CN117624582A - Fluorine-containing DSPE-PEG2000, application thereof, nanoparticle containing fluorine-containing DSPE-PEG2000 and application of fluorine-containing DSPE-PEG2000 - Google Patents

Fluorine-containing DSPE-PEG2000, application thereof, nanoparticle containing fluorine-containing DSPE-PEG2000 and application of fluorine-containing DSPE-PEG2000 Download PDF

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CN117624582A
CN117624582A CN202311103951.7A CN202311103951A CN117624582A CN 117624582 A CN117624582 A CN 117624582A CN 202311103951 A CN202311103951 A CN 202311103951A CN 117624582 A CN117624582 A CN 117624582A
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lipid
peg2000
cancers
dspe
substance
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顾臻
李洪军
张会鹏
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Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention discloses fluorine-containing DSPE-PEG2000, application thereof, nanoparticles containing the same and application thereof. The invention provides a compound shown as a formula I, wherein n is an integer of 0-10, R is a group consisting of 1, 2 or 3 CF 3 Substituted methyl. The compound shown in the formula I can be used for preparing lipid nano-particles. The lipid nanoparticle prepared by the invention has higher endosome escape efficiency in cells, and can obviously improve the expression quantity of the mRNA, thereby improving the immune effect.

Description

Fluorine-containing DSPE-PEG2000, application thereof, nanoparticle containing fluorine-containing DSPE-PEG2000 and application of fluorine-containing DSPE-PEG2000
Technical Field
The invention relates to the technical field of biomedical materials and drug delivery, in particular to novel fluorine-containing DSPE-PEG2000, application thereof, nanoparticles containing the fluorine-containing DSPE-PEG2000 and application thereof.
Background
Currently, nucleic acid vaccines are widely used worldwide as a major means of treating viral infections or tumors, and the messenger ribonucleic acids carried by them can be translated into the antigen of interest via antigen presenting cells in vivo to elicit humoral and cellular immunity to eliminate viral particles or tumor cells. There are many lipid nanoparticle designs such as cationic lipid type or ionizable lipid type lipid nanoparticle, but cationic lipid nanoparticle may cause higher cytotoxicity in cells due to its quaternary amine functional head, and insufficient escape efficiency of ionizable lipid nanoparticle in intracellular inclusion body may reduce its immune effect, however, increasing administration dose may also cause increased cytotoxicity.
Disclosure of Invention
The invention aims to solve the problem of insufficient escape efficiency of the existing ionizable lipid nanoparticle in a cell endosome, and provides a fluorine-containing DSPE-PEG2000, an application thereof, a nanoparticle containing the fluorine-containing DSPE-PEG2000 and an application thereof. The fluorinated end groups exposed on the surfaces of the novel lipid nanoparticles have the characteristics of bioinert property, low surface energy, strong electronegativity, hydrophobicity, oleophobicity and the like, so that the endosome escape efficiency of the novel lipid nanoparticles in cells is increased, the amount of messenger ribonucleic acid escaping to cytoplasm can be increased under the condition of the same administration dosage, the expression amount of corresponding proteins is increased, and the immune effect is remarkably improved.
The above object of the present invention is achieved by the following technical solutions:
the invention provides a compound shown as a formula I:
wherein n is an integer of 0 to 10, R is a group consisting of 1, 2 or 3 CF 3 Substituted methyl.
In one embodiment, n is an integer from 1 to 5, e.g., n is 1.
In one embodiment, R is-CH 2 CF 3 。
In one embodiment, the compound of formula I is the following:
the invention also provides application of the compound shown in the formula I in preparing the mass nano-particles.
The invention also provides a lipid carrier, which comprises a substance Z, wherein the substance Z is a compound shown as the formula I.
In a preferred embodiment, the lipid carrier further comprises a helper phospholipid.
In a preferred embodiment, the helper phospholipid may be a helper phospholipid conventional in the art, which is an amphoteric helper molecule that aids in the fusion of the lipid particle and cell membrane. The helper phospholipid may be a phospholipid-like molecule having a charged polar end and a fatty chain non-polar end, such as 1, 2-distearoyl-SN-glycero-3-phosphorylcholine (DSPC) or 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine (DOPE), preferably 1, 2-distearoyl-SN-glycero-3-phosphorylcholine (DSPC).
In a preferred embodiment, the lipid carrier further comprises an ionizable lipid.
In a preferred embodiment, the ionizable lipid is selected from one or more of 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecoxy) hexyl ] amino ] -octanoate (SM-102), 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester (Dlin-MC 3-DMA) and ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) (ALC-0315), for example 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecoxy) hexyl ] amino ] -octanoate or 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester.
In a preferred embodiment, the lipid carrier further comprises cholesterol.
In a preferred embodiment, the lipid carrier further comprises distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000).
In the present invention, the molar content means that a content of a substance is a percentage of the total mass of the lipid carrier, and the sum of the molar contents of the components in the lipid carrier is not more than 100%.
In a preferred embodiment, the molar content of the auxiliary phospholipid is about 5% to 15%, preferably 10%.
In a preferred embodiment, the ionizable lipid is present in a molar amount of about 45% to 60%, preferably 50%.
In a preferred embodiment, the cholesterol is present in a molar amount of about 30% to 40%, preferably 38.5%.
In a preferred embodiment, the DSPE-PEG2000 is present in a molar amount of less than about 5%, such as 0.5-1%, such as 0.5%, 1.0%, or 1.5%.
In a preferred embodiment, the molar content of the substance Z is about 0.01% to 5%, preferably 0.5-1.5%, for example 0.5%, 1.0% or 1.5%.
In a preferred embodiment, the sum of the molar content of the substance Z and the molar content of the DSPE-PEG2000 is 1.5% or more, preferably 1.5%.
In a preferred embodiment, the molar ratio of the substance Z to the DSPE-PEG2000 may be (1-2): (2-1).
In a preferred embodiment, the molar ratio of the ionizable lipid to the substance Z may be 50 (0.01-1.5), such as 50:0.5, 50:1, and 50:1.5.
In a preferred embodiment, the molar ratio of the co-phospholipid to the substance Z may be 10 (0.01-1.5), such as 10:0.5, 10:1 and 10:1.5.
In a preferred embodiment, the molar ratio of cholesterol to substance Z may be 38.5 (0.01-1.5), such as 38.5:0.5, 38.5:1 and 38.5:1.5.
In a preferred embodiment, the volume ratio of the ionizable lipid to the helper phospholipid is (2-2.5): 1, preferably (2-2.3): 1.
In a preferred embodiment, the volume ratio of the ionizable lipid to the cholesterol is (2-2.5): 1, preferably (2.1-2.4): 1.
In a preferred embodiment, the volume ratio of the ionizable lipid to the DSPE-PEG2000 is (3-13): 1.
In a preferred embodiment, the volume ratio of the ionizable lipid to the substance Z is (3-13): 1.
In a preferred embodiment, said lipid carrier consists of said substance Z, said ionizable lipid, said helper phospholipid, said DSPE-PEG2000 and said cholesterol.
In a preferred embodiment, the lipid carrier comprises, in mole percent: 50% of said ionizable lipid, 10% of said helper phospholipid, 35-38.5% of said cholesterol, 1.5% -5% of a pegylated lipid (said pegylated lipid being DSPE-PEG2000 and said substance Z, or said substance Z).
The present invention also provides a lipid nanoparticle comprising a therapeutic and/or prophylactic agent and the aforementioned lipid carrier.
In a preferred embodiment, the therapeutic agent may be a mRNA.
In a preferred embodiment, the prophylactic agent may be a mRNA.
In a preferred embodiment, the molar ratio of the nitrogen atoms in the ionizable lipid to the phosphorus atoms in the therapeutic and/or prophylactic agent (mRNA) in the lipid nanoparticle is 1:3 or 1:6.
In a preferred embodiment, the lipid nanoparticle has a total lipid mass (ionizable lipid + helper phospholipid + cholesterol + PEG lipid) to therapeutic and/or prophylactic agent mass ratio of 1:10 or 1:20.
In a preferred embodiment, in the lipid nanoparticle, the lipid carrier encapsulates the therapeutic and/or prophylactic agent.
The invention also provides a composition comprising a substance Z, wherein the substance Z is a compound shown as the formula I.
In a preferred embodiment, the composition further comprises one or more of the ionizable lipid, helper phospholipid, DSPE-PEG2000, cholesterol, and therapeutic and/or prophylactic agents.
In a preferred embodiment, the composition wherein the ionizable lipid, helper phospholipid, DSPE-PEG2000, cholesterol, and therapeutic and/or prophylactic agent are as described above.
In a preferred embodiment, in the composition, the substance Z forms a lipid carrier with one or more of the ionizable lipid, helper phospholipid, DSPE-PEG2000, and cholesterol as described above.
In a preferred embodiment, in the composition, the lipid carrier forms lipid nanoparticles as described above with the therapeutic and/or prophylactic agent.
In a preferred embodiment, the encapsulation efficiency of the therapeutic and/or prophylactic agent in the composition is at least 80%, preferably at least 85%.
The invention also provides application of the compound shown in the formula I, the lipid carrier, the lipid nanoparticle and the composition in preparation of medicines or vaccines for treating related diseases.
In a preferred embodiment, the related disease comprises a viral infection, an inflammatory disease, HIV, an autoimmune disease, a tumor.
In a preferred embodiment, the tumor comprises a melanoma cell, a hematological tumor, a lymphoid cancer, a brain cancer, a liver cancer, a lung cancer, an esophageal cancer, a gastric cancer, a breast cancer, a pancreatic cancer, a thyroid cancer, a nasopharyngeal cancer, an ovarian cancer, an endometrial cancer, a renal cancer, a prostate cancer, a bladder cancer, a colon cancer, a rectal cancer, a testicular cancer, or a head and neck cancer.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
Compared with the existing lipid nanoparticle, the invention has the beneficial effects that:
the invention designs a novel fluorine-containing DSPE-PEG2000, and the novel fluorine-containing DSPE-PEG2000 is self-assembled with other lipid monomers and mRNA to form the lipid nanoparticle, so that the endosome escape efficiency in cells is increased compared with that of the lipid nanoparticle which only contains non-fluorinated DSPE-PEG2000 and participates in formation. Furthermore, the lipid nanoparticle can obviously improve the expression quantity of the mRNA, thereby improving the immune effect.
Drawings
FIG. 1a is a schematic diagram of DSPE-PEG2000-NH according to the present invention 2 Is a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF).
FIG. 1b shows DSPE-PEG2000-NHCOCH according to the present invention 2 CH 2 CF 3 Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) profile (FPD).
FIG. 2a is a schematic diagram of DSPE-PEG2000-NH according to the present invention 2 Nuclear magnetic carbon spectrum of (a).
FIG. 2b shows DSPE-PEG2000-NHCOCH according to the present invention 2 CH 2 CF 3 Nuclear magnetic carbon spectrum of (FPD).
FIG. 3a shows FPD and DSPE-PEG2000-NH according to the present invention 2 Nuclear magnetic hydrogen spectrum of (2).
FIG. 3b shows DSPE-PEG2000-NHCOCH according to the present invention 2 CH 2 CF 3 Nuclear magnetic hydrogen spectrum of (FPD).
FIG. 4 shows DSPE-PEG2000-NHCOCH according to the present invention 2 CH 2 CF 3 Nuclear magnetic fluorine spectrum of (FPD).
FIG. 5 is a schematic representation of the preparation of fluorinated lipid nanoparticles of the present invention.
FIG. 6 is a frozen transmission electron micrograph of lipid nanoparticles containing 50% SM-102 with varying degrees of fluorination provided by an example of the present invention; wherein DSPE-PEG2000-NHCOCH in 0% FPD 2 CH 2 CF 3 (FPD) the molar ratio of lipid in lipid nanoparticle was 0%, FP in 0.5% FPDThe lipid molar ratio of D in the lipid nanoparticle was 0.5%, the lipid molar ratio of FPD in the lipid nanoparticle was 1.0% in 1% FPD, and the lipid molar ratio of FPD in the lipid nanoparticle was 1.5%.
FIG. 7 is a graph showing the particle size, polydispersity and potential statistics of lipid nanoparticles containing 50% SM-102 of varying degrees of fluorination provided by examples of the present invention; wherein the meanings of 0% FPD, 0.5% FPD, 1% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 8a shows the effect of lipid nanoparticles containing 50% SM-102 with different degrees of fluorination on the cell viability of B16F10 cells at different concentrations, as provided by the examples of the present invention.
Figure 8b shows the effect of lipid nanoparticles containing 50% sm-102 with different degrees of fluorination on cell viability of DC2.4 cells at different concentrations as provided by the examples of the present invention.
FIG. 9a shows the ratio of enhanced green fluorescent protein positive cells to average fluorescence intensity statistics of B16F10 cells detected using flow cytometry after lipid nanoparticles containing 50% SM-102 of varying degrees of fluorination provided by the examples of the present invention were co-incubated with B16F10 cells for 24 hours. Statistical analysis was assessed using GraphPad Prism (9.0). Statistical significance was calculated using unpaired student t-test (comparison between two groups) and one-way anova and Tukey post-hoc test (comparison between groups). Data are expressed as mean ± SD, P values of 0.05 or less are considered significant. In the figure, P <0.05, P <0.01, and P <0.001.
FIG. 9B is a photograph of fluorescence taken using a laser confocal microscope after 24 hours of co-incubation of lipid nanoparticles containing 50% SM-102 with B16F10 cells at varying degrees of fluorination; wherein the meanings of 0% FPD, 0.5% FPD, 1% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 9c shows the enhanced green fluorescent protein positive cell fraction and average fluorescence intensity statistics of HEK293T cells detected using flow cytometry after 50% SM-102 containing lipid nanoparticles of varying degrees of fluorination provided by the examples of the present invention were co-incubated with HEK293T cell lines for 24 hours. In the figure, P <0.05 is indicated.
FIG. 9d is a fluorescence photograph taken using a laser confocal microscope after 24 hours of incubation of lipid nanoparticles containing 50% SM-102 with HEK293T cell lines at varying degrees of fluorination; wherein the meanings of 0% FPD, 0.5% FPD, 1% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 9e shows the ratio of enhanced green fluorescent protein positive cells to average fluorescence intensity statistics of HEK293T cells detected using flow cytometry after 50% Dlin-MC3-DMA containing lipid nanoparticles of varying degrees of fluorination provided in the examples of the present invention were co-incubated with HEK293T cell lines for 24 hours. In the figure, P <0.05 is indicated.
FIG. 9f shows the enhanced green fluorescent protein positive cell fraction and average fluorescence intensity statistics of DC2.4 cells detected using flow cytometry after 50% SM-102 containing lipid nanoparticles of varying degrees of fluorination provided by the examples of the present invention were co-incubated with DC2.4 cell lines for 24 hours. In the figure, P <0.01 is indicated.
FIG. 9g shows the ratio of enhanced green fluorescent protein positive cells to average fluorescence intensity statistics of DC2.4 cells detected using flow cytometry after lipid nanoparticles of varying degrees of fluorination containing 50% Dlin-MC3-DMA provided in the examples of the present invention were co-incubated with DC2.4 cell lines for 24 hours. In the figure, P <0.001 is indicated.
FIG. 9h is a representative flow analysis scatter plot of B16F10, HEK293T and DC2.4 cells detected using flow cytometry after lipid nanoparticles containing 50% SM-102 of varying degrees of fluorination provided in the examples of the present invention were co-incubated with B16F10, HEK293T and DC2.4 cell lines for 24 hours.
FIG. 9i is a representative flow analysis scatter plot of HEK293T and DC2.4 cells detected using flow cytometry after 50% Dlin-MC3-DMA containing lipid nanoparticles of varying degrees of fluorination provided in the examples of the present invention were co-incubated with HEK293T and DC2.4 cell lines for 24 hours.
FIG. 9j shows the enhanced green fluorescent protein positive cell fraction and the average fluorescence intensity statistics of BMDC cells detected using flow cytometry after 50% SM-102 containing lipid nanoparticles of varying degrees of fluorination were co-incubated with bone marrow-derived dendritic cells (BMDC) for 24 hours as provided in the examples of the present invention. In the figure, P <0.001 is indicated.
Figure 9k is a representative flow chart of analysis of flow cytometry for detection of BMDC cells after 24 hours of co-incubation of lipid nanoparticles containing 50% sm-102 with BMDC cells as provided in the examples of the present invention.
FIG. 10a shows the ratio of enhanced green fluorescent protein positive cells to average fluorescence intensity statistics of B16F10 cells detected using flow cytometry after 24 hours of co-incubation of lipid nanoparticles containing 50% SM-102 at various concentrations and varying degrees of fluorination with B16F10 cells, as provided by the examples of the present invention. In the figure, P <0.001 is indicated.
FIG. 10B is a fluorescence image of B16F10 cells taken using a fluorescence microscope after 24 hours of co-incubation of lipid nanoparticles containing 50% SM-102 at various concentrations and varying degrees of fluorination with B16F10 cells, as provided by the examples of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 11 shows statistics of the proportion of enhanced green fluorescent protein positive cells of B16F10 cells detected using flow cytometry after storage of lipid nanoparticles containing 50% SM-102 of varying degrees of fluorination for 24 hours with B16F10 cells under 4℃conditions for 0,7,17,27,60 days as provided in the examples of the present invention. In the figure, P <0.05, P <0.01, and P <0.001.
FIG. 12a shows the flow cytometry analysis of the proportion of enhanced green fluorescent protein positive cells and the average fluorescence intensity statistics of B16F10 cells after 24 hours of co-incubation of lipid nanoparticles containing 50% SM-102 with B16F10 cells treated with different cell uptake inhibitors, as provided by the examples of the present invention. In the figure, P <0.05, P <0.01, and P <0.001.
FIG. 12B is a representative flow-based analytical scatter plot of B16F10 cells detected using flow cytometry after 24 hours of co-incubation of lipid nanoparticles containing 50% SM-102 of varying degrees of fluorination with B16F10 cells treated with different cell uptake inhibitors provided by the examples of the present invention.
FIG. 13a is a fluorescence photograph taken using a laser confocal microscope after 2,4,6,8 hours of co-incubation of lipid nanoparticles containing 50% SM-102 with B16F10 cells according to the present invention; wherein lipid nanoparticles of different degrees of fluorination (Cy 5-mRNA-0% -F-LNPs and Cy5-mRNA-1.5% -F-LNPs) were labeled with Cy5 fluorescent markers mRNA, lysosomes were labeled with Lysotracker Green, and nuclei were labeled with Hoechst.
FIG. 13B is an enlarged fluorescence photograph taken using a laser confocal microscope after 4 hours of co-incubation of lipid nanoparticles with B16F10 cells containing 50% SM-102 at various FPD levels provided in the examples of the present invention; wherein lipid nanoparticles of different degrees of fluorination (Cy 5-mRNA-0% -F-LNPs and Cy5-mRNA-1.5% -F-LNPs) were labeled with Cy5 fluorescent markers mRNA, lysosomes were labeled with Lysotracker Green, and nuclei were labeled with Hoechst.
FIG. 13c is a photograph of fluorescence taken using a laser confocal microscope after 4 hours of co-incubation of lipid nanoparticles containing 50% SM-102 with B16F10 cells provided in the examples of the present invention; wherein lipid nanoparticles (Cy 5-mRNA-0% -F-LNPs and Cy5-mRNA-1.5% -F-LNPs) with different degrees of fluorination are labeled with Cy5 fluorescent marker mRNA, lysosomes are labeled with Lysotracker Green, and cell nuclei are labeled with Hoechst; 1-6 are respectively the co-localization curve statistics of lysosomes and fluorinated lipid nanoparticles in the line segment part of the confocal fluorescence image.
FIG. 14a is a photograph of a living organism luminescence taken using a small animal living imaging system (IVIS Spectrum) after 1,2,3.5,5,6.5 hours of intraperitoneal injection of lipid nanoparticles containing 50% SM-102 in different degrees of fluorination into C57BL/6J mice, as provided by the example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 14b is a graph showing the quantitative plot of the systemic mean bioluminescence intensity counted using the in vivo small animal imaging system (IVIS Spectrum) after 1,2,3.5,5,6.5 hours of intraperitoneal injection of lipid nanoparticles containing 50% SM-102 in different degrees of fluorination in C57BL/6J mice, as provided by the example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6. In the figure, P <0.05, P <0.01, and P <0.001.
FIG. 14C is a bioluminescence image of each isolated organ (heart, liver, spleen, lung, kidney) taken using the in vivo small animal imaging system (IVIS Spectrum) 8 hours after intraperitoneal injection of lipid nanoparticles containing 50% SM-102 in different degrees of fluorination into C57BL/6J mice provided by the example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 14d is a graph showing the quantification of the mean bioluminescence intensity of isolated organs (liver, spleen, lung) counted using the in vivo small animal imaging system (IVIS Spectrum) 8 hours after intraperitoneal injection of lipid nanoparticles containing 50% SM-102 in different degrees of fluorination into C57BL/6J mice, as provided by the example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6. In the figure, P <0.05, P <0.01, and P <0.001.
FIG. 15a shows an example of the present invention providing a mean volume to melanoma-bearing tumor of 200mm 3 Is 8 hours after intratumoral injection of lipid nanoparticles containing 50% sm-102 in different degrees of fluorination using a bioluminescence picture of an ex vivo tumor taken with an in vivo small animal imaging system (IVIS Spectrum); wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 15b shows an average melanoma-bearing volume of 200mm according to an example of the present invention 3 Is a quantitative plot of the mean bioluminescence intensity of isolated tumors counted using the small animal in vivo imaging system (IVIS Spectrum) 8 hours after intratumoral injection of lipid nanoparticles containing 50% sm-102 of varying degrees of fluorination; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6. In the figure, P is represented by<0.05。
FIG. 16a is a photograph of a living organism luminescence taken using a living animal imaging system (IVIS Spectrum) after 1,2,3.5,5,6.5 hours of intravenous injection of lipid nanoparticles containing 50% SM-102 to the tail of C57BL/6J mice provided by an example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 16b is a graph showing the quantitative plot of the systemic mean bioluminescence intensity counted using the in vivo imaging system (IVIS Spectrum) of a small animal after 1,2,3.5,5,6.5 hours of intravenous injection of lipid nanoparticles containing 50% SM-102 to the tail of C57BL/6J mice provided by the example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6. In the figure, P <0.05, P <0.01, and P <0.001.
FIG. 16C is a bioluminescence image of each isolated organ (heart, liver, spleen, lung, kidney, pancreas) taken using the in vivo imaging system (IVIS Spectrum) of a small animal 8 hours after intravenous injection of lipid nanoparticles containing 50% SM-102 to the tail of a C57BL/6J mouse provided by an example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 16d is a graph showing quantification of mean bioluminescence intensity of isolated livers using in vivo imaging system (IVIS Spectrum) statistics of animals 8 hours after intravenous injection of lipid nanoparticles containing 50% SM-102 to the tail of C57BL/6J mice provided by the present examples; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6. In the figure, P <0.01 is indicated.
FIG. 17 is an H & E slice of each isolated organ taken 5 days after intravenous injection of lipid nanoparticles containing 50% SM-102 to the tail of C57BL/6J mice provided by the example of the present invention; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 18a is an H & E slice of each isolated organ taken 5 days after subcutaneous injection of lipid nanoparticles containing 50% SM-102 with different degrees of fluorination into C57BL/6J mice provided by the present example; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
FIG. 18b is an H & E slice of a subcutaneous injection site taken 5 days after the subcutaneous injection of lipid nanoparticles containing 50% SM-102 in lipid nanoparticles of varying degrees of fluorination into C57BL/6J mice provided by the present example; wherein the meanings of 0% FPD and 1.5% FPD are as defined in FIG. 6.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
SM-102: 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate; (Ai Weita (Shanghai) pharmaceutical technology Co., ltd., CAS: 2089251-47-6)
Dlin-MC3-DMA:4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester; (Shanghai Langxu Biotech Co., ltd., CAS: 1224606-06-7)
DSPE-PEG2000: distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (Shanghai Langxu Biotech Co., ltd., CAS: 147867-65-0)
ALC-0315: ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate); (CAS: 2036272-55-4)
DSPC:1, 2-distearoyl-sn-glycero-3-phosphorylcholine; (Shanghai Langxu Biotech Co., ltd., CAS: 816-94-4)
DOPE:1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine; (CAS: 4004-05-1)
Cholesterol: cholest-5-en-3 beta-ol. (Ai Weita (Shanghai) pharmaceutical technologies Co., ltd., CAS: 57-88-5)
Distearoyl phosphatidyl acetamide-polyethylene glycol 2000-amine: (Shanghai Langxu Biotech Co., ltd., CAS: 474922-26-4)
4, 4-trifluoro-butyric acid: (Aladin, CAS: 406-93-9)
Hydroxy pyrrolidine-2, 5-dione: (Merck, CAS: 6066-82-6)
4-dimethylaminopyridine: (Merck, CAS: 1122-58-3)
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide: (Merck, CAS: 1892-57-5)
CCK-8: cell Counting Kit-8 cell proliferation toxicity detection kit CCK-8 (CCK 8) (DOJINDO Cat No: CK 04)
The structure of each material is shown in table 1 below:
TABLE 1
EXAMPLE 1DSPE-PEG2000-NHCOCH 2 CH 2 CF 3 Synthesis of (FPD)
4, 4-trifluoro-butyric acid and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 1-hydroxypyrrolidine-2, 5-dione were prepared according to 1:1:1 in a molar ratio of 1 in dichloromethane, slowly dropwise adding 0.2 equivalent of 4-dimethylaminopyridine, stirring and reacting for two hours under ice bath, then adding 0.2 equivalent of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amine, stirring and reacting for 12 hours at normal temperature to obtain a final product DSPE-PEG2000-NHCOCH 2 CH 2 CF 3 (FPD), dialyzing and freeze-drying to obtain purified product, and confirming the product by matrix-assisted laser analysis ionization time-of-flight mass spectrum, nuclear magnetic carbon spectrum, nuclear magnetic hydrogen spectrum and nuclear magnetic fluorine spectrum (see figures 1-4).
Raw material DSPE-PEG2000-NH for analyzing ionization time-of-flight mass spectrogram by contrast matrix auxiliary laser 2 And the product peak position can be known, the product peak position is relative to DSPE-PEG2000-NH 2 The overall shift to the right of molecular weight 124.84, which corresponds to M (DSPE-PEG 2000-NH) 2 +FPD)-M(DSPE-PEG2000-NH 2 ) Molecular weight differences of (a).
Raw material DSPE-PEG2000-NH by contrasting nuclear magnetic carbon spectrum 2 And the product peak positions were found to be-CF at 123.64, 125.83, 128.03, 129.49 3 A signal.
EXAMPLE 2 preparation of fluorinated lipid nanoparticles
(1) Preparation of lipid monomer mother liquor
Each lipid monomer was dissolved in ethanol phase to prepare mother liquor of the following concentration:
1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate or 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester: 10mg/mL;
1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 5mg/mL;
cholest-5-en-3 beta-ol: 10mg/mL;
DSPE-PEG2000 or FPD:5mg/mL.
(2) N1 methyl pseudouridine modified base and common ACG synthesized mRNA mother liquor containing Cap1 Cap were prepared.
Dissolving mRNA in 10mM sodium citrate solution at pH 4 to obtain 0.072 μg/μl sodium citrate buffer
(3) Accurately pipetting each lipid monomer mother liquor by using pipetting gun
1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate: 45.48 μl,1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, distearoyl phosphatidylethanolamine-polyethylene glycol 2000: 10.52. Mu.L, and 56.36. Mu.L absolute ethanol were added;
1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate: 45.48 μl,1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, distearoyl phosphatidylethanolamine-polyethylene glycol 2000:7.00 μl, FPD: 3.72. Mu.L, and 56.16. Mu.L absolute ethanol were added;
1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate: 45.48 μl,1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, distearoyl phosphatidylethanolamine-polyethylene glycol 2000:3.52 μl, FPD: 7.44. Mu.L, and 55.92. Mu.L of absolute ethanol were added;
1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate: 45.48 μl,1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, FPD: 11.16. Mu.L, and 55.72. Mu.L of absolute ethanol were added;
4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester: 41.12. Mu.L, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, distearoyl phosphatidylethanolamine-polyethylene glycol 2000: 10.52. Mu.L, and 60.72. Mu.L of absolute ethanol were added;
4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester: 41.12. Mu.L, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, distearoyl phosphatidylethanolamine-polyethylene glycol 2000:7.00 μl, FPD: 3.72. Mu.L, and 60.52. Mu.L absolute ethanol were added;
4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester: 41.12. Mu.L, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, distearoyl phosphatidylethanolamine-polyethylene glycol 2000:3.52 μl, FPD: 7.44. Mu.L, and 60.28. Mu.L of absolute ethanol were added;
4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester: 41.12. Mu.L, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine: 20.52 μl, cholest-5-en-3 β -ol: 19.12 μl, FPD: 11.16. Mu.L, and 60.08. Mu.L of absolute ethanol were added.
(4) Preparation of lipid nanoparticles (F-LNPs)
Each monomer mother liquor except polyethylene glycol lipid (50% ionizable lipid: 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate or 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester, 10% helper phospholipid: after mixing 1, 2-distearoyl-sn-glycero-3-phosphorylcholine and 38.5% cholest-5-en-3β -ol, 1.5% of a polyethylene glycol lipid mother liquor (distearoyl phosphatidylethanolamine-polyethylene glycol 2000 or FPD) was finally added, the final total lipid concentration was kept at 8mM after mixing, the mixed volume ratio of Cap1 Cap-containing messenger ribonucleic acid solution (ffalogena biotechnology limited, L/N: mRNA-22-220712) synthesized with N1 methyl pseudouridine was 1:3, the syringe was aspirated and fixed to a syringe pump (longenrpump, model: LSP 02-2B), turbulent flow and sufficient mixing were generated through a microfluidic chip (micro-core start science instruments limited, in su state), lipid nanoparticles were formed by self-assembly, and a uniform quality lipid nanoparticle was removed using a dialysis cup (micro dialysis cup cutoff amount was 10000) (feiter, china) and a pH profile was prepared as shown in fig. 5.
The prepared lipid nanoparticles have uniform spherical structure (figure 6) observed by a Talos F200C 200kV low-temperature transmission electron microscope (FEI), and the particle sizes of the lipid nanoparticles with different degrees of fluorination are obtained by dynamic light scattering (DLS, malvern Zetasizer), and the results are shown in Table 2 (see figure 7 for details).
TABLE 2
Conclusion: from the above data, the particle size distribution of the lipid nanoparticles with different degrees of fluorination is 110-150nm, the polydispersity is less than 0.2, and the electric potential is 15-21mV, which indicates that the lipid nanoparticles are uniform and the physical properties are uniform and stable.
EXAMPLE 3 delivery of fluorinated lipid nanoparticles
Administering cells by mixing a serum-reduced medium (Zhejiang Senrui Biotechnology Co., ltd.) with a lipid nanoparticle solution or administering the lipid nanoparticle PBS solution by intraperitoneal injection, tail vein injection, intratumoral injection, subcutaneous injection, etc., and administering the animal by Qubit TM The detection procedure of ribonucleic acid high sensitivity kit (Simer Feichi technologies (China) Co.) quantifies the mRNA encapsulation efficiency (as shown in Table 3 below) and the drug administration concentration.
TABLE 3 encapsulation efficiency of mRNA of lipid nanoparticles with different degrees of fluorination
In the administration of lipid nanoparticles, the biocompatibility must be considered, and the cell viability cannot be affected. Lipid nanoparticles formed by assembling different lipid monomer ratios (including fluorinated or unfluorinated distearoyl phosphatidylethanolamine-polyethylene glycol 2000) were tested for proliferation activity of DC2.4 cells and B16F10 cells using CCK-8. Specific embodiment B16F10 or DC2.4 cells are expressed as 5X 10 3 Density of individuals/wells was pre-incubated in 96-well plates for 24h, then DC2.4 cells and B16F10 cells were incubated with different F-LNPs containing 50% SM-102 at a total lipid concentration in the F-LNPs of 5-120. Mu.g/mL. After 24h incubation, the medium was discarded, 100. Mu.L of fresh medium containing 10. Mu.L of CCK-8 solution was added to each well, and absorbance was measured at 450nm after 2h incubation. All ofCells were incubated in a humidified incubator (ThermoFisher), 37℃with 5% CO 2 . Lipid nanoparticles of different degrees of fluorination were found to have good biocompatibility by incubating the cells for 24 hours at total lipid concentrations of 5, 10, 20, 40, 80, 120 μg/mL without affecting cell viability, as compared to the placebo group (cell viability results are detailed in figures 8a-8 b).
HEK293T (cell bank of China academy of sciences), DC2.4 (Shanghai enzyme research biosciences Co., ltd.), B16F10 (cell bank of China academy of sciences) were prepared by 5×10 in advance 4 The density of individual cells was incubated in 24 well plates for 24h, after which the original medium was discarded and transfected with 500. Mu.L of Opti-MEM medium containing freshly prepared different F-LNPs with 0%, 0.5%, 1% or 1.5% FPD, respectively. The final concentration of enhanced green fluorescent protein (eGFP mRNA) was 1.5. Mu.g/mL. Meanwhile, in order to verify the influence of different ionizable lipids on the properties of the lipid nanoparticles, two types of ionizable lipids of SM-102 or Dlin-MC3-DMA are selected to prepare the lipid nanoparticles with different degrees of fluorination. After 4 hours incubation, the Opti-MEM medium was discarded and the cells were gently washed 2 times with PBS. mu.L of fresh complete medium (RPMI 1640 or DMEM medium, 10% foetal calf serum, 1% double antibody) was added, transfection was continued for 20h, then cells were digested with trypsin, centrifuged, resuspended in cell staining buffer (PBS (biosharp Cat No: BL 302A) +2% foetal calf serum). By flow cytometry (Beckman CytoFlex S or BD FACSCelesta TM ) Or the laser confocal microscope detects the expression efficiency and intensity. Cells were incubated in a humidified incubator (ThermoFisher), 37℃with 5% CO 2 . The results showed that compared with the experimental group incubated with lipid nanoparticles without fluorinated material, the lipid nanoparticles containing 1.5% of fluorinated material FPD had significant differences in both the number of transfected positive cells and the average fluorescence intensity of enhanced green fluorescent protein, indicating that the lipid nanoparticles can effectively increase the expression level of protein at the same mRNA concentration. Meanwhile, according to cytotoxicity and in vitro different cell transfection results, lipid nanoparticles containing 1.5% of fluorinated material FPD prepared by taking SM-102 as ionizable lipid are also prepared by taking Dlin-MC3-DMA as ionizable lipidThe delivery efficiency of lipid nanoparticles of FPD was significantly improved, so next lipid nanoparticles containing 0% or 1.5% of fluorinated material FPD prepared with SM-102 as ionizable lipid were selected for the exploration of delivery efficiency (HEK 293T, DC 2.4.4, B16F10 cell transfection efficiency results are detailed in fig. 9a-9 i).
BMDC cells (bone marrow derived dendritic cells, extracted from mouse femur and tibia) were prepared by washing bone marrow isolated from femur and tibia of C57BL/6J mice with RPMI 1640 medium, filtering off muscle and bone fragments with 70 μm filter, centrifuging, and resuspending cells with erythrocyte lysate. After 5 minutes the cells were centrifuged again and resuspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 1%o double antibody (Gibco, invitrogen, cat No: P1400), 20ng/mL GM-CSF (Biolegend) and 20ng/mL IL-4 (bioleged) at a cell density of 1X 10 6 /mL. Media was changed on day 2, day 4, cells were digested with trypsin on day 7, CD16/32 blocked non-specific sites, and stained with anti-CD 11c PE. Differentiation of BMDCs was analyzed using flow cytometry. Lipid nanoparticles containing 0% or 1.5% fluorinated material FPD prepared with SM-102 as ionizable lipid were then co-incubated with BMDC cells for in vitro transfection. The results show that the delivery efficiency of the lipid nanoparticles after fluorination is significantly improved compared to the non-fluorinated group. BMDC cells were cultured in a humidified incubator (ThermoFisher) at 37℃with 5% CO 2 And (5) incubating. (the results of BMDC cell transfection efficiency are shown in FIGS. 9j-9 k).
In addition, the correlation between the concentration of different mRNA-F-LNPs administered and the efficiency of delivery was further investigated. Specifically, B16F10 cells were pre-cultured at 5X 10 per well 4 Density of individual cells were cultured in 24 well plates for 24h and then replaced with 500. Mu.L of Opti-MEM containing 0% or 1.5% FPD of freshly prepared different F-LNPs, respectively. The final concentration of eGFP mRNA was 0.1,0.2,0.375,0.75,1.5. Mu.g/mL. After 4 hours incubation, the Opti-MEM medium was discarded and the cells were gently washed 2 times with PBS. mu.L of fresh complete medium (RPMI 1640,10% FBS,1% P/S) was added and transfection was continued for 20h, followed by digestion of the cells with trypsin, centrifugation, and re-weighting with cell staining buffer And (5) newly suspending. Flow cytometry (BD FACSCelesta) TM ) The expression efficiency was examined. Cells were incubated in a humidified incubator (ThermoFisher), 37℃with 5% CO 2 . The results showed that the cell positive rate and MFI of the mRNA-1.5% -F-LNPs (mRNA, 0.2. Mu.g/mL) treated B16F10 cells were 3% and 42% higher, respectively, than that of the mRNA-0% -F-LNPs (mRNA, 1.5. Mu.g/mL) treated group; and mRNA-1.5% -F-LNPs still achieved similar delivery efficiency to the control group at the required dose reduced to 1/7 of the control group (see FIGS. 10a-b for details).
Since most LNPs rapidly lose transfection ability under the conventional aqueous solution preservation method, the change of delivery efficiency of mRNA-1.5% -F-LNPs and mRNA-0% -F-LNPs at different storage time points was tracked by flow cytometry under the preservation condition of 4 ℃, and the stability of mRNA-1.5% -F-LNPs was primarily verified. Specifically, B16F10 cells were cultured at 5X 10 4 The density of individuals/wells was pre-inoculated in 24-well plates for 24 hours, and then different F-LNPs freshly prepared or stored at 4 degrees for 7, 17, 27, 60 days, respectively, were dispersed in Opti-MEM medium and dosed. After 24 hours incubation, cells were trypsinized, centrifuged, resuspended in cell staining buffer, and then purified by flow cytometry (BD FACSCelesta TM ) The positive rate and average fluorescence intensity were measured. Cells were incubated in a humidified incubator (ThermoFisher), 37℃with 5% CO 2 . The results show that mRNA-1.5% -F-LNPs still have comparable delivery capacity at day 7 and day 0 of storage. Furthermore, the decrease in expression efficiency at day 60 was still 26% less for the mRNA-1.5% -F-LNPs treated group compared to the mRNA-0% -F-LNPs treated group (see FIG. 11 for details).
The cellular internalization pathways of different preparations of mRNA-F-LNPs were explored by treating cells with different cell uptake inhibitors. Various cellular uptake inhibitors were used to study the internalization pathways of F-LNPs. Specifically, B16F10 cells were cultured at 5X 10 4 Density of individuals/wells was previously cultured on 24-well plates for 24 hours, and then incubated with lignan (Gen, 200. Mu.M), chlorpromazine (CPM, 30. Mu.M), amiloride (Ami, 5 mM) or 4℃for 1 hour, respectively, followed by addition of 500. Mu.L of Opti-MEM containing F-LNPs, respectively. The final concentration of Cy5-mRNA was 1.5. Mu.g/mL. After 4 hours incubation, the medium was discarded and the cells were gently washed twice with PBS. The position of the partThe conditioned cells were trypsinized, centrifuged, resuspended in cell staining buffer, flow cytometry (BD FACSCelesta TM ) Cell uptake efficiency was measured. The results show that cellular internalization is significantly limited following low temperature (4 ℃), ami and CPM treatments. Internalization of mRNA-1.5% -F-LNPs was demonstrated primarily through membrane fluidity dependence, clathrin-mediated endocytosis and macrophagia pathways (see FIGS. 12a-b for details).
Furthermore, to confirm whether a significant increase in mRNA expression was related to the number and rate of mRNA-1.5% -F-LNPs escaping from lysosomes, the rate of endosomal escape of F-LNPs was observed using confocal microscopy. The method is implemented by observing the co-localization of lysosomes and F-LNPs by using a laser confocal microscope (Nikon, model: ECLIPSE Ti 2-E). Before transfection, B16F10 cells were grown at 2X 10 5 The density of individual cells was pre-incubated in a confocal dish for 24h and then treated with Cy 5-labeled mRNA-F-LNPs containing 0% or 1.5% FPD for 2, 4, 6 and 8h, respectively. After 4 hours incubation, the Opti-MEM medium containing the different Cy 5-labeled F-LNPs was discarded and the cells were gently washed twice with PBS. mu.L of fresh complete medium (RPMI 1640,10% FBS,1% P/S) was added and transfection continued. Prior to observation, cells were incubated with 50nM LysoTrackerTM Green DND-26 (thermosusher, catalog number: L7526) in Opti-MEM for 40 min, tracking lysosomes and Hoechst 33342 (Beyotime, catalog number: C1025) for 10 min to track nuclei. Thereafter, the cells were gently rinsed twice with PBS and observed under a confocal microscope. The results showed that at 4 hours after treatment, the intracellular Cy 5-labeled mRNA-0% -F-LNPs remained almost co-localized with the lysosome, while most of the Cy 5-labeled mRNA-1.5% -F-LNPs had escaped from the lysosome and were evenly distributed in the cytoplasm. In addition, in the magnified confocal images taken at 4h of co-incubation, cy 5-labeled mRNA-1.5% -F-LNPs had a 3.2-fold higher intensity in the cells over time than Cy 5-labeled mRNA-0% -F-LNPs (see 13a-c for details).
Subsequently, the delivery efficiency of mRNA-1.5% -F-LNPs was verified on female C57BL/6J mice, while mRNA-0% -F-LNPs were introduced as controls. Specific implementation was performed by continuously monitoring the bioluminescence intensity of mice at 1,2,3.5,5,6.5h by a small animal In Vivo Imaging System (IVIS) after intraperitoneal injection of different Luci-mRNA-F-LNPs (0.25 mg/kg per mouse, mRNA) into C57BL/6J mice. From hour 2 to the end of the monitoring, the average bioluminescence intensity of mRNA-1.5% -F-LNPs treated mice was significantly higher than that of the control group. After 8 hours of intraperitoneal administration, the mice were euthanized and the bioluminescence intensity of each organ was measured ex vivo. From the bioluminescence image, the average bioluminescence intensity of liver, spleen, lung of mRNA-1.5% -F-LNPs treated group was increased about 4-fold, 1.7-fold and 1.9-fold, respectively, compared to control group (see FIGS. 14a-d for details).
In addition, the differences in delivery efficiency after intratumoral injection of different mRNA-F-LNPs were further compared. Is specifically implemented by 1X 10 6 The B16F10 cells were seeded on the right thigh of C57BL/6J mice. When the volume of melanoma increases to 200mm 3 At this time, different mRNA-F-LNPs were injected into the tumor of the C57BL/6J mice at an mRNA dose of 0.25mg/kg, and the in vivo bioluminescence was monitored at various time points using a small animal in vivo imaging system. Each mouse was intraperitoneally injected with 200 mu L D-fluorescein (Thermo Scientific Pierce) in D-PBS (15 mg/ml) for 10 minutes and then automatically imaged with IVIS. The relevant area was quantified using the live Image software. The results showed that the mRNA expression was also increased by about 2.7-fold at 8h in the mRNA-1.5% -F-LNPs treated group compared to the control group (see FIGS. 15a-b for details).
Meanwhile, the difference of the expression efficiency of different mRNA-F-LNPs was also explored by intravenous injection into the tail of C57BL/6J mice. From the 1 st hour of monitoring to the end of monitoring, the expression level of mRNA-1.5% -F-LNPs was significantly higher than that of the control group. After administration via the tail vein, most of the targeted proteins are expressed in the liver and spleen. In addition, both dosage forms were slightly expressed in the lung. The average luminous intensity of each isolated organ was measured 8 hours after intravenous injection. The results showed that mRNA-1.5% -F-LNPs treated liver showed significantly better protein expression than the control group by a factor of about 2.95 (see FIGS. 16a-d for details).
Subsequently, the biosafety of 0% and 1.5% FPD mRNA-F-LNPs was assessed by hematoxylin and eosin (H & E) staining. Specifically, PBS and mRNA-F-LNPs containing 0% or 1.5% FPD were administered to female C57BL/6J mice (8-10 weeks) at a dose of 0.25mg/kg, respectively, by tail vein or subcutaneously. After 5 days of injection, mice were euthanized and sections of the skin and major viscera at the injection site were H & E stained. The results showed that, consistent with the results of in vitro cytotoxicity experiments, after 5 days of subcutaneous or tail intravenous injection, no significant damage or inflammation was seen in major organs or skin at the injection site compared to PBS group, indicating good biological tolerance of mRNA-1.5% -F-LNPs (see fig. 17, 18a-b for details). Currently, representative fluorinated PEG-lipids (FPDs) are synthesized and purified by efficient condensation reactions, and F-LNP formulations are proposed that are effective in delivering mRNA. Unlike conventional high throughput component screening strategies, the present strategy alters the surface properties of LNP by simple fluorine modification to increase mRNA delivery efficiency. mRNA-1.5% -F-LNPs are prepared by using FPD, so that the internalization level and lysosome escape capacity of cells can be remarkably improved, and the utilization capacity of mRNA is remarkably improved. In addition, mRNA-1.5% -F-LNPs increased the in vivo expression level of the target protein at least 3-fold at a dose of 0.25mg/kg in various modes of administration, and all had good in vivo biosafety. In summary, fluorine modified FPDs have been validated as a representative strategy to improve the feasibility of LNP performance.

Claims (12)

1. A compound of formula I:
wherein n is an integer of 0 to 10, R is a group consisting of 1, 2 or 3 CF 3 Substituted methyl.
2. The compound of formula I according to claim 1, wherein the compound of formula I satisfies one or both of the following conditions:
(1) n is an integer from 1 to 5, for example n is 1;
and (2) R is-CH 2 CF 3 。
3. The compound shown in the formula I as claimed in claim 1, wherein the compound shown in the formula I is as follows:
4. use of a compound of formula I as defined in any one of claims 1 to 3 for the preparation of lipid nanoparticles.
5. A lipid carrier comprising a substance Z which is a compound of formula I according to any one of claims 1 to 3.
6. The lipid carrier of claim 5, wherein the lipid carrier satisfies one or more of the following conditions:
(1) The lipid carrier further comprises a helper phospholipid;
(2) The lipid carrier further comprises an ionizable lipid;
(3) The lipid carrier further comprises cholesterol;
and (4) the lipid carrier further comprises DSPE-PEG2000;
preferably, the lipid carrier satisfies one or more of the following conditions:
(1) The auxiliary phospholipid is a phospholipid molecule with charged polar end and fatty chain nonpolar end, such as 1, 2-distearoyl-SN-glycero-3-phosphorylcholine or 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine, preferably 1, 2-distearoyl-SN-glycero-3-phosphorylcholine;
(2) The ionizable lipid is selected from one or more of 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate, 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester and ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), for example 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate or 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester;
(3) The molar content of the auxiliary phospholipids is 5% to 15%, preferably 10%;
(4) The molar content of the ionizable lipid is 45% to 60%, preferably 50%;
(5) The cholesterol is present in a molar amount of about 30% to 40%, preferably 38.5%;
(6) The molar content of DSPE-PEG2000 is less than 5%, e.g., 0.5-1%, further e.g., 0.5%, 1.0%, or 1.5%;
(7) The molar content of the substance Z is from 0.01% to 5%, preferably from 0.5 to 1.5%, for example 0.5%, 1.0% or 1.5%;
(8) The sum of the molar content of the substance Z and the molar content of the DSPE-PEG2000 is more than or equal to 1.5%, preferably 1.5%;
(9) The molar ratio of the substance Z to the DSPE-PEG2000 is (1-2): (2-1);
(10) The molar ratio of the ionizable lipid to the substance Z is 50 (0.01-1.5), e.g. 50:0.5, 50:1 and 50:1.5;
(11) The molar ratio of the auxiliary phospholipid to the substance Z is 10 (0.01-1.5), such as 10:0.5, 10:1 and 10:1.5;
(12) The molar ratio of cholesterol to substance Z is 38.5 (0.01-1.5), such as 38.5:0.5, 38.5:1 and 38.5:1.5;
(13) The volume ratio of the ionizable lipid to the helper phospholipid is (2-2.5): 1, preferably (2-2.3): 1;
(14) The volume ratio of the ionizable lipid to the cholesterol is (2-2.5): 1, preferably (2.1-2.4): 1;
(15) The volume ratio of the ionizable lipid to the DSPE-PEG2000 is (3-13): 1;
(16) The volume ratio of the ionizable lipid to the substance Z is (3-13): 1;
and (17) the lipid carrier consists of the substance Z, the ionizable lipid, the helper phospholipid, the DSPE-PEG2000, and the cholesterol;
more preferably, the lipid carrier comprises, in mole percent: 50% of said ionizable lipid, 10% of said helper phospholipid, 35-38.5% of said cholesterol, 1.5% -5% of a pegylated lipid; the PEGylated lipid is DSPE-PEG2000 and the substance Z, or the substance Z.
7. Lipid nanoparticle, characterized in that it comprises a therapeutic and/or prophylactic agent and a lipid carrier according to claim 5 or 6.
8. The lipid nanoparticle of claim 7, wherein the lipid nanoparticle satisfies one or more of the following conditions:
(1) The therapeutic agent is messenger ribonucleic acid;
(2) The prophylactic agent is messenger ribonucleic acid;
(3) In the lipid nanoparticle, the molar ratio of the nitrogen atoms in the ionizable lipid to the phosphorus atoms in the therapeutic and/or prophylactic agent is 1:3 or 1:6;
(4) In the lipid nanoparticle, the ratio of the total lipid mass of the lipid nanoparticle to the therapeutic and/or prophylactic agent mass is 1:10 or 1:20;
and (5) in the lipid nanoparticle, the lipid carrier encapsulates the therapeutic and/or prophylactic agent.
9. A composition comprising a substance Z which is a compound of formula I according to any one of claims 1 to 3.
10. The composition of claim 9, further comprising one or more of the ionizable lipid, a helper phospholipid, DSPE-PEG2000, cholesterol, and a therapeutic and/or prophylactic agent;
Preferably, in the composition, the ionizable lipid, helper phospholipid, DSPE-PEG2000 and cholesterol are as described in claim 6; and/or, the therapeutic and/or prophylactic agent according to claim 8;
further preferably, in the composition, the substance Z forms a lipid carrier according to claim 5 or 6 with one or more of the ionizable lipids, helper phospholipids, DSPE-PEG2000 and cholesterol; and/or, in the composition, the therapeutic and/or prophylactic agent has an encapsulation efficiency of at least 80%, preferably at least 85%;
more preferably, in the composition, the lipid carrier forms a lipid nanoparticle according to claim 7 or 8 with the therapeutic and/or prophylactic agent.
11. Use of a compound according to any one of claims 1-3 as shown in formula I, a lipid carrier according to claim 5 or 6, a lipid nanoparticle according to claim 7 or 8, a composition according to claim 9 or 10 for the preparation of a medicament or vaccine for the treatment of a related disease, including viral infection, inflammatory disease, HIV, autoimmune disease, tumor.
12. The use of claim 11, wherein the neoplasm comprises melanoma cells, hematological tumors, lymphoid cancers, brain cancers, liver cancers, lung cancers, esophageal cancers, gastric cancers, breast cancers, pancreatic cancers, thyroid cancers, nasopharyngeal cancers, ovarian cancers, endometrial cancers, renal cancers, prostate cancers, bladder cancers, colon cancers, rectal cancers, testicular cancers or head and neck cancers.
CN202311103951.7A 2022-08-29 2023-08-29 Fluorine-containing DSPE-PEG2000, application thereof, nanoparticle containing fluorine-containing DSPE-PEG2000 and application of fluorine-containing DSPE-PEG2000 Pending CN117624582A (en)

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