CN118634207A

CN118634207A - Nucleic acid-lipid nanoparticle for atomization inhalation and preparation method and application thereof

Info

Publication number: CN118634207A
Application number: CN202411125688.6A
Authority: CN
Inventors: 于飞; 张凤; 宋更申; 孙振龙; 王环宇; 李玥; 王帅; 马海秋; 穆树花
Original assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Current assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Priority date: 2024-08-15
Filing date: 2024-08-15
Publication date: 2024-09-13

Abstract

The present disclosure provides a nucleic acid-lipid nanoparticle for aerosol inhalation, which includes a lipid, a nucleic acid encapsulated within the lipid, and a dispersion solution to which a water-soluble excipient is added, and a method of preparing the same and use thereof. The nucleic acid-lipid nanoparticle can deliver the target gene in an aerosol mode through a vibrating screen atomizer or a soft mist inhaler, the stability of particle size, PDI and encapsulation efficiency can be ensured in the atomization process, efficient delivery of the lung can be realized, the particle size and encapsulation efficiency are not obviously changed when the nucleic acid-lipid nanoparticle is refrigerated and stored for 6 months at the temperature of 2-8 ℃, and the stability is good.

Description

Nucleic acid-lipid nanoparticle for atomization inhalation and preparation method and application thereof

Technical Field

The present disclosure relates to the field of biological medicine, and in particular, to a nucleic acid-lipid nanoparticle pharmaceutical composition for aerosol inhalation, and a preparation method and application thereof.

Background

Antisense oligonucleotides (ANTISENSE OLIGODEOXYNUCLEOTIDES, ASO) are artificially synthesized oligonucleotide fragments, which are 15-30 nucleotides in length, and interfere transcription and translation of target genes mainly through a base complementary pairing principle, so that targeted treatment of genes is realized. ASO has the advantages of rich candidate targets, reasonable and designed orientation, high in-vivo and in-vitro effects, capability of being synthesized manually on a large scale and the like, and is considered as a gene therapy drug with great potential.

The antisense oligonucleotide alone has poor stability and no specific tissue targeting, so that the antisense oligonucleotide is easily degraded by nuclease in vivo and cannot perform the therapeutic effect well. In recent years, with the development of new delivery technologies, the development of antisense oligonucleotide drugs has made a breakthrough progress. Nucleic acid delivery vectors are numerous and include viral vectors, lipid nanoparticles, lipid-polymer hybrid nanoparticles, nanostructured lipid vectors, cationic polymers, exosome nanovesicles, and protein vectors, among others. Currently, lipid nanoparticles (LNP, lipid nanoparticle) are one of the more widely used delivery systems for nucleic acid drug research, LNP is capable of delivering nucleic acids safely and efficiently, and generally consists of four lipids with similar molar ratios: including cationic lipids, phospholipids, cholesterol, and PEG-lipids.

Compared with other delivery modes, the inhaled LNP can maximally increase the concentration of the nucleic acid medicine in the lung, and can be used for treating various diseases, such as Cystic Fibrosis (CF), tuberculosis, chronic obstructive pulmonary disease, lung cancer and the like. However, successful development of inhaled LNP faces many difficulties and challenges because whatever form of nebulization, such as ultrasonic nebulization, compressed air nebulization, vibrating screen nebulization, soft mist inhaler nebulization, etc., LNP is subject to the damaging effects of shear forces during nebulization, which may lead to an increase in LNP particle size and PDI, leakage of nucleic acid drugs, ultimately affecting its transfection efficiency, whether LNP can be nebulized to maintain its function, whether antisense oligonucleotides can be delivered into target cells, and protein expression in cells, etc., is a technical problem to be solved.

Disclosure of Invention

It is an object of the present disclosure to provide an LNP pharmaceutical composition comprising a nucleic acid which is suitable for delivering DNA or therapeutic genes targeted for gene therapy to the lungs in a nebulized inhalation, and which is capable of remaining intact during nebulization, such that the nucleic acid drug effectively performs its function after inhalation.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a lipid, a nucleic acid encapsulated within the lipid, and a dispersion solution with the addition of a water-soluble excipient;

the lipid comprises 40-60 mole% of non-ionizable cationic lipid, 10-20 mole% of neutral lipid, 1-5 mole% of long circulating lipid, and 30-50 mole% of cholesterol;

The non-ionizable cationic lipid is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride (DOTAP), the neutral lipid is distearoyl lecithin (DSPC), and the long-circulating lipid is dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2 k).

The pharmaceutical composition provided by the disclosure has higher universality, and can be ensured to endure the shearing force action in the atomization process when different lipid dosages and formulations are used, so that the consistency of granularity, dispersity (PDI) and Encapsulation Efficiency (EE) before and after atomization is realized.

Alternatively, the non-ionizable cationic lipid is present in an amount ratio to said nucleic acid expressed as an N/P ratio, said N/P ratio being (1:1) - (30:1), preferably (3:1) - (10:1).

Alternatively, the non-ionizable cationic lipid refers to a lipid molecule capable of permanently positively charging, and may be any one selected from the group consisting of dioleoyl propyl trimethylammonium chloride (DOTMA) and 1, 2-dioleyl-3-dimethylamino-propane (dotap);

optionally, the neutral lipid may be at least one selected from dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylethanolamine (DOPE);

Alternatively, the nucleic acid is any one selected from the group consisting of mRNA, siRNA, DNA, miRNA, antisense oligonucleotides and non-coding RNAs, preferably any one of siRNA and antisense oligonucleotides, more preferably antisense oligonucleotides.

Preferably, the nucleic acid is CT102, and the nucleotide sequence of CT102 is 5'-TCCTCCGGAGCCAGACTTCA-3'.

Alternatively, the particle size of the pharmaceutical composition is 50-300nm, preferably 50-200nm, and the polydispersity index of the pharmaceutical composition is not more than 0.3.

Optionally, the dispersion solution is at least one selected from Tris buffer, DPBS buffer, HEPES buffer, PBS buffer, citrate buffer, phosphate buffer, carbonate buffer, and acetate buffer.

Optionally, the water-soluble excipient comprises at least one of sucrose, trehalose, polyvinyl alcohol, tween 80, tween 20, poloxamer 188, triton, sodium dodecyl sulfate, sodium chloride, potassium chloride, and glycine; preferably at least one of tween 80, poloxamer 188 and Triton;

if the excipient is Tween 80, the excipient is used at a concentration of 0.01-0.5% (w/v), preferably 0.05-0.15% (w/v);

if the excipient is poloxamer 188, the excipient is used at a concentration of 0.05-1% (w/v), preferably 0.3-0.7% (w/v);

If the excipient is Triton, the excipient is used at a concentration of 0.01-0.5% (w/v), preferably 0.05-0.15% (w/v).

The pharmaceutical composition containing the non-ionizable cations, which is prepared by the method, has better atomization stability, and further, the lipid in the prepared pharmaceutical composition can resist the shearing force in the atomization process by adding the excipient into a dispersion system, and the granularity, PDI and EE are kept unchanged before and after atomization; the particle size of the pharmaceutical composition is increased within 3 nm after being refrigerated for 6 months at the temperature of 2-8 ℃, PDI is not obviously changed, the encapsulation efficiency is reduced by within 3%, the change is not obvious, the stability is good, and the technical problems that the market aerosol inhalation LNP cannot tolerate the atomizing shearing force and the stability is poor are effectively solved.

In another aspect, the present disclosure provides a method of preparing the above pharmaceutical composition, comprising the steps of:

S1, dissolving the lipid in an organic solvent to obtain a first dispersion liquid;

S2, dissolving the nucleic acid in a first dispersion system solution to obtain a second dispersion liquid;

S3, mixing the first dispersion liquid and the second dispersion liquid by using a microfluidic method to obtain a lipid nanoparticle intermediate solution;

And S4, performing ultrafiltration on the lipid nanoparticle intermediate solution by using a second dispersion system solution, removing the organic solvent, adding the water-soluble excipient, and mixing until uniform.

Optionally, the organic solvent is any one selected from ethanol, acetone and N, N-dimethylformamide.

Optionally, the first dispersion solution is at least one selected from a citrate buffer and an acetate buffer;

the second dispersion solution is at least one selected from Tris buffer, DPBS buffer and HEPES buffer.

On the other hand, the present disclosure provides an application of the above pharmaceutical composition and/or the pharmaceutical composition prepared by the above method in preparing an anti-tumor drug, where the administration method of the anti-tumor drug is aerosol inhalation;

The aerosol inhalation device used in the present disclosure is commercially available, and the aerosol inhalation device is any one selected from the group consisting of a compression atomizer, a vibrating screen atomizer, and a soft mist inhaler; preferably a vibrating screen atomizer or a soft mist inhaler.

The term "RNA therapy" in this disclosure refers to the treatment or prevention of diseases using RNA-based molecules, whose targeted drugs mainly include small molecule drugs targeting RNA, antisense oligonucleotides (ASOs), small interfering RNAs (sirnas), micrornas (mirnas), RNA aptamers (aptamers), short activating RNAs (sarnas), genome editing, exogenously expressed delivered mRNA, and the like.

The term "delivery system" in this disclosure refers to a formulation or composition that modulates the spatial, temporal and dose distribution of a bioactive ingredient within an organism.

Through the technical scheme, the medicine composition containing the non-ionizable cations has good atomization stability, can realize effective deposition of the lung after atomization inhalation, has remarkable proliferation inhibition effect on lung cancer cells, has good biocompatibility, is not easy to generate inflammation and irritation, and has good safety.

Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a graph of particle size results before and after atomization of a model sample of example 1 of the present invention;

FIG. 2 is a graph of particle size results before and after atomization for different DOTAP prescriptions in examples 2-3 of the present invention;

FIG. 3 is a graph showing the particle size stability at 2-8deg.C for the formulation of example 1 of the present invention;

FIG. 4 is a graph showing the results of weight change after aerosol inhalation administration in mice model sample of example 1 of the present invention;

FIG. 5 is a graph showing the results of tumor suppression after aerosol inhalation administration of mice model sample of example 1 of the present invention.

Detailed Description

The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

Wherein the non-ionizable cationic lipid is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride, the neutral lipid is distearoyl phosphatidylcholine, and the long-circulating lipid is dimyristoyl glycerol-3-methoxypolyethylene glycol 2000.

Alternatively, the non-ionizable cationic lipid may be any one selected from the group consisting of dioleoyl propyl trimethylammonium chloride and 1, 2-dioleoyl-3-dimethylamino-propane;

The neutral lipid may be at least one selected from dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine.

On the other hand, the present disclosure provides an application of the above pharmaceutical composition and/or the pharmaceutical composition prepared by the above method in preparing an anti-tumor drug, where the administration mode of the anti-tumor drug is aerosol inhalation;

the aerosol inhalation device is any one selected from a compression atomizer, a vibrating screen atomizer and a soft mist inhaler.

Term definitions of reagents used in the present disclosure:

DOTAP: chinese name: (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt; english name: (2, 3-Dioleoyloxy-propyl) -trimethylammonium-chloride, CAS:132172-61-3, the chemical structural formula is shown as follows:

DOTMA: chinese name: di-oleoyl propyl trimethylammonium chloride, english name: n, N, N-trimethyl-2,3-bis (octadec-9-en-1-yloxy) propan-1-aminium chloride, CAS:104872-42-6;

DODMA: chinese name: 1, 2-dioleyl-3-dimethylamino-propane, english name: 1,2-dioleyloxy-3-dimethylaminopropane, CAS:104162-47-2;

DSPC: chinese name: distearyl lecithin, english name: distearoyl Phosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine, CAS:816-94-4;

DOPE: chinese name: 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine; english name: 1,2-dioleoyl sn-glycero-3-phosphoethanolamine, CAS:4004-05-1;

DPPC: chinese name: dipalmitin phosphatidylcholine; english name: 1,2-dipalmitoy 1-sn-glycero-phosphocholine, CAS:63-89-8;

DMG-PEG2k: chinese name: dimyristoylglycerol-3-methoxypolyethylene glycol 2000; english name: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, CAS:160743-62-4;

PEG: chinese name: polyethylene glycol; english name: polyethylene glycol;

Tris buffer: chinese name: tris (hydroxymethyl) aminomethane; english name: 2-Amino-2- (hydroxyymethyl) -1,3-propanediol, CAS no: 77-86-1;

DPBS: du's phosphate buffer;

PBS buffer: the main components are Na ₂HPO₄、KH₂PO₄, naCl and KCl;

HEPEs buffer: chinese name: 4-hydroxyethyl piperazine ethane sulfonic acid; english name: 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid, CAS number: 7365-45-9;

Citrate buffer: the citrate buffer solution comprises citric acid and sodium citrate as main components;

LNP are lipid nanoparticles; the N/P ratio is the carrier amine nitrogen and antisense oligonucleotide phosphate ratio; t80 is Tween 80; p188 is poloxamer 188; PDI is the polydispersity index; EE% is encapsulation efficiency; RT is room temperature.

The present disclosure is further illustrated in detail by the following examples. The starting materials used in the examples are all available commercially. The experimental data P values <0.05 for the comparison between groups in this example below, the differences are statistically significant.

Example 1

This example was used to prepare the pharmaceutical composition CT102-LNP.

(1) The encapsulated antisense oligonucleotide drug is CT102, the nucleotide sequence of which is: 5'-TCCTCCGGAGCCAGACTTCA-3' SEQ ID NO.1; CT102 is a thiodeoxyoligonucleotide sodium salt with the length of 20 nucleotides, takes human insulin-like growth factor type 1 receptor (IGF 1R) messenger ribonucleic acid (mRNA) as a target point, inhibits the activity of IGF1R through antisense action, thereby leading tumor cells to apoptosis or inducing differentiation thereof and achieving the purpose of treating diseases.

(2) The preparation method comprises the following steps:

Cationic lipid DOTAP (Ai Weita (Shanghai) pharmaceutical technologies Co., ltd.), neutral lipid DSPC (Nippon FINE CHEMICAL, CO., LTD.) and cholesterol (Ai Weita (Shanghai) pharmaceutical technologies Co., ltd.) and PEG lipid DMG-PEG2k (SINOPEG) were formulated into lipid ethanol solutions at a molar ratio of 50/10/38.5/1.5, the antisense oligonucleotide drug was diluted with 100mM acetate buffer (pH 4.0), and the aqueous and alcoholic phases were mixed at a volume ratio of 1:3 using a microfluidic device, and the resulting LNP solution had an antisense oligonucleotide drug concentration of 0.2mg/mL and an N/P ratio of 6:1. After encapsulation on microfluidic devices, ethanol was removed by ultrafiltration with Tris buffer, tween 80 was added to give an excipient concentration of 0.1% (w/v) in the dispersion solution to prepare an LNP solution.

This example further measures the mass of CT102-LNP in the pharmaceutical composition after aerosolization.

(3) And (3) atomizing: CT102-LNP prepared in example 1, containing 0.1% T80 (w/v) as excipient, was used as model sample, and the sample was atomized using a vibrating screen atomizer (mist VAPO) at RT conditions to collect aerosol droplets by condensation in a sample tube.

(4) Physical and chemical property measurement:

determination of particle size and PDI:

Particle size and PDI of CT102-LNP samples before and after atomization were detected by Dynamic Light Scattering (DLS) in a nanometer laser particle sizer (manufacturer: MALVERN; model: ZSU 3305), and the detection parameters were set as shown in Table 1 below:

Table 1 parameters of detection

(5) Encapsulation efficiency measurement:

The sample was diluted to 2.8. Mu.g/mL, one portion was mixed with an equal volume (50. Mu.L) of Triton X-100, and the demulsified well for determining total antisense nucleic acid concentration, and the other portion was mixed with an equal volume (50. Mu.L) of TE (Tris-EDTA) for determining free unencapsulated antisense nucleic acid concentration. After incubating the samples respectively added with TE (Tris-EDTA) and Triton X-100 at 37 ℃ for a certain period of time, adding Ribogreen reagent diluted 200 times by 100 mu L, centrifuging to remove bubbles, and measuring fluorescence values by using a multifunctional enzyme-labeled instrument under the conditions of excitation wavelength 485, nm and emission wavelength 528 nm, and reading the plates, wherein the experimental results are shown in Table 2 and FIG. 1.

Table 2 CT102-LNP particle size, PDI, and encapsulation efficiency results before and after atomization.

As shown in the table, in the presence of 0.1% (w/v) Tween 80, particle size of DOTAP cationic lipid LNP is smaller than 200nm after atomization, and encapsulation efficiency is basically maintained unchanged, so that the DOTAP cationic lipid LNP has good stability.

Examples 2 to 3

In examples 2 and 3, the pharmaceutical compositions were confirmed to be versatile by varying the amounts of the lipids in the pharmaceutical compositions.

LNPs with different lipid levels were prepared and samples of LNPs were nebulized according to the method of example 1, and the experimental results are shown in table 3 and fig. 2 (wherein the left graph is example 2 and the right graph is example 3):

TABLE 3 LNP component proportions, particle size before and after atomization, PDI and encapsulation efficiency results

As can be seen from Table 3, DOTAP was used in the range of 40-60mol%, the particle size of LNP was less than 200 nm after atomization, the encapsulation efficiency was maintained, and the applicability of the cationic lipid was high.

Examples 4 to 5

This example 4, 5 is identical to the preparation of example 1, with the only difference that the N/P ratio is different from that of the pharmaceutical composition prepared in example 1.

LNP solutions were prepared according to the same formulation ratio and method as in example 1, and LNP samples were atomized, and experimental results are shown in table 4.

Table 4 LNP component ratio, particle size before and after atomization, PDI and encapsulation efficiency results

As can be seen from the above table, when the DOTAP prescription N/P ratio is 3:1 and 15:1, the particle size of LNP after atomization is less than 200nm, the encapsulation efficiency is maintained unchanged, and the cationic lipid in the pharmaceutical composition has stable atomization effect in the dosage range of 40% -60% and the N/P ratio in the range of 3:1-15:1 by combining the results of examples 1 and 4-5.

Examples 6 to 7

The preparation method of the present examples 6 and 7 is the same as that of the example 1, except that the pharmaceutical composition of the present example contains different amounts of PEG lipid and cholesterol.

LNP solution was prepared in the same manner as in example 1 and LNP samples were atomized, and experimental results are shown in table 5.

Table 5 LNP Components, particle size before and after atomization, PDI and encapsulation efficiency results

It can be seen from the above table that the pharmaceutical composition has a stable nebulization effect in combination with the results of example 1, having a PEG lipid content in the range of 1.5-5 mol%.

Examples 8 to 12

The pharmaceutical compositions of examples 8-12 were prepared in the same manner as in example 1, except that the excipient content in the dispersion was varied, and the particle size, PDI and encapsulation efficiency of the prepared pharmaceutical compositions were as shown in Table 6.

Comparative example 1

This comparative example was identical to the preparation of the pharmaceutical composition of example 1, except that no excipient was added to the sample of this comparative example, and the sample was atomized according to the method of example 1, and the measurement results are shown in Table 6.

TABLE 6 comparative example 1 LNP particle size before and after atomization, PDI and encapsulation efficiency results

As is clear from the above table, the samples prepared in the above comparative examples were not added with excipient, and the particle size and PDI after atomization were significantly increased, and in examples 1 and 8 to 12, the particle size of LNP after addition of excipient 0.1% (w/v) Tween 80, 0.5% (w/v) poloxamer 188 or 0.1% (w/v) Triton was reduced, PDI was less than 0.3, and the combination of excipients 0.05% (w/v) T80&0.05% (w/v) P188 had an atomization stabilizing effect almost identical to that of 0.1% (w/v) T80, and the stabilizing effect of Tween 80 was better in the overall.

Stability test:

The CT102-LNP solution without tween 80 prepared in comparative example 1 was left for 6 months at 2-8 ℃ and the particle size was significantly increased during 1 month, the particle size was not significantly changed during 1-6 months, the results are shown in table 8, and the CT102-LNP sample prepared in example 1 was similarly treated in the same manner to determine the stability, and the results are shown in table 7 and fig. 3, the particle size and the encapsulation efficiency were not significantly changed, the stability was good, specifically: at the temperature of 2-8 ℃, the particle size is increased within 3nm from 0 days to 6 months, PDI is not obviously changed, the encapsulation efficiency is reduced within 3%, and the change is not obvious. Example 1 sample stability, the addition of 0.1% (w/v) T80 to the dispersion was beneficial to prevent LNP aggregation and stabilize particle size.

TABLE 7 example 1 stability results of T80 added samples at 2-8deg.C

TABLE 8 results of stability at 2-8deg.C for the sample of comparative example 1 without T80 addition

Comparative examples 2 to 5

This comparative example 2-5 was prepared in the same manner as in example 1, except that the non-ionizable cationic lipid DOTAP was replaced with an ionizable cationic lipid (YK-009, MC3, SM-102, ALC-0315). And the sample was atomized as in example 1, and the experimental results are shown in table 9.

TABLE 9 sample Components, particle size before and after atomization, PDI, and encapsulation efficiency results for this comparative example

As can be seen from the table, in comparative example 1, the encapsulation efficiency of the ionizable cationic lipid LNP is significantly reduced after atomization in the presence of tween 80, whereas the encapsulation efficiency of DOTAP cationic lipid LNP in example 1 remains substantially unchanged after atomization, and the permanent cationic lipid has better stability.

Comparative examples 6 to 8

This comparative example was prepared as in example 1, except that LNP samples were prepared using different cationic lipid DOTMA or neutral lipid DPPC, DSPC, and the samples were atomized as in example 1, and the experimental results are shown in Table 10.

TABLE 10 LNP sample composition, particle size before and after atomization, PDI, and encapsulation efficiency results for this comparative example

As can be seen from the above table, the encapsulation efficiency after LNP atomization was reduced by 20% by replacing neutral lipid with DPPC in comparative example 6, compared to example 1; in comparative example 7, when the cationic lipid is replaced by DOTMA and the neutral lipid is DSPC, the particle size and PDI are obviously increased after atomization, and the encapsulation efficiency is reduced by less than 10%; comparative example 8 neutral lipid was changed to DPPC, and the encapsulation efficiency was reduced by 30% after LNP atomization; in comparative example 1, DOTAP is cationic lipid, LNP of the formulation of DSPC is neutral lipid, particle diameter is less than 200nm after atomization, encapsulation efficiency is basically maintained unchanged, and the formulation of example 1 has better atomization stability.

Example 13

In vitro cell transfection experiments

Sample to be tested: different concentrations example 1 model samples were nebulized in solution.

Cell culture: and taking the subcultured 95D, NCI-H4460, GLC-82 and A549 cell strains, diluting the logarithm growing cells into cell suspension with the concentration of 2-2.5X10 ⁴/ml by using 10% fetal calf serum DMEM culture solution (100 kU/L for supplementing penicillin and streptomycin respectively), adding 3000-4000 cells per well into a 96-well cell culture plate, culturing to be adhered to the wall of the cells per well by 100 mu L.

Transfection: culturing in a 96-well plate until the cell abundance reaches 40-60%, absorbing culture solution in each well, transfecting CT102-LNP in serum-free and antibiotic-free culture solution, adding test samples atomized at different concentrations into each well according to the design amount, simultaneously setting a positive control (cisplatin) group and a cell control group, wherein the adding volume of each group of solution is 100 mu L, and setting 3 parallel wells. After 6h of transfection, the culture medium from each well was aspirated, 100. Mu.L of normal medium containing serum was added to each well, and the culture was continued in a 5% CO ₂ incubator at 37℃for 72h.

Cell viability detection: the MTT assay was used to examine the growth inhibition of lung cancer cells by the test samples, and the results are shown in Table 11.

TABLE 11 determination of proliferation inhibition of lung cancer cells by model samples of example 1

As can be seen from Table 11, the atomized CT102-LNP has a strong proliferation inhibition effect on four lung cancer cells, and the inhibition effect is equivalent to that of positive control cisplatin at 0.2 mg/mL.

Example 14

Animal in vivo test

(1) Establishing a mouse lung cancer transplantation tumor model: and starting tumor grafting when the NCI-H446 human small lung cancer cells grow to 70-80% density in the culture flask. The subcultured tumor cells are digested into suspension under aseptic condition, the suspension is resuspended into suspension after washing by sodium chloride injection, 1mL of the cell suspension is sucked by a syringe, 200 mu L/of NCI-H446 lung cancer cell suspension is injected subcutaneously on the right chest of a C57BL/6 mouse by the axillary line, and the mice are routinely fed into an SPF-class mouse feeding chamber after tumor inoculation is completed.

(2) Experimental grouping and treatment scheme: after successful modeling, the mice were randomly divided into 3 groups, each group being a blank model control group (physiological saline), a CT102-LNP treated group prepared in example 1, and a positive drug (cisplatin) treated group, each group being 11 mice. Mice were administered by aerosol inhalation for 15 minutes with a total of 28 days at 24 hour intervals. The mice of each group were dosed by tail vein injection, and the body weights of the mice were measured and recorded on days 7, 14, 21 and 28 of dosing. After the experimental period was completed, the remaining mice were sacrificed, tumor tissues were peeled off, tumor mass was weighed, and tumor inhibition rate was calculated, and the results are shown in table 12.

Table 12 weight change after mice were dosed

As can be seen from table 12, the weight of the mice in the blank group tended to rise, and the weight was increased by 3.0% on day 28 over that on day 7; the mice in each dosing group had a reduced weight, the average weight of the mice on day 28 was reduced by 5.3% compared to the placebo group, and the CT102-LNP treated group was reduced by 5.5% (see fig. 4). This shows that the aerosol inhalation antisense oligonucleotide can effectively inhibit the growth of lung cancer tumor, and the inhibition effect is equivalent to that of positive control cisplatin.

TABLE 13 quality of transplanted tumors after mice were dosed

As shown in Table 13, the average tumor weight of the transplanted tumor of the blank control group is 0.150+/-0.016 g, the positive control cisplatin group is 0.052+/-0.014 g, the CT102-LNP treatment group is 0.057+/-0.013 g (see figure 5), compared with the blank model control group, the tumor inhibition rate of the positive control cisplatin group is 65.34%, the tumor inhibition rate of the CT102-LNP treatment group is 62.00%, the tumor weight of the administration group is obviously reduced, the CT102-LNP treatment group is equivalent to the positive control cisplatin, and the atomized inhalation CT102-LNP can inhibit the growth of human NCI-H446 small cell lung cancer tumor.

The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations are not described further in this disclosure in order to avoid unnecessary repetition.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Claims

1. A pharmaceutical composition comprising a lipid, a nucleic acid encapsulated within the lipid, and a dispersion solution with a water-soluble excipient added thereto;

2. The pharmaceutical composition of claim 1, wherein the non-ionizable cationic lipid to said nucleic acid usage ratio is expressed as an N/P ratio, said N/P ratio being (1:1) - (30:1).

3. The pharmaceutical composition of claim 2, wherein the N/P ratio is (3:1) - (10:1).

4. The pharmaceutical composition of claim 1, wherein the nucleic acid is any one selected from the group consisting of mRNA, siRNA, DNA, miRNA, antisense oligonucleotides, and non-coding RNAs.

5. The pharmaceutical composition of claim 4, wherein the nucleic acid is any one of the group consisting of siRNA and antisense oligonucleotide.

6. The pharmaceutical composition of claim 4, wherein the nucleic acid is an antisense oligonucleotide.

7. The pharmaceutical composition of claim 4, wherein the antisense oligonucleotide is CT102, the nucleotide sequence of CT102 is: 5'-TCCTCCGGAGCCAGACTTCA-3'.

8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has a particle size of 50-300nm and a polydispersity index of not greater than 0.3.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition has a particle size of 50-200nm.

10. The pharmaceutical composition of claim 1, wherein the dispersion solution is at least one selected from Tris buffer, DPBS buffer, HEPES buffer, PBS buffer, citrate buffer, phosphate buffer, carbonate buffer, and acetate buffer.

11. The pharmaceutical composition of claim 1, wherein the water-soluble excipient comprises at least one of sucrose, trehalose, polyvinyl alcohol, tween 80, tween 20, poloxamer 188, triton, sodium dodecyl sulfate, sodium chloride, potassium chloride, and glycine.

12. The pharmaceutical composition of claim 11, wherein the water-soluble excipient is at least one of tween 80, poloxamer 188, and Triton;

If the excipient is Tween 80, the excipient is used at a concentration of 0.01-0.5% (w/v);

if the excipient is poloxamer 188, the excipient is used at a concentration of 0.05-1% (w/v);

if the excipient is Triton, the excipient is used at a concentration of 0.01-0.5% (w/v).

13. A method of preparing a pharmaceutical composition according to any one of claims 1 to 12, characterized in that the method comprises the steps of:

14. The method according to claim 13, wherein the organic solvent is any one selected from ethanol, acetone and N, N-dimethylformamide;

the first dispersion system solution is at least one selected from citrate buffer and acetate buffer;

15. Use of a pharmaceutical composition according to any one of claims 1-12 and/or a pharmaceutical composition prepared by a method according to any one of claims 13-14 for the preparation of an anti-tumor drug, characterized in that the anti-tumor drug is administered by aerosol inhalation;