNA, micro-RNA) both in vitro and in vivo.

[0005] BACKGROUND OF THE INVENTION

[0006]

[0003] Despite extensive research, there are challenges faced during the later stages of drug development typically due to safety and efficacy concerns that fundamentally arise from high accumulation in off-target organs or poor accumulation in target organs, respectively. This has been a major bottleneck in the translation of potent drug candidates, which inherently possess excellent potential but fail to demonstrate significant clinical impact due to dose-related toxicities and / or dose-limited efficacies due to off-target effects.

[0007]

[0004] Nanoparticles have been developed to target therapies to specific targets. It is understood that modulation of physicochemical properties such as size and charge could improve nanoparticles’ targeting to specific tissues (Maldonado, R.A., LaMothe, R.A., Ferrari, J.D., Zhang, A.H., Rossi, R.J., Kolte, PN, Griset, A.P., O ’Neil, C., Altreuter, D.H., Browning, E., et al. (2015). Proc. Natl. Acad. Sci. USA 112, E156- -E165). However, nanoparticles also face biological barriers that impede their targeting capabilities (Blanco, E., Shen, H., and Ferrari, M. (2015). Nat. Biotechnol. 33, 941-951).

[0008]

[0005] Lipid nanoparticles (LNPs) then emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutic agents. Advanced therapeutic modalities are increasingly using lipid nanoparticles (LNP) as the preferred mode of delivering drug product. Lipids including ionizable lipids, cationic lipids, sterols, helper lipids, PEGylated lipids and or conjugated PEGylated lipids are components of LNPs which can be modified to guide the delivery of the LNPs to different tissues and cell types in the body.

[0006] LNPs have been successful in effectively protecting and transporting numerous therapeutic products such as small molecules, nucleic acids, peptides, antibodies, and cellbased strategies to cells. Thus, the application of Lipid nanoparticles (LNPs) has extended to other fields, such as medical imaging, cosmetics, nutrition, agriculture, and other innovative areas such as nanoreactors.

[0009]

[0007] However, lipid nanoparticles tend to naturally localize in the liver. The currently available entities lack high level selective tissue tropism resulting in lowering the efficiency in targeted delivery and bioavailability.

[0010]

[0008] Currently available nanoparticle formulations lack selective tissue tropism the presence of which improves therapeutic efficiency in addition to having lower toxicity and higher stability. Further, there is a shortage of entities that target organs other than the liver.

[0011]

[0009] Drug delivery entities as available currently use adjuvants that not only increase the potency but also increase the toxicity of the LNPs in vivo.

[0012]

[0010] There is ongoing research in exploring the conjugation of the LNPs with antibodies to increase their cell specificity. Although antibodies could increase specificity, the downstream process for characterization of the antibody-conjugated-LNPs and manufacturing at scale becomes very challenging. The antibody-conjugated-LNPs also very sensitive to storage conditions, which is a major concern.

[0013] [Oil] Thus there is a need for drug delivery entities that can be used to deliver drug candidates to different organs and tissues in vivo, particularly in the human body specifically avoiding the liver. This is because it has further been observed that extra hepatic delivery is highly beneficial in the field of non-viral drug delivery as most commercially available LNPs have the tendency to localize in the liver.

[0014]

[0012] The surface charges of LNPs are generally determined by the lipid head groups, which may be either positively or negatively charged or zwitterionic. The surface potential, which depends on the surface charge density, controls the interactions between particles and the adsorption of counterions and hence the stability of the nanoparticles. Uncharged particles or particles with low charge densities tend to aggregate over time, while more highly charged particles repel each other, preventing aggregation.

[0015]

[0013] Cationic LNPs, are widely used as nonviral delivery systems. A large number of cationic lipid amphiphiles have been synthesized and tested for use as nucleic acid carriers. The molecular architecture of the cationic lipids is similar to that of natural lipids, except for the presence of an ionizable (cationic) head group instead of the zwitterionic or anionic head group of the natural lipids. They comprise a hydrophobic part with two alkyl chains or a cholesterol moiety, a positively charged polar head group, and a linker connecting the polar group with the hydrophobic moiety. Ionizable lipids which are positively charged only inside the cell and uncharged in the bloodstream due to a change in pH value are preferred because they are less toxic than nonionizable cationic lipids.

[0016]

[0014] While cationic lipids offer great promise as carriers for the delivery of fragile compounds such as nucleic acids, some cationic lipids cause cytotoxicity. In some cases, cationic lipids reduce mitosis in cells, form vacuoles in the cytoplasm’s of cells, and cause detrimental effects on key cellular proteins such as protein kinase C. Thus, there is a requirement to arrive at drug delivery systems which are able to make targeted extra hepatic delivery while being minimally cytotoxic with high efficiency in achieving organ / tissue specific delivery.

[0017] SUMMARY OF THE INVENTION

[0018]

[0015] The present invention discloses a cationic lipid of Formula (I) or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof with the following structure: wherein:

[0019] R1 is -(CH2)aQ, or -(CO)C(-OCH2)b -ORA, Q is hydrogen, -OH, -NRB, C3-CS, RB, heterocyclic ring optionally substituted by straight chain or branched Ci-Cis alkyl or C3-C8 heterocyclic ring optionally substituted by straight chain or branched Ci-Cis alkylene;

[0020] RA is hydrogen or Ci-Cis alkyl;

[0021] RB is hydrogen, C3-C8 saturated or unsaturated carbocyclic ring optionally substituted with Ci- C>, alkyl, amino, oxo; or RB along with the N forms a 3 to 10 membered heterocyclic ring; C9- C14 fused heterocycle optionally substituted with Ci-Ce alkyl, amino, oxo.

[0022] R2 and R3 are same or different and is independently selected from

[0023] -hydrogen; Ci-Cis alkyl; -(CH2)dC(Rc)(Rc )OH, -(CH2)eCOORG;

[0024] -(CH2)fOCO(O)h(CH2)jCHRcRc’; -(CH2)kCOO(CH2)mCH(RD)(RD’), -(CH2)sN(COCi-i8alkyl)[(CH(CH2)pCOO(RD)(RD’))]2; where * denotes the point of attachment; -(CH2)rCOO(CH2)t(S)xRG; - (CH2)UCOO(CH2)VC(RC)3; R2 and R3 together form a C3-C8 N-containing ring optionally substituted with 0(RG)O-IS; wherein Rc and Rc’ are independently straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; -(CH2)kOCORG, or -(CH2)mOCO(CH2)eCH(ORG)2; wherein RD and RD’ are same or different and independently -(CH2)fCOO RG, straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; and wherein RG is straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; and wherein a, b, d, e, f, h, j, k, m, s, p, q, r, t, u, v and x are 0-18.

[0025] R4 is a saturated heterocyclic ring selected from and

[0026] X is Cl, Br or I.

[0027]

[0016] The present invention further discloses a cationic lipid of Formula II or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof with the following structure: wherein:

[0028] K is selected from N or N+(R4).

[0029] R2 and R3 are same or different and independently selected from: hydrogen; Ci-Ci8alkyl ; -(CH2)dOCO RG; -(CH2)eCOO(CH2)f(S)hRG ;

[0030] -(CH2)kN+[(CH2)mCONH RG]2; -(CH2)SC(OH)( RG); -(CH2)PCOO(RG);

[0031] -(CH2)qCONH RG;

[0032] Wherein RG is independently a straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; wherein d, e, f, h, k, m, s, p, and q are 0-18;

[0033] Z is selected from trialkylamino having at least one bond with R4, so as to form a quaternary nitrogen; or

[0034] Z along with R3-N+and K-R3, together forms a C5- C10 heterocyclic ring,

[0035] R4 is saturated heterocyclic rings selected from and

[0036] X is Cl, Br or I.

[0037]

[0017] In an embodiment, the present invention relates to a Lipid nanoparticle (LNP) formulation comprising at least one cationic lipid of Formula I or Formula II, at least one PEGylated lipid, and cholesterol. The formulation can comprise 30 to 70% by weight of cationic lipid, 10 to 30% by weight of cholesterol, 15 to 30% by weight of PEGylated lipid.

[0038]

[0018] In some embodiments, the formulation of the present invention further comprises at least one helper lipid or an ionizable lipid or a combination thereof.

[0039]

[0019] In another embodiment, the formulation of the present invention comprises at least one cationic lipid, at least one PEGylated lipid, cholesterol and at least one helper lipid. The formulation comprising 30 to 60% by weight of cationic lipid, 35 to 50% by weight of cholesterol, 1 to 5% by weight of PEGylated lipid, 7 to 10% by weight of helper lipid.

[0040]

[0020] In yet another embodiment, the formulation of the present invention comprises at least one cationic lipid, at least one PEGylated lipid, cholesterol and at least one ionizable lipid.

[0041]

[0021] In a different another embodiment, the formulation of the present invention comprises at least one cationic lipid, at least one PEGylated lipid, cholesterol, at least one helper lipid and at least one ionizable lipid. The formulation comprising 10 to 40% by weight of cationic liquid, 35 to 50% by weight of cholesterol, 1 to 5% by weight of PEGylated lipid, 7 to 10% by weight of helper lipid, 10 to 40% by weight of Ionizable lipid.

[0042]

[0022] In yet another embodiment, the formulation of the present invention comprises the cationic lipid and the ionizable lipid in a suitable ratio.

[0023] In an embodiment the present invention provides a process for the preparation of a formulation of the present invention comprising at least one cationic lipid of formula I or Formula II by a method comprising the steps of: a) preparing the cationic lipid of Formula I or Formula II; b) preparing an ethanolic mixture comprising ionizable lipid, cholesterol and PEG wherein the ionizable lipid, cholesterol and PEG are in a suitable ratio; c) adding buffer; d) adding the aqueous phase rapidly to the ethanolic lipid mixture in a ratio of 3: 1 aqueous: ethanol v / v; e) mixing the solution of step (a) and step (b) and then leaving it undisturbed to obtain the formulation.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044]

[0024] Fig. 1 shows lung tropism as achieved by formulation F.4 comprising Lipid 1, as disclosed in the present invention in comparison with the commercially available MC3 lipid- based formulation which accumulates in the liver.

[0045]

[0025] Fig. 2 shows kidney tropism as achieved by formulation F.6 comprising Lipid 2 as disclosed in the present invention in comparison with the commercially available ALC-0315 lipid-based formulation which accumulates in the liver.

[0046]

[0026] Fig. 3 shows spleen tropism as achieved by formulation F.9 comprising Lipid 2 as disclosed in the present invention in comparison with the commercially available ALC-0315 lipid-based composition which accumulates in the liver.

[0047]

[0027] Fig. 4a shows the kidney tropism of formulation F.8-2 LNP (0.5 mg / kg) comprising Lipid 1 in comparison with F3-1 LNP (0.5 mg / kg), which is the commercially available ALC-0315 lipid based composition, administered intravenously. Fig. 4b shows the graphical quantification of luciferase expression in mice with F.8-2 formulation comprising Lipid 1 in comparison with the commercially available ALC-0315 lipid based composition. Fig. 4c, 4d and 4e show the in-vivo organ wise biodistribution of formulations F.ll, F.12 and F.13 respectively and their quantification.

[0028] Fig. 5a shows the graphical comparison of encapsulation efficiencies of formulation F.8- 2 comprising Lipid 1 and F3-l(Control). Fig. 5b shows the comparison of encapsulation efficiency of inventive formulations F.ll, F.12 and F.13 with F3-1 (Control)

[0048]

[0029] Fig. 6 shows the biodistribution of formulation F.8-2 LNP (0.4 mg / kg) administered via retrograde ureteral route in W.T. mice, at 6 hours post-injection, and its corresponding radiance values.

[0049]

[0030] Fig. 7 shows the biodistribution of formulation F.8-2 (0.4 mg / kg) via retrograde ureteral route in PKD1 knockout model (a representative preclinical model for Autosomal Dominant Polycystic Disease) at 12 weeks age, assessed using IVIS (in-vivo) imaging at 6 hours postinjection, and and its corresponding radiance values.

[0050]

[0031] Fig. 8 shows the cell-specific uptake analysis of F8-2 lipid nanoparticles (0.4 mg / kg), delivered via retrograde ureteral injection, in PKD1 knockout mouse model at 13-14 weeks of age. Uptake in collecting ducts and proximal tubular epithelial cells was assessed 6 hours postinjection using immunofluorescence. Left kidney sections were stained with fLuc+AQP2 (for collecting ducts) and fLuc+LRP2 (for proximal tubular epithelial cells)

[0051]

[0032] Fig. 9 Quantification of gene editing by inventive formulation F.8-2 -mediated dual gRNA transfection in WT 9-7 cells (ADPKD patient-derived immortalized cell line).

[0052]

[0033] Fig. 10 shows 90-Day stability data of formulation F.8-2 at 4° C and encapsulation efficiency.

[0053]

[0034] Fig. 11 shows the comparative cytotoxicity of mCherry encapsulated F8-2 and F3-1 LNPS at 2 pg / well concentration in WT9-7 cells observed at 6 h and 24 h.

[0054]

[0035] Fig. 12a shows the immunohistochemistry (IHC) images of kidney, liver and spleen cross-sections of C57BL / 6 mice (6-8 weeks, male) 7 days post retrograde ureteral injection of F8-2 LNP compared to non-injected control. Fig. 12b shows that Differential interference contrast (DIC) images of kidney sections after renal artery injection in BALB / c mice, showing lower toxicity with F8-2 compared to F3-1 LNP. Uninjected control included for reference. DETAILED DESCRIPTION OF THE INVENTION

[0055]

[0036] Definitions: For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0056]

[0037] As used herein, the articles including "a" and "an" when used in a claim, are understood to mean one or more of what is claimed or described.

[0057]

[0038] As used herein, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0058]

[0039] As used herein, the terms "include," "includes," and "including," are meant to be nonlimiting and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0059]

[0040] As used herein, the terms “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0060]

[0041] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0061]

[0042] An “ionizable lipid” refers to a class of lipid molecules which remain neutral at physiological pH, but are protonated at low pH, making them positively charged. As used herein, the term “ionizable lipid” refers to a synthetic amphiphilic lipid, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug, or oxide thereof, comprising (i) an ionizable headgroup containing at least one primary, secondary, or tertiary amine capable of acquiring a positive charge under acidic conditions (e.g., during lipid nanoparticle formulation or within endosomal compartments) and remaining substantially neutral at physiological pH; (ii) one or more hydrophobic moieties, such as linear, branched, or unsaturated fatty acid chains, which facilitate self-assembly into lipid nanoparticles and enhance membrane interaction; and (iii) optionally, one or more linkers, such as ester, carbonate, or other biodegradable linkages, to enable metabolic degradation and enhance systemic clearance. Exemplary ionizable lipids include ALC-0315, C12-200, cKK-E12, LP01, BP Lipid 142, SM-102 and DLin-MC3-DMA. Such ionizable lipids and their derivatives are suitable for the encapsulation, stabilization, and intracellular delivery of nucleic acid payloads, including but not limited to messenger RNA (mRNA), small interfering RN or other oligonucleotides.

[0062]

[0043] As used herein, the term “cationic lipid” refers to a lipid molecule capable of carrying a positive charge, either permanently or in a pH-dependent manner. Exemplary cationic lipids include those containing one or more amine groups (primary, secondary, tertiary, or quaternary) that bear or acquire a positive charge. In some embodiments, the cationic lipid is ionizable, such that it can exist in either a positively charged or a neutral state depending on environmental pH. The ionization behavior of such lipids affects the surface charge of the lipid nanoparticle (LNP) under different pH conditions, influencing key parameters such as plasma protein adsorption, blood clearance, biodistribution, and tissue targeting (see, e.g., Semple, S.C., et al., Adv. Drug Deliv. Rev. 32, 3-17 (1998)). Moreover, these charge-dependent properties facilitate the formation of endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Then 8, 1188— 1196 (2001)), which are critical for efficient intracellular release of nucleic acid payloads.

[0063]

[0044] As used herein, the term “pH-sensitive cationic lipid” refers to a cationic lipid that remains positively charged at neutral pH and undergoes pH-triggered degradation or ionization under acidic or basic conditions. For example, in some embodiments, a lipid containing an acetal-based backbone can degrade into diols under specific pH conditions, significantly extending the circulation half-life and improving pharmacokinetic performance. The dynamic ionization and degradation profiles of such pH-sensitive lipids modulate nanoparticle surface charge and stability, thereby influencing plasma interactions, biodistribution, and intracellular trafficking of the lipid nanoparticles.

[0064]

[0045] The term “lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the cationic lipids of formula (I) or Formula II. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the present disclosure comprise a nucleic acid. Such lipid nanoparticles typically comprise a cationic lipid of Formula (I) or Formula II and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0065]

[0046] As used herein, the term “nucleic acid payload” refers to any natural, synthetic, or chemically modified nucleic acid molecule, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, derivative, conjugate, or complex thereof, that is encapsulated, associated, or delivered using a lipid nanoparticle (LNP) or other delivery system. Exemplary nucleic acid payloads include, but are not limited to: messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), guide RNAs (gRNAs) for genome editing, CRISPR-associated ribonucleoprotein complexes (CRISPR RNPs), plasmid DNA (pDNA), DNA oligonucleotides, ribozymes, aptamers, and combinations thereof. The nucleic acid payload may be unmodified or chemically modified, such as by incorporation of modified nucleosides, phosphorothioate linkages, cap analogs, base modifications, or conjugation with targeting ligands, to enhance stability, translation efficiency, nuclease resistance, cellular uptake, or tissue specificity, thereby optimizing therapeutic performance.

[0066]

[0047] As used herein, the term “pharmaceutically acceptable salt” refers to any salt form of an ionizable or cationic lipid that is non-toxic, physiologically compatible, and suitable for use in a pharmaceutical composition, including lipid nanoparticles (LNPs) for nucleic acid delivery. Such salts retain the lipid’s functional ability to encapsulate, protect, and deliver nucleic acid payloads, while modulating solubility, stability, or formulation properties. Exemplary pharmaceutically acceptable salts of ionizable or cationic lipids include acid addition salts formed with inorganic acids (e.g., hydrochloric, hydrobromic, sulfuric, phosphoric, nitric acids) or organic acids (e.g., acetic, citric, fumaric, maleic, tartaric, methanesulfonic, p- toluenesulfonic, or benzenesulfonic acids), as well as base addition salts formed with inorganic bases (e.g., sodium, potassium, calcium, magnesium, or ammonium hydroxides) or organic bases (e.g., ethanolamine, diethanolamine, tromethamine, or lysine). This definition also encompasses solvates, hydrates, polymorphs, stereoisomers, tautomers, and other pharmaceutically acceptable derivatives of such salts.

[0067]

[0048] As used herein, the term “helper lipid” refers to a lipid component that, when included in a lipid nanoparticle (LNP) or other lipid-based delivery system, enhances particle stability, structural integrity, or delivery efficiency of the formulation without necessarily carrying a therapeutic payload itself. Helper lipids may include, but are not limited to, neutral, zwitterionic, or structural phospholipids such as l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), phosphatidylserine, phosphatidylglycerol, cholesterol, and derivatives thereof. In certain embodiments, helper lipids facilitate membrane fusion, endosomal escape, lipid nanoparticle self-assembly, and modulation of pharmacokinetics, biodistribution, or tissue targeting. The term encompasses natural or synthetic lipids, as well as pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, and derivatives thereof, and may be used in combination with ionizable, cationic, or other lipid classes within a composition.

[0068]

[0049] As used herein, the term “PEGylated lipid” refers to a lipid molecule that is covalently conjugated to a polyethylene glycol (PEG) moiety or a derivative thereof, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, or derivative thereof. PEGylated lipids are typically included in lipid nanoparticles (LNPs) or other lipid-based delivery systems to modulate particle size, improve colloidal stability, reduce aggregation, and extend circulation time in vivo by providing a steric barrier that decreases nonspecific protein adsorption and recognition by the mononuclear phagocyte system. Exemplary PEGylated lipids include, but are not limited to, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(poly ethylene glycol)] (DMPE-PEG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and other phospholipid-PEG conjugates, as well as PEGylated cholesterol derivatives and other synthetic or natural lipid-PEG conjugates. In certain embodiments, the PEG moiety has a molecular weight in the range of about 500 Da to about 5 kDa, including commonly used PEGs of 1 kDa, 2 kDa, or 5 kDa, although higher or lower molecular weights may also be employed depending on formulation requirements,

[0069]

[0050] As used herein, the term “cholesterol” refers to a sterol or sterol derivative that can be incorporated into lipid nanoparticles (LNPs) or other lipid-based delivery systems to modulate membrane fluidity, stability, and structural integrity of the particle. Cholesterol and its derivatives can influence lipid packing, particle rigidity, encapsulation efficiency, biodistribution, and intracellular delivery of nucleic acid payloads. Exemplary cholesterol derivatives include, but are not limited to, cholesteryl esters, cholesteryl ethers, cholesteryl hemi succinate, and other modified or functionalized cholesterol molecules. The term also encompasses natural or synthetic sterols, or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, and derivatives thereof.

[0070]

[0051] As used herein, the term “composition” refers to any formulation comprising one or more active agents and pharmaceutically acceptable excipients, carriers, or auxiliary agents suitable for administration to a subject. In certain embodiments, the composition comprises a lipid nanoparticle (LNP) or other lipid-based delivery system that encapsulates, complexes, or associates with a nucleic acid payload, including messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), antisense oligonucleotides (ASOs), CRISPR-associated guide RNAs (gRNAs), or ribonucleoprotein complexes (RNPs). The composition may comprise multiple lipid components, including ionizable lipids that facilitate nucleic acid encapsulation and endosomal release; cationic lipids that promote electrostatic interactions and membrane fusion; helper lipids, such as phospholipids (e.g., DOPE, DOPC, DSPC, DSPE) and cholesterol, which enhance structural integrity, membrane fluidity, and particle stability; PEGylated lipids, which provide steric stabilization, reduce aggregation, and modulate circulation time (with PEG moieties typically ranging from 500 Da to 5 kDa); and optionally, peptide-conjugated lipids or targeting ligands, incorporated either pre- or post-peptide conjugation to enhance tissue specificity, receptor targeting, or intracellular trafficking. In some embodiments, the composition is a 4-component LNP, comprising ionizable lipid, helper lipid, cholesterol, and PEGylated lipid, or a 5- component LNP, which additionally includes a cationic or peptide-conjugated lipid. The composition may be provided in any pharmaceutically acceptable form, including solutions, suspensions, emulsions, or lyophilized powders, and may further include buffers, stabilizers, surfactants, or other excipients. The term encompasses salts, solvates, hydrates, stereoisomers, tautomers, derivatives, and mixtures thereof.

[0071]

[0052] The present disclosure relates to novel cationic lipids of Formula I or Formula II including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can be used alone or in combination with other lipid components such as ionizable lipids, sterols, PEGylated lipids, helper lipids and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents.

[0072]

[0053] In one embodiment, the cationic lipid has the structure of Formula I:

[0073] R4 x-

[0074] R3 - N+- m

[0075] R2

[0076] (I) wherein:

[0077] R1 is -(CH2)aQ, or -(CO)C(-OCH2)b -ORA,

[0078] Q is hydrogen, -OH, -NRB, C3-CS, RB, heterocyclic ring optionally substituted by straight chain or branched Ci-Cis alkyl or Cri-Cx heterocyclic ring optionally substituted by straight chain or branched Ci-Cis alkylene;

[0079] RA is hydrogen or Ci-Cis alkyl;

[0080] RB is hydrogen, Cx-Cx saturated or unsaturated carbocyclic ring optionally substituted with Ci- C , alkyl, amino, oxo; or RB along with the N forms a 3 to 10 membered heterocyclic ring; C9- C14 fused heterocycle optionally substituted with Ci-Ce alkyl, amino, oxo.

[0081] R2 and R3 are same or different and is independently selected from:

[0082] -hydrogen; Ci-Cis alkyl; -(CH2)dC(Rc)(Rc )OH, -(CH2)eCOORG; -(CH2)fOCO(O)h(CH2)jCHRcRc’; -(CH2)kCOO(CH2)mCH(RD)(RD), -(CH2)sN(COCi-i8alkyl)[(CH(CH2)pCOO(RD)(RD’))]2; where * denotes the point of attachment; -(CH2)rCOO(CH2)t(S)xRG; -

[0083] (CH2)UCOO(CH2)VC(RC)3; R2 and R3 together form a C3-C8 N-containing ring optionally substituted with 0(RG)O-18. wherein Rc and Rc’ are independently straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; -(CH2)k OCORG, or -(CH2)mOCO(CH2)eCH(O Ro ; wherein RD and RD’ are same or different and independently -(CH2)fCOO RG, straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; and wherein RG is straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; and wherein a, b, d, e, f, h, j, k, m, s, p, q, r, t, u and v are 0-18.

[0084] R4 is C1-C14 alkyl chain, C2-Ci4 alkenyl, C3-C14 alkynyl, C3-C8 carbocyclic ring, aryl, C3-C14 heterocyclic rings; wherein the C1-C14 alkyl chain is optionally substituted with saturated or unsaturated C3-C8 carbocyclic ring; aryl ring, or C3-C14 heterocyclic rings; wherein C3-C8 carbocyclic ring is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings;

[0085] C3-C14 heterocyclic rings is aliphatic heterocyclic or aromatic heterocyclic rings;

[0086] C3-C14 aliphatic heterocyclic ring is selected from

[0087]

[0054] In a preferred embodiment, R4 in compound of formula (I) is a saturated heterocyclic ring selected from and

[0088] X is Cl, Br or I.

[0089]

[0055] In another embodiment the cationic lipid has the structure of Formula H: wherein:

[0090] K is selected from N or N+(R4);

[0091] R2 and R3 are same or different and independently selected from hydrogen; Ci-Ci8alkyl ; -(CH2)dOCO RG; -(CH2)eCOO(CH2)f(S)hRG ; -(CH2)kN+[(CH2)mCONH RG]2; -(CH2)SC(OH)( RG); -(CH2)PCOO(RG); -(CH2)qCONH RG;

[0092] Wherein RG is independently a straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; wherein d, e, f, h, k, m, s, p, and q are 0-18;

[0093] Z is selected from trialkylamino having at least one bond with R4, so as to form a quaternary nitrogen; or

[0094] Z along with R3-N+and K-R3, together forms a C5- C10 heterocyclic ring,

[0095] R4 is C1-C14 alkyl chain, C2-C14 alkenyl, C3-C14 alkynyl, C3-C8 carbocyclic ring, aryl, C3-C14 heterocyclic rings; wherein the C1-C14 alkyl chain is optionally substituted with saturated or unsaturated C3-C8 carbocyclic ring; aryl ring, or C3-C14 heterocyclic rings; wherein C3-C8 carbocyclic ring is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings;

[0096] C3-C14 heterocyclic rings is aliphatic heterocyclic or aromatic heterocyclic rings;

[0097] C3-C14 aliphatic heterocyclic ring is selected from

[0098] C3-C14 aromatic heterocyclic ring is selected from

[0099]

[0056] In a preferred embodiment, R4 in compound of formula (II) is a saturated heterocyclic ring selected from and

[0100] X is Cl, Br or I.

[0057] The cationic lipid of Formula I or Formula II may also be in the form of a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0101]

[0058] Some non-limiting exemplary embodiments of the cationic lipids of Formula (I) or Formula (II) of the present disclosure are described in further detail below in Table 1.

[0102] TABLE 1

[0103] wherein R4 in compound of formula (I) or formula (II) is a saturated heterocyclic ring selected from

[0104]

[0059] It is understood that any embodiment of the cationic lipids of Formula I or Formula II, as set forth above, and any specific substituent and / or variable in the cationic lipids of Formula I or Formula II, as set forth above, may be independently combined with other embodiments and / or substituents and / or variables of cationic lipids of Formula I or Formula II to form embodiments of the inventions not specifically set forth above. It is also understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in stable lipids and formulations.

[0060] Further non-limiting exemplary embodiments of the cationic lipids of Formula I are provided herewith:

[0105]

[0061] Lipid-1 analogues are:

[0106]

[0062] Preferred Lipid-1 analogues are

[0107]

[0108]

[0063] Lipid-2 analogues are

[0064] Preferred Lipid-2 analogues are

[0065] Lipid-3 analogues are

[0109]

[0066] Preferred Lipid-3 analogues are

[0110]

[0111]

[0067] Lipid-4 analogues are

[0068] Preferred lipid-4 analogues are

[0112]

[0069] Preferred lipid-5 analogues are

[0113]

[0114]

[0070] Lipid-6 analogues are:

[0071] Preferred lipid-6 analogues are:

[0115]

[0072] Preferred lipid-7 analogues are:

[0116]

[0117]

[0073] Lipid-8 analogues are

[0074] Preferred lipid -8 analogues are

[0118]

[0075] Preferred lipid -9 analogues are

[0076] Lipid-10 analogues are

[0119] 5

[0077] Preferred lipid-10 analogues are

[0120]

[0121]

[0078] Preferred lipid-11 analogues are

[0122]

[0079] Preferred lipid -12 analogues are

[0123]

[0124]

[0080] Lipid- 13 analogues are

[0125] 5

[0081] Preferred lipid- 13 analogues are

[0126]

[0127]

[0128]

[0084] Preferred lipid -16 analogues are

[0129]

[0130]

[0131]

[0085] Preferred lipid-17 analogues are

[0132]

[0133]

[0086] Preferred cationic lipids of formula (I) are

[0134]

[0135]

[0087] In a more preferred embodiment, the cationic lipid of formula (I) is

[0136] “Lipid-1-1”;

[0137]

[0088] In an aspect of the present invention a process for the preparation of the cationic lipid of formula I or Formula II is disclosed wherein the process comprises the steps of: a) preparing a precursor LNP to Formula I or Formula II; b) reacting the precursor with a suitable ligand R4; wherein:

[0138] R4 is C1-C14 alkyl chain, C2-C14 alkenyl, C3-C14 alkynyl, C3-C8 carbocyclic ring, aryl, C3-C14 heterocyclic rings;

[0139] Wherein the C1-C14 alkyl chain is optionally substituted with saturated or unsaturated C3-C8 carbocyclic ring; aryl ring, or C3-C14 heterocyclic rings; wherein C3-C8 carbocyclic ring is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings;

[0140] C3-C14 heterocyclic rings is aliphatic heterocyclic or aromatic heterocyclic rings;

[0141] C3-C14 aliphatic heterocyclic ring is selected from C3-C14 aromatic heterocyclic ring is selected from

[0142]

[0089] In a preferred embodiment of the process for the preparation of the cationic lipid of formula I or Formula II, R4 is a saturated heterocyclic ring selected from and

[0143] X is Cl, Br or I.

[0144]

[0090] In an embodiment, a Lipid nanoparticle (LNP) formulation of the present invention comprises at least one cationic lipid of Formula I or Formula II, at least one PEGylated lipid, and cholesterol. In an embodiment, the formulation comprises 30 to 70% by weight of cationic lipid, 10 to 30% by weight of cholesterol, 15 to 30% by weight of PEGylated lipid.

[0145]

[0091] In a specific embodiment, the formulation of the present invention further comprises at least one helper lipid or an ionizable lipid or a combination thereof.

[0146]

[0092] In another embodiment, the formulation of the present invention comprises at least one cationic lipid, at least one PEGylated lipid, cholesterol and at least one helper lipid. In an embodiment, the formulation comprises 30 to 60% by weight of cationic lipid, 35 to 50% by weight of cholesterol, 1 to 5% by weight of PEGylated lipid, 7 to 10% by weight of helper lipid.

[0093] In an embodiment, the LNP formulation may be at least a 3- component, 4- component or a 5- component system.

[0147]

[0094] In yet another embodiment, the formulation of the present invention comprises at least one cationic lipid, at least one PEGylated lipid, cholesterol and at least one ionizable lipid.

[0148]

[0095] In a different another embodiment, the formulation of the present invention comprises at least one cationic lipid, at least one PEGylated lipid, cholesterol, at least one helper lipid and at least one ionizable lipid.

[0149]

[0096] In an embodiment, the formulation comprises 10 to 40% by weight of cationic lipid, 35 to 50% by weight of cholesterol, 1 to 5% by weight of PEGlyated lipid, 7 to 10% by weight of helper lipid, 10 to 40% by weight of Ionizable lipid.

[0150]

[0097] In yet another embodiment, the formulation of the present invention comprises the cationic lipid and the ionizable lipid in a suitable ratio.

[0151]

[0098] In an embodiment the process for the preparation of a formulation of the present invention includes preparing a formulation comprising at least one cationic lipid of formula I or Formula II by a method with the steps of: a. preparing the cationic lipid of Formula I or Formula II; b. preparing an ethanolic mixture comprising ionizable lipid, cholesterol and PEG wherein the ionizable lipid, cholesterol and PEG are in a suitable ratio; c. adding buffer; d. adding the aqueous phase rapidly to the ethanolic lipid mixture in a ratio of 3: 1 aqueous: ethanol v / v; e. mixing the solution of step a and step b and then leaving it undisturbed to obtain the formulation.

[0152]

[0099] In other embodiments, the present disclosure provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition of lipid nanoparticles comprising the cationic lipids of Formula I or Formula II and a therapeutic agent and delivering the composition to the patient.

[0100] In an aspect of the invention, the present disclosure pertains to novel cationic lipids of Formula I or Formula II including stereoisomers, pharmaceutically acceptable salts or tautomers thereof that are pH sensitive cationic lipids and unexpectedly achieve selective organ tropism.

[0153]

[0101] In a preferred embodiment, the cationic lipids of the present disclosure are active in the range of pH 3.5-7. The novel cationic lipids of Formula I or Formula II demonstrate selective tissue tropism in vivo. Preferably, the present disclosure demonstrates selective tissue tropism for cell types in the kidney, lungs, spleen, heart and other organs. In another aspect, the cationic lipids of the present disclosure show localization in the desired target tissue with high efficacy without unwanted hepatic accumulation.

[0154]

[0102] In an embodiment, the cationic lipids of Formula I or Formula II comprise of at least one quaternary ammonium nitrogen.

[0155]

[0103] The lipid nanoparticle compositions of the present disclosure achieve targeted extra hepatic delivery while being minimally cytotoxic with high efficiency in achieving organ / tissue / cell specific delivery.

[0156]

[0104] The present invention relates to cationic lipid nanoparticles directed towards extra hepatic delivery and further organ / tissue tropism. When delivering drug products, nucleic acids and other therapeutics to specific organs and tissues, the cationic lipid nanoparticles of the present invention successfully prevent accumulation of the therapeutic products in the liver and are able to direct them to specific target organs or tissues. Thus the cationic lipid nanoparticles of the present invention demostrate tissue and organ tropism specifically detargeting the liver.

[0157]

[0105] The present invention further discloses LNP formulations comprising at least one cationic lipid, along with a helper lipid or an ionozable lipid, cholesterol and at least one PEGylated lipid. The LNP formulations also exhibit unexpected extra hepatic delivery tissue / organ tropism specifically detargeting the liver. The accumalation of drug products, and other therapeutics in the liver is prevented by the LNPs and their formulations as dislcosed in the present invention. This is an unexpected improvement over LNPs of prior art which have a tendency to localize in the liver. The formulations of the present invention have demonstrated localization in kidneys, lung, spleen among other organs and tissues.

[0106] In an aspect of the present invention the LNP formulations have siginificant encapsulation efficicency. The average encapsulation of the LNP formulations of the present invention is more than 80%. The inclusion of cationic lipids of the present invention, with its positive surface charge, enhances electrostatic interactions with the mRNA cargo, thereby improving encapsulation efficiency, resulting in a higher payload per particle and more stable nanoparticles that better protect the cargo until release. The LNP formulations of the present invention enable efficient intracellular payload delivery.

[0158]

[0107] In another aspect of the present invention the LNP formulations are not cytotoxic. Where severe morphological changes and extensive cell debris is common for conventional LNP carriers the LNP formulations of the present invention preserve cell integrity and viability under similar conditions. The low cytotoxicity of the inventive formulation stems from the headgroups, which are more hydrophilic and less reactive than the tertiary amine and ester linkages present in ALC-based conventional lipids. This design limits non-specific interactions with cell membranes, making the lipids of the invention more biocompatible and less fusogenic. The inventive lipid architecture reduces membrane-disruptive toxicity in vivo. This advantage again stems from the optimized headgroups, thereby limiting nonspecific interactions while maintaining efficient intracellular release. Moreover, under acidic conditions after endocytosis, the cationic lipids degrade and neutralize their charge, further reducing membrane interactions while maintaining efficient intracellular delivery contributing to effective release of payload.

[0159]

[0108] Various non-limiting LNP formulations as per the present invention are provided herewith in Table 2:

[0160] TABLE 2 General scheme of preparation:

[0161]

[0109] The present invention is directed towards the process of preparing the cationic lipids of Formula I or Formula II The present invention further discloses a process of preparing the lipid nanoparticle formulations.

[0162]

[0110] In an aspect of the present invention, the lipid nanoparticles were prepared by the manual pipetting method. To prepare the three-component system, the ethanolic lipid mixture was prepared including cationic lipid, cholesterol and PEG. For the four-component system the ethanolic lipid mixture was prepared where the molar ratios of the component were cationic lipid, cholesterol, helper Lipid and PEG. Sodium acetate buffer (pH4, 20mM-35mM), the aqueous phase was rapidly added to the ethanolic lipid mixture in the ratio of 3: 1 aqueous: ethanol (v / v) and thoroughly mixed by pipetting and left undisturbed at room temperature for 15 minutes. The resulting formulations were diluted with DPBS (IX), subjected to buffer exchange, and finally concentrated using centrifugal concentrators.

[0163]

[0111] In an aspect of the present invention, pH-sensitive lipids behave as cationic lipids that results in higher encapsulation of the therapeutic payload (>95% encapsulation).

[0164]

[0112] The present disclosure successfully delivers drug candidates or therapeutic agents to different organs and tissues in vivo, particularly in the human body specifically avoiding the liver.

[0165]

[0113] The present disclosure may also be used for co-delivery of more than one drug candidate or therapeutic agent to different organs and tissues in vivo, particularly in the human body specifically avoiding the liver.

[0166]

[0114] Encapsulation by the Lipid nanoparticle formulation of the present disclosure: The encapsulation of nucleic acids within LNPs is achieved through manual pipette mixing method. During this process, ethanolic mixture of lipids is rapidly mixed with an aqueous buffer (pH = 3.0-4.0) containing gene editing machinery (Cas9 mRNA and guide RNAs) at a ratio of 1 :3 (v / v). This rapid mixing results in the spontaneous self-assembly of lipids into nanoparticles, effectively encapsulating the nucleic acids within a lipid bilayer. Usually, acetate and citrate buffers are commonly used as aqueous phase during the formulation of LNPs to maintain a mildly acidic environment (pH close to 4). This acidic pH ensures that ionizable lipids are protonated, enhancing their positive charge. The protonated state of the lipids at this pH promotes strong electrostatic interactions with negatively charged phosphate backbone of the mRNA, facilitating effective encapsulation. This interaction is essential for encapsulating the mRNA within the lipid nanoparticle. Apart from electrostatic interactions, the hydrophobic tails of these lipids help in forming the lipid bilayer or monolayer structure around the mRNA. This structure creates a protective environment, shielding the mRNA from enzymatic degradation and promoting its stability during delivery.

[0167]

[0115] During LNP formation, ionizable cationic lipids, helper lipids (like DSPC- distearoylphosphatidylcholine), cholesterol, and polyethylene glycol (PEG) lipids come together in the presence of mRNA to form a nanoparticle. The positively charged ionizable lipids condense the mRNA into the core of the LNP by forming a complex with it, ensuring efficient encapsulation and delivery.

[0168]

[0116] After the LNPs are formed, they are subjected to a buffer exchange step to replace acidic buffer with a neutral pH buffer, preferably DPBS, for storage and subsequent use. At physiological pH, the ionizable lipids become neutral, reducing their interaction with the mRNA and minimizing any potential cytotoxicity. The end product of this process is a uniform nanoparticle suspension of RNA core in lipid shell, typically ranging from 100 - 150 nm in size, zeta potential +10 mV to -10 mV, PDI < 0.2, and 60-90% encapsulation efficiency, which is compatible with physiological pH and suitable for cellular uptake. The combination of electrostatic interactions, self-assembly, structural stability provided by cholesterol and DSPC, PEGylation, and efficient endosomal escape facilitated by ionizable cationic lipids ensures the effective encapsulation and delivery of gene editing machinery for genome editing applications.

[0169]

[0117] Once the LNPs are formulated and the nucleic acids are encapsulated, they are administered to the target cells where they release their cargo through several steps. The LNPs are primarily taken up by the cells via endocytosis, after which they are trapped in endosomes. The ionizable cationic lipids within the LNPs become protonated in the acidic environment of the endosome, causing the nanoparticles to destabilize the endosomal membrane and facilitate the release of the nucleic acid cargo into the cytoplasm. Once released, Cas9 mRNA is translated into the Cas9 protein which is guided towards the target DNA sequence with the help of guide RNAs, thus, enabling a precise gene editing event.

[0118] In other different embodiments, the disclosure is directed to a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing any of the foregoing compositions and administering the composition to the patient.

[0170]

[0119] For the purposes of administration, the cationic lipids of the present disclosure (typically in the form of lipid nanoparticles in combination with a therapeutic agent) may be administered as a raw chemical or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present disclosure comprise cationic lipids of Formula I or Formula II and one or more pharmaceutically acceptable carrier, diluent or excipient. The cationic lipids of Formula I or Formula II is present in the composition in an amount which is effective to form a lipid nanoparticle formulation and deliver the therapeutic agent, e.g., for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one skilled in the art.

[0171]

[0120] Administration of the compositions of the disclosure can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the disclosure may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. Pharmaceutical compositions of the disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of cationic lipid of the disclosure in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of cationic lipid of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this invention.

[0172] EXAMPLES

[0173]

[0121] Example 1 : Preparation of Lipid-1-1

[0174] To a stirred solution of compound MC3 (80 mg, 0.124 mmol) in acetonitrile (2.5 mL), dimethyl dioxolane iodide (191 mg, 0.747 mmol) and sodium carbonate (Na2COs) (26 mg, 0.24 mmol) were added under a nitrogen atmosphere in a sealed tube. The reaction mixture was stirred at 65 °C for 6 hours. After completion, the reaction mixture was concentrated under high vacuum using a rotary evaporator, maintaining the bath temperature at room temperature (RT). The resulting crude product was purified by aluminum oxide neutral chromatography, eluted with 2-3% methanol in dichloromethane (DCM: MeOH-9: l, stain: Iodine) to obtain pure ALS-UB-MC3-D / Lipid-l (105 mg) as a colorless, thick oily compound. NMR (400 MHz, CDC13) 5: ppm: 5.41-5.29 (m, 8H); 4.84 (m, 1H); 4.19 (m,2H);3.94-3.87 (m, 1H); 3.78-3.68 9m, 3H);2.77 (t, 3H);2.51 (t, 2 H); 2.07-2.01 (m, 9H); 1.52-1.27 (m, 43H); 0.89(m, 6H)

[0175]

[0122] Example 2: Preparation of Lipid-2-1

[0176] To a stirred solution of compound ALC0315 (200mg, 0.261mmol) in acetonitrile (4ml), dimethyl dioxolane iodide (267mg, 1.04mmol) and sodium carbonate (ISfeCCE) (55mg, 0.52mmol) were added under nitrogen atmosphere in a seal tube. The reaction mixture was stirred at 80°C for 48 hours. After completion, the reaction mixture was then concentrated under high vacuum using a rotary evaporator, maintaining the bath temperature at room temperature (RT). The resulting crude product was purified by aluminium oxide neutral chromatography, eluted with 2-3% methanol in dichloromethane (DCM: MeOH - 9: 1, stain: Iodine) to obtain pure ALS-UB-ALC0315-D / Lipid-2 (160 mg) as a colourless thick oily compound. NMR (400 MHz, CDC13) 5: ppm: 4.22-4.17 (m, 2H)4.06 (t, 4H); 3.75 (m, 3H); 3.56(m, 3H); 3.41-3.32 (m, 5H);2.34-2.27(m, 3H); 1.94-1.25 (m, 75H); 0.87 (m, 12H).

[0177]

[0123] Example 3: Preparation of Lipid-3-1

[0178] To a stirred solution of compound SM102 (75mg, 0.105mmol) in acetonitrile (5ml), dimethyl di oxolane iodide (107mg, 0.417mmol) and sodium carbonate (Na2CC>3) (22mg, 0.207mmol) were added under nitrogen atmosphere in a seal tube. The reaction mixture was stirred at 80°C for 63hours. After completion, the reaction mixture was then concentrated under high vacuum using a rotary evaporator, maintaining the bath temperature at room temperature (RT). The resulting crude product was purified by aluminium oxide neutral chromatography eluted with 2-3% methanol in di chloromethane (DCM: MeOH - 9:1, stain: Iodine) to obtain pure ALS-UB- SM102-D / Lipid-3 (35 mg) as a colourless thick oily compound. NMR (400 MHz, CDC13) 5: ppm: 4.85 (m, 1H); 4.18-4.13 (m, 4H); 4.05 (t, 2H); 3.72-3.31(m, 8H); 2.36-2.24 (m, 4 H); 1.81-1.25 (m, 72 H); 0.87 (t, 9H).

[0179] Example 4: Biological Data:

[0180]

[0124] Experimental data with respect to organ tropism achieved by the LNP of the present disclosure

[0181]

[0125] Materials: Cationic lipids were designed and synthesized in house. Cholesterol, 1,2- distearoyl-sn-glycero-3-phosphocholine, 16:0 PEG2000 PE and DiR dye were purchased from Medchem Express. DPBS and Nuclease free water were procured from Invitrogen, and lOOkDa centrifugal filters were purchased from Merck.

[0126] (4A) Formulation of lipid nanoparticles: The lipid nanoparticles were prepared by the manual pipetting method. Lipid mixture containing cationic lipid, cholesterol, helper lipid and PEG was prepared with 2mol% DiR dye to label the LNPs followed by rapidly mixing with the aqueous phase (Sodium acetate buffer; pH 4, 20mM-35mM) in the ratio of 1 :3 v / v (aqueous: ethanol). After mixing, LNPs were left undisturbed at room temperature for 15 minutes. The resulting formulations were diluted 3 times with DPBS (IX) followed by buffer exchange using centrifugal filters. Formulated LNPs were stored at 4°C until use.

[0182]

[0127] (4B) Characterization of lipid nanoparticles: Lipid nanoparticles were characterized for size and surface zeta potential using Anton Paar Kalliope Litesizer 500. Prior to analysis, the LNPs were diluted 40-fold in PBS. Three technical replicates were conducted on each sample for both size and surface zeta potential. LNPs were found to be in the range of 150-200nm in size with PDIs less than 0.2. Zeta-potential of the formulations and were in the range of -4 to +4mV.

[0183]

[0128] (4C) In vivo biodistribution of lipid nanoparticles: 6-8-week-old BALB / c mice weighing 20-25 gm were administered with LNPs containing DiR dye (0.16mg / Kg body weight) via tail vein injection. After 6 h, in vivo fluorescence imaging (IVIS Lumina III, Perkin Elmer) was carried out for DiR signals. Mice were euthanized, and major organs were harvested (lungs, liver, spleen, heart, and kidneys) and imaged for ex vivo fluorescence.

[0184]

[0129] Observation from the ex-vivo imaging show organ tropism being achieved by the formulations of the present invention. In Figure 1, the results demonstrate that formulation F.4 containing Lipid 1 have achieved lung tropism in figure 1. The Lipid 1 is a specialized lipid used in lipid nanoparticle (LNP) formulations was engineered to enhance the delivery and absorption of therapeutic agent specifically by the lungs. The targeting mechanism of quaternary charged Lipid 1 involves its unique surface properties, which include an optimized charge, pKa of the LNP >8, and a finely tuned hydrophilic / hydrophobic balance — that facilitate interaction with lung epithelial cells and alveolar macrophages. The highly hydrophobic linoleic acid chains, with conjugated double bonds (sp2hybridized), stabilize the bilayer core, supporting deep lung penetration and prolonged circulation. These features, along with the hydrophilic headgroup surface properties, enable efficient accumulation in the lungs while limiting premature hepatic or renal clearance.

[0130] The linoleic acid chains, being highly hydrophobic and furthermore SP2 Hybridized on this chain length, help to stabilize the lipid bilayer by promoting strong hydrophobic interactions within the core of the nanoparticle. They are better suited for deep lung penetration and longer circulation times, which increases their chances of reaching and accumulating in the lungs. Furthermore, Lipid 1 hydrophilic surface properties enhance the stability and dispersion of LNPs in the bloodstream, preventing premature clearance by the liver and kidneys. These combined attributes make Lipid 1 a critical component in LNP formulations designed for effective lung-targeted drug delivery.

[0185]

[0131] In figure 2, formulation F.6 containing Lipid 2 showed specificity for kidney tropism. The F.6 formulation, a 4-component LNP containing 46.3% Lipid 2, exhibited distinct kidney tropism in vivo, as confirmed by ex vivo biodistribution imaging in Fig. 2. Unlike MC3- or ALC-0315-based controls that preferentially accumulate in the liver, incorporation of Lipid 2 redirected delivery to the kidney. Notably, these LNPs demonstrated extended circulation times, enabling them to persist even after partial hepatic absorption, thereby evading rapid hepatic clearance and facilitating accumulation in extra-hepatic tissues, including the kidney. The unique composition of 4C forms a unique protein corona to be available for organs beyond the liver, and the optimized size, charge and pKa enable uptake by the kidney.

[0186]

[0132] The formulation F.9 containing Lipid 2 in the cationic lipid as well as ionizable lipid in a ratio of 70:30 demonstrated spleen tropism as depicted in Fig. 3. The formulation F.9, containing Lipid 2 cationic lipid in combination with ALC-0315 ionizable lipid at a 70:30 ratio, demonstrated distinct spleen tropism. While ALC-0315-based formulations typically exhibit strong liver accumulation without organ selectivity, incorporation of Lipid 2 altered the biodistribution profile to favor spleen targeting. This shift arises from the cationic lipid’s influence on surface charge and hydrophilic-hydrophobic balance, which enhances uptake by spleen-resident immune cells such as macrophages and dendritic cells. In addition, particle size and morphology — modulated by the lipid composition — facilitate splenic retention, as the spleen preferentially filters nanoparticles within a defined size window. Once in circulation, the formation of a unique protein corona avoids ApoE protein interaction and thereby accumulation in the liver, which further directs the particles toward splenic compartments, highlighting the role of rational cationic lipid design in tuning organ tropism.

[0133] The formulation F.8-2 containing Lipid 1 as well as an ionizable lipid as per Table 2 demonstrated kidney tropism as depicted in Fig. 4a Systemic IV delivery of F8-2 formulation, containing 4.63% of Lipid 1 and 46.3% of ionizable lipid, encapsulating mRNA demonstrated robust kidney localization, with reduced hepatic signal compared to F3-1 formulation (Control) containing corresponding amounts of DOTAP and ALC0315 in the place of Lipid 1 and ionizable lipid.

[0187]

[0134] Fig. 4b further reflects the quantification of luciferase expression post intravenous injection. It is observed that F8-2 achieved near-equal biodistribution between kidney and liver, whereas F3-1 exhibited significantly higher liver accumulation, reinforcing efficacy of F.8-2 (Lipid 1) in kidney targeting and minimizing off-target biodistribution to the liver. Lipid 1, a pH-sensitive cationic lipid used herein in F.8-2, remains neutral in circulation, reducing nonspecific interactions and clearance by the reticuloendothelial system, especially in the liver. This reduces opsonization and uptake by hepatocytes, allowing more nanoparticles to circulate and reach the kidney. Formulation F8-2 displays the combination of the optimal size, charge, and pKa essential for its uptake by the kidney, which has a different pKa (pKa =7-8) in comparison to that of the liver (pKa = 6-7).

[0188] Organ-wise biodistribution of inventive formulations

[0189]

[0135] In vivo biodistribution of three formulations F.11, F.12 and F.13 containing a commercial ionizable lipid with different inventive cationic lipids was evaluated in Balb / c mice using IVIS imaging following intravenous injection of firefly luciferase mRNA-LNPs (0.5 mg / kg; 6-hour timepoint).

[0190] Formulation F.ll (Lipid 2 cationic lipid): Signal was broadly distributed across major organs, with highest accumulation in the spleen, followed by lung and liver (Fig. 4c).

[0191] Formulation F.12 (Lipid 3 cationic lipid): Distribution was again strongest in spleen and lung, but with comparatively higher uptake in kidney and heart. (Fig. 4d).

[0192] Formulation F.13 (Lipid 1 cationic lipid): The spleen remained the dominant site of signal, alongside lung and liver. (Fig. 4e)

[0193]

[0136] Together, these data highlight how substituting the cationic lipids of the present invention alter organ tropism thereby playing a critical role in extrahepatic delivery. Example 5: Translational efficacy of the LNP of the present invention

[0194] Encapsulation Efficiency (E8-2, kidney)

[0195]

[0137] Encapsulation efficiency was assessed for inventive formulations F8-2 and F3-1 (Control). Ribogreen assay demonstrated that F8-2 achieved an average encapsulation efficiency of 83.99%, significantly higher than F3-1, which averaged 42.5%, owing to the unique structural design of the cationic Lipid 1 in the F8-2 formulation. Fig. 5a is the graphical comparison of the encapsulation efficiencies.

[0196]

[0138] Encapsulation efficiency was assessed for other inventive formulations F. ll, F. 12 and F.13 as well comprising Lipids 1-3 respectively. Fig. 5b shows that the inventive formulations demonstrated higher encapsulation efficiency (57-67%) compared to the F3-1 (DOTAP containing Control). It was observed that replacement of DOTAP with rationally designed cationic lipids (Lipids 1-3) increased encapsulation efficiency to >55%, compared to 42% for the control. The inventive lipids improved nucleic acid packaging and nanoparticle stability, enabling higher payload per particle.

[0197] Example 6: Routes of administration (F.8-2, kidney)

[0198]

[0139] Administration of inventive formulation F8-2 via the retrograde ureteral (RU) route resulted in highly localized kidney accumulation, with negligible liver distribution. Fig. 6a reflects the in vivo and ex vivo imaging at 6 h post-injection confirming strong renal tropism, thereby validating the utility of F8-2 for localized kidney delivery. This route-dependent specificity highlights not only the targetability of the F8-2 LNP, but also its robustness in withstanding direct injection into a high-pressure organ environment such as the kidney.

[0199] Example 7: Biodistribution of inventive formulation in Disease Mice Model

[0200]

[0140] In the PKD1 knockout model (Pkdlfl / fl; Cdhl6-CreERT2ki / wt) of ADPKD, inventive formulation F8-2 was administered at 12 weeks of age, when kidneys are highly cystic and the disease phenotype is advanced. Both intravenous (IV) and retrograde ureteral (RU) injections were performed, and organ-level biodistribution was assessed 6 h post-administration. In both routes, kidney delivery was observed, with RU yielding markedly higher renal accumulation. Fig. 7 shows that inventive formulation F8-2 retains its kidney-targeting capacity even under conditions of severe structural distortion (enlarged and heavily cystic kidneys), underscoring its translational relevance for treating advanced kidney disease. The ability to maintain encapsulation, avoid cytotoxicity, and demonstrate controlled release under such physiologically demanding conditions underscores its translational potential for one-time or infrequently redosed therapies such as cell and gene therapies.

[0201] Example 8: Cell-specific Uptake in the Disease Mice Model

[0202]

[0141] Cell-specificity analysis of F8-2 (0.4 mg / kg) administered via the retrograde ureteral route in the PKD 1 knockout model (Pkdlfl / fl; Cdhl6-CreERT2ki / wt) at 13-14 weeks of age demonstrated homogenous renal epithelial uptake. Immunofluorescence at 6 h post-injection revealed strong reporter expression in both collecting duct (AQP2) and proximal tubular (LRP2) epithelial cells as reflected in Figure 8. The optimized cationic lipid composition in F8- 2 enhances targeting tubular epithelial cells, and also enables delivery to cyst lining cells and cysts themselves. The unique composition of the inventive LNP F8-2 displays an optimal size, charge, and pKa that allow for longer interaction of the LNPs with the tubular epithelial cells even in the scenario of the retrograde ureteral injection, which is against the flow of urine and hence can lead to washing away of the LNPs before uptake by the target cells. This cell-specific uptake underscores the therapeutic relevance of the inventive formulations for kidney diseases rooted in renal tubular epithelial dysfunction.

[0203] Example 9: In vivo Tolerability of F8-2 (5C with Lipid 1-1)

[0204]

[0142] Retrograde ureteral (RU) injection: BALB / c mice were administered F8-2 via the RU route, and kidneys were collected 24 h later for histopathological analysis. Examination of both major organs and the injected kidney revealed no evidence of moderate to severe tissue damage in the immunohistochemistry images (H4C) (Figure 12a). This favorable safety profile aligns with the preserved cell viability observed in vitro, confirming the biocompatibility of the optimized formulation under physiologically demanding conditions.

[0205]

[0143] Renal artery (RA) injection: Tissue toxicity was assessed 24 h post-renal artery administration. Differential interference contrast (DIC) imaging of cryosections showed that F3-1 (ALC-0315 + DOTAP) induced notable cytotoxicity in the medullary region, whereas F8-2 maintained tissue integrity with minimal adverse changes (Fig. 12b). Together, these findings extend the earlier in vitro observations (Figure 11) by confirming that the F8-2 architecture reduces membrane-disruptive toxicity in vivo. This advantage stems from the optimized Lipid structure, which is more hydrophilic and less reactive than tertiary amineester systems, thereby limiting nonspecific interactions while maintaining efficient intracellular release Example 10: Effective genome editing

[0206]

[0144] Functional validation of delivery by the inventive formulations was confirmed by genome editing experiments in patient-derived ADPKD kidney epithelial cells (WT9-7). F8-2 LNP-mediated CRISPR Cas9 mRNA and gRNA co-delivery resulted in efficient editing of the target, as assessed by T7E1 endonuclease assay and Sanger sequencing. Fig. 9 compares the editing rates were comparable to those obtained with benchmark liposomal controls (MessengerMax) while maintaining superior cell viability. These results establish that the inventive formulation not only achieves cell-type specific delivery but also supports functional genome modification, thereby extending its utility beyond reporter expression to therapeutic gene editing applications.

[0207] Example 11 : Stability of LNP formulations

[0208]

[0145] Formulation F.8-2 LNPs maintained particle size and encapsulation efficiency for up to 90 days at 4°C (Figure 10), demonstrating robust stability under standard refrigeration. This addresses a key limitation of current mRNA-LNPs that require ultra-cold storage, enabling practical deployment and broader therapeutic use. The inventive cationic lipids, such as Lipid 1, enable to stabilize lipid-cargo interactions during circulation and uptake. This design maintains stability across temperature variations, preventing aggregation and lipid fusion and ensuring the nanoparticles remain monodisperse, with size and the polydispersity index preserved during above-zero storage.

[0209] Example 12: Cytotoxicity of LNP formulations

[0210]

[0146] Cytotoxicity was evaluated in WT9-7 kidney epithelial cells for Formulation F.8-2 and F3-1 (Control) at 6 hours and 24 hours after delivery. It was observed that Formulation F.8-2 preserved cell integrity and viability whereas F3-1 (Control) induced severe morphological changes and extensive cell debris by 24 h, under identical conditions (Figure 11). The low cytotoxicity of the F.8-2 formulation is due to the headgroups of the inventive Lipid, which is more hydrophilic and less reactive than the tertiary amine and ester linkages present in ALC- based lipids. This limits non-specific interactions with cell membranes, making the inventive Lipid more biocompatible and less fusogenic. Moreover, under acidic conditions after endocytosis, the cationic lipids degrade and neutralize their charge, further reducing membrane interactions while maintaining efficient intracellular delivery contributing to effective release of payload.

Claims

Claims :

1. A cationic lipid or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof represented by a structure of formula (I):R4XR3 - N+- R1R2 wherein:R1 is -(CH2)aQ, or -(CO)C(-OCH2)b -ORA,Q is hydrogen, -OH, -NRB, C3-CS, RB, heterocyclic ring optionally substituted by straight chain or branched Ci-Cis alkyl or C3-C8 heterocyclic ring optionally substituted by straight chain or branched Cl -Cl 8 alkylene;RA is hydrogen or Ci-Cis alkyl;RB is hydrogen, C3-C8 saturated or unsaturated carbocyclic ring optionally substituted with Ci- C>, alkyl, amino, oxo; or RB along with the N forms a 3 to 10 membered heterocyclic ring; C9- C14 fused heterocycle optionally substituted with Ci-Ce alkyl, amino, oxo.R2 and R3 are same or different and are independently selected from:-hydrogen; Ci-Ci8alkyl; -(CH2)dC(Rc)(Rc )OH, -(CH2)eCOORG;-(CH2)fOCO(O)h(CH2)jCHRcRc’; -(CH2)kCOO(CH2)mCH(RD)(RD’), -(CH2)sN(COCi-i8alkyl)[(CH(CH2)pCOO(RD)(RD’))]2;where * denotes the point of attachment; -(CH2)rCOO(CH2)t(S)xRG; - (CH2)UCOO(CH2)VC(RC)3; R2 and R3 can together form a C3-C8 N-containing ring optionally substituted with 0(RG)O-18.wherein Rc and Rc’ are independently straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; -(CH2)k OCORG, or -(CH2)mOCO(CH2)e CH(0RG)2; wherein RD and RD’ are same or different and independently -(CH2)fCOO RG, straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; and wherein RG is straight chain or branched Ci-Cis alkyl or straight chain or branched Ci-Cis alkylene; and wherein a, b, d, e, f, h, j, k, m, s, p, q, r, t, u, v and x are 0-18.R4 is a saturated heterocyclic ring selected fromandX is Cl, Br or I.

2. The cationic lipid as claimed in claim 1, wherein R1 is -(CH2)aQ, or -(CO)C(-OCH2)b -ORA, wherein Q is hydrogen, OH, -NRB, CX-CX. RB; RA and RB are as defined in claim 1 for formula I; wherein R2 and R3 are same or different and is independently selected from - hydrogen; Ci-Ci8alkyl; -(CH2)eCOORG; -(CH2)fOCO(O)h(CH2)jCHRcRc’; (CH2)kCOO(CH2)mCH(RD)(RD’). wherein R4 is selected from:

3. The cationic lipid as claimed in claim 1, wherein R1 is -(CTLX OH; R2 and R3 are independently selected from -(CH2)CCOORG, -(CH2)fOCO(O)h(CH2)jCHRcRc’; (CH2)kCOO(CH2)mCH(RD)(RD’).

4. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) iswherein R4 in compound of formula (I) is a saturated heterocyclic ring selected from5. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) iswherein R4 is selected from:

6. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) iswherein R4 is selected from:

7. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) iswherein R4 is selected from:

8. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) is9. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) is10. The cationic lipid as claimed in claim 1, wherein the compound of formula (I) is11. A lipid formulation comprising a cationic lipid of formula (I) as claimed in any one of the preceding claims, a cholesterol, PEG or PEGylated lipid.

12. The formulation as claimed in claim 10, comprising 30-70% by weight of cationic lipid, 10-30% by weight of cholesterol, 15-30% by weight of PEGylated lipid.

13. The formulation as claimed in claim 10, wherein the formulation comprises helper lipid and / or an ionizable lipid.

14. The formulation as claimed in claim 12, comprising 30-60% by weight of cationic lipid, 35-50% by weight of cholesterol, 1 -5% by weight of PEGylated lipid, 7-10% by weight of helper lipid.

15. The formulation as claimed in claim 12, comprising 10-40% by weight of cationic lipid, 35-50% by weight of cholesterol, 1-5% by weight of PEGlyated lipid, 7-10% by weight of helper lipid, 10-40% by weight of Ionizable lipid.

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