Drug delivery system based on long-chain fatty acid nano-emulsion as well as preparation method and application of drug delivery system

Abstract

The invention discloses a drug delivery system based on a long-chain fatty acid nano-emulsion and a preparation method and application thereof, the system is composed of long-chain fatty acid, a surfactant, lipid and a hydrophobic drug, and the stable nano-emulsion is prepared through a solvent injection method; experiments show that the nano-emulsion can significantly improve the water solubility and bioavailability of the hydrophobic drug taprazopalide, and meanwhile, the anti-tumor effect of the taprazopalide is significantly improved by utilizing the biocompatibility of the fatty acid nano-emulsion, and the nano-emulsion does not have significant toxic or side effects of the drug.

Description

Technical Field

[0001] This invention relates to a drug delivery system, particularly to a drug delivery system based on long-chain fatty acid nanoemulsions, and also to a method for preparing the above-mentioned drug delivery system and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide. Traditional treatments include surgical resection, radiotherapy, chemotherapy, and targeted therapy. However, chemotherapy drugs generally suffer from poor water solubility and low bioavailability, and are prone to causing systemic toxic side effects (such as bone marrow suppression and cardiotoxicity). Furthermore, the complexity of the tumor microenvironment makes it difficult for drugs to effectively accumulate at the lesion site, further limiting efficacy. Therefore, developing efficient and low-toxicity delivery systems has become a key need in breast cancer treatment.

[0003] In existing technologies, PARP (polyadenylated diphosphate ribose polymerase) inhibitors can selectively kill...

[0004] Breast cancer cells with BRCA gene mutations. This mechanism utilizes defects in tumor cell DNA repair, blocking the PARP-mediated single-strand repair pathway, leading to the accumulation of DNA double-strand breaks and ultimately inducing apoptosis. Although PARP inhibitors (such as tapolaparib) have shown potential in clinical practice, their hydrophobicity results in low oral bioavailability, and their systemic distribution can easily cause side effects such as hematologic toxicity. Therefore, there is an urgent need for novel delivery systems to improve drug performance.

[0005] Naturally derived lipids (such as phospholipids and fatty acids) are widely used in nanomedicine delivery systems due to their high biocompatibility and biodegradability. For example, distearylphosphatidylcholine (DSPC) can form a stable lipid bilayer, enhancing drug loading capacity. Lipid metabolism, especially the synthesis of fatty acids (FAs), is an important cellular process that converts nutrients into metabolic intermediates for membrane biosynthesis, energy storage, and signal molecule generation. Long-chain fatty acids are more easily integrated into biological membranes and participate in signal transduction, thus becoming targets for metabolic therapy. Current research suggests preparing oil-in-water nanoemulsions by neutralizing fatty acids (such as oleic acid and palmitic acid) with NaOH / KOH to form soap-like surfactants, combining them with water-miscible liquids such as glycerol, and using high-shear mixing and single-stage high-pressure homogenization. The neutralized fatty acids spontaneously form an interfacial film, and glycerol reduces interfacial tension, achieving thermodynamic stability without the need for traditional surfactants. Other methods utilize specific N-acyl derivatives containing amino acid salts as emulsifiers (accounting for more than 50% of the aqueous phase), combined with C8-C18 fatty acids (accounting for 0.5-10% of the emulsion), to prepare nanoemulsions containing petrolatum or triglycerides through high-pressure homogenization (above 5000 psi). The electrostatic repulsion and steric hindrance of amino acid surfactants, along with the reduction of interfacial energy by fatty acids, stabilize the oil droplets. However, the preparation process of lipid formation and delivery systems is complex and costly: many existing technologies rely on high-pressure homogenization and high-shear mixing equipment, which not only has high investment costs but also high energy consumption; some preparation methods have stringent requirements for operating conditions (such as precise control of temperature, pH value, and the proportion of each component), increasing the technical difficulty and quality control costs in the production process. In addition, there are significant deficiencies in drug loading capacity and drug delivery efficiency: the drug loading capacity of existing fatty acid nanoemulsions often cannot meet the needs of clinical treatment. Due to the limited solubility of drugs in fatty acids, the amount of drug loaded in nanoemulsions is relatively small, which may require increasing the dosage or frequency of administration, causing inconvenience to patients. At the same time, the release rate and release site of drugs from fatty acid nanoemulsions are difficult to control precisely, and the release may be too fast or too slow, resulting in the drug loading system not being effectively absorbed by the target cells and failing to meet the treatment needs in a timely manner. Summary of the Invention

[0006] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention provides a drug delivery system based on long-chain fatty acid nanoemulsions to improve the utilization of hydrophobic drugs, and also provides a preparation method and application of the above drug delivery system.

[0007] Technical solution: The drug delivery system based on long-chain fatty acid nanoemulsion of the present invention is composed of long-chain fatty acids, surfactants, lipids and hydrophobic drugs. The hydrophobic regions of long-chain fatty acids and lipids combine to form a nanoscale hydrophobic core, which is internally coated with a hydrophobic drug. The hydrophobic end of the surfactant is embedded in the outer layer of the hydrophobic core, and the hydrophilic end faces the aqueous phase to form an interface film, which coats the hydrophobic core, forming a hierarchical structure of hydrophobic drug, hydrophobic core and surfactant interface film coating.

[0008] The delivery system structure is as follows: In an oil-in-water nanoemulsion structure, the hydrophobic alkyl chains of long-chain fatty acids and lipids (such as the long-chain saturated fatty acid chains of DSPC) aggregate through hydrophobic interactions and van der Waals forces to form a spherical or near-spherical nanoscale hydrophobic core, with a disordered or semi-ordered liquid / semi-solid "network" matrix inside. The hydrophobic drug, due to its strong hydrophobicity, dissolves or disperses inside the hydrophobic core through hydrophobic interactions and van der Waals forces, becoming a component of the core. The hydrophobic end of the surfactant is embedded in the outer layer of the hydrophobic core and combines with the hydrophobic regions of the long-chain fatty acids and lipids through hydrophobic bonds. The hydrophilic end faces the aqueous phase to form an interfacial film, which encapsulates the entire hydrophobic core, making the core stably suspended in the aqueous phase, forming a hierarchical structure in which the hydrophobic drug is encapsulated by the hydrophobic core and then coated by the surfactant interfacial film.

[0009] The long-chain fatty acid is oleic acid, linoleic acid, stearic acid, squalene, or squalane; the surfactant is one of Tween-80, sophorolipid, poloxamer 188, or polyethylene glycol-15-hydroxystearate (Solutol HS15); and the lipid is one of distearylphosphatidylcholine (DSPC), hydrogenated soybean lecithin (HSPC), dipalmitoylphosphatidylcholine (DPPC), or dioleoylphosphatidylethanolamine (DOPE).

[0010] The hydrophobic drug is one of the following PARP inhibitors: tapolarib (TZL), paclitaxel, docetaxel, etoposide, and irinotecan.

[0011] The molar ratio of lipids, surfactants, long-chain fatty acids, and hydrophobic drugs is 30–40:10–20:20–60:5–20. Lipid molecules: distearylphosphatidylcholine (DSPC), accounting for 30%–40%, is used to form a stable bilayer and improve stability; surfactant: Tween-80, accounting for 10%–20%, improves emulsion stability and prolongs blood circulation time; fatty acids: oleic acid, linoleic acid, or stearic acid, accounting for 20%–60%, serve as a hydrophobic core to encapsulate the drug; loaded drug: tapolazoparib (TZL), accounting for 5%–20%.

[0012] The preparation method of the above-mentioned drug delivery system based on long-chain fatty acid nanoemulsions includes the following steps:

[0013] (1) Prepare a lipid ethanol solution by mixing distearate phosphatidylcholine DSPC, Tween-80 and long-chain fatty acids with anhydrous ethanol.

[0014] (2) Prepare a hydrophobic drug solution using dimethyl sulfoxide (DMSO); mix the lipid ethanol solution with the hydrophobic drug solution to prepare a lipid drug mixture solution, inject it into phosphate-buffered saline (PBS) under vortex conditions, and after ultrasonic disruption, obtain a drug delivery system based on long-chain fatty acid nanoemulsion.

[0015] In step (1), when the long-chain fatty acid is oleic acid, the mass ratio of oleic acid, DSPC and Tween-80 is (200-150):(60-30):(60-30), preferably 150:(60-30):(60-30), and more preferably 150:(60-30):(30-15);

[0016] When the long-chain fatty acid is linoleic acid, the mass ratio of linoleic acid:DSPC:Tween-80 is (300~100):(30~10):(60~30), preferably 150:(60~30):(60~30), and more preferably 150:(60~30):(30~15);

[0017] When the long-chain fatty acid is stearic acid, the mass ratio of stearic acid:DSPC:Tween-80 is (175-50):(50-10):(60-30), preferably 150:(50-10):(60-30), and more preferably 150:(50-10):(30-15).

[0018] When the long-chain fatty acid is squalene, the mass ratio of squalene:DSPC:Tween-80 is (150-50):(50-20):(30-10), preferably 100:(50-20):(30-10), and more preferably 100:(50-20):(20-10).

[0019] When the long-chain fatty acid is squalane, the mass ratio of squalane:DSPC:Tween-80 is (175-50):(50-20):(30-10), preferably 100:(50-20):(30-10), and more preferably 100:(50-20):(20-10).

[0020] In step (2), when the long-chain fatty acid is oleic acid, the mass ratio of oleic acid to the hydrophobic drug is (200-150):(60-30), preferably 150:(60-30), and more preferably 150:(30-15).

[0021] When the long-chain fatty acid is linoleic acid, the mass ratio of linoleic acid to the hydrophobic drug is (300-100):(30-10), preferably 150:(30-10), and more preferably 150:(15-10).

[0022] When the long-chain fatty acid is stearic acid, the mass ratio of stearic acid to the hydrophobic drug is (175-50):(50-10), preferably 150:(50-10), and more preferably 150:(30-10).

[0023] When the long-chain fatty acid is squalene, the mass ratio of squalene to the hydrophobic drug is (150-50):(50-20), preferably 100:(50-20), and more preferably 100:(50-30).

[0024] When the long-chain fatty acid is squalane, the mass ratio of squalane to the hydrophobic drug is (175-50):(50-20), preferably 100:(50-20), and more preferably 100:(50-30).

[0025] In step (2), the vortex conditions are: power 20W, high-speed vortex 60s; the ultrasonic conditions are: power 150W, 5% setting, on for 3s and off for 2s, ultrasonic for 60s.

[0026] The drug delivery system based on long-chain fatty acid nanoemulsions described above can also be used in the preparation of antitumor drugs.

[0027] The tumors mentioned include breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, and non-small cell lung cancer. The drugs are injectable or topical formulations. The drug nanoemulsion enhances drug accumulation in MCF-7 cells, improving drug bioavailability and showing potential application in breast cancer treatment. Specifically, drug delivery systems containing paclitaxel are used to treat breast cancer, non-small cell lung cancer, ovarian cancer, and pancreatic cancer. Docetaxel-containing drug delivery systems are used to treat breast cancer, prostate cancer, gastric cancer, and non-small cell lung cancer. Etoposide-containing drug delivery systems are used to treat small cell lung cancer, testicular cancer, ovarian cancer, and lymphoma. Irinotecan-containing drug delivery systems are used to treat colorectal cancer, gastric cancer, and pancreatic cancer.

[0028] Invention Principle: The drug delivery system based on long-chain fatty acid nanoemulsion of the present invention uses a combination of lipids, surfactants and fatty acids to enhance the stability of the nanodrug delivery system, ensuring the successful preparation of the nanodrug delivery system and its therapeutic effect in vivo, thereby enabling the nanodrug delivery system to have ideal therapeutic effects and achieve high loading capacity and anti-tumor effect for the hydrophobic drug taporabide.

[0029] Taking stearic acid nanoemulsion as a specific example: From the perspective of structural formation, the two 18-carbon saturated stearoyl chains of DSPC and the 18-carbon alkyl chain of stearic acid are tightly intertwined through hydrophobic interactions and van der Waals forces due to their matching chain lengths, forming a hydrophobic core matrix that combines rigidity and stability. As a hydrophobic drug, tapolaparib not only embeds itself into this core through hydrophobic interactions, but also forms weak hydrogen bonds with the polar groups of the phospholipid head of DSPC, further enhancing its binding with the core. The hydrophobic end of Tween-80 (the long-chain alkyl group of oleic acid linked by ester bonds of dehydrated sorbitol) is precisely embedded in the outer layer of the hydrophobic core, anchored to the hydrophobic regions of DSPC and stearic acid through hydrophobic bonding, while the hydrophilic end (polyoxyethylene chain) extends into the aqueous phase to form a monolayer interfacial film, which reduces the oil-water interfacial tension and prevents core aggregation through steric hindrance. In this structure, the phospholipid bilayer properties of DSPC and the long-chain structure of stearic acid form a core framework of "rigid-flexible balance". The interface modification of Tween-80 achieves a stable connection between the hydrophobic core and the aqueous phase. It is a unique system formed by the complementary structure of each component, rather than a simple combination of single lipids or surfactants in the existing technology.

[0030] From the perspective of improved efficacy, this specific structure brings multi-dimensional synergistic effects: In terms of physicochemical properties, the matching chain lengths of DSPC and stearic acid result in uniform core particle size, and the Tween-80 interfacial membrane resists serum protein adsorption; the particle size change rate is <1% after 30 days of storage at 4℃, demonstrating significantly better stability than single lipid or surfactant systems; regarding encapsulation efficiency, due to the dual interactions (hydrophobic interactions and hydrogen bonds) between tapolarib and the core, the encapsulation efficiency reaches over 70%; in vitro release exhibits sustained-release characteristics, with a cumulative release rate exceeding 80% over 24 hours, avoiding the burst release problem of free drug. This is attributed to the rigid matrix of the DSPC-stearic acid core regulating drug diffusion and the barrier effect of the Tween-80 interfacial membrane; uptake experiments show that the uptake effect of the nanoemulsion by cancer cells is 3 times that of free tapolarib; cancer cell toxicity experiments show that after 72 hours of incubation, the nanoemulsion significantly reduces the IC50 concentration of breast cancer cells. 50 It is 0.0918 μM, which is lower than that of free TZL (IC). 50 =3.22μM) decreased by 97%; apoptosis experiments showed that stearic acid nanoemulsion increased the apoptosis rate by 88% compared with free tprazoleparib. Furthermore, animal experiments confirmed that in live animals, the tumor growth inhibition rate of stearic acid nanoemulsion with the same dose of tprazoleparib (TGI = 52.76 ± 7.96%) was 16% higher than that of free tprazoleparib (TGI = 45.39 ± 9.12%).

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The drug delivery system of the present invention has strong stability (particle size change rate <1% after 30 days of storage at 4℃), exhibits sustained release characteristics in vitro, and has a cumulative release rate of over 80% in 24 hours. It not only avoids the problem of sudden release of free drugs, but also has excellent release effect on the weakly acidic tumor microenvironment; (2) The preparation method of the drug delivery system of the present invention is solvent injection method, which does not require complex equipment such as high pressure homogenizer. It simply involves rapidly injecting an organic phase (ethanol phase) containing DSPC, Tween-80, stearic acid, and tapolaparib into a PBS solution. An emulsion can be formed, and then it can be prepared by simple vortexing and ultrasound, avoiding the destruction of lipid molecular structure by traditional high-pressure processes; at the same time, the reaction conditions are mild, namely at room temperature 25-30℃ and normal pressure, without the need for high temperature or extreme pH, which can protect the chemical stability of tapazolidone (its pyrazolopyridine structure is easily degraded at high temperature), and solve the problem of reduced drug activity caused by high-temperature processes; (3) In anti-tumor drugs, the biocompatibility of fatty acid nanoemulsions can significantly improve the anti-tumor effect of tapazolidone; cancer cell toxicity experiments showed that after 72 hours of incubation, stearic acid nanoemulsions significantly reduced the IC50 of breast cancer cells. 50 It is 0.0918 μM, which is lower than that of free TZL (IC). 50=3.22μM) decreased by 97%; cell apoptosis experiments showed that the apoptosis rate of stearic acid nanoemulsion increased by 88% compared with that of free taprazoride. Furthermore, animal experiments confirmed that in live animals, the tumor growth inhibition rate of stearic acid nanoemulsion at the same dose of taprazoride (TGI = 52.76 ± 7.96%) was 16% higher than that of free taprazoride (TGI = 45.39 ± 9.12%); and blood biochemistry and HE tissue sections demonstrated that the nanoemulsion did not have significant drug toxicity. Attached Figure Description

[0032] Figure 1 Schematic diagram of nanoemulsion structure: A is oleic acid nanoemulsion; B is linoleic acid nanoemulsion; C and D are stearic acid nanoemulsions.

[0033] Figure 2 The diagram shows the particle size stability of nanoemulsions. A represents oleic acid nanoemulsion; B represents linoleic acid nanoemulsion; C represents stearic acid nanoemulsion; and D represents a composite particle size diagram of the three emulsions.

[0034] Figure 3 The diagram shows the drug loading and encapsulation efficiency of the nanoemulsion particles (A) and the standard curve for TZL drug detection (B).

[0035] Figure 4 In vitro drug release curves (release rate exceeds 85% at pH 6.5 after 24 hours). A, B, and C are in vitro release curves of oleic acid, linoleic acid, and stearic acid at 4℃ and different pH conditions, respectively; D, E, and F are in vitro release curves of oleic acid, linoleic acid, and stearic acid at 37℃ and different pH conditions, respectively.

[0036] Figure 5 The graph shows the cytotoxicity of nanoemulsions on MCF-7 cells. Figure A shows the results of the DiO dye experiment; Figure B shows the TZL autofluorescence results.

[0037] Figure 6 The graphs show the cytotoxicity of different nanoemulsions. A represents the results after 48 hours of incubation; B represents the results after 72 hours of incubation; and C represents the results of the blank stearic acid group after 48 hours and 72 hours of incubation.

[0038] Figure 7 The graphs are from flow cytometry. A represents the control group; B represents the free TZL group; C represents the oleic acid group; D represents the linoleic acid group; E represents the stearic acid group; F represents the difference in apoptosis rate between the control group and other groups; G represents the difference in apoptosis rate between free TZL and each emulsion group.

[0039] Figure 8 This is a diagram showing the results of RT-qPCR in vitro.

[0040] Figure 9The graphs show the results of mouse body weight, tumor volume, and tumor growth inhibition rate. A represents the change in mouse tumor volume; B represents the change in mouse body weight; C represents the tumor growth inhibition rate; and D represents the solid tumor removed after the mouse was sacrificed.

[0041] Figure 10 This is a graph showing the biochemical results of mouse blood. Figure 11 Staining diagram of H&E section. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be purchased through conventional means.

[0043] Example 1

[0044] The drug delivery system based on long-chain fatty acid nanoemulsion of the present invention uses distearylphosphatidylcholine (DSPC) as lipid molecule; Tween-80 as surfactant; oleic acid, linoleic acid or stearic acid as long-chain fatty acid; and tapolaparib (TZL) as loaded drug.

[0045] The preparation method includes the following steps:

[0046] (1) Weigh 3mg DSPC, 1mg Tween-80, and 1mg oleic acid / linoleic acid / stearic acid, and dissolve them in 120μL ethanol;

[0047] (2) Dissolve 0.5 mg of TZL in 20 μL of DMSO and inject it into the above organic phase; after vortex mixing, sonicate to obtain a drug delivery system based on long-chain fatty acid nanoemulsion, and name it according to the long-chain fatty acid used.

[0048] Example 2

[0049] The drug delivery system based on long-chain fatty acid nanoemulsion of the present invention uses distearate phosphatidylcholine (DSPC) as a lipid molecule; Tween-80 as a surfactant; squalene or squalane as a long-chain fatty acid; and tapolazoparib (TZL) as a loaded drug.

[0050] The preparation method includes the following steps:

[0051] (1) Weigh 3 mg of DSPC, 1 mg of Tween-80, and 1.6 mg of squalene / squalane, and dissolve them in 120 μL of ethanol;

[0052] (2) Dissolve 0.5 mg of TZL in 20 μL of DMSO and inject it into the above organic phase; after vortex mixing, sonicate to obtain a drug delivery system based on long-chain fatty acid nanoemulsion, and name it according to the long-chain fatty acid used.

[0053] The particle sizes of the prepared drug delivery system nanoemulsions were as follows: oleic acid nanoemulsion with a particle size of 118.13 nm; linoleic acid nanoemulsion with a particle size of 66.46 nm; stearic acid nanoemulsion with a particle size of 118.45 nm; squalene nanoemulsion with a particle size of 631.53 nm; and squalane nanoemulsion with a particle size of 638.79 nm.

[0054] Table 1. Particle size details of fatty acid nanoemulsions

[0055]

[0056] Comparative Example 1

[0057] A nanoemulsion, compared with Example 1, uses hexanoic acid, octanoic acid, arachidonic acid and arachidonic acid as the fatty acid phases in the nanoemulsion system, and the preparation method is the same as in the Example.

[0058] The particle sizes of the prepared nanoemulsions were as follows: hexanoic acid nanoemulsion with a particle size of 536.29 nm; n-octanoic acid nanoemulsion with a particle size of 479.45 nm; arachidonic acid nanoemulsion with a particle size of 230.62 nm; and arachidonic acid nanoemulsion with a particle size of 262.4 nm.

[0059] The particle size results show significant differences in the nanoemulsion particle size formed by different oils under this preparation method. The average particle size of the emulsions formed by arachidonic acid and arachidonic acid is relatively small, at 230.62 nm and 262.4 nm, respectively; the average particle size of squalane and squalene is relatively large, at 631.53 nm and 638.79 nm, respectively; the average particle size of octanoic acid and hexanoic acid is between the two, at 536.29 nm and 479.45 nm, respectively.

[0060] The main reasons for these differences are related to the structure of the oils themselves and their interactions with other components in the system. Unsaturated fatty acids such as arachidonic acid contain multiple double bonds, resulting in highly flexible molecular chains that entangle more tightly with the hydrophobic chains of DSPC. Under the mild conditions of solvent injection, the slow diffusion of the organic phase allows for more orderly self-assembly of the components, forming smaller core particles. In contrast, long-chain saturated hydrocarbons such as squalane and squalene have more rigid molecular chains and relatively weaker interactions with other hydrophobic components, making it difficult to form a compact structure during self-assembly, thus resulting in larger particle sizes. Octanoic acid and hexanoic acid are medium- to short-chain fatty acids, with chain lengths and polarities between unsaturated fatty acids and long-chain saturated hydrocarbons. Their compatibility with other components in the system is moderate, resulting in intermediate particle sizes. Furthermore, the solvent injection method, by precisely controlling the diffusion rate of the organic phase, can adaptively regulate the assembly process according to the structural characteristics of the oils, enabling the formation of stable nanoemulsions from different oils. This further demonstrates the adaptability of this method to different oils and the precision of its structural control.

[0061] Stability and encapsulation efficiency tests were conducted on the fabricated drug delivery system.

[0062] After storage at 4℃ for 30 days, the particle size of oleic acid nanoemulsion increased from 118.13 nm to 184.19 nm; the particle size of linoleic acid nanoemulsion increased from 66.46 nm to 135.24 nm; and the particle size of stearic acid nanoemulsion increased from 118.45 nm to 128.64 nm. The experimental results are as follows: Figure 2 As shown in the figure, stearic acid nanoemulsion exhibits the best stability among the three drug-loaded nanoemulsions prepared. The drug loading was determined using dialysis combined with Nanodrop. The encapsulation efficiency of oleic acid nanoemulsion was 68.77±4.51%; that of linoleic acid nanoemulsion was 72.69±2.26%; and that of stearic acid nanoemulsion was 71.42±3.17%. The experimental results are as follows: Figure 3 As shown.

[0063] After storage at 4°C for 30 days, the stearic acid nanoemulsion showed the smallest increase in particle size (from 118.45 nm to 128.64 nm), significantly better than the oleic acid (66.06 nm increase) and linoleic acid (68.78 nm increase) systems. This is because stearic acid is an 18-carbon saturated fatty acid with no double bonds in its molecular chain, exhibiting high rigidity and chemical stability. It forms a tightly wound, rigid core with the 18-carbon saturated chain of DSPC through hydrophobic interactions and van der Waals forces. The hydrophobic end of Tween-80 (the long oleic acid chain) can stably embed itself in the outer layer of this core, forming a dense interfacial film that effectively resists droplet aggregation. In contrast, the unsaturated chains of oleic acid (containing one double bond) and linoleic acid (containing two double bonds) are highly flexible and prone to molecular rearrangement during low-temperature storage, leading to a loose hydrophobic core, decreased interfacial film stability, and consequently, increased particle size.

[0064] Encapsulation efficiency results showed that the encapsulation efficiency of the linoleic acid (72.69±2.26%) and stearic acid (71.42±3.17%) systems was slightly higher than that of oleic acid (68.77±4.51%). The multiple double bond structure of linoleic acid enhances the binding of the drug to the hydrophobic core through π-π stacking and interaction with the aromatic ring of tprazole. Although stearic acid lacks double bonds, its saturated long chain has a higher compatibility with DSPC, and the compactness of the core structure reduces drug leakage, resulting in smaller encapsulation efficiency fluctuations, demonstrating the positive regulation of encapsulation effect by structural stability. Oleic acid, due to its moderate chain flexibility caused by single and double bonds, has weaker interaction with the drug than linoleic acid, and its core stability is lower than that of stearic acid, hence its lowest encapsulation efficiency and greater fluctuations. This difference indicates that stearic acid, through the rigidity of its saturated chain and the synergistic effect formed by DSPC and Tween-80, significantly improves system stability while ensuring a high encapsulation efficiency. This balanced effect cannot be achieved through single fatty acids or conventional processes.

[0065] In vitro drug release experiments were conducted on the prepared drug delivery system:

[0066] PBS solutions with pH values ​​of 5.0, 6.5, and 7.4 were prepared as in vitro release media. Each emulsion was placed in a dialysis membrane (3500 kDa), sealed with a special clamp, and then immersed in PBS solutions of different pH values. Samples were taken at 0.25, 0.75, 2, 4, 6, 12, and 24 hours. The TZL content in the samples at different time points was detected using a Nanodrop ultraviolet spectrophotometer to calculate the cumulative drug release rate. The calculation formula is as follows, where V is the volume of the release medium, and C... i Let be the concentration of TZL during the i-th sample collection, and M be the total content of TZL. In vitro drug release curves for each drug-loaded nanoemulsion are obtained by plotting time on the x-axis and cumulative release rate on the y-axis.

[0067] Table 2. Release rates of various nanoemulsions at different temperatures and pH values

[0068]

[0069] Experimental results are as follows Figure 4 As shown in Table 2, the nanoemulsions can achieve sustained drug release. Comparing the release results at the same pH, the difference in drug release rate is very small regardless of the temperature (4℃ or 37℃), indicating that temperature has little effect on the drug release rate, indirectly proving that the prepared nanoemulsions have good thermal stability. Comparing the drug release results at different pH values, the release rate of the three emulsions at pH 5.0 is significantly lower than that at pH 6.5 and 7.4. This may be because the overly acidic environment has a certain inhibitory effect on the release behavior of the nanoemulsions. In addition, the release rate of all three emulsions is the highest at pH 6.5, reaching 85%, indicating that a weakly acidic environment is more conducive to drug release, which is very beneficial for the release of nanoemulsions in the weakly acidic tumor microenvironment.

[0070]

[0071] Cellular uptake experiments were conducted on the prepared drug delivery system:

[0072] MCF-7 breast cancer cells were used as an in vitro cell model system for studying oleic acid nanoemulsions, linoleic acid nanoemulsions, stearic acid nanoemulsions, and free TZL. MCF-7 cells were cultured at 5 × 10⁶ cells per well. 3Cells were seeded into 96-well plates and incubated overnight. Free TZL and each drug-loaded emulsion were stained with DiO2 dye and then added to the wells to achieve a drug concentration of 30 μM. After incubation in the dark for 48 h, the culture medium was aspirated, and the cells were washed twice with cold PBS. Then, 100 μL of PBS was added, and fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Simultaneously, cell uptake was assessed using the fluorescence properties of TZL. The same number of cells were seeded and incubated overnight. Free TZL and each drug-loaded emulsion were added to achieve a drug concentration of 30 μM in the wells, and the cells were incubated in the dark for 24 h, 48 h, and 72 h, and treated in the same manner. Fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The relative fluorescence intensity was calculated using the following formula, where I... C The mean I represents the fluorescence intensity of the cells within the pores. C For I C The average value of the group, I M+C The fluorescence intensity of each drug-loaded emulsion and cell pore was increased.

[0073] Experimental results are as follows Figure 5 As shown in Figure 1, the DiO dye uptake results (A) indicate that the stearic acid group showed the highest uptake rate compared to free TZL, while the linoleic acid group showed the least satisfactory results, possibly due to the poor stability of linoleic acid. The TZL autofluorescence detection results (B) show that the oleic acid group performed better than free TZL alone, while the linoleic acid group exhibited instability with increasing incubation time. The stearic acid group showed the best uptake performance, which was positively correlated with incubation time.

[0074]

[0075] Cytotoxicity experiments were conducted on the prepared drug delivery system:

[0076] MCF-7 cells were spaced at 5 × 10⁶ cells per well. 3 Cells were seeded into 96-well plates and incubated overnight to allow cell adhesion. The culture medium was then aspirated, and fresh medium containing oleic acid nanoemulsion, linoleic acid nanoemulsion, stearic acid nanoemulsion, and free TZL was added to each well to achieve final drug concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 7.5 μM, 10 μM, 50 μM, 100 μM, and 200 μM. The 96-well plates were incubated for 48 h and 72 h. After 48 h and 72 h of incubation, the culture medium was discarded, and 100 μL of the basal medium and CCK-8 reagent were mixed at a 10:1 ratio and added to the 96-well plates. After incubation for 2 h, the absorbance at 450 nm was measured using a microplate reader, and the IC50 was calculated based on the fluorescence intensity. 50 Numerical value.

[0077] Experimental results are as follows Figure 6 As shown, the IC50 values ​​of oleic acid nanoemulsion, linoleic acid nanoemulsion, stearic acid nanoemulsion, and free TZL in MCF-7 cells after 48 h are... 50 The concentrations were 31.35 μM, 35.29 μM, 43.50 μM, and 152 μM; IC50 for 72 hours. 50 The concentrations were 0.1930 μM, 0.9096 μM, 0.0918 μM, and 3.22 μM. Based on the comprehensive experimental results, the stearic acid nanoemulsion showed the best cytotoxic effect. Considering the effect of stearic acid itself on cancer cells, the cytotoxicity of the blank stearic acid emulsion against MCF-7 cells was evaluated. The results showed that after 48 h and 72 h of incubation, the IC50 of SANE-E against the MCF-7 cell line was [not specified in the original text]. 50 The values ​​were 213.9 and 85.07 μM, respectively. Compared with the results of stearic acid nanoemulsion, this demonstrates that stearic acid can not only serve as an excellent carrier for loading drugs, but also has certain toxic effects, indicating that stearic acid has great potential for application in cancer treatment.

[0078] Apoptosis experiments were conducted on the prepared drug delivery system:

[0079] MCF-7 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates and incubated overnight in complete growth medium at 37°C and 5% CO2. The medium was then aspirated and replaced with fresh medium containing 30 μM oleic acid nanoemulsion, linoleic acid nanoemulsion, stearic acid nanoemulsion, and free TZL. A control group with standard medium was added. After 24 hours of incubation, the medium was removed, cells were washed twice with PBS, digested with trypsin without EDTA, centrifuged, and the cells were collected. The cells were washed three times with cold PBS and resuspended in 500 μL Binding Buffer to form a single-cell suspension. 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide working solution were added, mixed, and the cells were incubated at room temperature in the dark for 5–10 minutes. Finally, the cells were analyzed by flow cytometry.

[0080] Table 3. Correlation Comparison and Increase Rate of Each Group

[0081]

[0082] Experimental results are as follows Figure 7As shown in Table 3, oleic acid nanoemulsion, linoleic acid nanoemulsion, and stearic acid nanoemulsion showed increases of 81.55%, 44.58%, and 88.12% respectively compared to the free TZL group, and increases of 195.36%, 135.20%, and 206.04% respectively compared to the control group. The results demonstrate that, compared to free TZL and the control group, the percentage of apoptotic cells was significantly increased when cells were treated with a series of nanoemulsions, and stearic acid nanoemulsion showed the best apoptosis-inducing effect. Combined with the previously demonstrated cell uptake and toxicity effects of stearic acid nanoemulsion, from the perspective of tumor cell interaction mechanism analysis, the saturated long chain of stearic acid is more easily recognized by lipid transport proteins (such as CD36) highly expressed on the surface of tumor cells, and enters the cells efficiently through receptor-mediated endocytosis. At the same time, its stable interfacial membrane (Tween-80 hydrophilic end) can reduce non-specific binding with normal cells, thus focusing on inducing tumor cell apoptosis. The single and double bond structure of oleic acid enhances the fusion ability of nanoemulsions with cell membranes (unsaturated chains are more likely to insert into the phospholipid bilayer), promoting drug entry into cells. However, the slightly lower system stability leads to premature release of some drugs, and the increase in apoptosis rate (81.55%) is slightly lower than that of stearic acid. Linoleic acid, with its multiple double bonds, is easily oxidized, which not only damages the core structure leading to drug leakage but may also affect the targeting of the carrier due to oxidation products. Therefore, it has the lowest increase in apoptosis rate (44.58%).

[0083] RT-qPCR sequence validation of the developed drug delivery system

[0084] To validate RNA-seq data, RT-qPCR analysis was performed using RNA samples from post-drug-administered cells. RNA concentration was determined using NanoDrop, and cDNA was synthesized using 1 μg of RNA via HiScript III All-in-one RT SuperMix reverse transcriptase. Gene expression was detected using a SYBR Green ultramix real-time quantitative PCR system; gene primers (sequences shown in Table 4) were ordered from KGI Biotechnology. Values ​​were normalized to the internal control gene GAPDH, 2 -ΔΔCt The method was used to assess relative gene expression, and the experimental results are as follows: Figure 8 As shown.

[0085] Table 4. Primer Sequences

[0086]

[0087]

[0088] Different fatty acid carriers for tapazolidone delivery systems exhibit varying effects on gene expression regulation. Stearic acid nanoemulsions, due to their high stability, result in higher intracellular drug concentrations in tumor cells, significantly inhibiting transcriptional changes in the cell cycle regulator CCND-1, triggering a stronger repair response in the DNA crosslinking damage repair gene FANCI, and significantly inhibiting the expression of homologous recombination repair genes BRCA1 and RAD51, while also significantly increasing the expression of the drug-sensitizing gene SLFN11. Linoleic acid nanoemulsions activate the base excision repair gene PARP1, possibly related to the effect of linoleic acid-regulated membrane fluidity-enhancing drugs on single-chain damage repair pathways. Oleic acid and linoleic acid nanoemulsion carriers promote the expression of the epithelial-mesenchymal transition inhibitory gene E-cadherin, presumably related to its unsaturated structure regulating the tumor microenvironment, while the stearic acid carrier has no significant effect. These gene expression changes are related to the chain length and saturation of the carrier fatty acids, reflecting differences in the anti-tumor mechanisms of different carrier systems and providing a molecular basis for carrier optimization.

[0089] In vivo validation of the developed drug delivery system in mice.

[0090] Based on the results of in vitro experiments, stearic acid nanoemulsion was selected for animal experiments. The in vivo therapeutic effect of stearic acid nanoemulsion was evaluated in mice implanted with MDA-MB-231 cells. Treatment was initiated when the tumor diameter was 4-5 mm, with a treatment cycle of 20 days. Mice with confirmed tumors were randomly divided into 6 treatment groups: control group (physiological saline, IV, N=6), free TZL (IV, 0.33 mg / kg, N=6), blank stearic acid group (IV, N=6), low-dose stearic acid group (IV, 0.11 mg / kg, N=6), medium-dose stearic acid group (IV, 0.33 mg / kg, N=6), and high-dose stearic acid group (IV, 1.0 mg / kg, N=6). All nanoemulsions were diluted to working concentration with PBS. Free TZL was dissolved in DMSO and diluted with saline (DMSO content in the final solution was less than 1%). Treatment was administered every two days. All mice were weighed and tumor size was measured using calipers before each injection.

[0091] Table 5. Comparison of tumor volume correlation and tumor inhibition rate among different groups

[0092]

[0093] Results of tumor volume changes in mice are as follows Figure 9As shown in Figure A, compared with the control group, all treatment methods slowed tumor growth, but the stearic acid group showed the best therapeutic effect, which was positively correlated with the administered dose. Notably, there was a significant difference between the same dose of free TZL and the medium-dose stearic acid group (*p = 0.0119). This indicates that nanoemulsions, as drug carriers, do indeed improve drug utilization compared to free drugs. Figure 9 Figure B shows the changes in mouse body weight; no weight loss was observed, and the mice's weight increased over time. However, in the high-dose stearic acid group, weight gain ceased at the end of treatment. Figure 9 In the figure, C represents the tumor growth inhibition rate calculated based on tumor weight, indicating that the blank stearic acid group had a certain inhibitory effect on tumor growth. The inhibition rate of the stearic acid group was positively correlated with the drug loading dose. Figure 9 Figure D shows the solid images of tumors in each group of mice after treatment. The formula for calculating the tumor growth inhibition rate is as follows: m 对照组 This is the tumor weight in the control group, m 实验组 This refers to the tumor weight in the experimental group.

[0094]

[0095] Analysis of blood biochemical parameters in mice based on the developed drug delivery system

[0096] After treatment, blood was collected from the mice's eyes. Whole blood samples were left at room temperature for 2 hours, then centrifuged, and the supernatant was immediately used for analysis. Results were as follows: Figure 10 As shown in the figure. To assess potential tissue toxicity after treatment, heart, liver, spleen, lung, kidney, and tumor tissues were collected from all groups of animals after the last administration. H&E staining was used to assess related toxicity, and the results are shown in the figure. Figure 11 As shown.

[0097] The results from both parts were analyzed: HE section analysis showed that stearic acid nanoemulsion at all dosage groups did not cause significant acute damage to normal tissues such as the heart, liver, spleen, lungs, and kidneys, consistent with blood biochemistry results (normal ALT, AST, BUN, and CRE), confirming its good safety profile. In tumor tissue, stearic acid nanoemulsion induced necrosis in a dose-dependent manner, with the high-dose group showing the largest necrosis area. This corresponded to the 60.21% tumor inhibition rate and the gene-level proliferation inhibition and apoptosis activation results, validating the carrier synergistic effect.

[0098] Blood biochemistry analysis further supports its safety: stable lipid metabolism indicators (TG, T-CHO) ruled out metabolic interference; normal LDH suggests that the drug targets tumors rather than causing widespread damage to normal tissues. Elevated CK but normal CK-MB, combined with the absence of myocardial damage in HE sections, suggests that the change is due to membrane permeability changes accompanying the immune response. In summary, stearic acid nanoemulsion enhances antitumor efficacy while maintaining safety, providing multidimensional evidence for its clinical translation.

Claims

1. A drug delivery system based on long-chain fatty acid nanoemulsions, characterized in that, The drug delivery system comprises an oil-in-water nanoemulsion structure consisting of a hydrophobic drug, long-chain fatty acids, lipids, and surfactants. The hydrophobic regions of the long-chain fatty acids and lipids combine to form a nanoscale hydrophobic core, which encapsulates the hydrophobic drug. The hydrophobic ends of the surfactants are embedded in the outer layer of the hydrophobic core, while the hydrophilic ends face the aqueous phase to form an interfacial film that encapsulates the hydrophobic core, thus forming a hierarchical structure of hydrophobic drug, hydrophobic core, and surfactant interfacial film.

2. The drug delivery system according to claim 1, characterized in that, The long-chain fatty acid is oleic acid, linoleic acid, stearic acid, squalene, or squalane; the surfactant is one of Tween-80, sophorolipid, poloxamer 188, or polyethylene glycol-15-hydroxystearate; the lipid is one of distearylphosphatidylcholine (DSPC), hydrogenated soybean lecithin (HSPC), dipalmitoylphosphatidylcholine (DPPC), or dioleoylphosphatidylethanolamine (DOPE).

3. The drug delivery system according to claim 1, characterized in that, The hydrophobic drug is one of the following PARP inhibitors: tapolarib (TZL), paclitaxel, docetaxel, etoposide, and irinotecan.

4. The nanoemulsion according to claim 1, characterized in that, The molar ratio of the lipid, surfactant, long-chain fatty acid and hydrophobic drug is 30-40:10-20:20-60:5-20.

5. A method for preparing a drug delivery system based on long-chain fatty acid nanoemulsion as described in claim 1, characterized in that, Includes the following steps: (1) Prepare a lipid ethanol solution by mixing distearate phosphatidylcholine DSPC, Tween-80 and long-chain fatty acids with anhydrous ethanol. (2) Prepare a hydrophobic drug solution using dimethyl sulfoxide (DMSO); mix the lipid ethanol solution with the hydrophobic drug solution to prepare a lipid drug mixed solution, and inject it into phosphate-buffered saline (PBS) under vortex conditions to obtain a drug delivery system based on long-chain fatty acid nanoemulsion.

6. The manufacturing method according to claim 5, characterized in that, In step (1), when the long-chain fatty acid is oleic acid, the mass ratio of oleic acid:DSPC:Tween-80 is 200-150:60-30:60-30; when the long-chain fatty acid is linoleic acid, the mass ratio of linoleic acid:DSPC:Tween-80 is 300-100:30-10:60-30; when the long-chain fatty acid is stearic acid, the mass ratio of stearic acid:DSPC:Tween-80 is 175-50:50-10:60-30; when the long-chain fatty acid is squalene, the mass ratio of squalene:DSPC:Tween-80 is 150-50:50-20:30-10; when the long-chain fatty acid is squalane, the mass ratio of squalane:DSPC:Tween-80 is 175-50:50-20:30-10.

7. The manufacturing method according to claim 5, characterized in that, In step (2), when the long-chain fatty acid is oleic acid, the mass ratio of oleic acid to the hydrophobic drug is 200-150:60-30; when the long-chain fatty acid is linoleic acid, the mass ratio of linoleic acid to the hydrophobic drug is 300-100:30-10; when the long-chain fatty acid is stearic acid, the mass ratio of stearic acid to the hydrophobic drug is 175-50:50-10; when the long-chain fatty acid is squalene, the mass ratio of squalene to the hydrophobic drug is 150-50:50-20; when the long-chain fatty acid is squalane, the mass ratio of squalane to the hydrophobic drug is 175-50:50-20.

8. The application of the drug delivery system based on long-chain fatty acid nanoemulsion as described in claim 1 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumors are breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer.

10. The application according to claim 8, characterized in that, The drug is an injectable preparation or a topical preparation.

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