WO2019052049A1

WO2019052049A1 - Cationic polymer/tdns drug-loading complex and preparation method therefor

Info

Publication number: WO2019052049A1
Application number: PCT/CN2017/116300
Authority: WO
Inventors: 林云锋; 田陶然; 蔡潇潇; 林世宇; 石思容
Original assignee: 四川大学
Priority date: 2017-09-12
Filing date: 2017-12-14
Publication date: 2019-03-21
Also published as: CN109364255B; CN109364255A; CN107432935A

Abstract

A cationic polymer/TDNs drug-loading complex. A preparation method for the cationic polymer/TDNs drug-loading complex comprises the following steps: mixing a PEI solution and a TDNs solution according to the ratio of N/P = 0.2-7.5, and conducting incubation at the room temperature for 15-30 min to obtain the cationic polymer/TDNs drug-loading complex. According to the method, the PEI solution with a specific concentration and the TDNs with a specific concentration are mixed according to the specific N/P value, the obtained drug-loading complex can effectively improve the biocompatibility of cations, and moreover, degradation of TDNs by DNAses can be avoided, TDNs are promoted to enter cells, and therefore effective drug loading can be realized.

Description

Cationic polymer/TDNs drug-loading composite and preparation method thereof

Technical field

The invention belongs to the technical field of DNA drug loading, and in particular relates to a cationic polymer/TDNs drug-loading compound and a preparation method thereof.

Background technique

In recent years, thanks to the excellent processing properties and biocompatibility of nucleic acids, a variety of DNA nanostructures have been designed for use in various fields such as drug delivery and bioprobe. However, due to the biochemical properties of the nucleic acid itself, there are problems such as degradation by DNase and renal clearance in the systemic circulation, thereby limiting its application in vivo. In addition, although some specially designed DNA structures (such as DNA tetrahedrons, TDNs) can enter and enter the nucleus to some extent in recent years, due to the negatively charged nature of nucleic acids, it is difficult for DNA to autonomously pass through lipids. The bilayer thus enters the cell. Therefore, in specific applications, various methods are often used to assist nucleic acids in entering or transfecting cells. Common methods include cationic liposome, virus, electroporation and the like, and cationic liposomes have been widely used due to their high transfection efficiency and high speed. Polyethylenimine (PEI) is a cationic polymer that binds to nucleic acids by electrostatic forces and encapsulates DNA to attenuate the effects of non-specific scavenging mechanisms in the body on the nucleic acid carrying, using the positive charge on the surface to bind to the cell membrane. Enter the cell. Although PEI has obvious advantages and wide application, PEI as a cationic polymer has obvious cytotoxicity at a certain concentration, and is also not suitable for wide application in vivo.

Summary of the invention

In view of the above problems existing in the prior art, the present invention provides a cationic polymer/TDNs drug-loading compound and a preparation method thereof, which can effectively solve the problem that TDNs are easily degraded by DNase in the prior art, and the amount of simple TDNs is small. , the problem of high toxicity of PEI.

In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problem thereof is:

A cationic polymer/TDNs drug-loading complex, the preparation method thereof comprises the following steps: PEI The solution is mixed with TDNs solution in a ratio of N/P=0.2-7.5, and incubated at room temperature for 15-30 min.

Further, the PEI solution was mixed with the TDNs solution in a ratio of N/P=1.

Further, the concentration of the PEI solution was 1 mg/mL.

Further, the concentration of the TDNs solution was 1 μmol/L.

Further, TDNs are prepared by the following methods:

(1) Dissolve four single strands of DNA tetrahedron with ddH ₂ O to a concentration of 1 nmol/μL, and then determine the absorbance of DNA at wavelengths of 260 nm and 330 nm by UV quantification, and then calculate according to the following formula. 100 μL, volume of each single strand in a 1 μM system:

V=100/[(A260-A330)×10 ⁵ /(15.2×number of single-chain adenine+7.4×number of cytosine in single chain+11.4×number of guanine in single chain+8.3× single-chain thymus Number of pyrimidines)]

(2) According to the result calculated in the step (1), the dissolved DNA tetrahedron four single-chain solutions are taken up, and then mixed with TM buffer to make the total volume of 100 μL, vortex vibration mixed, and finally placed in the PCR instrument, The temperature was rapidly raised to 95 ° C for 10 min, then cooled to 4 ° C for 20 min, and finally stored at -20 ° C.

Further, the TM buffer in the step (3) is 5-10 mM Tris-HCl, 5-50 mM MgCl ₂ , pH 8.0.

Further, the TM buffer in the step (3) is 10 mM Tris-HCl, 50 mM MgCl ₂ , pH 8.0.

Four chains of DNA tetrahedra:

S1:

S2:

S3:

S4:

The invention provides a cationic polymer/TDNs drug-loaded composite and a preparation method thereof, which have the following beneficial effects:

The TDNs prepared by the invention have stable structure and can pass through the cell membrane alone. PEI binds to TDNs by electrostatic force, and encapsulates TDNs to weaken the effect of non-specific scavenging mechanism in vivo on carrying TDNs, and the positive charge on the surface is combined with the cell membrane. Entering the cell, although PEI can protect ordinary nucleic acids from entering the cell, the entry speed is slow, and some complexes are still blocked outside the cell, and the present invention can effectively enhance the complex by interacting with the prepared TDNs by PEI. After entering the cell and passing rate, PEI can also promote its entrapment of TDNs to escape lysosome through its "proton sponge" effect.

When the amount of PEI is large, it will lead to excessive cytotoxicity, and when it is too low, it will not protect. Therefore, screening a suitable ratio of PEI and TDNs can control the toxicity of PEI and maintain the PEI in the drug-loading system. Transport and protection of TDNs are not dissolved by lysosomes.

The present invention mixes a specific concentration of PEI solution with a specific concentration of TDNs according to a specific N/P, and the obtained drug-loading complex can effectively improve the biocompatibility of the cation, and at the same time, can prevent the degradation of TDNs by DNase and promote its Enter the cell, thus playing the role of effective drug loading.

DRAWINGS

Figure 1 is a TEM representation of the cationic polymer/TDNs drug-loaded complex prepared in Example 4.

2 is a potential diagram of the cationic polymer/TDNs drug-loaded complex prepared in Example 4.

Figure 3 is a particle size diagram of the cationic polymer/TDNs drug-loaded complex prepared in Example 4.

Figure 4 is a graph showing the toxicity results of cationic polymer/TDNs drug-loaded complexes on fibroblasts (L929) after 24 hours of culture.

Figure 5 is a graph showing the toxicity of cationic polymer/TDNs drug-loaded complex on fibroblasts (L929) after 72 hours of culture.

Figure 6 is an electrophoresis pattern of DNaseI added to TDNs solution.

Figure 7 is an electropherogram of a complex of different N/P ratios.

Figure 8 is a laser confocal microscope image of L929 cells and A549 cells.

Figure 9 is a graph showing the fluorescence signal intensity of cells detected by flow cytometry; the left side is L929 cells and the right side is A549 cells.

Detailed ways

Example 1

A cationic polymer/TDNs drug-loading compound, the preparation method comprising the steps of: mixing a PEI solution having a concentration of 1 mg/mL with 1 μmol/L of TDNs according to a ratio of N/P=0.2, and incubating at room temperature for 30 min, be made of.

Among them, 1 ug of TDNs represents 3 nmol of phosphoric acid (P), 1 μL of PEI solution contains 10 nmol of amino nitrogen (N), and the amount of TDNs (P) and PEI(N) added to the complex solution determines the N/ of synthetic TNDs/PEI. P ratio.

TDNs are prepared by the following methods:

(2) According to the calculation result in the step (1), the dissolved DNA tetrahedron is subjected to four single-chain solutions, and then mixed with TM buffer (10 mM Tris-HCl, 50 mM MgCl ₂ , pH 8.0) to make a total volume of 100 μL. The vortex was mixed and finally placed in the PCR machine. The temperature was rapidly raised to 95 ° C for 10 min, then cooled to 4 ° C for 20 min, and finally stored at -20 ° C.

Example 2

A cationic polymer/TDNs drug-loading compound, the preparation method comprising the steps of: mixing a PEI solution having a concentration of 1 mg/mL with 1 μmol/L of TDNs according to a ratio of N/P=5, and incubating at room temperature for 30 minutes, be made of.

The preparation method of TDNs is the same as in Example 1.

Example 3

A cationic polymer/TDNs drug-loading compound, the preparation method comprising the steps of: mixing a PEI solution having a concentration of 1 mg/mL with 1 μmol/L of TDNs according to a ratio of N/P=7.5, and incubating at room temperature for 30 min, be made of.

Among them, 1 μg of TDNs represents 3 nmol of phosphoric acid (P), 1 μL of PEI solution contains 10 nmol of amino nitrogen (N), and the amount of TDNs (P) and PEI(N) added to the complex solution determines the N/ of synthetic TNDs/PEI. P ratio.

The preparation method of TDNs is the same as in Example 1.

Example 4

A cationic polymer/TDNs drug-loading compound, the preparation method comprising the steps of: mixing a PEI solution having a concentration of 1 mg/mL with 1 μmol/L of TDNs according to a ratio of N/P=1, and incubating at room temperature for 30 minutes, be made of.

The preparation method of TDNs is the same as in Example 1.

We also optimized the concentration of PEI solution and the concentration of TDNs solution. The concentration of PEI solution was 0.1-10mg/mL, which was increased by 0.5mg/mL. The concentration of TDNs was 0.1-10mg/mL to 0.5mg/ The mL was increased as a gradient, and the rest of the process was the same as in Example 4, and then orthogonal test, such as toxicity test, nucleic acid protection test and cell-injection experiment, showed that when the concentration of PEI solution was 1 mg/mL, the concentration of TDNs solution was 1 μmol. /L works best. The toxicity test, the nucleic acid protection test, and the cell-injection experiment were also performed in the same manner as in Example 1-4, and Example 4 was the best.

We also did the following experiment: PEI and ordinary DNA were combined, and the proportional relationship was the same as that in Example 4. Although some of the complexes could enter the cells, the rate of entry into the cells was slow, and only part of them could enter, and the number of cells entering the cells was low. Some of the complexes that are blocked from the cell, or that enter the cell, are degraded by the enzyme due to DNA structural instability.

Characterization and toxicity experiments, nucleic acid protection experiments and cell-injection experiments of the complex obtained in Example 4 were as follows:

The TEM characterization, potential map and particle size diagram of the cationic polymer/TDNs drug-loaded complex prepared by the above method are shown in Fig. 1, Fig. 2 and Fig. 3, respectively.

It can be seen from Fig. 1 that the drug-loading compound is in the form of particles, and the apparent diameter is in the range of 200-400 μm. Fine fiber connection, PEI.

As can be seen from Fig. 2, when N/P is 5 or 7.5, the charge carried by the drug-loading complex is positive; when N/P = 1, the charge of the complex is negative. Positively charged particles are more toxic to cells than negatively charged particles, and reversal of complex charge by the addition of TNDs may be a cause of reduced cytotoxicity of the complex.

It can be seen from Fig. 3 that as the proportion of PEI increases, the DLS diameter of the composite particles gradually decreases. It should be noted that the DLS diameter is the hydraulic diameter of the particles in the solution, and the value may be different from the TEM observation.

Experimental Example 1 Toxicity test of complex on fibroblasts

Taking fibroblasts (L929) as the research object, firstly, appropriate amount of fibroblasts were inoculated into 96-well plates (n=6, ie, 6 replicate wells were set), cultured for 24 hours, and then serum culture was lowered to reduce serum concentration. When cultured at 8% for 12h, then reduced to 2% for 12h, then reduced to serum for 0h and then equilibrated for 1h. According to different groups, add 100μL of blank medium (RPMI), blank medium and 250nM TDNs. (75 μL of blank medium, 25 μL of TDNs), a mixture of blank medium and complexes with different N/P ratios (7.5, 5, 1, 0.2) (75 μL of blank medium, 25 μL of complex), and cultured for 24 hours under the same conditions. After 72 hours, the medium was aspirated, and the CCK-8 toxicity test was performed according to the CCK-8 kit.

After 24 h and 72 h of culture, the toxicity results of the complex on fibroblasts (L929) are shown in Figure 4 and Figure 5, respectively.

It is reported in the literature that pure PEI has a transient toxicity of about 30 minutes for cells and a long-term toxicity of about 24 hours. As can be seen from Fig. 4 and Fig. 5, the cationic polymer/TDNs drug-loaded complex prepared by the method provided by us, fibroblasts (L929) on the first and third days of treatment, the cells are not only not subjected to cationic toxicity. The effect was inhibited, and a statistically significant proliferation was obtained, indicating that the complex has good biocompatibility.

Experimental Example 2 Protection of nucleic acids by complexes

250nM TDNs solution was digested with different concentrations of DNaseI (0 units, 10 units and 20 units) for 3 min, then the mixed solution was added to a 1% agarose gel for electrophoresis experiments, and the nucleic acid was observed to be significantly degraded by 20 units of DNaseI. Take a photo.

The same 20 units of DNase I were added to the complexes of different N/P ratios (Examples 5-8), and electrophoresis experiments were carried out for 3 minutes, and the results are shown in Fig. 6 and Fig. 7, respectively.

It can be seen from Fig. 6 that after the pure TDNs are mixed with DNase, the nucleic acid is degraded to different degrees with different concentrations of DNase. When the DNase concentration is 20 U, the nucleic acid can be completely degraded.

After the addition of the cation, the DNA strip was able to maintain good morphology and brightness after DNase treatment, and it was found that the nucleic acid was well protected in the complex of the present invention.

Experimental Example 3 DNA nanostructure entry assay

Flow cytometry and Confocal immunofluorescence were used to determine the conclusion that the complex of the invention promotes the entry of DNA tetrahedral structures into cells.

TLDs were labeled with CY5 fluorescent molecules using L929 cells and A549 cells as targets. The cells were cultured in a special glass dish for 24 hours, and the serum was equilibrated to zero serum for 1 h. The medium containing 100 nM TDNs and N/P=1:1 complex (Example 5) was added and cultured for 6 hours. Thereafter, the cells were fixed with 4% paraformaldehyde, and the nuclei and cytoskeleton were stained with DAPI and phalloidin, and finally photographed using a laser confocal microscope (TCS SP8; Leica, Wetzlar, Germany), and the results are shown in 8.

After CY5 fluorescent labeling of TDNs, the cells were seeded in 6-well plates, cultured for 24 hours, and then serum cultured. The serum concentration was reduced to 8% for 12 hours, then decreased to 2% for 12 hours, and then decreased to serum. After the content is 0, the equilibrium is 1 h. The medium was replaced with blank medium, 100 nM TDNs, N/P=1:1 complex, and after 6 hours of culture, the cells were digested and centrifuged to prepare a cell suspension using flow cytometry. The instrument (FC500Beckman, IL USA) probed the fluorescence signal intensity of the cells and performed quantitative analysis. The results are shown in Figure 9 (direct flow diagram) and Table 1 (positive rate of threshold).

Table 1 Fluorescence signal intensity of cells

	对照Control	单纯核酸Simple nucleic acid	阳离子复合物Cationic complex
A549A549	0.18±0.020.18±0.02	18.00±0.6018.00±0.60	96.57±1.2796.57±1.27
L929L929	0.20±0.030.20±0.03	0.19±0.020.19±0.02	93.97±0.1593.97±0.15

Flow cytometry was used to detect the entry of fluorescently labeled nucleic acid nanostructures into cells. As shown in Figure 9 and Table 1, in A549 and L929 cells, the proportion of cells using cationic composite nucleic acids into cells was much greater than that of simple nucleic acids. Fluorescence can be visually seen that fluorescently labeled nucleic acids enter the cell in large amounts after complexation by cations.

Claims

The invention relates to a method for preparing a cationic polymer/TDNs drug-loading compound, which comprises the steps of: mixing a PEI solution with a TDNs solution according to a ratio of N/P=0.2-7.5, and incubating at room temperature for 15-30 min. Got it.
The method for preparing a cationic polymer/TDNs drug-loaded complex according to claim 1, wherein the PEI solution and the TDNs solution are mixed in a ratio of N/P=1.
The method for preparing a cationic polymer/TDNs drug-loaded complex according to claim 1, wherein the PEI solution has a concentration of 1 mg/mL.
The method for producing a cationic polymer/TDNs drug-loaded complex according to claim 1, wherein the TDNs solution has a concentration of 1 μmol/L.
The method for preparing a cationic polymer/TDNs drug-loaded complex according to claim 1, wherein the TDNs are prepared by the following method:

(1) Dissolve four single strands of DNA tetrahedron with ddH 2 O to a concentration of 1 nmol/μL, and then determine the absorbance of DNA at wavelengths of 260 nm and 330 nm by UV quantification, and then calculate according to the following formula. 100 μL, volume of each single strand in a 1 μM system:

V=100/[(A260-A330)×10 5 /(15.2×number of single-chain adenine+7.4×number of cytosine in single chain+11.4×number of guanine in single chain+8.3× single-chain thymus Number of pyrimidines)]

(2) According to the result calculated in the step (1), the dissolved DNA tetrahedron four single-chain solutions are taken up, mixed with TM buffer to 100 μL, vortexed and mixed, and finally placed in the PCR machine, and the temperature is rapidly raised to 95. °C is stable for 10min, then cooled to 4 °C for 20min, and finally stored at -20 °C.
The method for preparing a cationic polymer/TDNs drug-loaded complex according to claim 5, wherein the TM buffer in the step (3) is 5-10 mM Tris-HCl, 5-50 mM MgCl 2 , pH 8.0.
The method for preparing a cationic polymer/TDNs drug-loaded complex according to claim 6, wherein the TM buffer in the step (3) is 10 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0.
The cationic polymer/TDNs drug-loaded complex prepared by the method of any of claims 1-7.