Rigidity-adjustable nano-liposome hydrogel composite system and preparation method thereof

Abstract

The invention relates to a rigidity-adjustable nano-liposome hydrogel composite system and a preparation method thereof. According to the composite system, a shell is formed by DOPC and beta-sitosterol, and a core is prepared from sodium alginate or sodium alginate and Ca < 2 + >. According to the method, the rigidity of the liposome is controlled by controlling the crosslinking degree of sodium alginate and Ca < 2 + > of the core of the composite nano-liposome, so that the application stability of the composite liposome and the cell absorption efficiency of the medicine are improved. The preparation process is efficient, simple and convenient, and compared with traditional lipidosome, the liposome has good stability and biocompatibility and can be widely applied to transport carriers of drugs and nutrients.

Description

Technical Field

[0001] The invention relates to the technical field of nano liposomes, and in particular to a nano liposome hydrogel composite system with adjustable rigidity and a preparation method thereof. Background Art

[0002] Nanoliposomes are drug delivery systems used to control drug distribution and release. They can simultaneously encapsulate hydrophilic, lipophilic, and amphiphilic active compounds. Nanoliposomes are mainly bilayer vesicles composed of phospholipid molecules. The typical structure is a lamellar vesicle with an internal water core surrounded by one or more concentric phospholipid bilayers. Hydrophilic, hydrophobic, or amphiphilic bioactive compounds can be encapsulated therein, thereby stabilizing the encapsulated active substances, making them resistant to a series of environmental and chemical changes, and improving the stability and delivery efficiency of encapsulated drugs. Due to their unique characteristics, liposomes have attracted great attention to the controlled release or directional release of various drugs and diagnostic drugs.

[0003] Despite much research, liposomes have not yet fully realized their potential. Traditional liposomes have some disadvantages, such as low encapsulation rate, instability and drug leakage, which limit the development of drug-based products. The instability of liposomes may cause them to be quickly cleared from the vesicle circulation before reaching the target. In order to solve the above problems, scientists have tried to modify liposomes with a variety of modifiers to enhance the mechanical properties of liposomes. Several key mechanical properties include rigidity, hardness and strength. The release of active substances encapsulated in nanoliposomes and the circulation time in the blood are related to the mechanical properties of liposomes. Among them, the rigidity of nanoparticles significantly affects the cellular absorption efficiency, cellular internalization mode, system circulation life and biodistribution of nanoparticles. Generally, nanoparticles with moderate rigidity have a longer system circulation life than nanoparticles with high rigidity and higher cellular absorption efficiency.

[0004] The invention provides a nano liposome hydrogel composite system with adjustable rigidity, small particle size, high encapsulation rate and good stability, namely composite liposome. Summary of the invention

[0005] The present invention aims to provide a nanoliposome hydrogel composite system with adjustable rigidity and a preparation method thereof, which not only shortens the preparation process and is highly efficient, but also provides a composite liposome with good stability and adjustable rigidity. The shell of the composite liposome is composed of DOPC and β-sitosterol, and the core is composed of sodium alginate or sodium alginate and Ca 2+ Prepared from sodium alginate and Ca 2+ The degree of cross-linking controls the rigidity. Compared with traditional liposomes, the composite liposome has a higher encapsulation rate, good stability, and uniform morphology. It can be used as a drug transport carrier and broadens its application range.

[0006] The specific technical solutions are as follows:

[0007] A method for preparing a nanoliposome hydrogel composite system with adjustable rigidity comprises the following steps:

[0008] (1) DOPC and β-sitosterol were mixed with chloroform and evaporated under reduced pressure using a rotary evaporator to remove the solvent and form a thin lipid film.

[0009] (2) The thin lipid film was resuspended in a DMSO:EtOH (7:3, v:v) solution and added dropwise to a sodium alginate aqueous solution under vigorous stirring.

[0010] (3) After freeze-thaw cycles and nanoporous membrane extrusion, preliminary nanoliposomes are formed.

[0011] (4) The obtained solution containing the preliminary nanoliposomes is dialyzed in a dialysis bag to remove impurities.

[0012] (5) adding an ionic crosslinking agent CaCl2 solution to the dialyzed nanoliposome solution to form crosslinked composite liposomes. After incubation, dialyzing is performed again to obtain the final composite liposomes with adjustable rigidity.

[0013] The present invention forms a lipid membrane using DOPC through rotary evaporation, and after cooling to room temperature, undergoes freeze-thaw treatment, and then uses sodium alginate and Ca 2+ The rigidity is controlled by the degree of cross-linking, making it a drug-loaded composite liposome with small particle size, high encapsulation rate and good stability.

[0014] Preferably, in step (1), the molar ratio of DOPC to β-sitosterol is 0.65:0.35 to 0.75:0.25.

[0015] Preferably, the volume ratio of the total amount of DOPC and β-sitosterol to the DMSO:EtOH solution is 30-50 μmol:0.5-1.5 mL.

[0016] Preferably, in the freeze-thaw cycle in step (3), the number of freeze-thaw cycles is preferably 5 times, the heating temperature and time are 60-70°C and 2-3 minutes, and the freezing temperature and time are -80°C and 15-20 seconds.

[0017] Preferably, in step (3), the diameter of the nanoporous membrane is 100 nm.

[0018] Preferably, in step (4), the dialysis time is 12 h and the temperature is 4°C.

[0019] Preferably, in step (5), the mass of CaCl2 in every 10 mL of the ionic crosslinking agent is 0-10 mmoL, and the volume ratio of the ionic crosslinking agent to the volume of the nanoliposome solution is 1:1.

[0020] Preferably, in step (5), the incubation temperature is 37°C, the dialysis time is 24 to 48 hours, and the dialysis temperature is 4°C.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses DOPC and sodium alginate as composite raw materials, which have good biocompatibility, wide sources, low preparation cost, simple and portable preparation method, and energy saving. 2+ The dosage is used to adjust the sodium alginate and Ca 2+ The degree of cross-linking can be precisely controlled to achieve the rigidity of nanoliposomes.

[0023] (2) The composite liposomes obtained by the present invention have good biocompatibility and stability, are suitable as drug delivery carriers, and have excellent mechanical properties, stability and uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The following are appearance diagrams of the composite liposome solutions of Examples 1-5 and Comparative Example 1.

[0025] Figure 2 The transmission electron microscope (TEM) images of Examples 1-5 and Comparative Example 1 are shown.

[0026] Figure 3 The encapsulation efficiency and drug loading of Examples 1-5 and Comparative Example 1 are shown.

[0027] Figure 4 It is the infrared spectra of Examples 1-5 and Comparative Example 1.

[0028] Figure 5 The storage modulus (G') and loss modulus (G") curves of Examples 1-5 are shown in FIG. DETAILED DESCRIPTION

[0029] The present invention is further explained below in conjunction with specific examples. The present invention is implemented according to the technical solution of the invention, and detailed implementation methods and operating steps are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions.

[0030] Example 1

[0031] A method for preparing a nanoliposome hydrogel composite system with adjustable rigidity, the specific steps are as follows:

[0032] (1) 40 μmol of DOPC and β-sitosterol (0.75:0.25) were mixed with 5 ml of chloroform, and the mixture was placed in a rotary evaporator and evaporated under reduced pressure at 50°C until a thin lipid film (phospholipid film) was formed on the wall of the rotary evaporator. The vacuum evaporation was continued to remove the residual solvent and stored in a vacuum desiccator overnight. The resulting phospholipid film was resuspended in 1 mL of DMSO:EtOH (7:3, v:v). The solution was added dropwise to 9 mL of sodium alginate aqueous solution through a syringe under vigorous stirring. The resulting solution was subjected to 5 freeze-thaw cycles, frozen at -80°C for 20 s, heated at 60°C for 3 min, and repeatedly extruded through a 100 nm polycarbonate track-etched nanoporous membrane for 5 times.

[0033] (2) The solution obtained in (1) was dialyzed overnight in a dialysis bag (1000 kDa) in pure water at 4°C. After dialysis, an ionic crosslinker containing 10 mmol of CaCl2 was added dropwise to the liposome solution (1:1, v:v) to crosslink the sodium alginate monomers in the liposome membrane (the concentration of the sodium alginate aqueous solution was 1 mg / mL, but the sodium alginate content in the liposome core was very small, so CaCl2 was added dropwise to the liposome solution (1:1, v:v). 2+ The concentration of liposomes should not be too high, otherwise the liposomes may rupture after the two are cross-linked at the core of the liposomes). After incubation at 37°C for 1 hour, the liposome solution was dialyzed in a PBS solution using a dialysis bag (1000 kDa) at 4°C for 24 hours to obtain a composite liposome solution.

[0034] The composite liposome solution prepared above was taken for index determination. The results are shown in Table 1. Figure 1-4 shown.

[0035] Example 2

[0036] This example is exactly the same as Example 1 except that the amount of CaCl2 used as the ionic crosslinking agent in step (2) is 2.5 mmol.

[0037] Example 3

[0038] This example is exactly the same as Example 1 except that the amount of CaCl2 used as the ionic crosslinking agent in step (2) is 5 mmol.

[0039] Example 4

[0040] This example is exactly the same as Example 1 except that the amount of CaCl2 used as the ionic crosslinking agent in step (2) is 1 mmol.

[0041] Example 5

[0042] This example is exactly the same as Example 1 except that the mass of CaCl2 as the ionic crosslinking agent in step (2) is 0 mmol.

[0043] Comparative Example 1

[0044] A method for preparing a nanoliposome hydrogel composite system with adjustable rigidity, the specific steps are as follows:

[0045] (1) 40 μmol of DOPC and β-sitosterol (0.75:0.25) were mixed with 5 ml of chloroform, and the mixture was placed in a rotary evaporator and evaporated under reduced pressure at 50°C until a thin lipid film (phospholipid film) was formed on the wall of the rotary evaporator. The vacuum evaporation was continued to remove the residual solvent and stored in a vacuum desiccator overnight. The resulting phospholipid film was resuspended in 1 mL of DMSO:EtOH (7:3, v:v). The solution was added dropwise to 9 mL of PBS solution through a syringe under vigorous stirring. The resulting solution was subjected to 5 freeze-thaw cycles, frozen at -80°C for 20 s, heated at 60°C for 3 min, and repeatedly extruded through a 100 nm polycarbonate track-etched nanoporous membrane for 5 times.

[0046] The liposome solution prepared above was taken and the indexes were measured. The results are shown in Table 1. Figure 1-4 shown.

[0047] Table 1 shows the particle size, polydispersity index (PDI) and Zeta potential of the composite liposomes of Examples 1-5 and Comparative Example 1.

[0048]

[0049] According to the above embodiments, comparative examples and Table 1, Figure 2 It can be seen that the particle size of the embodiment and the comparative example is between 106-160nm, PDI <0.3, and NLP and NLG are monodisperse. The absolute value of the Zeta potential is high, indicating that the surface of the nanoliposome has a high charge to prevent the aggregation of the nanoliposome. As the degree of cross-linking increases, the Zeta potential tends to increase. The embodiment has uniformity and stability. Compared with the comparative example, the embodiment has better stability.

[0050] Depend on Figure 3 It can be seen that the encapsulation efficiency and drug loading of Example 1 are the highest, reaching 80.91±4.33% and 14.14±0.37% respectively. 2+As the concentration increases, the encapsulation rate of β-sitosterol gradually increases. Since some methyl hydrogen atoms of β-sitosterol can be connected to the carboxyl group of sodium alginate, the hydrogel with a higher degree of cross-linking can better link β-sitosterol, thereby improving the encapsulation rate of β-sitosterol. The drug loading of the embodiment is significantly higher than that of the comparative example. This is because the polysaccharide network limits the Brownian diffusion of macromolecules and acts on the lipid layer to improve the stability of the lipid layer, indicating that the composite lipid system has good stability.

[0051] Depend on Figure 4 As shown, it is found through the Fourier infrared spectrum that the absorption peak of the sodium alginate in the example is covered, indicating that the sodium alginate is embedded in the complex liposome.

[0052] Depend on Figure 5 It can be seen that liposomes with different cross-linking degrees have different rigidities. G' is also called elastic modulus, which refers to the amount of energy stored in a material due to elastic reversible deformation when it is deformed, reflecting the elasticity of the material. Within the frequency range, G' of all Examples 1-4 is greater than the loss modulus G", and G' of Examples 1-4 is in the range of 1300-9900Pa, indicating that all samples exhibit the characteristic behavior and elasticity-dominated properties of solids, and exhibit a stable structural state at a fixed frequency, showing good cross-linking characteristics, but the storage modulus of Example 5 is only 0.1-0.5Pa. This is because Ca 2+ The increase in Ca 2+ The enhanced interaction with sodium alginate molecules can promote the cross-linking degree of the core, thereby increasing the gel strength, and the hydrogel core exhibits higher elasticity and rigidity.

Claims

1. A nanoliposome hydrogel composite system with adjustable rigidity, characterized in that: The composite liposome has an outer layer composed of dioleoylphosphatidylcholine (DOPC) and β-sitosterol, and a core composed of sodium alginate or sodium alginate and Ca 2+ It is prepared from sodium alginate and Ca 2+ The degree of cross-linking controls the rigidity of the composite liposomes.

2. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 1, characterized in that: The preparation method comprises the following steps: Step 1: DOPC and β-sitosterol were mixed with chloroform and evaporated under reduced pressure by a rotary evaporator to remove the solvent and form a thin lipid film; Step 2: resuspend the thin lipid film in a mixture of DMSO and EtOH (7:3, v:v) and dropwise add it into the sodium alginate aqueous solution under vigorous stirring; Step 3: After freeze-thaw cycles and nanoporous membrane extrusion, preliminary nanoliposomes are formed; Step 4: dialyzing the resulting solution containing the preliminary nanoliposomes in a dialysis bag to remove impurities; Step 5: Add an ionic crosslinking agent CaCl2 solution to the dialyzed nanoliposome solution to form a crosslinked composite liposome. After incubation, dialyze again to obtain a nanoliposome hydrogel composite system with adjustable rigidity.

3. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The molar ratio of DOPC to β-sitosterol in step 1 is 0.65:0.35 to 0.75:0.

25.

4. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The volume ratio of the total amount of DOPC and β-sitosterol to the mixed solution of DMSO and EtOH is 30-50 μmol: 0.5-1.5 mL.

5. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The volume ratio of DMSO to EtOH in the mixed solution is 7:

3.

6. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The freeze-thaw cycle described in step 3 is to alternately freeze and heat the sample, with the heating temperature and time being 60°C-70°C and 2-3 minutes, and the freezing temperature and time being -80°C and 15-20 seconds.

7. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The diameter of the nanoporous membrane in step 3 is 100 nm.

8. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The dialysis time in step 4 is 12 h and the temperature is 4°C.

9. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity as claimed in claim 2, characterized in that: The mass of CaCl2 in the ionic crosslinking agent in step 5 is 0-10 mmol / 10 mL, and the volume ratio of the ionic crosslinking agent to the volume of the nanoliposome solution is 1:

1.

10. The method for preparing the nanoliposome hydrogel composite system with adjustable rigidity according to claim 2, characterized in that: The incubation temperature in step 5 is 37°C, the dialysis time is 24 to 48 hours, and the dialysis temperature is 4°C.

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