DESC:RELATED PATENT APPLICATION:

This application claims the priority to and benefit of Indian Patent Application No. 202341025033 filed on April 01, 2023; the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION:

The present invention relates to novel lipid compounds of structural formula of VII and XII, a process for synthesis, intermediates used in the synthesis and use thereof. The invention discloses use of the novel lipids for preparing self-assembling lipid nanoparticle compositions having efficacy for delivery of therapeutic agents including nucleic acids (NA) into cells.

More specifically invention provides composition comprising cationic lipids: Dendritic cells (DCs)-targeting lipids that contain both transfection-enhancing guanidine functionality and DC-targeting mannose-mimicking shikimoyl- functionality in their polar head-group region, endosome-disrupting histidinylated lipids; co-lipids; and one or more pharmaceutically acceptable excipients for delivering the nucleic acids in the Antigen Presenting Cells (APCs) via mannose receptors for inducing effective immune response against the various diseases including the infectious disease.

BACKGROUND OF THE INVENTION:

An update from WHO on December 6, 2022, revealed more than 6.6 million deaths worldwide due to corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). The dreaded disease led to a severe global economic crisis. This extraordinary situation prompted entities in government, industry, and academia to work together at unprecedented speed to develop safe and effective vaccines (Krammer F. 2020. SARS-CoV-2 vaccines in development. Nature 586:516–27). A worldwide race has been witnessed to tame Covid-19. Vaccines of multiple types have been developed in record time, many have been evaluated in clinical trials, and many more are in late-stage clinical development (Li Y et al. 2021. A Comprehensive Review of the Global Efforts on COVID-19 Vaccine Development. ACS Cent. Sci. 7: 512-533). A total of 256 COVID-19 vaccine candidates have been developed by the end of February 2021 out of which 74 are in clinical trials and 182 in preclinical studies. Out of the 74 clinical candidates, 16 are undergoing further validations in Phase 3 or Phase 4 clinical trials with large numbers of volunteers in terms of their safety and efficacy. (Draft landscape of COVID-19 candidate vaccines. https:// www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines (accessed December 11, 2020)).

Design strategies for vaccines are based on various existing approaches including live attenuated disease-causing microorganisms or inactivated vaccines, recombinant protein-based vaccines (protein subunit vaccines, virus-like particles (VLP)), viral vector vaccines, and nucleic acid vaccines (DNA and mRNA-based vaccines). Among these strategies, mRNA vaccines are showing great promises and are entering into clinical trials at an unprecedented speed. (Pardi N, 2018. mRNA vaccines—a new era in vaccinology. Nat. Rev. Drug. Discov. 17:261–79; Pardi N et al. 2020. Recent advances in mRNA vaccine technology. Curr. Opin. Immunol. 65:14–20).

The most distinguishing advantages of this vaccine type over traditional platforms include its synthetic nature and sequence-independent manufacturing process which allows fast and flexible vaccine design and production. Compared to DNA vaccines which need to access to the cell nucleus, mRNA vaccines only need access to the cytoplasmic ribosomal translation machinery, and therefore, does not risk any genomic integration. Compared to both proteins and viral systems, mRNA manufacturing is cell-free, faster, and the protein product bears native glycosylation and conformational properties.
For instance, SARS-CoV-2 mRNA-based vaccine “mRNA-1273” was designed and produced through good manufacturing practice (GMP)-quality for human trials only 42 days after obtaining the nucleotide sequence of the target antigen. Besides fast design and production features, another unique feature of the SARS-CoV-2 mRNA vaccines is high efficacy (~90% at =6 months of follow-up) shown in phase III clinical trials and in the general population. (Polack FP et al. 2020. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383:2603–15; Baden LR et al. 2021.Efficacy and safety of the mRNA 1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 384:403–16).

Further the mRNA vaccines have received widespread approval for human use and immunization through mRNA vaccines has significantly mitigated COVID-19 related morbidity and mortality worldwide. Such unprecedented fast track record for mRNA vaccine development has propelled mRNA vaccines into the spotlight among the global scientific community and the general public as well. (Dagan N et al. 2021. BNT162b2 mRNA Covid-19 vaccine in a nationwide mass vaccination setting. N. Engl. J. Med. 384:1412–23; Thomas SJ, et al. 2021. Safety and efficacy of the BNT162b2 mRNA Covid 19 vaccine through 6 months. N. Engl. J. Med. 385:1761–73).

Several SARS-CoV-2 antigens can potentially induce immune responses, and thereby, can provide protection from viral infection. The spike (S) surface glycoprotein of SARS-CoV-2 virus responsible for mediating viral attachment and entry via the cellular receptor angiotensin converting enzyme 2 (ACE-2) is the most widely used antigens by mRNA vaccine industries. The S protein has two subunits: S1 (contains the receptor binding domain (RBD) and multiple neutralizing epitopes) and S2 (responsible for viral fusion with the host cell membrane). mRNA vaccine industries have designed and produced many S-encoding mRNAs including the wild-type (WT) full-length S (a trimerized soluble version of RBD) and a membrane-bound full-length construct named S-2P. Proline substitutions in the S2 subunit (K986P and V987P) induces stabilization of S-2P in the prefusion conformation which presumably leads to superior neutralizing antibody responses compared to its WT counterpart. The S2-P construct has been used in both the approved SARS-CoV-2 mRNA vaccines. mRNA vaccines provide protection against SARS-CoV-2 by inducing S-specific IgG and neutralizing antibodies. Another preclinical study showed that mice immunized with S-encoding modified mRNA-LNP vaccines develop strong CD8+ T cell responses in the lungs, suggesting that these cells might be primed to home to sites of infection to control SARS-CoV-2 replication. Both the approved mRNA-based vaccines induced some level of CD8+ T cell and CD4+ T cell response. (Tarke A et al. 2021. Impact of SARS-CoV-2 variants on the total CD4+ and CD8+ T cell reactivity in infected or vaccinated individuals. Cell Rep. Med. 2:100355; Sahin U et al. 2021. BNT162b2 vaccine induces neutralizing antibodies and polyspecific T cells in humans. Nature 595:572–77).

Despite the above-mentioned distinct advantages in using mRNA vaccines, there are a number of up-hill challenges inherent to the mRNA vaccine platform. First, mRNAs possess intrinsic immunogenicity. Secondly, they are susceptible to enzymatic degradation. Third, the degrees of cellular uptake for naked mRNA are almost negligible. The innate immunogenicity of mRNA owes its origin to the cellular detection of single- and double-stranded RNAs by toll like receptors (TLRs)), helicase receptors, including retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and others. The challenges of abrogating the inherent immunogenicity of mRNAs are being overcome by substituting naturally occurring nucleosides such as 1-methylpseudouridine and other nucleosides present in transfer and ribosomal RNA (but not typically in mRNA) into the mRNA sequence. Such substitutions render the modified mRNA undetectable via these innate immune sensors. The second and the third challenges are addressed by entrapping a nucleoside-modified or sequence-engineered mRNA into a delivery system that protects the mRNA from assault by RNA degrading enzymes (such as RNases) and facilitates cellular uptake as well.

There are number of recently disclosed reports on using LNPs prepared from ionizable lipids for inducing immune response against antigen encoded genetic vaccines (both mRNA and DNA vaccines). WO2017075531A1 disclosed self-assembled lipid nanoparticles formulations containing novel series of ionizable lipids in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids to facilitate the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) into cells under both in vitro and in vivo conditions. Prior LNPs formulation: Stabilized plasmid–lipid particle (SPLP) forerunner of today’s LNPs was prepared by combining the fusogenic ionizable phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with a quaternized cationic lipid dioleyldimethylammonium chloride (DODAC). (Pfizer-BioNTech Covid-19 Vaccine FDA EAU Letter of Authorization. https://www.fda.gov/media/144412; ModernaTX, Inc.mRNA-1273-P301-Protocol.: https://www.modernatx.com/sites/default/files/mRNA-1273-P301-Protocol.pdf).

The positively charged lipid mixture electrostatically bind and encapsulate plasmid DNA to form lipid: DNA complexes (lipoplexes). The resulting lipoplexes are finally coated with hydrophilic polyethylene glycol (PEG) to stabilize it in aqueous media and to limit protein and cell interactions upon in vivo administration. DOPE gets protonated in the endosome after endocytotic cellular uptake, and since DOPE is a cone-shaped molecule, it presumably forms an endosomolytic ion pair with endosomal phospholipids to facilitate endosomal release of nucleic acids, a critical event for successful delivery of genetic materials. To encapsulate nucleic acids, SPLP was subsequently modified to Stabilized Nucleic Acid Lipid Particle (SNALP) containing four lipids: an ionizable rather than quaternized cationic lipid, a saturated bilayer forming quaternized zwitterionic lipid namely, Distearoylphosphatidylcholine (DSPC), cholesterol and a PEG–lipid.

The ionizable lipid in the SNALPs plays the role of a fusogenic lipid after becoming protonated in the endosome. The protonated ionizable lipids form a membrane-destabilizing ion pair with an endosomal phospholipid which results into release of nucleic acids from the endosomes to cell cytoplasm. DSPC forms a stable bilayer underneath the PEG surface. Cholesterol seals the gaps in the particles, in limiting LNP–protein interactions, and possibly, in promoting membrane fusion. Formulation optimization studies involving 300 ionizable lipids and thousands of formulations revealed an optimal mole ratio of 50:10:38.5:1.5 for MC3 (an ionizable lipid): DSPC: Cholesterol: PEG–lipid for in vivo delivery of therapeutic nucleic acid (siRNA). This optimal formulation showed 200-fold increase in potency and a corresponding reduction in the effective dose in order to achieve durable suppression of the target gene by >80% leading to clinical approval of Onpattro™ in 2018. (Akinc A et al. 2019. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 14: 1084–1087; Evers MJW, Kulkarni JA et al. 2018. State-of-the-Art Design and Rapid-Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery. Small Methods 2: 1700375).

This clinically approved siRNA delivery formulation formed the basis for developing many approved LNPs based formulations for in vivo delivery of SARSCoV-2 mRNA vaccines. Prior art also reported preclinical and clinical studies using MC3 in the formulation described above in order to deliver nucleoside-modified mRNA-encoded immunogens. Subsequently, to replace MC3 (due to its slow degradability), a new class of ionizable lipids aimed at increasing their potency by enabling greater branching in their alkyl tails were developed. This new class of lipids contain an ethanolamine ionizable head group which is connected to both a single saturated tail containing a primary degradable ester and a second saturated tail that branches after seven carbons into two saturated C8 tails using a less degradable secondary ester. (Sabnis S, et al. 2018. A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-Human Primates. Mol. Ther. 26: 1509–1519).

Increased branching presumably creates an ionizable lipid with a more cone shaped structure which leads to more endosomal release of mRNA while pairing with the anionic phospholipids of the endosomal membranes. Lipid ALC-0315 combined with DSPC, cholesterol and a PEG–lipid was used as the delivery system in the SARS-COV-2 trials of BioNTech (Cheng X et al 2016. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery. Adv. Drug Deliv. Rev. 99: 129–137).

Another mRNA LNP (CVnCoV) uses a non-chemically modified, sequence-engineered mRNA encoding a diproline stabilized full-length S protein which was delivered possibly with Acuitas LNP containing the ionizable lipid ALC-0315. The number of weeks between two doses was varied from 1 to 4 when using 2 µg doses in mice. Longer intervals produced higher titers and T cell responses and a balanced Th1/Th2 response in Balb/c mice. (Rauch S, Roth N, et al. 2020. mRNA Based SARS-CoV-2 Vaccine Candidate CVnCoV Induces High Levels of Virus Neutralizing Antibodies and Mediates Protection in Rodents. bioRxiv).

Another prior art disclosed use of a non-modified mRNA encoding a double mutant form of the diproline stabilized spike protein delivered in an LNP containing ionizable lipids. (WO2020214946A1; Reichmuth AM et al. 2016. mRNA Vaccine Delivery Using Lipid Nanoparticles; and US20200237671A1). In Balb/c mice, 0.2–10 µg dose range resulted in binding and neutralization titers well above convalescent levels. In non-human primates, 15, 45 and 135 µg doses all generated Th1 biased titers exceeding the human convalescent panel. (Kalnin KV et al. Immunogenicity of Novel mRNA COVID-19 Vaccine MRT5500 in Mice and Non-Human Primates. bioRxiv 2020).

Further Prior Art exploited a self-amplifying, full-length, unmodified mRNA encoding a pre-fusion SARS-CoV-2 full-length spike protein which was encapsulated in an LNP containing an ionizable lipid. In this ionizable lipid a thioester functionality was used to link the amine-bearing headgroup to lipid tails via two additional ester groups. (Rajappan K et al. 2020. Property-Driven Design and Development of Lipids for Efficient Delivery of siRNA. J. Med. Chem. 63: 12992–13012). A distinguishing feature of self-amplifying mRNA was observed. While luciferase reporter expression was maintained at a fairly constant level beyond one week of IM administration, conventional mRNA expression levels were found to be reduced much faster. (De Alwis R et al. 2020. A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity in Mice. bioRxiv). Arcturus completed a phase 1 clinical trial with doses from 1–10 µg and has chosen 7.5 µg for its phase 3 trial. (Arcturus Therapeutics Announces Positive Interim ARCT-021 (LUNAR-COV19) Phase 1/2 Study Results for Both Single Shot and Prime-Boost Regimens, and Up to $220 Million in Additional Financial Commitments from Singapore Arcturus Therapeutics, Inc. Available online:interim-arct-021-lunar). Study by Imperial College London used a self-amplifying mRNA-encoded prefusion-stabilized spike protein and delivered it with Acuitas LNP. Remarkably high and dose-dependent antibody and neutralizing titters were observed after two injections in the 0.01-10 µg doses range in Balb/c mice. (US10221127B2; McKay PF et al. 2020. Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nat. Commun. 11: 3523).

Chulalongkorn University, in collaboration with the University of Pennsylvania, reported development of a native spike immunogen nucleoside-modified mRNA LNP (WO2020219941A1). Further Providence therapeutics has been granted a Health Canada notice of authorization for human clinical trials using their PTXCOVID-19B mRNA LNP vaccine. (Providence Therapeutics COVID-19 Vaccine Receives Health Canada Authorization to Begin Clinical Trials. Available online: http://www.providencetherapeutics.com/providence-therapeutics-covid-19-vaccine-receives-health-canada-authorizationto-begin-clinical-trials).

Preclinical studies were conducted using three mRNA candidates encoding the receptor-binding domain, the full-length spike with or without a mutation in the furin cleavage site using a dose of 20 µg in C57BL6 mice following a prime-boost regimen. (Providence Therapeutics Reports Supportive Preclinical Data for its COVID-19 Vaccine Candidate (PTX-COVID19-B). supportive-preclinical-data-for-its-covid 19-vaccine-candidate-ptx-covid19-b (accessed on 10 January 2021)). Preclinical data using an undisclosed lipid from Genevant revealed robust neutralization titters against the full length and the formulated payloads. (Laczkó D, et al. 2020. A Single Immunization with Nucleoside Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity 53: 724–732.e7).

Further, a number of lipid-like entities, termed lipidoids (initially developed for siRNA delivery) have been developed for mRNA delivery. For instance, a previously disclosed ionizable lipid C12-200 (a small molecule dendrimer with five alkyl chains and five nitrogen atoms out of which three nitrogen atoms are protonable) was found to be efficient for delivering mRNA to liver by reducing the percentage of ionizable lipid to 35% but increasing the weight ratio of ionizable lipid to nucleic acid from 5 to 10 and replacing DSPC with the fusogenic unsaturated DOPE. (Kauffman KJ et al. 2015. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 15: 7300–7306).

Interestingly, presence of an additional single carbon branch at the terminus of each of the four alkyl chains of this small, three-nitrogen lipidoids increased the potency of mRNA expression in liver by more than 10-fold compared to other lipidoids in this class. (Hajj KA et al. 2019. Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo Due to Enhanced Ionization at Endosomal PH. Small 15: 1805097). The MC3 LNP has been used for intranasal delivery of a 4.5 kb nucleoside-modified sequence encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in mice. (Robinson E et al. 2018. Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis. Mol. Ther. 26: 2034–2046).

A luciferase reporter was successfully expressed in the lungs by pipetting a 12-µg dose into the nostrils for spontaneous inhalation. Then, in a transgenic CFTR knockout mouse, application of CFTR mRNA LNPs restored CFTR-mediated chloride secretion to conductive airway epithelia for at least 14 days. Interestingly, there are not many prior reports on developing self-assembled LNPs that enter body’s antigen presenting cells via mannose receptors. A nucleoside-modified mRNA encoding the influenza antigen H3N2-HA was delivered using DOTAP/DOPE/PEG– lipid LNPs containing mannose as a ligand to facilitate uptake by macrophages and dendritic cells. The sizes of these LNPs were around 200 nm, they were positively charged with around 15 mV zeta potential, and they were able to express luciferase in the lungs following intranasal instillation of a 12-µg dose. (Zhuang X et al. 2020. mRNA Vaccines Encoding the HA Protein of Influenza A H1N1 Virus Delivered by Cationic Lipid Nanoparticles Induce Protective Immune Responses in Mice. Vaccines 8: 123). Dendritic cells in vitro have been transfected using a mannosylated liposome (Perche F et al. 2011. Selective gene delivery in dendritic cells with mannosylated and histidylated lipopolyplexes. J. Drug Target. 19: 315–325). Higher throughput screening methods are now being employed to identify ligands targeting specific cell types and such methods may find use in future for the targeting of specific dendritic cell subsets. (Veiga N et al. 2018. Cell Specific Delivery of Modified mRNA Expressing Therapeutic Proteins to Leukocytes. Nat. Commun. 9: 4493. 79; Kedmi R et al. 2018. A modular platform for targeted RNAi therapeutics. Nat. Nanotechnol. 13: 214–219).

Among the numerous mRNA delivery systems developed including lipids, lipid-like materials, polymers and protein derivatives, lipid nanoparticles (LNPs) based mRNA delivery systems have been most thoroughly investigated and have successfully entered the clinic. The mRNA vaccines use lipid nanoparticles (LNPs) containing encapsulated mRNA that carry the genetic instruction for producing the antigenic proteins. Upon vaccination, LNP-associated mRNA is first delivered to the cytosol of the host cells, and thereafter, the mRNA functions as a template for protein antigen synthesis. In many COVID-19 mRNA vaccine candidates, the genetic code of the full-length S protein is delivered and translated into S protein using the host cells’ protein-making machinery within the cytosol. Like DNA vaccines, the mRNA vaccines possess the capability of inducing both antibody production and T cell responses once the mRNA encoded antigen is produced in the cells of the vaccinated person. Furthermore, antigen expression post mRNA vaccination is transient, limiting its persistence in the body. All these distinguishing features explain why mRNA is being used as a robust platform for vaccine development. Effective humoral and cellular immune responses against deadly disease-causing microorganisms can be induced by targeting antigenic protein encoded mRNA vaccines to dendritic cells (DCs), body’s most potent antigen presenting cells. However, non-availability of efficacious and biologically safe systems capable of directly delivering antigen encoded DNA and mRNA vaccines or peptide/protein-based antigens to DCs under in vivo settings are impeding the clinical success of dendritic cell (DC) based immunotherapy.

? Majority of the conventional liposomal preparation processes are energy demanding, and hence, are not upscalable for their industrial production.
? Conventional liposome preparations involve use of water immiscible chlorinated organic solvents (such as chloroform) which are removed later first with gentle nitrogen flow followed by subjecting the resulting dried lipid films for 6-8 h under high vacuum (thus the conventional processes of liposome preparation are energy demanding).
? Moreover, presence of even trace amount of residual chlorinated solvent may cause toxicity to human, a possible risk for human applications, complete removal of which is very difficult.
? Complexes of the antigen encoded mRNA: self-assembled LNPs often need to be prepared mostly at pH 4-5 to ensure hydrolytic stability of the mRNA.
? Targeting the complexes of antigen encoded mRNA & self-assembled LNPs to antigen presenting cells (APCs) and accomplishing good gene expression remain up-hill challenges.
? The complexes of antigen encoded genetic vaccines and LNPs enter body’s cell cytoplasm usually via a process called endocytosis where part of the plasma membrane of body’s cells engulf the complexes to form endosomes. The genetic vaccines then need to be released from the endosomes into the cell cytoplasm. Inefficient endosomal release process is one of the major draw backs for conventional LNPs (e.g. liposomes).

Thus, attentions are now being directed for developing up-scalable self-assembling, and therefore not energy demanding, lipid-nanoparticles for delivering genetic materials to body’s antigen presenting cells.

The present invention discloses development of up-scalable self-assembled formulations of LNPs for targeting antigen encoded mRNA/DNA vaccines to antigen presenting cells. Present invention overcomes the prior art drawbacks and technical advance over the prior arts by providing the self-assembled formulations of LNPs prepared using cationic lipids: such as novel DC (dendritic cell) targeting lipids that contain both transfection-enhancing guanidine functionality and DC-targeting mannose-mimicking shikimoyl-functionality in their polar head-group region, endosome-disrupting histidinylated lipids, co-lipids and one or more pharmaceutically acceptable excipients. These selective liposomal nucleic acid (NA) vaccine carriers using liposomes are prepared for delivering DNA vaccines directly to dendritic cells under in vivo conditions.
OBJECTS OF THE INVENTION:

The primary object of the present invention is to provide novel PEGylated DC targeting lipids and PEGylated endosome disrupting lipids.

Another object of the invention is to provide novel compounds of structural formula of VII as PEGylated endosome disrupting lipids and XII as PEGylated DC targeting lipids.

Another object of the invention is to provide a process for preparation of novel PEGylated DC targeting lipids and PEGylated endosome disrupting lipids.

Another object of the invention is to provide a process for synthesis, intermediates used in the synthesis and use thereof.

Another object of the invention is to provide the self-assembling cationic lipid-based composition for effective transfection of nucleic acids.

One more object of the invention is to provide novel cationic lipids: comprising PEGylated DC targeting lipids and PEGylated endosome disrupting lipids for pharmaceuticals and vaccine formulations.

Further object of the invention is to provide a lipid-based composition, formulation and use for nucleic acid vaccine delivery and preparation thereof.

One further object of the invention is to provide a non-energy demanding up-scalable self-assembled non-cytotoxic lipid-based composition/formulation LNPs formulations for delivering nucleic acid materials to body’s antigen presenting cells.

Another object of the invention is to provide a self-assembling nanoparticle composition for delivery of therapeutic agents.

One further object of the invention is to provide the self-assembled LNPs to deliver mRNA/DNA vaccines to antigen presenting cells via targeting mannose receptors.

Another object of the invention is to provide the method of delivering the nucleic acid to the antigen presenting cells for inducing effective immune response against the various diseases including the infectious disease.

The present invention discloses development of a new up-scalable self-assembled non-cytotoxic lipid-based formulation for delivering genetic mRNA/DNA vaccines to body’s antigen presenting cells.

Another object of the instant invention is to utilize the endosome-disrupting histidinylated lipid in the presently described lipid-based formulation, due to its endosome-disrupting histidinylated lipid component, to ensure efficient endosomal release of the mRNA/DNA vaccines.

SUMMARY OF THE INVENTION:

Accordingly, the invention discloses the novel cationic pegylated lipid compounds of structural formula: VII and XII and composition comprising the novel pegylated lipids to form non-cytotoxic self-assembling LNPs formulation to ensure efficient endosomal release of the mRNA/DNA vaccines.

In one aspect present invention discloses a cationic PEGylated lipid compound for non-cytotoxic self-assembling LNPs formulation, facilitating efficient endosomal release of mRNA/DNA vaccines, comprising:
a. a positively charged endosome disrupting PEGylated histidinylated lipid, represented by the structural formula VII:

General structural formula VII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl; or

b. a DC targeting PEGylated lipid, represented by the structural formula XII:

General structural formula XII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl.

The positively charged endosome disrupting PEGylated histidinylated lipid of structural formula VII of the present invention is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride:
.

The said PEGylated histidinylated compound of structural formula VII is characterized by HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976. The said compound is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).

The DC targeting PEGylated lipid of structural formula XII of the present invention is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride:
.

The said DC targeting PEGylated lipid of structural formula XII is characterized by HRMS(m/z): [M+H]+ = 2140 and [M+1]+/2 =1070. The said compound is further characterized by 1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 – 7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

In another aspect present invention provides a process for the synthesis of an endosome disrupting PEGylated histidinylated lipid of structural formula VII, comprising the following steps:

General structural formula VII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl;
a. reacting the ethane-1,2-diamine in presence of tert-butoxycarbonyl (Boc) anhydride, DCM to obtain intermediate I of structure:

wherein intermediate I is tert-butyl (2-aminoethyl carbamate) characterized by: ESI MS: [M]+ =161;
wherein the intermediate I is further characterized by 1H NMR (400 MHz, Chloroform-d): d 4.90 (s, 1H), 3.16 (q, J = 5.8 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 1.43 (s, 9H);

b. reacting the intermediate I, tert-butyl (2-aminoethyl carbamate) in presence of ethyl acetate, K2CO3, 1-bromo-hexadecane to obtain intermediate II of structural formula:

wherein intermediate II is tert-butyl (2- (dihexadecylamino)ethyl) carbamate characterized by: HRMS: [M]+= 609;
wherein the intermediate II is further characterized by 1H NMR (500 MHz, Chloroform-d): d 5.02 (s, 1H), 3.51 (s, 2H), 3.20 – 3.12 (m, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.39 (m, 2H), 1.47 (s, 9H), 1.42 – 1.23 (m, 56H), 0.90 (t, J = 6.8 Hz, 6H);

c. reacting the intermediate II, tert-butyl (2-(dihexadecylamino)ethyl) carbamate in presence of TFA/DCM to obtain intermediate III of structural formula:

wherein R is n-C16H33,
wherein intermediate III is dihexadecylethane-1,2-diamine characterized by: ESI-MS: [M]+ = 509;
wherein the intermediate III is further characterized by 1H NMR (500 MHz, Chloroform-d) d 4.07 (m, 1H), 3.40 – 3.16 (m, 1H), 2.92 – 2.73 (m, 2H), 2.63 – 2.49 (m, 2H), 2.45 – 2.39 (m, 4H), 1.47 – 1.18 (m, 56H), 0.89 (t, J = 6.9 Hz, 6H);

d. reacting the intermediate III, dihexadecylethane-1,2-diamine with BocNH-(PEG)27-COOH in presence of HATU, DIPEA, DCM, to obtain intermediate IV of structural formula:

wherein R is n-C16H33,
wherein intermediate IV is tert-butyl(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) carbamate characterized by HRMS (m/z): [M+1]+ = 1914;
wherein the intermediate IV is further characterized by 1H NMR (500 MHz, Chloroform-d): d 6.53 (s, 1H), 5.08 (s, 1H), 3.73 (t, J = 5.5 Hz, 112H), 3.37 – 3.21 (m, 4H), 2.65 – 2.29 (m, 8H), 1.44 (s, 9H), 1.25 (s, 56H), 0.87 (t, J = 6.7 Hz, 6H);

e. reacting the intermediate IV, tert-butyl(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) carbamate in presence of TFA/DCM to obtain intermediate V of structural formula:

wherein R is n-C16H33,
wherein intermediate V is 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide characterized by: HRMS (m/z): [M+1]+ =1814;
wherein the intermediate is further characterized by 1H NMR (400 MHz, Chloroform-d): d 6.91 – 6.28 (m, -1H), 3.64 (s, 112H), 3.49 – 3.26 (m, 2H), 3.09 – 2.97 (m, 1H), 2.60 – 2.51 (m, 2H), 2.45 (q, J = 9.1, 7.6 Hz, 5H), 1.47 – 1.37 (m, 4H), 1.32 – 1.21 (m, 56H), 0.88 (d, J = 13.7 Hz, 6H);

f. reacting the intermediate V, 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide with N,N di-Boc L-histidine in presence of HATU, DIPEA, DMF to obtain intermediate VI of structural formula:

wherein R is n-C16H33,
wherein intermediate VI is tert-butyl (S)-5-(2-((tert-butoxycarbonyl) amino)-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontyl)-1H-imidazole-1-carboxylate characterized by: HRMS (m/z): [M+Na]+ =2173;
wherein the intermediate is further characterized by 1H NMR (500 MHz, Chloroform-d) d 8.18 – 8.05 (m, 2H), 7.20 – 7.14 (m, 1H), 7.09 – 7.04 (m, 1H), 6.63 – 6.58 (m, 1H), 5.15 – 5.09 (m, 1H), 3.81 – 3.75 (m, 2H), 3.74 (t, J = 6.0 Hz, 2H), 3.69 – 3.60 (m, 108H), 3.57 – 3.54 (m, 1H), 3.52 – 3.46 (m, 2H), 3.43 – 3.37 (m, 1H), 2.52 – 2.48 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.60 (s, 9H), 1.44 – 1.39 (m, 9H), 1.25 (s, 60H), 0.88 (t, J = 6.4 Hz, 6H); and

g. reacting the intermediate VI, tert-butyl (S)-5-(2-((tert-butoxycarbonyl) amino)-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontyl)-1H-imidazole-1-carboxylate in presence of TFA/DCM to obtain endosome disrupting PEGylated histidine lipid of structural formula VIII:

VIII
wherein R is n-C16H33,
wherein the endosomal disrupting lipid is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride is characterized by: HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976;
wherein the intermediate is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).

Yet in another aspect present invention provides a process for the synthesis of the cationic DC targeting PEGylated lipid of structural formula XII, comprising the steps:
a. reacting the intermediate V, 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide, obtained from steps a-e as claimed in claim 8, with (R, E)-1,10-dioxo-12-phenyl-11-oxa-3,9-diaza-1-boradodec-2-ene-8-carboxylic acid in presence of HATU, DIPEA, DCM in to obtain intermediate VI of structural formula:

VI
wherein R is n-C16H33,
wherein Z is C6H5CH2OCO-,
wherein intermediate VI is benzyl (S)-(23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamate characterized by: HRMS(m/z): [M+H]+= 2176;
wherein the compound is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.60 – 8.53 (m, 1H), 8.32 – 8.21 (m, 1H), 8.11 (dd, J = 7.4, 3.5 Hz, 1H), 7.35 (d, J = 14.1 Hz, 5H), 6.97 – 6.90 (m, 1H), 5.72 – 5.65 (m, 1H), 5.20 – 5.06 (m, 3H), 4.80 – 4.70 (m, 1H), 4.22 – 4.15 (m, 1H), 3.66 (s, 108H), 3.47 (dd, J = 4.7, 2.0 Hz, 2H), 3.22 (td, J = 8.5, 6.9, 3.1 Hz, 2H), 3.15 – 3.00 (m, 6H), 2.67 – 2.61 (m, 1H), 2.58 – 2.53 (m, 2H), 1.60 – 1.48 (m, 4H), 1.44 (s, 9H), 1.26 (d, J = 13.7 Hz, 60H), 0.90 (t, J = 6.3 Hz, 6H);

b. reacting the intermediate VI, benzyl (S)-(23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamate in presence of MeOH and Pd/Charcoal into obtain intermediate VII of structural formula:

VII
wherein R is n-C16H33,
wherein intermediate VII is (S)-6-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-2-amino-N-(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) hexanamide characterized by: HRMS(m/z): [M+H]+ = 2042;
wherein the compound is further characterized by 1H NMR (500 MHz, DMSO-d6): d 8.38 – 8.32 (m, 1H), 8.31 – 8.25 (m, 1H), 8.15 – 8.10 (m, 1H), 7.65 (td, J = 6.4, 3.4 Hz, 1H), 7.46 – 7.22 (m, 1H), 7.19 –7.11 (m, 1H), 6.76 – 6.67 (m, 1H), 3.50 (s, 112H), 3.10 – 3.03 (m, 2H), 2.90 –2.84 (m, 2H), 2.63 (s, 1H), 2.41 – 2.32 (m, 6H), 2.27 (t, J = 6.3 Hz, 2H), 1.64 –1.46 (m, 2H), 1.29 (d, J = 66.4 Hz, 69H), 0.84 (t, J = 6.3 Hz, 6H);

c. reacting the intermediate VII (S)-6-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-2-amino-N-(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) hexanamide with (3S,4R,5S)-3,4,5-triacetoxycyclohex-1-ene-1-carboxylic acid in presence of HATU, DIPEA, DCM to obtain the intermediate VIII:

VIII
wherein R is n-C16H33,
wherein intermediate VIII is (1S,2R,3S)-5-(((S)-23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+ Na]+= 2346;
wherein the compound is further characterized by: 1H NMR (500 MHz, Chloroform-d): d 8.06 (s, 1H), 7.17 (s, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.36 (s, 1H), 5.71 (s, 1H), 5.24 (s, 1H), 5.14 – 4.89 (m, 1H), 4.79 (s, 1H), 4.51 – 4.39 (m, 1H), 3.78 (s, 1H), 3.74 (t, J = 6.1 Hz, 2H), 3.71 – 3.58 (m, 108H), 3.55 (s, 1H), 3.51 – 3.41 (m, 2H), 3.17 (s, 2H), 3.03 – 2.91 (m, 4H), 2.52 (t, J = 6.2 Hz, 2H), 2.41 (dd, J = 17.9, 5.0 Hz, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.69 (d, J = 14.3 Hz, 4H), 1.53 – 1.47 (m, 2H), 1.43 (s, 9H), 1.27 (d, J = 15.1 Hz, 60H), 0.87 (d, J = 7.0 Hz, 6H);

d. reacting the intermediate VIII (1S,2R,3S)-5-(((S)-23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of TFA/DCM to obtain the intermediate IX:

IX
wherein R is n-C16H33,
wherein intermediate IX is (1S,2R,3S)-5-(((S)-1-amino-20-hexadecyl-6,16-dioxo-10,13-dioxa-7,17,20-triazahexatriacontan-5-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+H]+ = 2224;
wherein the compound is further characterized by 1H NMR (500 MHz, DMSO-d6): d 8.12 – 7.88 (m, 1H), 7.71 – 7.51 (m, 2H), 6.42 (s, 1H), 5.65 – 5.51 (m, 1H), 5.23 – 4.93 (m, 2H), 4.25 (s, 1H), 3.50 (s, 112H), 3.14 – 2.93 (m, 4H), 2.92 – 2.80 (m, 2H), 2.80 – 2.68 (m, 3H), 2.65 – 2.58 (m, 3H), 2.37 – 2.32 (m, 5H), 2.06 – 1.97 (m, 6H), 1.90 – 1.37 (m, 8H), 1.23 (s, 58H), 0.87 – 0.80 (m, 6H);

e. reacting the intermediate IX (1S,2R,3S)-5-(((S)-1-amino-20-hexadecyl-6,16-dioxo-10,13-dioxa-7,17,20-triazahexatriacontan-5-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of HgCl2, Di-BOC-thiourea, Et3N, dry DCM/DMF to obtain the intermediate X:

X
wherein R is n-C16H33,
wherein intermediate X is 1S,2R,3S)-5-(((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+H]+ = 2466;
wherein the compound is further characterized by: 1H NMR (500 MHz, DMSO-d6): d 11.49 (s, 1H), 8.25 (t, J = 5.1 Hz, 1H), 8.16 – 7.84 (m, 2H), 6.46 – 6.36 (m, 1H), 5.60 (d, J = 3.5 Hz, 1H), 5.14 (d, J = 3.7 Hz, 2H), 4.24 (q, J = 8.2 Hz, 2H), 3.92 (d, J = 5.7 Hz, 2H), 3.68 – 3.62 (m, 2H), 3.61 – 3.57 (m, 3H), 3.50 (s, 118H), 3.40 (d, J = 5.8 Hz, 3H), 3.21 (ddd, J = 26.6, 13.2, 6.4 Hz, 5H), 3.10 – 2.91 (m, 1H), 2.90 – 2.81 (m, 1H), 2.63 (s, 1H), 2.42 – 2.35 (m, 1H), 2.33 (d, J = 7.0 Hz, 2H), 2.26 (t, J = 7.3 Hz, 1H), 2.05 (s, 2H), 2.04 (s, 3H), 2.00 (s, 3H), 1.67 – 1.58 (m, 3H), 1.51 (s, 2H), 1.47 (s, 9H), 1.38 (s, 9H), 1.23 (s, 60H), 0.84 (d, J = 4.0 Hz, 6H);

f. reacting the intermediate X 1S,2R,3S)-5-(((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of NaOMe, MeOH to obtain the intermediate XI:

XI
wherein R is n-C16H33,
wherein intermediate XI is (3S,4R,5S)-N-((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide characterized by: HRMS(m/z): [M+ Na]+= 2362;
wherein the compound is further characterized by: 1H NMR (500 MHz, DMSO-d6): d 11.50 (s, 1H), 9.49 (s, 1H), 8.36 (s, 1H), 8.27 (t, J = 5.5 Hz, 1H), 8.10 (s, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.71 – 7.61 (m, 2H), 7.56 – 7.45 (m, 1H), 7.35 (dd, J = 18.3, 8.1 Hz, 1H), 7.22 – 7.06 (m, 1H), 6.88 (s, 1H), 6.35 (dd, J = 3.4, 1.7 Hz, 1H), 4.24 (s, 1H), 4.19 (s, 1H), 3.93 (d, J = 5.7 Hz, 1H), 3.86 – 3.80 (m, 2H), 3.51 (s, 108H), 3.25 (d, J = 6.5 Hz, 1H), 3.07 (d, J = 7.2 Hz, 1H), 2.68 – 2.52 (m, 3H), 2.48 – 2.44 (m, 1H), 2.41 – 2.33 (m, 4H), 2.27 (q, J = 7.3, 6.9 Hz, 3H), 1.64 (d, J = 7.8 Hz, 2H), 1.55 – 1.50 (m, 2H), 1.49 – 1.36 (m, 18H), 1.24 (s, 60H), 0.87 – 0.84 (m, 6H); and

g. reacting the intermediate XI (3S,4R,5S)-N-((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide in presence of TFA/DCM to obtain the DC targeting PEGylated lipid (XII):

XII
wherein R is n-C16H33,
wherein obtained compound (lipid) is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride characterized by: HRMS(m/z): [M+H]+= 2140, [M+1]+/2 = 1070;
wherein the compound is further characterized by 1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 – 7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

In further aspect present invention provides a lipid-based composition for nucleic acid vaccine delivery, comprising:
a. cationic lipids, including endosome disrupting lipids and DC targeting lipids;
b. co-lipids and their combinations thereof;
c. various excipients mixed in different ratios;
wherein said cationic lipids are pegylated or non-pegylated, and
wherein the composition is formulated to target and deliver antigen-encoding nucleic acids utilizing self-assembled LNPs (Lipid Nanoparticles) to antigen-presenting cells (APC) via mannose receptors, facilitating efficient endosomal release of nucleic acids in the antigen-presenting cells for effective nucleic acid vaccine delivery.

In the lipid-based composition the co-lipid is selected from:
a. a steroid lipid which may be sterol and is selected from cholesterol, ergosterol, stigmasterol, sitosterol, campesterol, stigmastanol like phytosterols and other steroids such as dexamethasone, prednisolone, triamcinolone, and/or mixture thereof; and
b. a phospholipid selected from dioleoyl phosphatidylethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-distearoyl-snglycero-3-phosphoethanolamine [DSPE], 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dipalmitoyl-sn-glycero-3-phosphocholone [DPPC], hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

Preferably the co-lipid is dioleoyl phosphatidylethanolamine [DOPE].

Accordingly, the said lipid-based composition for nucleic acid vaccine delivery comprises endosome disrupting lipids, DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients, wherein lipids are pegylated or non-pegylated.

The said lipid-based composition comprises non-PEGylated endosome disrupting lipids, PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

The pharmaceutically acceptable excipients are selected from buffers, adjuvants, diluents, lubricants, binders, stabilizers, preservatives, disintegrants, absorbents, colorants, surfactants, flavours, sweeteners, residuals, immune-boosting co-excipients, or a combination thereof.

In the said lipid-based composition the endosome disrupting lipids, DC-targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1 to 1:1:1.

Preferably the endosome disrupting lipids, PEGylated DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1.

The nucleic acid (NA) is a biologically active agent selected from ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), plasmid DNA (pDNA), a fragment of RNA, mRNA, DNA, pDNA, miRNA, or any chimeric or fusion thereof.

The said lipid-based composition is formulated as a lipid-nanoparticle (LNP) formulation or a liposomal formulation encapsulating the biologically active agent.

The present composition is formulated as a lipoplex formulation formed by complexing the liposomal preparation with the biologically active agent.

The in vitro transfection is achieved at a N/P charge ratio (Lipid: Nucleic acid) of 1:1 to 4:1.

In another aspect, present invention provides a method for the preparation of a lipid-based composition, comprising the steps of:
a. taking lipids and other co-lipids and excipients in a vial and thoroughly mixing them in the vial;
b. diluting the mixture in an ethanolic solution; and
c. injecting the resulting ethanolic lipid mixture into nuclease-free water (pH ~7) under stirring conditions for 15 minutes to form a lipid-based formulation at room temperature.

Yet in another aspect, present invention provides a vaccine formulation comprising nucleic acid as a vaccine antigen for the prophylaxis of virus-mediated diseases, said vaccine formulation comprising:
a. nucleic acid (NA) selected from ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), plasmid DNA (pDNA), fragment of RNA, mRNA, DNA, pDNA, miRNA, or any chimeric or fusion thereof;
b. DC targeting lipids;
c. endosome disrupting lipids; and
d. co-lipids selected from:
i. a steroid lipid, which may be sterol and is selected from cholesterol, ergosterol, stigmasterol, sitosterol, campesterol, stigmastanol like phytosterols, and other steroids such as dexamethasone, prednisolone, triamcinolone, and/or a mixture thereof;
ii. a phospholipid selected from dioleoyl phosphatidylethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [DSPE], 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dipalmitoyl-sn-glycero-3-phosphocholone [DPPC], hydrogenated soy phosphatidylcholine (HSPC), or mixtures thereof; and
e. optionally, one or more pharmaceutically acceptable excipients.

In the said vaccine formulation, the co-lipid is dioleoyl phosphatidylethanolamine [DOPE].

The vaccine formulation of the present invention comprises nucleic acid, PEGylated endosome disrupting lipids, non-PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

Alternatively, the vaccine formulation of the present invention comprises nucleic acid, non-PEGylated endosome disrupting lipids, PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

The pharmaceutically acceptable excipients are selected from buffers, adjuvants, diluents, lubricants, binders, stabilizers, preservatives, disintegrants, absorbents, colorants, surfactants, flavours, sweeteners, residuals, immune-boosting co-excipients, or a combination thereof.

In the said vaccine composition, the endosome disrupting lipids, DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1 to 1:1:1.

Preferably, the endosome disrupting lipids, PEGylated DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1.

In one aspect present invention provides a vaccine formulation comprising the lipid-based composition of the present invention for the prophylaxis and/or treatment of disease including virus mediated disease in a human subject.

The said vaccine formulation comprises of lipid-based composition along with the biologically active Nucleic Acid (NA) to provide a pharmaceutical formulation with or without one or more pharmaceutically acceptable excipient to form Lipid-Nanoparticle (LNP) formulation or a Liposomal formulation or Lipoplex formulation.

The said formulation performs in vitro transfection at a N/P charge ratio (Lipid: Nucleic acid) of 1:1 to 4:1.

Yet in another aspect present invention provides a method for the preparation of a vaccine formulation, comprising the steps of:
a. preparing nucleic acid constructs;
b. complexing the lipid-based formulation as claimed in claim 10 with nucleic acid constructs at fixed mole ratios to form liposomes or lipoplex; and
c. obtaining the vaccine formulation and preserving it at room temperature or 4 °C.

Present invention discloses use of a lipid-based composition of the present invention for:
a. delivery of nucleic acid to body antigen presenting cells (APC);
b. targeting the antigen encoding nucleic acid and self-assembled LNPs to antigen presenting cells;
c. preparing the LNPs without the use of chlorinated organic solvents and energy-demanding processes;
d. preparation of pharmaceutical formulation, including vaccine formulation; and
e. prophylaxis, treatment, and management of diseases, including infectious diseases.

BRIEF DISCRIPTION OF THE FIGURES

Figure 1: Representative TEM image (A), surface potentials (B, measured by zetasizer) and sizes (C, measured by DLS) for self-assembled LNPs containing 1:1:1 mole ratio of non-PEGylated endosome disrupting histidine lipid, non-PEGylated DC-targeting lipid and commercially available co-lipid DOPE, respectively.

Figure 2: Representative sizes (A, measured by DLS), surface potentials (B, measured by zetasizer) and SEM images (C) for self-assembled LNPs containing 1:1:0.01 mole ratio of endosome disrupting non-PEGylated histidine lipid, commercially available co-lipid DOPE and PEGylated DC-targeting lipid respectively. This self-assembled LNPs formulation of PEGylated DC-targeting lipid showed promising in vitro GFPmRNA delivery efficiency in RAW 264.7 cells.

Figure 3: Representative mRNA transfection efficiency of self-assembled LNPs containing non-PEGylated DC-targeting lipid: endosome disrupting Histidine Lipid: DOPE at 1:1:1 mole ratio electrostatically complexed with GFPmRNA having 2:1 +/- charge ratio.

Figure 4: Representative mRNA transfection efficiencies of the self-assembled PEGylated LNPs qualitatively containing DOPE: PEGylated DC-targeting lipid at 1:0.01 mole ratio by flow cytometry techniques in RAW 264.7 cells (mouse macrophage cells, widely used as model antigen presenting cells).

Figure 5: Representative Epiflourescence images of RAW 264.7 cells (2.5 × 104cells) transfected by the GFPmRNAplexes of the non-PEGylated DC-targeting lipid: DOPE (1:1 mole ratio) LNPs (2:1 +/- charge ratio) in presence of increasing concentrations (0-300 µg) of mannan. A) untreated cells; B) Cells pre-saturated with 75 µg of mannan; C) Cells pre-saturated with 150 µg of mannan; D) Cells pre-saturated with 300 µg of mannan.

Figure 6: Representative Epifluorescence images of RAW 264.7 cells (2.5 × 104 cells) transfected by the GFP mRNAplexes of the non-PEGylated DC-targeting lipid: non-PEGylated Histidine lipid: DOPE (1:1:1 mole ratio) LNPs (2:1 +/- charge ratio) in presence of increasing concentrations (0-300 µg) of mannan. A) untreated cells; B) Cells pre-saturated with 75 µg of mannan; C) Cells pre-saturated with 150 µg of mannan; D) Cells pre-saturated with 300 µg of mannan.

Figure 7: Representative Epiflourescence images of RAW 264.7 cells (2.5 × 104 cells) transfected with the GFPmRNAplexes of the PEGylated DC-targeting lipid: non-PEGylated His: DOPE (0.01:1:1 mole ratio) LNPs (4:1 +/- charge ratio) in presence of increasing concentrations (0-300 µg) of mannan. A) untreated cells; B) Cells pre-saturated with 75 µg of mannan; C) Cells pre-saturated with 150 µg of mannan; D) Cells pre-saturated with 300 µg of mannan.

Figure 8: Representative transfection efficiency optimizations for self-assembled His:DOPE:DCD (1:1:0.01 mole ratio) lipid formulations in RAW 264.7 cells by epifluorescence microscopy using a range of GFPmRNA:PS ratios: (A) mRNA without complex with PS; (B) mRNA complex with PS (10:1); (C) mRNA complex with PS (20:1); (D) mRNA complex with PS (40:1); (E) mRNA complex with PS (80:1).

Figure 9: Representative transfection efficiency optimizations for self-assembled His:DOPE:DCD (1:1:0.01 mole ratio) lipid formulations in RAW 264.7 cells by epifluorescence microscopy using a range of GFPmRNA:PS ratios: (A) mRNA without complex with PS (0.5:1); (B) mRNA complex with PS (1:1); (C) mRNA complex with PS (2:1); (D) mRNA complex with PS (3:1); (E) mRNA complex with PS (6:1).

Figure 10: Representative time course optimization for optimal transfection efficiency for self-assembled His:DOPE:DCD (1:1:0.01 mole ratio) lipid formulations in RAW 264.7 cells by epifluorescence microscopy using GFPmRNA:PS ratios: (A) Cells with PS (1:1) 6 h; (B) mRNA complex with PS (1:1) 6h; (C) mRNA complex with PS (1:1) 12h; (D) mRNA complex with PS (1:1) 24h.

Figure 11: Representative transfection efficiencies by Histidine:DOPE (1:1) mRNAPlexes for self-assembled lipid formulations in RAW 264.7 cells by epifluorescence microscopy using a GFPmRNA:PS ratios: (A) Control untransfected RAW264.7 cells; (B) RAW264.7 cells transfected with GFPmRNAplexes of liposomes of DOPE and His (at 1 mM each, total lipid concentration: 2 mM).

Figure 12: Representative efficiencies of the mRNAplexes of two different self-assembled lipid formulations in transfecting RAW264.7 cells measured by flow cytometry: (A) Control RAW264.7 untransfected cells; (B) mRNAplexes of His:DOPE:DCD in 16:1 +/- ratio (37.1%); (C) mRNAplexes of His:DOPE in 16:1 +/- ratio (51.2%).

DESCRIPTION OF THE INVENTION:

Nucleic acid-based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism in order to realize this potential. Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme.

In one aspect present invention relates to novel cationic lipids that can be used in combination with other lipid components, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g. oligonucleotides, messenger RNA).

In another aspect the invention relates to lipid compounds and their use, for example for the transport of biologically active substances or molecules in cells.

In one more aspect invention discloses, cationic lipids such as: PEGylated lipid compounds of structural formula VII and XII and composition comprising the novel PEGylated lipids to form non-cytotoxic self-assembling LNPs formulation to ensure efficient endosomal release of the mRNA/DNA vaccines.

Present invention discloses a cationic lipid-based composition comprising cationic lipids such as Endosome disrupting lipids, DC targeting lipids; co-lipids; their combinations therefore along with various excipients mixed in different ratios.

Further the invention describes schemes of synthesis of Endosome disrupting lipids, novel DC targeting lipids.

CATIONIC PEGYLATED LIPID COMPOUNDS AND PREPARATION METHOD THEREOF:

In one aspect, the invention provides novel positively charged endosome disrupting PEGylated histidinylated lipid as shown in the structural formula -VII:

General Structural formula VII

Wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl.

In one preferred embodiment when R is hexadecane (C16 H33), the compound of formula VII is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride as shown in the structure below:

Wherein R is n-C16H33.

In one aspect of the invention, the embodiment provides a novel endosome disrupting PEGylated histidinylated lipid for preparation of liposomes and lipoplexes to be utilized in pharmaceuticals and vaccine formulations.

In one embodiment of the invention, the compound is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride as shown in the structure below:

The PEGylated histidinylated compound is characterized by the lipid molecule, wherein compound is characterized by HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976.

The compound is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).

In one aspect of the invention, the embodiment provides the process of synthesis of the endosome disrupting histidinylated lipid of general formula VII comprising the steps (as shown in Scheme I):

General structural formula VII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl;

a. reacting the ethane-1,2-diamine in presence of tert-butoxycarbonyl (Boc) anhydride, DCM to obtain intermediate I of structure:

wherein intermediate I is tert-butyl (2-aminoethyl carbamate) characterized by: ESI MS: [M]+ =161;
wherein the intermediate I is further characterized by 1H NMR (400 MHz, Chloroform-d): d 4.90 (s, 1H), 3.16 (q, J = 5.8 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 1.43 (s, 9H);

b. reacting the intermediate I, tert-butyl (2-aminoethyl carbamate) in presence of ethyl acetate, K2CO3, 1-bromo-hexadecane to obtain intermediate II of structural formula:

wherein intermediate II is tert-butyl (2- (dihexadecylamino)ethyl) carbamate characterized by: HRMS: [M]+= 609;
wherein the intermediate II is further characterized by 1H NMR (500 MHz, Chloroform-d): d 5.02 (s, 1H), 3.51 (s, 2H), 3.20 – 3.12 (m, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.39 (m, 2H), 1.47 (s, 9H), 1.42 – 1.23 (m, 56H), 0.90 (t, J = 6.8 Hz, 6H);

c. reacting the intermediate II, tert-butyl (2-(dihexadecylamino)ethyl) carbamate in presence of TFA/DCM to obtain intermediate III of structural formula:

wherein R is n-C16H33,
wherein intermediate III is dihexadecylethane-1,2-diamine characterized by: ESI-MS: [M]+ = 509;
wherein the intermediate III is further characterized by 1H NMR (500 MHz, Chloroform-d) d 4.07 (m, 1H), 3.40 – 3.16 (m, 1H), 2.92 – 2.73 (m, 2H), 2.63 – 2.49 (m, 2H), 2.45 – 2.39 (m, 4H), 1.47 – 1.18 (m, 56H), 0.89 (t, J = 6.9 Hz, 6H);

d. reacting the intermediate III, dihexadecylethane-1,2-diamine with BocNH-(PEG)27-COOH in presence of HATU, DIPEA, DCM, to obtain intermediate IV of structural formula:

wherein R is n-C16H33,
wherein intermediate IV is tert-butyl(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) carbamate characterized by HRMS (m/z): [M+1]+ = 1914;
wherein the intermediate IV is further characterized by 1H NMR (500 MHz, Chloroform-d): d 6.53 (s, 1H), 5.08 (s, 1H), 3.73 (t, J = 5.5 Hz, 112H), 3.37 – 3.21 (m, 4H), 2.65 – 2.29 (m, 8H), 1.44 (s, 9H), 1.25 (s, 56H), 0.87 (t, J = 6.7 Hz, 6H);

e. reacting the intermediate IV, tert-butyl(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) carbamate in presence of TFA/DCM to obtain intermediate V of structural formula:

wherein R is n-C16H33,
wherein intermediate V is 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide characterized by: HRMS (m/z): [M+1]+ =1814;
wherein the intermediate is further characterized by 1H NMR (400 MHz, Chloroform-d): d 6.91 – 6.28 (m, -1H), 3.64 (s, 112H), 3.49 – 3.26 (m, 2H), 3.09 – 2.97 (m, 1H), 2.60 – 2.51 (m, 2H), 2.45 (q, J = 9.1, 7.6 Hz, 5H), 1.47 – 1.37 (m, 4H), 1.32 – 1.21 (m, 56H), 0.88 (d, J = 13.7 Hz, 6H);

f. reacting the intermediate V, 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide with N,N di-Boc L-histidine in presence of HATU, DIPEA, DMF to obtain intermediate VI of structural formula:

wherein R is n-C16H33,
wherein intermediate VI is tert-butyl (S)-5-(2-((tert-butoxycarbonyl) amino)-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontyl)-1H-imidazole-1-carboxylate characterized by: HRMS (m/z): [M+Na]+ =2173;
wherein the intermediate is further characterized by 1H NMR (500 MHz, Chloroform-d) d 8.18 – 8.05 (m, 2H), 7.20 – 7.14 (m, 1H), 7.09 – 7.04 (m, 1H), 6.63 – 6.58 (m, 1H), 5.15 – 5.09 (m, 1H), 3.81 – 3.75 (m, 2H), 3.74 (t, J = 6.0 Hz, 2H), 3.69 – 3.60 (m, 108H), 3.57 – 3.54 (m, 1H), 3.52 – 3.46 (m, 2H), 3.43 – 3.37 (m, 1H), 2.52 – 2.48 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.60 (s, 9H), 1.44 – 1.39 (m, 9H), 1.25 (s, 60H), 0.88 (t, J = 6.4 Hz, 6H); and

g. reacting the intermediate VI, tert-butyl (S)-5-(2-((tert-butoxycarbonyl) amino)-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontyl)-1H-imidazole-1-carboxylate in presence of TFA/DCM to obtain endosome disrupting PEGylated histidine lipid of structural formula VIII:

VIII
wherein R is n-C16H33,
wherein the endosomal disrupting lipid is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride is characterized by: HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976;
wherein the intermediate is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).

The scheme of the invention for Synthesis of endosome disrupting PEGylated histidine lipid of structural formula VII (lipid 1) is depicted below:

Scheme I:

Reagents: i). (BOC)2O, Et3N, DCM, 2h, 0 ºC; ii). K2CO3, n-C16H33Br, EtOAC, 70-80 ºC, Reflux, 48 h; iii). TFA/DCM (1:1, v/v), 24 h; iv). HATU, DIPEA, DCM, 48 h; v). TFA/DCM (1:1, v/v), 24 h; vi). HATU, DIPEA, DMF/DCM (1:2), 48 h, N2atm, vii). TFA/DCM (1:1, v/v), 24 h; viii). Amberlite IR 400 Cl-ion exchange resin, MeOH.

In another aspect, the invention provides novel DC targeting PEGylated lipid as shown in the structural formula -XII:

General Structural formula XII

Wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl.

In one preferred embodiment when R is n-hexadecyl (C16H33), the compound of formula XII is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride as shown in the structure below:

Wherein R is n-C16H33

In one aspect of the invention, the embodiment provides novel DC targeting PEGylated lipid for preparation of liposomes and lipoplexes to be utilized in pharmaceuticals and vaccine formulations.

In one embodiment of the invention, the compound is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride as shown in the structure below:

wherein R is n-C16H33

The said compound is characterized by HRMS(m/z): [M+H]+= 2140, [ M+1] +/2 =1070.

The compound is characterized by 1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 –7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

In one aspect of the invention, the embodiment provides the process of synthesis of the DC targeting PEGylated lipid of general formula XII comprising the steps (as shown in Scheme II):
a. reacting the intermediate V, 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide, obtained from steps a-e as shown above, with (R, E)-1,10-dioxo-12-phenyl-11-oxa-3,9-diaza-1-boradodec-2-ene-8-carboxylic acid in presence of HATU, DIPEA, DCM in to obtain intermediate VI of structural formula:

VI
wherein R is n-C16H33,
wherein Z is C6H5CH2OCO-,
wherein intermediate VI is benzyl (S)-(23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamate characterized by: HRMS(m/z): [M+H]+= 2176;
wherein the compound is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.60 – 8.53 (m, 1H), 8.32 – 8.21 (m, 1H), 8.11 (dd, J = 7.4, 3.5 Hz, 1H), 7.35 (d, J = 14.1 Hz, 5H), 6.97 – 6.90 (m, 1H), 5.72 – 5.65 (m, 1H), 5.20 – 5.06 (m, 3H), 4.80 – 4.70 (m, 1H), 4.22 – 4.15 (m, 1H), 3.66 (s, 108H), 3.47 (dd, J = 4.7, 2.0 Hz, 2H), 3.22 (td, J = 8.5, 6.9, 3.1 Hz, 2H), 3.15 – 3.00 (m, 6H), 2.67 – 2.61 (m, 1H), 2.58 – 2.53 (m, 2H), 1.60 – 1.48 (m, 4H), 1.44 (s, 9H), 1.26 (d, J = 13.7 Hz, 60H), 0.90 (t, J = 6.3 Hz, 6H);

b. reacting the intermediate VI, benzyl (S)-(23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamate in presence of MeOH and Pd/Charcoal into obtain intermediate VII of structural formula:

VII
wherein R is n-C16H33,
wherein intermediate VII is (S)-6-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-2-amino-N-(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) hexanamide characterized by: HRMS(m/z): [M+H]+ = 2042;
wherein the compound is further characterized by 1H NMR (500 MHz, DMSO-d6): d 8.38 – 8.32 (m, 1H), 8.31 – 8.25 (m, 1H), 8.15 – 8.10 (m, 1H), 7.65 (td, J = 6.4, 3.4 Hz, 1H), 7.46 – 7.22 (m, 1H), 7.19 –7.11 (m, 1H), 6.76 – 6.67 (m, 1H), 3.50 (s, 112H), 3.10 – 3.03 (m, 2H), 2.90 –2.84 (m, 2H), 2.63 (s, 1H), 2.41 – 2.32 (m, 6H), 2.27 (t, J = 6.3 Hz, 2H), 1.64 –1.46 (m, 2H), 1.29 (d, J = 66.4 Hz, 69H), 0.84 (t, J = 6.3 Hz, 6H);

c. reacting the intermediate VII (S)-6-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-2-amino-N-(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) hexanamide with (3S,4R,5S)-3,4,5-triacetoxycyclohex-1-ene-1-carboxylic acid in presence of HATU, DIPEA, DCM to obtain the intermediate VIII:

VIII
wherein R is n-C16H33,
wherein intermediate VIII is (1S,2R,3S)-5-(((S)-23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+ Na]+= 2346;
wherein the compound is further characterized by: 1H NMR (500 MHz, Chloroform-d): d 8.06 (s, 1H), 7.17 (s, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.36 (s, 1H), 5.71 (s, 1H), 5.24 (s, 1H), 5.14 – 4.89 (m, 1H), 4.79 (s, 1H), 4.51 – 4.39 (m, 1H), 3.78 (s, 1H), 3.74 (t, J = 6.1 Hz, 2H), 3.71 – 3.58 (m, 108H), 3.55 (s, 1H), 3.51 – 3.41 (m, 2H), 3.17 (s, 2H), 3.03 – 2.91 (m, 4H), 2.52 (t, J = 6.2 Hz, 2H), 2.41 (dd, J = 17.9, 5.0 Hz, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.69 (d, J = 14.3 Hz, 4H), 1.53 – 1.47 (m, 2H), 1.43 (s, 9H), 1.27 (d, J = 15.1 Hz, 60H), 0.87 (d, J = 7.0 Hz, 6H);

d. reacting the intermediate VIII (1S,2R,3S)-5-(((S)-23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of TFA/DCM to obtain the intermediate IX:

IX
wherein R is n-C16H33,
wherein intermediate IX is (1S,2R,3S)-5-(((S)-1-amino-20-hexadecyl-6,16-dioxo-10,13-dioxa-7,17,20-triazahexatriacontan-5-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+H]+ = 2224;
wherein the compound is further characterized by 1H NMR (500 MHz, DMSO-d6): d 8.12 – 7.88 (m, 1H), 7.71 – 7.51 (m, 2H), 6.42 (s, 1H), 5.65 – 5.51 (m, 1H), 5.23 – 4.93 (m, 2H), 4.25 (s, 1H), 3.50 (s, 112H), 3.14 – 2.93 (m, 4H), 2.92 – 2.80 (m, 2H), 2.80 – 2.68 (m, 3H), 2.65 – 2.58 (m, 3H), 2.37 – 2.32 (m, 5H), 2.06 – 1.97 (m, 6H), 1.90 – 1.37 (m, 8H), 1.23 (s, 58H), 0.87 – 0.80 (m, 6H);

e. reacting the intermediate IX (1S,2R,3S)-5-(((S)-1-amino-20-hexadecyl-6,16-dioxo-10,13-dioxa-7,17,20-triazahexatriacontan-5-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of HgCl2, Di-BOC-thiourea, Et3N, dry DCM/DMF to obtain the intermediate X:

X
wherein R is n-C16H33,
wherein intermediate X is 1S,2R,3S)-5-(((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+H]+ = 2466;
wherein the compound is further characterized by: 1H NMR (500 MHz, DMSO-d6): d 11.49 (s, 1H), 8.25 (t, J = 5.1 Hz, 1H), 8.16 – 7.84 (m, 2H), 6.46 – 6.36 (m, 1H), 5.60 (d, J = 3.5 Hz, 1H), 5.14 (d, J = 3.7 Hz, 2H), 4.24 (q, J = 8.2 Hz, 2H), 3.92 (d, J = 5.7 Hz, 2H), 3.68 – 3.62 (m, 2H), 3.61 – 3.57 (m, 3H), 3.50 (s, 118H), 3.40 (d, J = 5.8 Hz, 3H), 3.21 (ddd, J = 26.6, 13.2, 6.4 Hz, 5H), 3.10 – 2.91 (m, 1H), 2.90 – 2.81 (m, 1H), 2.63 (s, 1H), 2.42 – 2.35 (m, 1H), 2.33 (d, J = 7.0 Hz, 2H), 2.26 (t, J = 7.3 Hz, 1H), 2.05 (s, 2H), 2.04 (s, 3H), 2.00 (s, 3H), 1.67 – 1.58 (m, 3H), 1.51 (s, 2H), 1.47 (s, 9H), 1.38 (s, 9H), 1.23 (s, 60H), 0.84 (d, J = 4.0 Hz, 6H);

f. reacting the intermediate X 1S,2R,3S)-5-(((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of NaOMe, MeOH to obtain the intermediate XI:

XI
wherein R is n-C16H33,
wherein intermediate XI is (3S,4R,5S)-N-((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide characterized by: HRMS(m/z): [M+ Na]+= 2362;
wherein the compound is further characterized by: 1H NMR (500 MHz, DMSO-d6): d 11.50 (s, 1H), 9.49 (s, 1H), 8.36 (s, 1H), 8.27 (t, J = 5.5 Hz, 1H), 8.10 (s, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.71 – 7.61 (m, 2H), 7.56 – 7.45 (m, 1H), 7.35 (dd, J = 18.3, 8.1 Hz, 1H), 7.22 – 7.06 (m, 1H), 6.88 (s, 1H), 6.35 (dd, J = 3.4, 1.7 Hz, 1H), 4.24 (s, 1H), 4.19 (s, 1H), 3.93 (d, J = 5.7 Hz, 1H), 3.86 – 3.80 (m, 2H), 3.51 (s, 108H), 3.25 (d, J = 6.5 Hz, 1H), 3.07 (d, J = 7.2 Hz, 1H), 2.68 – 2.52 (m, 3H), 2.48 – 2.44 (m, 1H), 2.41 – 2.33 (m, 4H), 2.27 (q, J = 7.3, 6.9 Hz, 3H), 1.64 (d, J = 7.8 Hz, 2H), 1.55 – 1.50 (m, 2H), 1.49 – 1.36 (m, 18H), 1.24 (s, 60H), 0.87 – 0.84 (m, 6H); and

g. reacting the intermediate XI (3S,4R,5S)-N-((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide in presence of TFA/DCM to obtain the DC targeting PEGylated lipid (XII):

XII
wherein R is n-C16H33,
wherein obtained compound (lipid) is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride characterized by: HRMS(m/z): [M+H]+= 2140, [M+1]+/2 = 1070;
wherein the compound is further characterized by 1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 – 7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

In another embodiment of the present invention, the compound is a PEGylated DC-targeting lipid that is a DC-targeting double chain with a PEG attached as shown in the structure below:

The scheme of the invention for Synthesis of DC targeting PEGylated lipid of structural formula XII (lipid 2) is depicted below:

Scheme-IIA

Synthesis of PEGylated Lipid Precursor (V)
In one aspect, the invention provides Scheme IIA for Synthesis of intermediate V for the synthesis of DC targeting PEGylated lipid 2 (XII) is depicted below:

Reagents: i). (BOC)2O, Et3N, DCM, 2 h, 0 ºC; ii). K2CO3, n-C16H33Br, EtOAC, 70-80 ºC, Reflux, 48 h; iii). TFA/DCM (1:1, v/v), 24 h; iv). HATU, DIPEA, DCM, 48 h; v). TFA/DCM (1:1, v/v), 24 h

Scheme-IIB

Synthesis of the DC-targeting PEGylated Lipid 2 from Intermediate V
In one aspect, the invention provides Scheme IIB for Synthesis of DC -targeting PEGylated Lipid 2 (XII) from intermediate V is depicted below:

Reagents: i). HATU, DIPEA, DCM, 0 ºC-RT, 48 h, N2 atm; ii). Pd-Charcoal (10% Pd, w/w), MeOH, H2 atm, 14 h; iii). HATU, DIPEA, DCM, 0 ºC-RT, 48 h, N2 atm; iv). TFA/DCM (1:1, v/v), 24 h; v). HgCl2, Di-BOC-thiourea, Et3N, dry DCM/DMF (3:1, v/v) 1 h, N2 atm; vi). NaOMe, MeOH, 2 h, pH=10; vii). TFA/DCM (1:1, v/v), 24 h; viii). Amberlite IR 400 Cl- ion exchange resin, MeOH.

The schemes I and II, are further described in detail through examples.

LIPID-BASED COMPOSITION AND PREPARATION METHOD THEREOF:

The invention further discloses the novel cationic: PEGylated lipid compounds of structural formula VII and XII and composition comprising the novel PEGylated lipids to form non-cytotoxic self-assembling LNPs formulation to ensure efficient endosomal release of the mRNA/DNA vaccines.

In one embodiment present invention discloses a cationic lipid-based composition comprising cationic lipids such as Endosome disrupting lipids, DC targeting lipids; co-lipids; their combinations therefore along with various excipients mixed in different ratios to formulate a lipid composition with biologically active agents.

The said composition is formulated to target and deliver antigen-encoding nucleic acids utilizing self-assembled LNPs (Lipid Nanoparticles) to antigen-presenting cells (APC) via mannose receptors, facilitating efficient endosomal release of nucleic acids in the antigen-presenting cells for effective nucleic acid vaccine delivery.

The co-lipid is selected from:
a. a steroid lipid which may be sterol and is selected from cholesterol, ergosterol, stigmasterol, sitosterol, campesterol, stigmastanol like phytosterols and other steroids such as dexamethasone, prednisolone, triamcinolone, and/or mixture thereof; and
b. a phospholipid selected from dioleoyl phosphatidylethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-distearoyl-snglycero-3-phosphoethanolamine [DSPE], 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dipalmitoyl-sn-glycero-3-phosphocholone [DPPC], hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

In the present composition, preferably the co-lipid is dioleoyl phosphatidylethanolamine [DOPE].

In one of the preferred embodiments, said cationic lipid-based composition comprises endosome disrupting lipids, DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients, wherein lipids are pegylated or non-pegylated.

In one alternate embodiment, the present invention provides lipid-based composition comprising non-PEGylated endosome disrupting lipids, PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

In one embodiment, the lipid-based composition of the present invention for nucleic acid vaccine delivery comprises the endosome disrupting lipids, DC-targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] present in the mole ratio of 1:0.01:1 to 1:1:1.

In another embodiment, the lipid-based composition of the present invention for nucleic acid vaccine delivery comprises the endosome disrupting lipids, PEGylated DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] present in the mole ratio of 1:0.01:1.

In the said composition he nucleic acid (NA) is a biologically active agent selected from ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), plasmid DNA (pDNA), a fragment of RNA, mRNA, DNA, pDNA, miRNA, or any chimeric or fusion thereof.

The present composition is formulated as a lipid-nanoparticle (LNP) formulation or a liposomal formulation encapsulating the biologically active agent.

The said composition is formulated as a lipoplex formulation formed by complexing the liposomal preparation with the biologically active agent.

The present formulation comprising cationic lipids can be in form of LNPs (Lipid nanoparticles): Liposomal formulation and lipoplex formulation. These liposomal formulations are spherical, self-contained structures made of lipid bilayers that store part of the solvent in which they float. They can consist of one or more concentric membranes and their size is in the range from a few nanometers to a few dozen micrometers. Liposomes are mainly formed from amphiphilic molecules which are characterized by a hydrophilic (polar end) group and a hydrophobic group (non-polar end) in the same molecule. In most cases, liposome-forming molecules are not water-soluble. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The lipid/polymer emulsion formulation comprises lipid components, a nucleic acid and an aqueous carrier. The said emulsion formulation can be used in manufacturing of a medicament for the treatment of a condition, wherein the treatment comprises the delivery of a nucleic acid to the cells of the human or animal.

Lipoplexes are complexes formed among plasmid nucleic acid and cationic lipids. lipoplexes are liposome-based formulations that are formed by electrostatic interaction of cationic liposomes with anionic nucleic acids.

Lipid-based nanoparticle systems represent one of the most promising colloidal carriers for bioactive organic molecules. These nanoparticles can transport hydrophobic and hydrophilic molecules, display very low or no toxicity, and increase the time of drug action by means of a prolonged half-life and a controlled release of the drug.

The positive charged liposome or lipoplexes are able to contact with negative charged nucleic acids (NA) and entrap or encapsulate the nucleic acid (NA) for the delivery of nucleic acid in target cells.

The present invention utilizes the cationic DC targeting lipid which contains both transfection-enhancing guanidine functionality and DC-targeting mannose-mimicking shikimoyl- functionality in their polar head-group region leading to mannose receptor (MR)-selective, dendritic cell (DC)-targeting lipid nanoparticles (LNPs).

It transports of biologically active substances or molecules in cells. The biologically active substance of the present invention may be a therapeutic agent such as antigen. The term “antigen” means any substance that causes the body to make an immune response against that substance.

Antigens include toxins, chemicals, bacteria, viruses, or other substances that come from outside the body. Vaccines are examples of antigens in an immunogenic form, which are intentionally administered to a recipient to induce the memory function of the adaptive immune system towards antigens of the pathogen invading that recipient.

The composition may be formulated with or without one or more pharmaceutically acceptable excipient(s), suitable for composition or formulation to be administered in mammals through various routes of administration in suitable concentration, which may be selected from group comprising of adjuvants, buffers, diluents, lubricants, binders, stabilizers, preservatives, disintegrants, absorbents, colorants surfactants, flavours, sweeteners, residuals, immune-boosting co-excipients or combination thereof.

In one of embodiment, the liposomes or lipoplexes perform transfection at N/P charge ratio (Lipid: Nucleic acid) of 1:1 to 4:1.

In another aspect, present invention discloses a method for preparation of a lipid-based composition, comprising the steps of:
a. Taking lipids and other co-lipids and excipients in vial and mixing thoroughly in a vial;
b. diluting the mixture in ethanolic solution; and
c. injecting the resulting ethanolic lipid mixture in the nuclease free water (pH~7) under stirring conditions for 15 min to make lipid-based formulation in room temperature.

VACCINE FORMULATION AND PREPARATION METHOD THEREOF:

In another aspect, present invention is directed towards the vaccine formulation comprising the cationic lipid-based composition or formulation of the present invention for the prophylaxis and/or treatment of virus mediated disease in a human subject.

Accordingly, present invention discloses a vaccine formulation comprising biologically active agent as vaccine antigen along with a lipid-based composition for the prophylaxis and/or treatment of disease in a human subject.

Term “vaccine formulation”, is a substance used to stimulate the production of antibodies and provide immunity against one or several diseases. The vaccine provides prophylaxis and treatment of various infections which is capable of conferring immunity against such infections.

The term “antigen” means any substance that causes the body to make an immune response against that substance. Antigens include toxins, chemicals, bacteria, viruses, or other substances that come from outside the body. Vaccines are examples of antigens in an immunogenic form, which are intentionally administered to a recipient to induce the memory function of the adaptive immune system towards antigens of the pathogen invading that recipient.

The present invention discloses the Vaccine formulation with above-described novel cationic lipid-based composition or formulation formulated with biologically active agents not limited to hormones, antibodies, cholesterol, peptides, proteins, nucleic acid etc.

In one embodiment the Vaccine formulation of the present invention comprises a biologically active agent, wherein the biologically active agent is a nucleic acid. The said nucleic acid (NA) is selected from ribonucleic acid (RNA), messenger RNA (mRNA), Deoxyribonucleic acid (DNA), plasmid DNA (pDNA), fragment of RNA, mRNA, DNA, pDNA, miRNA or any chimeric or fusion thereof.

Accordingly, present invention provides a vaccine formulation comprising nucleic acid as a vaccine antigen for the prophylaxis of virus-mediated diseases, said vaccine formulation comprising:
a. nucleic acid (NA) selected from ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), plasmid DNA (pDNA), fragment of RNA, mRNA, DNA, pDNA, miRNA, or any chimeric or fusion thereof;
b. DC targeting lipids;
c. endosome disrupting lipids; and
d. co-lipids selected from:
i. a steroid lipid, which may be sterol and is selected from cholesterol, ergosterol, stigmasterol, sitosterol, campesterol, stigmastanol like phytosterols, and other steroids such as dexamethasone, prednisolone, triamcinolone, and/or a mixture thereof;
ii. a phospholipid selected from dioleoyl phosphatidylethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [DSPE], 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dipalmitoyl-sn-glycero-3-phosphocholone [DPPC], hydrogenated soy phosphatidylcholine (HSPC), or mixtures thereof; and
e. optionally, one or more pharmaceutically acceptable excipients.

Preferably, the co-lipid the said vaccine formulation is dioleoyl phosphatidylethanolamine [DOPE].

In one preferred embodiment, the said vaccine formulation comprises nucleic acid, PEGylated endosome disrupting lipids, non-PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

In another preferred embodiment, the said vaccine formulation comprises nucleic acid, non-PEGylated endosome disrupting lipids, PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.
The pharmaceutically acceptable excipients are selected from buffers, adjuvants, diluents, lubricants, binders, stabilizers, preservatives, disintegrants, absorbents, colorants, surfactants, flavours, sweeteners, residuals, immune-boosting co-excipients, or a combination thereof.

In the said vaccine composition, the endosome disrupting lipids, DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1 to 1:1:1.

Preferably, the endosome disrupting lipids, PEGylated DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1.

Present invention discloses a vaccine formulation comprising the lipid-based composition of the present invention for the prophylaxis and/or treatment of disease including virus mediated disease in a human subject.

The said vaccine formulation comprises of lipid-based composition along with the biologically active Nucleic Acid (NA) to provide a pharmaceutical formulation with or without one or more pharmaceutically acceptable excipient to form Lipid-Nanoparticle (LNP) formulation or a Liposomal formulation or Lipoplex formulation.

The said vaccine formulation performs in vitro transfection at a N/P charge ratio (Lipid: Nucleic acid) of 1:1 to 4:1.

In another aspect present invention discloses a method for preparation of vaccine formulation, comprising the steps of:
(a) preparing the nucleic acid constructs;
(b) complexing the lipid-based formulation of the present invention with nucleic acid constructs at fixed mole ratios to make liposomes or lipoplex;
(c) obtaining the same as vaccine formulation and preserving the same at room temperature or 4ºC.

USE:

In yet another aspect present invention discloses the use of lipid-based formulation for:
• delivery of Nucleic acid to body antigen presenting cells (APC);
• targeting the antigen encoding nucleic acid and self-assembled LNPs to antigen presenting cells.
• Preparing the LNPs without use of chlorinated organic solvents and energy demanding processes.
• in preparation of pharmaceutical formulation including vaccine formulation;
• in prophylaxis, treatment, and management of diseases including infectious diseases.

EXAMPLES:

The novel PEGylated lipids, their synthesis and novel intermediates used for synthesis and evaluation of these novel lipid for synthesis of lipid-based composition and formulation further be understood by following non-limiting examples and corresponding drawing figures.

Endosome disrupting Histidinylated lipid (labeled in the Figures as His). His was synthesized as described previously (Gene Therapy 2003, 10, 1206-1215.

Commercially available DOPE (Di-oleoyl-phosphatidyl ethanolamine) was used as co-lipid.

Example 1:

Scheme I: Schematic description of the synthetic routes used for preparing endosome-disrupting PEGylated histidinylated lipid.

Reagents: i). (BOC)2O, Et3N, DCM, 2 h, 0 0C; ii). K2CO3, n-C16H33Br, EtOAC, 70-80 0C, Reflux, 48 h; iii). TFA/DCM (1:1, v/v), 24 h; iv). HATU, DIPEA, DCM, 48 h; v). TFA/DCM (1:1, v/v), 24 h; vi). HATU, DIPEA, DMF/DCM (1:2), 48 h, N2 atm, vii). TFA/DCM (1:1, v/v), 24 h; viii). Amberlite IR 400 Cl- ion exchange resin, MeOH.

Experimental procedures:

Step 1: Preparation of Intermediate I (Scheme I):
5 g (83 mmol) ethylenediamine was dissolved in 5 mL DCM in an ice-cold condition and the solution was kept stirred. To this ice-cold solution, 3 g BOC-anhydride (14 mmol) was added dropwise for 10 min, and thereafter, 3.3 mL triethylamine was added. The reaction mix was kept under stirring condition for 2 h and diluted with 50 mL DCM. The resulting solution was washed with water (3x100 mL), the organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate upon rotatory evaporation and vacuum drying afforded intermediate I (1.35 g, 60% yield).
ESI MS: [M]+ =161.
1H NMR (400 MHz, Chloroform-d): d 4.90 (s, 1H), 3.16 (q, J = 5.8 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 1.43 (s, 9H).

Step 2: Preparation of Intermediate II (Scheme I):
0.36 g (2.25 mmol) intermediate I was dissolved in 10 mL ethyl acetate and the temperature of the oil bath was kept at 80 ºC. 1.24 g K2CO3 was added and the reaction mixture was kept stirred for 10 min. 1.7 g (5.56 mmol) 1-bromo-hexadecane was added and the reaction mixture was kept under reflux for 48 h. The reaction mixture was cooled, diluted with 50 mL ethyl acetate and washed with water (2 x 100 mL) followed by brine solution (3 x 100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate, filtered and the solvent from the filtrate was removed by rotary evaporation. The residue, upon purification by column chromatography over 100-200 mesh size silica using 10% EtOAC/Hexane (v/v), afforded intermediate II (1.2 g, 89% yield, Rf = 0.7 at 30% EtOAC/Hexane, v/v).
HRMS: [M]+= 609.
1H NMR (500 MHz, Chloroform-d): d 5.02 (s, 1H), 3.51 (s, 2H), 3.20 – 3.12 (m, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.39 (m, 2H), 1.47 (s, 9H), 1.42 – 1.23 (m, 56H), 0.90 (t, J = 6.8 Hz, 6H).

Step 3: Preparation of Intermediate III (Scheme I):
0.32 g (0.52 mmol) intermediate II was dissolved in 3 mL TFA/DCM (1:1, v/v) and the solution was kept under stirring condition for 24 h at room temperature. The reaction mixture was transferred to 20 mL DCM, extracted by distilled water (2x100 mL) followed by aqueous saturated NaHCO3 (2 x 100 mL) and brine solution (2x100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate and filtered. Rotatory evaporation of the solvent from the filtrate afforded intermediate III (0.23 g, 86% yield, Rf = 0.5 at 10% MeOH/CHCl3, v/v).
ESI-MS: [M]+ = 509.
The intermediate III is further characterized by 1H NMR (500 MHz, Chloroform-d) d 4.07 (m, 1H), 3.40 – 3.16 (m, 1H), 2.92 – 2.73 (m, 2H), 2.63 – 2.49 (m, 2H), 2.45 – 2.39 (m, 4H), 1.47 – 1.18 (m, 56H), 0.89 (t, J = 6.9 Hz, 6H).

Step 4: Preparation of Intermediate IV (Scheme I):
Boc-(PEG)27-COOH (0.45 g, 0.32 mmol) was dissolved in 3 mL dry DMF and HATU (0.18 g, 0.47 mmol) was added to the solution. The reaction mixture was left for 30 min at 0 0C under nitrogen atmosphere. Amine intermediate III (0.3 g, 0.59 mmol) was dissolved in dry DCM and added dropwise to the ice-cooled solution and thereafter, 0.2 mL DIPEA was added. The reaction was continued for 48 h at room temperature under nitrogen atmosphere. The reaction mixture was transferred to 50 mL DCM and the resulting solution was washed with cold 1N HCl (2x100 mL), followed by cold saturated sodium bicarbonate (2x100 mL) and brine solution (1x100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate, filtered and the solvent from the filtrate was removed by rotary evaporator. The residue, upon purification by column chromatography over activated neutral alumina using 0.2-0.3% MeOH/DCM (v/v), afforded intermediate IV (0.41 g, 66% yield, Rf = 0.5 at 5% MeOH/CHCl3, v/v).
HRMS (m/z): [M +1]+ = 1914.
1H NMR (500 MHz, Chloroform-d): d 6.53 (s, 1H), 5.08 (s, 1H), 3.73 (t, J = 5.5 Hz, 112H), 3.37 – 3.21 (m, 4H), 2.65 – 2.29 (m, 8H), 1.44 (s, 9H), 1.25 (s, 56H), 0.87 (t, J = 6.7 Hz, 6H).

Step 5: Preparation of Intermediate V (Scheme I):
Intermediate IV (0.18 g, 0.09 mmol) was dissolved in 3 mL TFA/DCM (1:1, v/v) and the solution was kept under stirring condition for 24 h at room temperature. The reaction mixture was then transferred to 20 mL DCM and extracted with distilled water (2x100 mL) followed by aqueous saturated NaHCO3 (2x100 mL) and brine solution (2x 100 mL). Finally, the organic layer was passed through anhydrous sodium sulphate and the filtrate, upon rotatory evaporation, afforded intermediate V (0.14 g, 89% yield, Rf = 0.4-0.3 at 5% MeOH/DCM, v/v).
HRMS (m/z): [M +1]+ =1814.
1H NMR (400 MHz, Chloroform-d): d 6.91 – 6.28 (m, -1H), 3.64 (s, 112H), 3.49 – 3.26 (m, 2H), 3.09 – 2.97 (m, 1H), 2.60 – 2.51 (m, 2H), 2.45 (q, J = 9.1, 7.6 Hz, 5H), 1.47 – 1.37 (m, 4H), 1.32 – 1.21 (m, 56H), 0.88 (d, J = 13.7 Hz, 6H).

Step 6: Synthesis of Intermediate VI (Scheme I):
N, N di-boc-L-histidine (0.056 g, 0.16 mmol) was dissolved in 3 mL dry DCM and HATU (0.12 g, 0.32 mmol) was added to it sequentially at 0 ºC under nitrogen atmosphere. The reaction was left for half an hour at this condition and then the amine intermediate V (0.14 g, 0.08 mmol dissolved in DCM) was added to it. DIPEA (0.1 ml) was added to the reaction mixture dropwise. The reaction was continued for 48 h at room temperature under nitrogen atmosphere. The reaction mixture was transferred in DCM (50 ml) and washed it by cold 1(N) HCl (2x100 mL), cold saturated sodium bicarbonate (2x100 mL), brine solution (1x100 mL). The organic layer was dried over anhydrous sodium sulphate, filtered, evaporated by rotary evaporator. The residue upon column chromatography over 100-200 mesh size silica using 5% MeOH/DCM (v/v) as mobile phase afforded pure intermediate VI (0.101 g, 59.5% yield, Rf = 0.7 at 10% MeOH/DCM (v/v).
HRMS (m/z): [M+Na]+ =2173
1H NMR (500 MHz, Chloroform-d) d 8.18 – 8.05 (m, 2H), 7.20 – 7.14 (m, 1H), 7.09 – 7.04 (m, 1H), 6.63 – 6.58 (m, 1H), 5.15 – 5.09 (m, 1H), 3.81 – 3.75 (m, 2H), 3.74 (t, J = 6.0 Hz, 2H), 3.69 – 3.60 (m, 108H), 3.57 – 3.54 (m, 1H), 3.52 – 3.46 (m, 2H), 3.43 – 3.37 (m, 1H), 2.52 – 2.48 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.60 (s, 9H), 1.44 – 1.39 (m, 9H), 1.25 (s, 60H), 0.88 (t, J = 6.4 Hz, 6H).

Step 7 & 8: Synthesis of Target endosome disrupting PEGylated histidine lipid 1:
The above prepared Intermediate VI (0.097 g, 0.045 mmol) was dissolved in 3 ml (1:1) TFA/DCM and the reaction mixture was stirred for 24 h at room temperature. The excess TFA was removed by nitrogen flushing and the final deprotected compound was chased by DCM several times to eliminate the excess TFA. Finally, the trifluoro acetate counterion of the residue was replaced by Cl- counterion using amberlite IR 400 Cl- ion exchange resin in MeOH. The resulting targeting lipid containing chloride counterion upon further purification (by first dissolving in MeOH/CHCl3 (1:1, v/v) and then precipitating by adding diethyl ether) afforded 0.06 g pure PEGylated histidine lipid 1 (yield 68.4%).
HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976.
1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).
Example 2:

Scheme-IIA: Schematic description of the synthetic routes used for preparing the PEGylated Lipid Precursor (V) (Scheme-II).

Step 1: Synthesis of lipid (Scheme IIA):
Preparation of Intermediate I (Scheme IIA): 5 g (83 mmol) ethylenediamine was dissolved in 5 mL DCM in an ice-cold condition and the solution was kept stirred. To this ice-cold solution, 3 g BOC-anhydride (14 mmol) was added dropwise for 10 min, and thereafter, 3.3 mL triethylamine was added. The reaction mix was kept under stirring condition for 2 h and diluted with 50 mL DCM. The resulting solution was washed with water (3x100 mL), the organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate upon rotatory evaporation and vacuum drying afforded intermediate I (1.35 g, 60% yield).
ESI MS: [M]+ =161.
1H NMR (400 MHz, Chloroform-d): d 4.90 (s, 1H), 3.16 (q, J = 5.8 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 1.43 (s, 9H).

Step 2: Preparation of Intermediate II (Scheme IIA):
0.36 g (2.25 mmol) intermediate I was dissolved in 10 mL ethyl acetate and the temperature of the oil bath was kept at 80 ºC. 1.24 g K2CO3 was added, and the reaction mixture was kept stirred for 10 min. 1.7 g (5.56 mmol) 1-bromo-hexadecane was added and the reaction mixture was kept under reflux for 48 h. The reaction mixture was cooled, diluted with 50 mL ethyl acetate and washed with water (2 x 100 mL) followed by brine solution (3 x 100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate, filtered and the solvent from the filtrate was removed by rotary evaporation. The residue, upon purification by column chromatography over 100-200 mesh size silica using 10% EtOAC/Hexane (v/v), afforded intermediate II (1.2 g, 89% yield, Rf = 0.7 at 30% EtOAC/Hexane, v/v).
HRMS: [M]+= 609.
1H NMR (500 MHz, Chloroform-d): d 5.02 (s, 1H), 3.51 (s, 2H), 3.20 – 3.12 (m, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.39 (m, 2H), 1.47 (s, 9H), 1.42 – 1.23 (m, 56H), 0.90 (t, J = 6.8 Hz, 6H).

Step 3: Preparation of Intermediate III (Scheme IIA):
0.32 g (0.52 mmol) intermediate II was dissolved in 3 mL TFA/DCM (1:1, v/v) and the solution was kept under stirring condition for 24 h at room temperature. The reaction mixture was transferred to 20 mL DCM, extracted by distilled water (2x100 mL) followed by aqueous saturated NaHCO3 (2 x 100 mL) and brine solution 17 (2x100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate and filtered. Rotatory evaporation of the solvent from the filtrate afforded intermediate III (0.23 g, 86% yield, Rf = 0.5 at 10% MeOH/CHCl3, v/v).
ESI-MS: [M]+ = 509.

Step 4: Preparation of Intermediate IV (Scheme IIA):
Boc-(PEG)27-COOH (0.45 g, 0.32 mmol) was dissolved in 3 mL dry DMF and HATU (0.18 g, 0.47 mmol) was added to the solution. The reaction mixture was left for 30 min at 0 0C under nitrogen atmosphere. Amine intermediate III (0.3 g, 0.59 mmol) was dissolved in dry DCM and added dropwise to the ice-cooled solution and thereafter, 0.2 mL DIPEA was added. The reaction was continued for 48 h at room temperature under nitrogen atmosphere. The reaction mixture was transferred to 50 mL DCM and the resulting solution was washed with cold 1N HCl (2x100 mL), followed by cold saturated sodium bicarbonate (2x100 mL) and brine solution (1x100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate, filtered and the solvent from the filtrate was removed by rotary evaporator. The residue, upon purification by column chromatography over activated neutral alumina using 0.2-0.3% MeOH/DCM (v/v), afforded intermediate IV (0.41 g, 66% yield, Rf = 0.5 at 5% MeOH/CHCl3, v/v).
HRMS (m/z): [M +1]+ = 1914.
1H NMR (500 MHz, Chloroform-d): d 6.53 (s, 1H), 5.08 (s, 1H), 3.73 (t, J = 5.5 Hz, 112H), 3.37 – 3.21 (m, 4H), 2.65 – 2.29 (m, 8H), 1.44 (s, 9H), 1.25 (s, 56H), 0.87 (t, J = 6.7 Hz, 6H).

Step 5: Preparation of Intermediate V (Scheme IIA):
Intermediate IV (0.18 g, 0.09 mmol) was dissolved in 3 mL TFA/DCM (1:1, v/v) and the solution was kept under stirring condition for 24 h at room temperature. The reaction mixture was then transferred to 20 mL DCM and extracted with distilled water (2x100 mL) followed by aqueous saturated NaHCO3 (2x100 mL) and brine solution (2x 100 mL). Finally, the organic layer was passed through anhydrous sodium sulphate and the filtrate, upon rotatory evaporation, afforded intermediate V (0.14 g, 89% yield, Rf = 0.4-0.3 at 5% MeOH/DCM, v/v).
HRMS (m/z): [M +1]+ =1814.
1H NMR (400 MHz, Chloroform-d): d 6.91 – 6.28 (m, -1H), 3.64 (s, 112H), 3.49 – 3.26 (m, 2H), 3.09 – 2.97 (m, 1H), 2.60 – 2.51 (m, 2H), 2.45 (q, J = 9.1, 7.6 Hz, 5H), 1.47 – 1.37 (m, 4H), 1.32 – 1.21 (m, 56H), 0.88 (d, J = 13.7 Hz, 6H).

Scheme-IIB: Schematic description of the synthetic routes used for preparing the DC-targeting PEGylated Lipid from Intermediate V (Scheme-IIB).

Step 6: Preparation of Intermediate VI (Scheme IIB):
N ? -Z-N ? -BOC-LLysine (0.075 g, 0.2 mmol) was dissolved in 3 mL DCM/DMF (3:1, v/v, dry DCM: dry DMF) and HATU (0.15 g, 0.4 mmol) was added to the solution. The reaction mixture was kept under stirring for 30 min at 0 ºC under nitrogen atmosphere. Amine intermediate V (0.18g, 0.1 mmol) was dissolved in dry DCM. The resulting solution was added to the ice-cooled solution and 0.1 mL DIPEA was added. The reaction was continued for 48 h at room temperature under nitrogen atmosphere. The reaction mixture was diluted with 50 mL DCM and washed by cold 1N HCl (2x100 mL) followed by cold saturated sodium bicarbonate (2x100 mL) and brine solution (1x100 mL). Finally, the organic layer was dried over anhydrous sodium sulphate, filtered, and the solvent from the filtrate was removed by rotary evaporator. The residue, upon purification by column chromatography over 100-200 mesh size silica using 5% MeOH/DCM (v/v), afforded pure intermediate VI (0.17 g, 80% yield, Rf = 0.6 at 10% MeOH/DCM, v/v).
HRMS(m/z): [M+H]+= 2176.
1H NMR (500 MHz, Chloroform-d): d 8.60 – 8.53 (m, 1H), 8.32 – 8.21 (m, 1H), 8.11 (dd, J = 7.4, 3.5 Hz, 1H), 7.35 (d, J = 14.1 Hz, 5H), 6.97 – 6.90 (m, 1H), 5.72 – 5.65 (m, 1H), 5.20 – 5.06 (m, 3H), 4.80 – 4.70 (m, 1H), 4.22 – 4.15 (m, 1H), 3.66 (s, 108H), 3.47 (dd, J = 4.7, 2.0 Hz, 2H), 3.22 (td, J = 8.5, 6.9, 3.1 Hz, 2H), 3.15 – 3.00 (m, 6H), 2.67 – 2.61 (m, 1H), 2.58 – 2.53 (m, 2H), 1.60 – 1.48 (m, 4H), 1.44 (s, 9H), 1.26 (d, J = 13.7 Hz, 60H), 0.90 (t, J = 6.3 Hz, 6H).

Step 7: Preparation of Intermediate VII (Scheme IIB):
Intermediate VI (0.26 g, 0.12 mmol) was dissolved in 5 mL MeOH and Pd/Charcoal (0.05 g 10% Pd, w/w) was added. The solution was degassed and purged with H2 gas. The reaction mixture was kept under stirring condition for 14 h under hydrogen atmosphere. The reaction mixture was filtered through celite, and the filtrate was dried over anhydrous sodium sulphate. Rotatory evaporation of the solvent from the afforded 0.24 g (97% yield) intermediate VII (Rf = 0.3 in 10% MeOH/DCM, v/v).
HRMS(m/z): [M+H]+= 2042.
1H NMR (500 MHz, DMSO-d6): d 8.38 – 8.32 (m, 1H), 8.31 – 8.25 (m, 1H), 8.15 – 8.10 (m, 1H), 7.65 (td, J = 6.4, 3.4 Hz, 1H), 7.46 – 7.22 (m, 1H), 7.19 – 7.11 (m, 1H), 6.76 – 6.67 (m, 1H), 3.50 (s, 112H), 3.10 – 3.03 (m, 2H), 2.90 – 2.84 (m, 2H), 2.63 (s, 1H), 2.41 – 2.32 (m, 6H), 2.27 (t, J = 6.3 Hz, 2H), 1.64 – 1.46 (m, 2H), 1.29 (d, J = 66.4 Hz, 69H), 0.84 (t, J = 6.3 Hz, 6H).

Step 8: Preparation of Intermediate VIII (Scheme IIB):
3,4,5 Triacetoxycyclohex-1-ene carboxylic acid (0.09 g, 0.29 mmol) was dissolved in 3 mL dry DMF and HATU (0.22 g, 0.58 mmol) was added to the solution. The reaction mixture was kept under stirring for 30 min at 0 ºC under nitrogen atmosphere. Amine intermediate VII (0.24 g, 0.12 mmol) was dissolved in dry DCM and the solution was added to the ice-cooled solution. 0.1 mL DIPEA was added, and the reaction was continued for 48 h at room temperature under nitrogen atmosphere. The reaction mixture was diluted with 50 mL DCM and washed with cold 1N HCl (2x100 mL) followed by sequential washings with cold saturated sodium bicarbonate (2x100 mL) and brine (1x100 mL) solutions. The organic layer was dried over anhydrous sodium sulphate, filtered and the solvent from the filtrate was removed by rotatory evaporation. The residue, upon purification by column chromatography over 234-400 mesh size silica using 10% MeOH/DCM (v/v), afforded pure intermediate VIII (0.14 g, 50% yield, Rf = 0.5 at 10% MeOH/DCM, v/v).
HRMS(m/z): [M+ Na]+= 2346.
1H NMR (500 MHz, Chloroform-d): d 8.06 (s, 1H), 7.17 (s, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.36 (s, 1H), 5.71 (s, 1H), 5.24 (s, 1H), 5.14 – 4.89 (m, 1H), 4.79 (s, 1H), 4.51 – 4.39 (m, 1H), 3.78 (s, 1H), 3.74 (t, J = 6.1 Hz, 2H), 3.71 – 3.58 (m, 108H), 3.55 (s, 1H), 3.51 – 3.41 (m, 2H), 3.17 (s, 2H), 3.03 – 2.91 (m, 4H), 2.52 (t, J = 6.2 Hz, 2H), 2.41 (dd, J = 17.9, 5.0 Hz, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.69 (d, J = 14.3 Hz, 4H), 1.53 – 1.47 (m, 2H), 1.43 (s, 9H), 1.27 (d, J = 15.1 Hz, 60H), 0.87 (d, J = 7.0 Hz, 6H).

Step 9: Preparation of Intermediate IX (Scheme IIB):
Intermediate VIII (0.14 g, 0.06 mmol) was dissolved in 3 ml TFA/DCM (1:1, v/v) and the solution was kept under stirring condition for 24 h at room temperature. The reaction mixture was then diluted with 20 mL DCM and sequentially extracted with distilled water (2 x100 mL), aqueous saturated NaHCO3 and brine solution (2x100 mL). The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate, upon rotary evaporation of solvent, afforded intermediate IX (0.11 g, 83% yield, Rf = 0.4 at 10% MeOH/DCM, v/v).
HRMS(m/z): [M+H]+= 2224.
1H NMR (500 MHz, DMSO-d6): d 8.12 – 7.88 (m, 1H), 7.71 – 7.51 (m, 2H), 6.42 (s, 1H), 5.65 – 5.51 (m, 1H), 5.23 – 4.93 (m, 2H), 4.25 (s, 1H), 3.50 (s, 112H), 3.14 – 2.93 (m, 4H), 2.92 – 2.80 (m, 2H), 2.80 – 2.68 (m, 3H), 2.65 – 2.58 (m, 3H), 2.37 – 2.32 (m, 5H), 2.06 – 1.97 (m, 6H), 1.90 – 1.37 (m, 8H), 1.23 (s, 58H), 0.87 – 0.80 (m, 6H).

Step 10: Preparation of Intermediate X (Scheme IIB):
Mercuric chloride (0.02 g, 0.07 mmol) was added to a 3 mL 1:2 (v/v) dry DMF: dry DCM. Intermediate IX (0.11 g, 0.05 mmol), bis-N-Boc-thiourea (0.02 g, 0.07 mmol) and triethylamine (0.2 µL) were sequentially added, and the reaction mixture was kept under stirring for 1 hour at 0 °C under nitrogen atmosphere. The reaction mixture solution was diluted with 20 mL ethyl acetate and filtered through a pad of Celite. The filtrate was sequentially washed with cold water (2 x 50 mL) and brine solution (2x50 mL), dried over anhydrous sodium sulphate, filtered and the solvent from the filtrate was removed by rotary evaporator. The residue, upon purification by column chromatography over 100-200 mesh silica using 5% methanol in DCM (v/v), afforded 0.08 g (yield 60%) of the pure intermediate X (Rf = 0.6, 10%methanol in DCM, v/v).
HRMS(m/z): [M+H]+= 2466.
1H NMR (500 MHz, DMSO-d6): d 11.49 (s, 1H), 8.25 (t, J = 5.1 Hz, 1H), 8.16 – 7.84 (m, 2H), 6.46 – 6.36 (m, 1H), 5.60 (d, J = 3.5 Hz, 1H), 5.14 (d, J = 3.7 Hz, 2H), 4.24 (q, J = 8.2 Hz, 2H), 3.92 (d, J = 5.7 Hz, 2H), 3.68 – 3.62 (m, 2H), 3.61 – 3.57 (m, 3H), 3.50 (s, 118H), 3.40 (d, J = 5.8 Hz, 3H), 3.21 (ddd, J = 26.6, 13.2, 6.4 Hz, 5H), 3.10 – 2.91 (m, 1H), 2.90 – 2.81 (m, 1H), 2.63 (s, 1H), 2.42 – 2.35 (m, 1H), 2.33 (d, J = 7.0 Hz, 2H), 2.26 (t, J = 7.3 Hz, 1H), 2.05 (s, 2H), 2.04 (s, 3H), 2.00 (s, 3H), 1.67 – 1.58 (m, 3H), 1.51 (s, 2H), 1.47 (s, 9H), 1.38 (s, 9H), 1.23 (s, 60H), 0.84 (d, J = 4.0 Hz, 6H).

Step 11: Preparation of Intermediate XI (Scheme IIB):
Intermediate X (0.06 g, 0.02 mmol) was dissolved in 2 mL MeOH and catalytic amount NaOMe (0.003 g, 0.06 mmol) was added to increase the pH of the reaction mixture to 10. The reaction mixture was stirred for 1 h, and thereafter, Dowex 50 resin was added to bring the pH of the reaction mixture to 7. The reaction mixture was then filtered. Rotatory evaporation of the solvent from the filtrate afforded 0.05 g intermediate XI (yield 100%, Rf = 0.3, 10% MeOH/DCM, v/v).
HRMS(m/z): [M+ Na]+= 2362.
1H NMR (500 MHz, DMSO-d6): d 11.50 (s, 1H), 9.49 (s, 1H), 8.36 (s, 1H), 8.27 (t, J = 5.5 Hz, 1H), 8.10 (s, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.71 – 7.61 (m, 2H), 7.56 – 7.45 (m, 1H), 7.35 (dd, J = 18.3, 8.1 Hz, 1H), 7.22 – 7.06 (m, 1H), 6.88 (s, 1H), 6.35 (dd, J = 3.4, 1.7 Hz, 1H), 4.24 (s, 1H), 4.19 (s, 1H), 3.93 (d, J = 5.7 Hz, 1H), 3.86 – 3.80 (m, 2H), 3.51 (s, 108H), 3.25 (d, J = 6.5 Hz, 1H), 3.07 (d, J = 7.2 Hz, 1H), 2.68 – 2.52 (m, 3H), 2.48 – 2.44 (m, 1H), 2.41 – 2.33 (m, 4H), 2.27 (q, J = 7.3, 6.9 Hz, 3H), 1.64 (d, J = 7.8 Hz, 2H), 1.55 – 1.50 (m, 2H), 1.49 – 1.36 (m, 18H), 1.24 (s, 60H), 0.87 – 0.84 (m, 6H).

Step 12: Synthesis of the Target Lipid 1 (Scheme IIB):
Intermediate XI (0.05 g, 0.02 mmol) was dissolved in 3 mL TFA/DCM (1:1, v/v) and the solution was kept under stirring condition for 24 h at room temperature. The excess TFA was removed by gentle nitrogen flow and the deprotected residue was chased by DCM several times to eliminate the remaining TFA. Finally, the trifluoroacetate counterion of the residue was replaced with chloride ion by chloride ion exchange chromatography over amberlite IR 400 Clion exchange resin using methanol as eluent. Methanol was removed by rotatory evaporation and the resulting residue was dissolved in MeOH/CHCl3 (1:1, v/v). Addition of anhydrous diethyl resulted into precipitation of the target compound which after filtering and vacuum drying afforded 0.04 g of lipid 1 (yield 93%).
HRMS(m/z): [M+H]+= 2140, [ M +1]+/2 =1070
1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 – 7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

Example 3:

Preparation and physico-chemical characterizations of lipid-based nanoparticles (LNPs) and their electrostatic complexes with GFPmRNA (mRNAplexes):

Appropriate amounts of endosome disrupting Histidinylated lipid, the dendritic cell (DC) targeting cationic amphiphile containing mannose-mimicking shikimoyl and transfection enhancing guanidine functionality in its polar head-group region and DOPE (commercially available co-lipid) from their stock solutions (in molecular biology grade absolute ethanol) were dissolved taken in a small glass vial and the mixture was diluted to 25 µL with absolute ethanol. The resulting ethanolic solution of lipids was rapidly injected into 75 µL nuclease free water (~pH 7) under stirring conditions for 15 min such that the concentrations of each of the three lipids in the 100 µL stock solution of the resulting self-assembled cationic LNPs reached at appropriate mole ratio.

Figure 1 represent the TEM image, surface potentials, and sizes for self-assembled LNPs containing 1:1:1 mole ratio of non-PEGylated endosome disrupting histidine lipid, non-PEGylated DC-targeting lipid and commercially available co-lipid DOPE, respectively. This self-assembled LNPs formulation showed promising in vitro GFPmRNA delivery efficiency in RAW 264.7 cells.

Figure 2 represent sizes, surface potentials, and SEM images for self-assembled LNPs containing 1:1:0.01 mole ratio of endosome disrupting non-PEGylated histidine lipid, commercially available co-lipid DOPE and PEGylated DC-targeting lipid respectively.

Example 4:

Preparation and physico-chemical characterizations of lipid-based nanoparticles (LNPs) and their electrostatic complexes with GFPmRNA (mRNAplexes):

Appropriate amounts of endosome disrupting Histidinylated lipid, the dendritic cell (DC) targeting cationic amphiphile containing mannose-mimicking shikimoyl and transfection enhancing guanidine functionality in its polar head-group region and DOPE (commercially available co-lipid) from their stock solutions (in molecular biology grade absolute ethanol) were dissolved taken in a small glass vial and the mixture was diluted to 25 µL with absolute ethanol. The resulting ethanolic solution of lipids was rapidly injected into 75 µL nuclease free water (~pH 7) under stirring conditions for 15 min such that the concentrations of each of the three lipids in the 100 µL stock solution of the resulting self-assembled cationic LNPs became 0.33 mM each (i.e. 1 mM total lipid concentration). GFPmRNAplexes (electrostatic complexes of self-assembled LNPs and GFPmRNAs) containing 2:1 and 4:1 charge ratios of cationic DC-targeting lipid: GFPmRNA were prepared by mixing appropriate volumes of stock solutions of LNPs (containing 1 mM total lipid concentration) and GFPmRNA so that both the resulting mRNAplexes contained 0.3 µg GFPmRNA.

Figure 3 represent mRNA transfection efficiency of self-assembled LNPs containing non-PEGylated DC-targeting lipid: non-PEGylated endosome disrupting Histidine Lipid: DOPE at 1:1:1 mole ratio electrostatically complexed with GFPmRNA having 2:1 +/- charge ratio.

Example 5:

In vitro Transfection protocol in RAW 264.7 cells (mouse macrophage as model antigen-presenting cells):
Appropriate amounts of DOPE (co-lipid), endosome-disrupting histidinylated lipid and the PEGylated dendritic cell (DC) targeting PEGylated lipid from their stock solutions (in Molecular Biology Grade absolute ethanol) were taken in a small glass vial and the mixture was diluted to absolute ethanol so that, final alcohol conc. should be 25%. The resulting ethanolic solution of lipids was rapidly injected into 75 µL nuclease free water (~pH 7) under stirring conditions for 15 min such that the concentrations of both the lipids in the resulting 100 µL liposome stock solution became 1 mM DOPE, 1mM histidinylated lipid, 0.01 mM PEGylated DC-targeting lipid (i.e. 2.01 mM total lipid concentration). RAW 264.7 cells were seeded on 24 well plates (105 cells per well) and incubated for 24 h in CO2 incubator. Cells were washed with 1x PBS and GFPmRNAplexes containing 4:1 +/- charge ratios using 0.3 µg of GFPmRNA. One positive charge for histidinylated lipid was taken into account when calculating the charge ratio of GFPmRNAplexes, and positive charge from the PEGylated DC-targeting lipid was ignored because it’s conc was significantly lower in liposome solutions. Appropriate amounts of liposome (4 µL from 2.01 mM total lipid conc.) were added after calculating it for 4:1 +/- charge ratios. After incubating the lipid/m-RNA mixture for 25-30 min at 25 0C, serum-free RPMI media (500 µL) were added to each well and the cells were incubated for 4 h at 37ºC. Media was then replaced with RPMI media containing 10% FBS and the transfection efficiencies were measured after 24 h by flow cytometry.

Figure 4 represent transfection efficiencies of the self-assembled of the PEGylated LNPs qualitatively measured by flow cytometry techniques in RAW 264.7 cells (mouse macrophage cells, widely used as model antigen presenting cells).

Example 6:

In vitro transfection protocol in RAW 264.7 cells preincubated with mannan:

RAW 264.7 cells were seeded on 96 well plates (2.5x104 cells per well) and incubated for 24 h in CO2 incubator. After 24 h incubation, RAW264.7 cells were preincubated for 30 min with increasing concentrations of (0-300 µg) of mannan (a natural ligand for mannose receptor) per well. Without washing, cells were transfected with: GFPmRNAplexes of non-PEGylated DC-targeting lipid & DOPE (1:1 mole ratio) and GFPmRNAplexes of non-PEGylated DC-targeting lipid, non-PEGylated histidine lipid & DOPE (1:1:1 mole ratio). The amount of GFPmRNA used in both the cases were 0.3 µg and the +/- charge ratios in both the cases were kept at 2:1. Serum free RPMI media was added so that the total solution volume in each well was 100 µL and the cells were incubated for 4 h at 37 ºC. Media was then replaced with complete RPMI media and the transfection efficiencies were measured after 24 h using epifluorescence microscope. The DC transfection efficiencies were adversely affected in presence of 300 µg of mannan.

Thus, the findings summarized in Figures 5 & 6 demonstrated mannose receptor-mediated DC transfection efficacies of the presently described self-assembled LNPs.

Example 7:

In vitro transfection protocol in RAW 264.7 cells preincubated with mannan:

RAW 264.7 cells were seeded on 96 well plates (2.5x104 cells per well) and incubated for 24 h in CO2 incubator. After 24 h incubation, RAW264.7 cells were preincubated with increasing concentrations of (0-300 µg) of mannan (a natural ligand for mannose receptor) per well. Without washing cells were transfected with GFPm-RNAplexes of: PEGylated DC-targeting lipid, DOPE & Histidine (0.01:1:1) mmol liposomes containing 4:1 +/- charge ratios (prepared by adding 0.3 µg GFPm-RNA and appropriate amounts of PEGylated DC-targeting liposome stock solutions). Serum free RPMI media was added (so that the total solution volume in each well was 100 µL) and the cells were incubated for 4 h at 37 ºC. Media was then replaced with 10% FBS containing RPMI media and the transfection efficiencies were measured after 24 h using epifluorescence microscope. The DC transfection efficiencies were adversely affected in presence of 300 µg of mannan.

Figure 7 demonstrates mannose receptor-mediated DC transfection efficacies of the presently described self-assembled LNPs.

Example 8:

Newly developed self-assembled formulation of His, DOPE and DCD for mRNA vaccine delivery to antigen presenting cells.

Appropriate amounts of DOPE (commercially available co-lipid), His and DCD from their stock solutions (in Molecular Biology Grade absolute ethanol) were taken in a small glass vial and the mixture was diluted with absolute ethanol to make the final volume 25 µL. The resulting ethanolic solution was rapidly injected into 75 µL nuclease free water (~pH 7) under stirring conditions and the stirring was continued for 15 min. The final concentrations of DOPE, His and the pegylated lipid DCD in the resulting liposomes became 1 mM, 1 mM and 0.01 mM, respectively (i.e. the final total lipid concentrations became 2.01 mM). In order to remove ethanol, the liposomes were centrifuged using Merck-Millipore Amicon centrifugal filter units with 3 kD cut off filter at 5000 rpm for 35 min. The volume of the concentrated liposomes was reduced in volume by approximately 4 times. 100 µL nuclease free water (~pH 7) was added to the resulting concentrated liposomes and spinning was carried out at 5000 rpm for 35 min. Finally, such concentration procedure after adding 100 µL nuclease free water (~pH 7) was repeated one more time to remove most of the ethanol from the resulting concentrated liposome solution.

The physicochemical characterizations of self-assembled lipid formulations of His, DOPE, DCD and their mRNAplexes for mRNA vaccine delivery to antigen presenting cells have been studied with respect to mole ratio, hydrodynamic diameters (nm) and Zeta potentials (?, mV). The Table 1 represents the physicochemical characterizations of different self-assembled lipid formulations as below:

Table 1: Physico-chemical characterizations of self-assembled lipid formulations.
Sr. No. Liposomal formulations Mole Ratio Hydrodynamic diameters (nm) Zeta potentials
(?, mV)
1. His: DOPE 1:1 171.5± 2 23.7 ± 1
2. His: DOPE:DCD 1:1:0.01 159.5± 1.4 22.0 ± 0.8
3. His:DOPE mRNAplexes 1:1:1 507.2± 3.2 -13.3± 1.1
4. His:DOPE:DCD mRNAplexes 1:1:0.01 467.2± 3.4 -13.8± 0.9

Example 9:

Self-assembled formulation of His and DOPE for mRNA vaccine delivery to antigen presenting cells.

Appropriate amounts of DOPE (commercially available co-lipid) and His from their stock solutions (in Molecular Biology Grade absolute ethanol) were taken in a small glass vial and the mixture was diluted with absolute ethanol to make the final volume 25 µL. The resulting ethanolic solution was rapidly injected into 75 µL nuclease free water (~pH 7) under stirring conditions and the stirring was continued for 15 min. The final concentrations of DOPE and His in the resulting liposomes became 1 mM and 1 mM, respectively (i.e. the final total lipid concentrations became 2 mM). In order to remove ethanol, the liposomes were centrifuged using Merck-Millipore Amicon centrifugal filter units with 3 kD cut off filter at 5000 rpm for 35 min. The liposome volume was reduced approximately 4 times. 100 µL nuclease free water (~pH 7) was added to the resulting concentration liposomes and spinning was carried out at 5000 rpm for 35 min. Finally, such concentration procedure after adding 100 µL nuclease free water (~pH 7) was repeated one more time to remove most of the ethanol from the resulting concentrated liposome solution.

Example 10:

In vitro Transfection protocol (by epifluorescence microscopy) in RAW 264.7 cells (mouse macrophage as model antigen-presenting cells) using GFPmRNA precondensed with protamine sulfate (PS):

RAW 264.7 cells were seeded in 96 well plates (~2.5 x 104 cells per well) and incubated for 24 h in CO2 incubator. In the meanwhile, 0.3 µg of GFPmRNA was precondensed with 0.3 µg of protamine sulphate (PS). This 1:1 (w/w) GFPmRNA:PS complex was incubated for 10 min. Serum free RPMI media was added to bring the total volume to 100 µL. 4 µL of Amicon concentrated liposomes (prepared above) was added to this pre-complex of GFPmRNA & PS so that the total lipid:GFPmRNA charge ratio became approximately 16:1 and the mixture (GFPmRNAplex) was incubated for 20 min. Serum free RPMI media was added to make total volume 200 µL. The resulting GFPmRNAplex (200 µL) was added to the above mentioned RAW 264.7 cells in 96 well plates, incubated for 24 h in CO2 incubator. Cells were washed with 1X PBS, serum free RPMI media was added to the washed cells to make the total volume 200 µL and the cells were incubated for 4 h. Media was then replaced with complete RPMI media and the transfection efficiencies were measured after 24 h using epifluorescence microscope.

The present invention includes various figures illustrating the transfection efficiency optimizations of self-assembled lipid formulations in RAW 264.7 cells using GFPmRNA:PS ratios with protamine sulfate (PS).

Figure 8 demonstrates the transfection efficiency optimization of His:DOPE:DCD (1:1:0.01 mole ratio) lipid formulations with GFPmRNA precondensed with PS at different wt/wt ratios (10:1, 20:1, 40:1, and 80:1) using epifluorescence microscopy.

Figure 9 showcases the transfection efficiency optimizations of His:DOPE:DCD (1:1:0.01 mole ratio) lipid formulations with GFPmRNA precondensed with PS at various wt/wt ratios (0.5:1, 1:1, 2:1, 3:1, and 6:1) using epifluorescence microscopy.

Figure 10 represents the time course optimization for optimal transfection efficiency of His:DOPE:DCD (1:1:0.01 mole ratio) lipid formulations in RAW 264.7 cells using GFPmRNA:PS ratios. The figure captures the transfection efficiency at 6-hour, 12-hour, and 24-hour intervals using epifluorescence microscopy.

Figure 11 shows transfection of Histidine:DOPE (1:1) mRNAPlexes in RAW 264.7 cells. (A) Control untransfected RAW264.7 cells; (B) RAW264.7 cells transfected with GFPmRNAplexes of liposomes of DOPE and His (at 1 mM each, total lipid concentration: 2 mM). Transfection efficiency was evaluated using epifluorescence microscopy, allowing for accurate assessment and observation of the transfection process in the cells.

Example 11:

Flow cytometric methods for studying in vitro transfection efficiencies in RAW 264.7 cells (mouse macrophage as model antigen-presenting cells) using GFPmRNA precondensed with protamine sulfate (PS):

1 µg of GFPmRNA was mixed with 1µg of PS (i.e. in 1:1 w/w ratio) and incubated for 10 min. Serum free RPMI media was added to make the total volume 100 µL. To it, 12 µL of amicon concentrated liposome solution were added so that the cationic lipid:GFPmRNA approximate charge ratio became 16:1. The mixture of GFPmRNAplexes was incubated for 20 min and the serum free RPMI media was added to make total volume 500 µL.

RAW 264.7 cells were seeded on 24 well plates (~105 cells per well) and incubated for 24 h in CO2 incubator. Cells were washed with 1XPBS, GFPmRNAplexes (prepared as described above) were added to the cells and incubated for 4 h at 37 ºC in CO2 incubator. Media was then replaced with RPMI media containing 10% FBS and the transfection efficiencies were measured after 24 h by flow cytometry. In the present invention, Figure 12 demonstrates transfection efficiencies using mRNAplexes of two different self-assembled lipid formulations in RAW264.7 cells. The lipid compositions involve Histidine, DOPE, and DCD in various charge ratios. Transfection efficiency results are depicted as percentages: 37.1% for His:DOPE:DCD (16:1 + /- ratio) mRNAplexes; 51.2% for His:DOPE (16:1 + /- ratio) mRNAplexes and Control RAW264.7 untransfected cells highlighting their potential in nucleic acid delivery into cells.
,CLAIMS:
1. A cationic PEGylated lipid compound for non-cytotoxic self-assembling LNPs formulation, facilitating efficient endosomal release of mRNA/DNA vaccines, comprising:
a. a positively charged endosome disrupting PEGylated histidinylated lipid, represented by the structural formula VII:

General structural formula VII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl; or

b. a DC targeting PEGylated lipid, represented by the structural formula XII:

General structural formula XII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl.

2. The cationic PEGylated lipid compound as claimed in claim 1, wherein the positively charged endosome disrupting PEGylated histidinylated lipid of structural formula VII is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride:
.

3. The cationic PEGylated lipid compound as claimed in claim 2, wherein the PEGylated histidinylated compound of structural formula VII is characterized by HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976.

4. The cationic PEGylated lipid compound as claimed in claim 2, wherein the compound is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).

5. The cationic PEGylated lipid compound as claimed in claim 1, wherein the DC targeting PEGylated lipid of structural formula XII is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride:
.

6. The cationic PEGylated lipid compound as claimed in claim 5, wherein the DC targeting PEGylated lipid of structural formula XII is characterized by HRMS(m/z): [M+H]+ = 2140 and [M+1]+/2 =1070.

7. The cationic PEGylated lipid compound as claimed in claim 5, wherein the compound is further characterized by 1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 – 7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

8. A process for the synthesis of an endosome disrupting PEGylated histidinylated lipid of structural formula VII, comprising the following steps:

General structural formula VII
wherein R is alkyl, aryl, substituted aryl, alkoxy alkyl;
a. reacting the ethane-1,2-diamine in presence of tert-butoxycarbonyl (Boc) anhydride, DCM to obtain intermediate I of structure:

wherein intermediate I is tert-butyl (2-aminoethyl carbamate) characterized by: ESI MS: [M]+ =161;
wherein the intermediate I is further characterized by 1H NMR (400 MHz, Chloroform-d): d 4.90 (s, 1H), 3.16 (q, J = 5.8 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 1.43 (s, 9H);
b. reacting the intermediate I, tert-butyl (2-aminoethyl carbamate) in presence of ethyl acetate, K2CO3, 1-bromo-hexadecane to obtain intermediate II of structural formula:

wherein intermediate II is tert-butyl (2- (dihexadecylamino)ethyl) carbamate characterized by: HRMS: [M]+= 609;
wherein the intermediate II is further characterized by 1H NMR (500 MHz, Chloroform-d): d 5.02 (s, 1H), 3.51 (s, 2H), 3.20 – 3.12 (m, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.39 (m, 2H), 1.47 (s, 9H), 1.42 – 1.23 (m, 56H), 0.90 (t, J = 6.8 Hz, 6H);

c. reacting the intermediate II, tert-butyl (2-(dihexadecylamino)ethyl) carbamate in presence of TFA/DCM to obtain intermediate III of structural formula:

wherein R is n-C16H33,
wherein intermediate III is dihexadecylethane-1,2-diamine characterized by: ESI-MS: [M]+ = 509;
wherein the intermediate III is further characterized by 1H NMR (500 MHz, Chloroform-d) d 4.07 (m, 1H), 3.40 – 3.16 (m, 1H), 2.92 – 2.73 (m, 2H), 2.63 – 2.49 (m, 2H), 2.45 – 2.39 (m, 4H), 1.47 – 1.18 (m, 56H), 0.89 (t, J = 6.9 Hz, 6H);

d. reacting the intermediate III, dihexadecylethane-1,2-diamine with BocNH-(PEG)27-COOH in presence of HATU, DIPEA, DCM, to obtain intermediate IV of structural formula:

wherein R is n-C16H33,
wherein intermediate IV is tert-butyl(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) carbamate characterized by HRMS (m/z): [M+1]+ = 1914;
wherein the intermediate IV is further characterized by 1H NMR (500 MHz, Chloroform-d): d 6.53 (s, 1H), 5.08 (s, 1H), 3.73 (t, J = 5.5 Hz, 112H), 3.37 – 3.21 (m, 4H), 2.65 – 2.29 (m, 8H), 1.44 (s, 9H), 1.25 (s, 56H), 0.87 (t, J = 6.7 Hz, 6H);

e. reacting the intermediate IV, tert-butyl(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) carbamate in presence of TFA/DCM to obtain intermediate V of structural formula:

wherein R is n-C16H33,
wherein intermediate V is 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide characterized by: HRMS (m/z): [M+1]+ =1814;
wherein the intermediate is further characterized by 1H NMR (400 MHz, Chloroform-d): d 6.91 – 6.28 (m, -1H), 3.64 (s, 112H), 3.49 – 3.26 (m, 2H), 3.09 – 2.97 (m, 1H), 2.60 – 2.51 (m, 2H), 2.45 (q, J = 9.1, 7.6 Hz, 5H), 1.47 – 1.37 (m, 4H), 1.32 – 1.21 (m, 56H), 0.88 (d, J = 13.7 Hz, 6H);

f. reacting the intermediate V, 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide with N,N di-Boc L-histidine in presence of HATU, DIPEA, DMF to obtain intermediate VI of structural formula:

wherein R is n-C16H33,
wherein intermediate VI is tert-butyl (S)-5-(2-((tert-butoxycarbonyl) amino)-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontyl)-1H-imidazole-1-carboxylate characterized by: HRMS (m/z): [M+Na]+ =2173;
wherein the intermediate is further characterized by 1H NMR (500 MHz, Chloroform-d) d 8.18 – 8.05 (m, 2H), 7.20 – 7.14 (m, 1H), 7.09 – 7.04 (m, 1H), 6.63 – 6.58 (m, 1H), 5.15 – 5.09 (m, 1H), 3.81 – 3.75 (m, 2H), 3.74 (t, J = 6.0 Hz, 2H), 3.69 – 3.60 (m, 108H), 3.57 – 3.54 (m, 1H), 3.52 – 3.46 (m, 2H), 3.43 – 3.37 (m, 1H), 2.52 – 2.48 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.60 (s, 9H), 1.44 – 1.39 (m, 9H), 1.25 (s, 60H), 0.88 (t, J = 6.4 Hz, 6H); and

g. reacting the intermediate VI, tert-butyl (S)-5-(2-((tert-butoxycarbonyl) amino)-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontyl)-1H-imidazole-1-carboxylate in presence of TFA/DCM to obtain endosome disrupting PEGylated histidine lipid of structural formula VIII:

VIII
wherein R is n-C16H33,
wherein the endosomal disrupting lipid is (S)-5-(2-ammonio-17-hexadecyl-3,13-dioxo-7,10-dioxa-4,14,17-triazatritriacontan-17-ium-1-yl)-1H-imidazol-3-ium chloride is characterized by: HRMS (m/z): [M]+ =1951, ([M +1]+/ 2) = 976;
wherein the intermediate is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.59 (s, 1H), 8.13 (s, 1H), 7.57-7.25 (m, 7H), 3.85-3.68 (m, 6H), 3.57 (s, 108H), 3.30-3.17(m, 2H), 2.92 (s, 2H), 2.52-2.37 (m, 7H), 1.74-1.67 (m, 4H), 1.5-1.10 (m, 56H), 0.82 (t, 6H).

9. A process for the synthesis of the cationic DC targeting PEGylated lipid of structural formula XII, comprising the steps:
a. reacting the intermediate V, 3-(2-(2-aminoethoxy) ethoxy)-N-(2(dihexadecylamino)ethyl) propenamide, obtained from steps a-e as claimed in claim 8, with (R, E)-1,10-dioxo-12-phenyl-11-oxa-3,9-diaza-1-boradodec-2-ene-8-carboxylic acid in presence of HATU, DIPEA, DCM in to obtain intermediate VI of structural formula:

VI
wherein R is n-C16H33,
wherein Z is C6H5CH2OCO-,
wherein intermediate VI is benzyl (S)-(23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamate characterized by: HRMS(m/z): [M+H]+= 2176;
wherein the compound is further characterized by 1H NMR (500 MHz, Chloroform-d): d 8.60 – 8.53 (m, 1H), 8.32 – 8.21 (m, 1H), 8.11 (dd, J = 7.4, 3.5 Hz, 1H), 7.35 (d, J = 14.1 Hz, 5H), 6.97 – 6.90 (m, 1H), 5.72 – 5.65 (m, 1H), 5.20 – 5.06 (m, 3H), 4.80 – 4.70 (m, 1H), 4.22 – 4.15 (m, 1H), 3.66 (s, 108H), 3.47 (dd, J = 4.7, 2.0 Hz, 2H), 3.22 (td, J = 8.5, 6.9, 3.1 Hz, 2H), 3.15 – 3.00 (m, 6H), 2.67 – 2.61 (m, 1H), 2.58 – 2.53 (m, 2H), 1.60 – 1.48 (m, 4H), 1.44 (s, 9H), 1.26 (d, J = 13.7 Hz, 60H), 0.90 (t, J = 6.3 Hz, 6H);

b. reacting the intermediate VI, benzyl (S)-(23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamate in presence of MeOH and Pd/Charcoal into obtain intermediate VII of structural formula:

VII
wherein R is n-C16H33,
wherein intermediate VII is (S)-6-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-2-amino-N-(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) hexanamide characterized by: HRMS(m/z): [M+H]+ = 2042;
wherein the compound is further characterized by 1H NMR (500 MHz, DMSO-d6): d 8.38 – 8.32 (m, 1H), 8.31 – 8.25 (m, 1H), 8.15 – 8.10 (m, 1H), 7.65 (td, J = 6.4, 3.4 Hz, 1H), 7.46 – 7.22 (m, 1H), 7.19 –7.11 (m, 1H), 6.76 – 6.67 (m, 1H), 3.50 (s, 112H), 3.10 – 3.03 (m, 2H), 2.90 –2.84 (m, 2H), 2.63 (s, 1H), 2.41 – 2.32 (m, 6H), 2.27 (t, J = 6.3 Hz, 2H), 1.64 –1.46 (m, 2H), 1.29 (d, J = 66.4 Hz, 69H), 0.84 (t, J = 6.3 Hz, 6H);

c. reacting the intermediate VII (S)-6-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-2-amino-N-(13-hexadecyl-9-oxo-3,6-dioxa-10,13-diazanonacosyl) hexanamide with (3S,4R,5S)-3,4,5-triacetoxycyclohex-1-ene-1-carboxylic acid in presence of HATU, DIPEA, DCM to obtain the intermediate VIII:

VIII
wherein R is n-C16H33,
wherein intermediate VIII is (1S,2R,3S)-5-(((S)-23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+ Na]+= 2346;
wherein the compound is further characterized by: 1H NMR (500 MHz, Chloroform-d): d 8.06 (s, 1H), 7.17 (s, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.36 (s, 1H), 5.71 (s, 1H), 5.24 (s, 1H), 5.14 – 4.89 (m, 1H), 4.79 (s, 1H), 4.51 – 4.39 (m, 1H), 3.78 (s, 1H), 3.74 (t, J = 6.1 Hz, 2H), 3.71 – 3.58 (m, 108H), 3.55 (s, 1H), 3.51 – 3.41 (m, 2H), 3.17 (s, 2H), 3.03 – 2.91 (m, 4H), 2.52 (t, J = 6.2 Hz, 2H), 2.41 (dd, J = 17.9, 5.0 Hz, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.69 (d, J = 14.3 Hz, 4H), 1.53 – 1.47 (m, 2H), 1.43 (s, 9H), 1.27 (d, J = 15.1 Hz, 60H), 0.87 (d, J = 7.0 Hz, 6H);

d. reacting the intermediate VIII (1S,2R,3S)-5-(((S)-23-hexadecyl-2-(?1-oxidanyl)-9,19-dioxo-1?1-13,16-dioxa-3,10,20,23-tetraaza-2-boranonatriacontan-8-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of TFA/DCM to obtain the intermediate IX:

IX
wherein R is n-C16H33,
wherein intermediate IX is (1S,2R,3S)-5-(((S)-1-amino-20-hexadecyl-6,16-dioxo-10,13-dioxa-7,17,20-triazahexatriacontan-5-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+H]+ = 2224;
wherein the compound is further characterized by 1H NMR (500 MHz, DMSO-d6): d 8.12 – 7.88 (m, 1H), 7.71 – 7.51 (m, 2H), 6.42 (s, 1H), 5.65 – 5.51 (m, 1H), 5.23 – 4.93 (m, 2H), 4.25 (s, 1H), 3.50 (s, 112H), 3.14 – 2.93 (m, 4H), 2.92 – 2.80 (m, 2H), 2.80 – 2.68 (m, 3H), 2.65 – 2.58 (m, 3H), 2.37 – 2.32 (m, 5H), 2.06 – 1.97 (m, 6H), 1.90 – 1.37 (m, 8H), 1.23 (s, 58H), 0.87 – 0.80 (m, 6H);

e. reacting the intermediate IX (1S,2R,3S)-5-(((S)-1-amino-20-hexadecyl-6,16-dioxo-10,13-dioxa-7,17,20-triazahexatriacontan-5-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of HgCl2, Di-BOC-thiourea, Et3N, dry DCM/DMF to obtain the intermediate X:

X
wherein R is n-C16H33,
wherein intermediate X is 1S,2R,3S)-5-(((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate characterized by: HRMS(m/z): [M+H]+ = 2466;
wherein the compound is further characterized by: 1H NMR (500 MHz, DMSO-d6): d 11.49 (s, 1H), 8.25 (t, J = 5.1 Hz, 1H), 8.16 – 7.84 (m, 2H), 6.46 – 6.36 (m, 1H), 5.60 (d, J = 3.5 Hz, 1H), 5.14 (d, J = 3.7 Hz, 2H), 4.24 (q, J = 8.2 Hz, 2H), 3.92 (d, J = 5.7 Hz, 2H), 3.68 – 3.62 (m, 2H), 3.61 – 3.57 (m, 3H), 3.50 (s, 118H), 3.40 (d, J = 5.8 Hz, 3H), 3.21 (ddd, J = 26.6, 13.2, 6.4 Hz, 5H), 3.10 – 2.91 (m, 1H), 2.90 – 2.81 (m, 1H), 2.63 (s, 1H), 2.42 – 2.35 (m, 1H), 2.33 (d, J = 7.0 Hz, 2H), 2.26 (t, J = 7.3 Hz, 1H), 2.05 (s, 2H), 2.04 (s, 3H), 2.00 (s, 3H), 1.67 – 1.58 (m, 3H), 1.51 (s, 2H), 1.47 (s, 9H), 1.38 (s, 9H), 1.23 (s, 60H), 0.84 (d, J = 4.0 Hz, 6H);

f. reacting the intermediate X 1S,2R,3S)-5-(((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl) carbamoyl) cyclohex-4-ene-1,2,3-triyl triacetate in presence of NaOMe, MeOH to obtain the intermediate XI:

XI
wherein R is n-C16H33,
wherein intermediate XI is (3S,4R,5S)-N-((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide characterized by: HRMS(m/z): [M+ Na]+= 2362;
wherein the compound is further characterized by: 1H NMR (500 MHz, DMSO-d6): d 11.50 (s, 1H), 9.49 (s, 1H), 8.36 (s, 1H), 8.27 (t, J = 5.5 Hz, 1H), 8.10 (s, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.71 – 7.61 (m, 2H), 7.56 – 7.45 (m, 1H), 7.35 (dd, J = 18.3, 8.1 Hz, 1H), 7.22 – 7.06 (m, 1H), 6.88 (s, 1H), 6.35 (dd, J = 3.4, 1.7 Hz, 1H), 4.24 (s, 1H), 4.19 (s, 1H), 3.93 (d, J = 5.7 Hz, 1H), 3.86 – 3.80 (m, 2H), 3.51 (s, 108H), 3.25 (d, J = 6.5 Hz, 1H), 3.07 (d, J = 7.2 Hz, 1H), 2.68 – 2.52 (m, 3H), 2.48 – 2.44 (m, 1H), 2.41 – 2.33 (m, 4H), 2.27 (q, J = 7.3, 6.9 Hz, 3H), 1.64 (d, J = 7.8 Hz, 2H), 1.55 – 1.50 (m, 2H), 1.49 – 1.36 (m, 18H), 1.24 (s, 60H), 0.87 – 0.84 (m, 6H); and

g. reacting the intermediate XI (3S,4R,5S)-N-((S)-4-(((?1-methyl) (?1-oxidanyl) boranyl) amino)-25-hexadecyl-2-(?1-oxidanyl)-11,21-dioxo-1?1-15,18-dioxa-3,5,12,22,25-pentaaza-2-borahentetracont-4-en-10-yl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide in presence of TFA/DCM to obtain the DC targeting PEGylated lipid (XII):

XII

wherein R is n-C16H33,
wherein obtained compound (lipid) is (S)-1-amino-N, N-dihexadecyl-1-iminio-8,18-dioxo-7-((3S,4R,5S)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamido)-12,15-dioxa-2,9,19-triazahenicosan-21-aminium chloride characterized by: HRMS(m/z): [M+H]+= 2140, [M+1]+/2 = 1070;
wherein the compound is further characterized by 1H NMR (400 MHz, DMSO-d6): d 10.50 (s, 1H), 8.34 – 8.24 (m, 1H), 8.06 – 7.99 (m, 1H), 7.95 – 7.87 (m, 1H), 7.68 – 7.61 (m, 1H), 7.44 – 7.35 (m, 1H), 7.35 – 7.20 (m, 3H), 7.18 – 7.10 (m, 1H), 6.31 (q, J = 2.4 Hz, 1H), 4.68 – 4.51 (m, 2H), 4.28 – 4.20 (m, 1H), 4.18 – 4.13 (m, 1H), 4.08 (dd, J = 5.8, 3.7 Hz, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.84 – 3.71 (m, 2H), 3.47 (s, 108H), 3.14 (s, 1H), 2.68 (d, J = 4.8 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.52 (dd, J = 3.6, 1.9 Hz, 1H), 2.44 – 2.08 (m, 8H), 1.61 – 1.58 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27 – 1.16 (m, 60H), 0.82 – 0.79 (m, 6H).

10. A lipid-based composition for nucleic acid vaccine delivery, comprising:
a. cationic lipids, including endosome disrupting lipids and DC targeting lipids;
b. co-lipids and their combinations thereof;
c. various excipients mixed in different ratios;
wherein said cationic lipids are pegylated or non-pegylated, and
wherein the composition is formulated to target and deliver antigen-encoding nucleic acids utilizing self-assembled LNPs (Lipid Nanoparticles) to antigen-presenting cells (APC) via mannose receptors, facilitating efficient endosomal release of nucleic acids in the antigen-presenting cells for effective nucleic acid vaccine delivery.

11. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the co-lipid is selected from:
a. a steroid lipid which may be sterol and is selected from cholesterol, ergosterol, stigmasterol, sitosterol, campesterol, stigmastanol like phytosterols and other steroids such as dexamethasone, prednisolone, triamcinolone, and/or mixture thereof; and
b. a phospholipid selected from dioleoyl phosphatidylethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-distearoyl-snglycero-3-phosphoethanolamine [DSPE], 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dipalmitoyl-sn-glycero-3-phosphocholone [DPPC], hydrogenated soy phosphatidylcholine (HSPC) or mixtures thereof.

12. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the co-lipid is dioleoyl phosphatidylethanolamine [DOPE].

13. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the said composition comprises endosome disrupting lipids, DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients, wherein lipids are pegylated or non-pegylated.

14. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the said composition comprises non-PEGylated endosome disrupting lipids, PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

15. The lipid-based composition for nucleic acid vaccine delivery as claimed in claims 10, 12 and 13, wherein the pharmaceutically acceptable excipients are selected from buffers, adjuvants, diluents, lubricants, binders, stabilizers, preservatives, disintegrants, absorbents, colorants, surfactants, flavours, sweeteners, residuals, immune-boosting co-excipients, or a combination thereof.

16. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the endosome disrupting lipids, DC-targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1 to 1:1:1.

17. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the endosome disrupting lipids, PEGylated DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1.

18. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the nucleic acid (NA) is a biologically active agent selected from ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), plasmid DNA (pDNA), a fragment of RNA, mRNA, DNA, pDNA, miRNA, or any chimeric or fusion thereof.

19. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the composition is formulated as a lipid-nanoparticle (LNP) formulation or a liposomal formulation encapsulating the biologically active agent.

20. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the composition is formulated as a lipoplex formulation formed by complexing the liposomal preparation with the biologically active agent.

21. The lipid-based composition for nucleic acid vaccine delivery as claimed in claim 10, wherein the in vitro transfection is achieved at a N/P charge ratio (Lipid: Nucleic acid) of 1:1 to 4:1.

22. A method for the preparation of a lipid-based composition, comprising the steps of:
a. taking lipids and other co-lipids and excipients in a vial and thoroughly mixing them in the vial;
b. diluting the mixture in an ethanolic solution; and
c. injecting the resulting ethanolic lipid mixture into nuclease-free water (pH ~7) under stirring conditions for 15 minutes to form a lipid-based formulation at room temperature.

23. A vaccine formulation comprising nucleic acid as a vaccine antigen for the prophylaxis of virus-mediated diseases, said vaccine formulation comprising:
a. nucleic acid (NA) selected from ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), plasmid DNA (pDNA), fragment of RNA, mRNA, DNA, pDNA, miRNA, or any chimeric or fusion thereof;
b. DC targeting lipids;
c. endosome disrupting lipids; and
d. co-lipids selected from:
i. a steroid lipid, which may be sterol and is selected from cholesterol, ergosterol, stigmasterol, sitosterol, campesterol, stigmastanol like phytosterols, and other steroids such as dexamethasone, prednisolone, triamcinolone, and/or a mixture thereof;
ii. a phospholipid selected from dioleoyl phosphatidylethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [DSPE], 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-dipalmitoyl-sn-glycero-3-phosphocholone [DPPC], hydrogenated soy phosphatidylcholine (HSPC), or mixtures thereof; and
e. optionally, one or more pharmaceutically acceptable excipients.

24. The vaccine formulation as claimed in claim 23, wherein the co-lipid is dioleoyl phosphatidylethanolamine [DOPE].

25. The vaccine formulation as claimed in claim 23, wherein the said vaccine formulation comprising nucleic acid, PEGylated endosome disrupting lipids, non-PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

26. The vaccine formulation as claimed in claim 23, wherein the said vaccine formulation comprising nucleic acid, non-PEGylated endosome disrupting lipids, PEGylated DC targeting lipids, dioleoyl phosphatidylethanolamine [DOPE], and optionally, one or more pharmaceutically acceptable excipients.

27. The vaccine formulation as claimed in claims 23, 25 and 26, wherein the pharmaceutically acceptable excipients are selected from buffers, adjuvants, diluents, lubricants, binders, stabilizers, preservatives, disintegrants, absorbents, colorants, surfactants, flavours, sweeteners, residuals, immune-boosting co-excipients, or a combination thereof.

28. The vaccine formulation as claimed in claim 23, wherein the endosome disrupting lipids, DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1 to 1:1:1.

29. The vaccine formulation as claimed in claim 23, wherein the endosome disrupting lipids, PEGylated DC targeting lipids, and dioleoyl phosphatidylethanolamine [DOPE] are present in the mole ratio of 1:0.01:1.

30. A vaccine formulation comprising the lipid-based composition as claimed in claim 10 for the prophylaxis and/or treatment of disease including virus mediated disease in a human subject.

31. The vaccine formulation as claimed in claim 30, wherein said vaccine formulation comprises of lipid-based composition along with the biologically active Nucleic Acid (NA) to provide a pharmaceutical formulation with or without one or more pharmaceutically acceptable excipient to form Lipid-Nanoparticle (LNP) formulation or a Liposomal formulation or Lipoplex formulation.

32. The vaccine formulation as claimed in claim 30, wherein the formulation performs in vitro transfection at a N/P charge ratio (Lipid: Nucleic acid) of 1:1 to 4:1.

33. A method for the preparation of a vaccine formulation, comprising the steps of:
a. preparing nucleic acid constructs;
b. complexing the lipid-based formulation as claimed in claim 10 with nucleic acid constructs at fixed mole ratios to form liposomes or lipoplex; and
c. obtaining the vaccine formulation and preserving it at room temperature or 4 °C.

34. Use of a lipid-based composition as claimed in claim 10 for:
a. delivery of nucleic acid to body antigen presenting cells (APC);
b. targeting the antigen encoding nucleic acid and self-assembled LNPs to antigen presenting cells;
c. preparing the LNPs without the use of chlorinated organic solvents and energy-demanding processes;
d. preparation of pharmaceutical formulation, including vaccine formulation; and
e. prophylaxis, treatment, and management of diseases, including infectious diseases.