Desoxyribonucleic acid-poly-L-lysine composite hydrogel and preparation method thereof

Abstract

The invention discloses a desoxyribonucleic acid-poly-L-lysine composite hydrogel and a preparation method thereof, the desoxyribonucleic acid-poly-L-lysine composite hydrogel comprises a DNA skeleton and poly-L-lysine wrapping the DNA skeleton, and the DNA skeleton contains a nucleic acid aptamer targeting exosome surface protein. By optimizing a cross-linking strategy (such as physical entanglement or chemical covalent bonds), the pore size and the stability of the gel are regulated and controlled, and the DNA aptamer capable of identifying the exosome surface protein in a targeted manner is introduced, so that efficient and specific exosome separation is expected to be realized, the key problems of low efficiency, poor specificity, insufficient stability and the like in exosome separation in the prior art are solved, and the method is suitable for large-scale industrial production. The method has wide application prospects in the fields of liquid biopsy, targeted therapy and the like, and a revolutionary technical platform is provided for clinical application of the exosome.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a deoxyribonucleic acid-poly-L-lysine composite hydrogel and its preparation method. Background Technology

[0002] In recent years, exosomes have attracted much attention due to their important role in disease diagnosis, drug delivery, and regenerative medicine. However, efficient separation and enrichment of exosomes remains a technical challenge. Traditional methods (such as ultracentrifugation and immunomagnetic beads) are complex, costly, and prone to damaging exosomes. Therefore, developing a mild, efficient, and tunable exosome separation material has significant scientific and clinical value.

[0003] Currently, hydrogel materials used for exosome separation mainly include the following technical solutions: 1. Natural polymer hydrogels (such as hyaluronic acid and collagen), which form porous network structures through physical cross-linking (temperature / pH triggering) or chemical cross-linking (glutaraldehyde / EDC-NHS). However, these methods suffer from low separation efficiency, lack of specific recognition sites, and uncontrollable degradation (>30 days). Giorgia Natalia Iaconisi's team and others discussed the low separation efficiency (dependent on passive diffusion) and uncontrollable degradation of hyaluronic acid hydrogels. Han Xianglong's research group also pointed out that collagen gels lack specific binding sites for exosomes and rely on non-specific adsorption (efficiency <40%).

[0004] 2. Synthesizing polymeric hydrogels (polyethylene glycol, polylactic acid-glycolic acid copolymer) involves constructing a cross-linked network through free radical polymerization. However, this method suffers from drawbacks such as reliance on UV light or high temperature for curing and lack of nuclease responsiveness. The polyethylene glycol hydrogel synthesized by Xie Huiqi's research group lacks nuclease responsiveness and cannot dynamically release isolated exosomes. Minchae Kim's team discovered that PLGA degradation products may acidify the microenvironment, potentially affecting exosome stability.

[0005] 3. Nucleic acid aptamer-modified materials, where aptamers (such as Apt CD63) are covalently coupled to the hydrogel backbone, suffer from the problem of aptamers being easily degraded by serum nucleases. Research by Lei Zheng's group has shown that unmodified DNA aptamers have a half-life of <2 hours in serum.

[0006] The common drawback of existing technologies is that efficiency and specificity cannot be achieved simultaneously. Natural materials rely on passive diffusion, synthetic materials lack biorecognition capabilities, and degradation relies on hydrolysis rather than physiological signals (such as nucleases), leading to uncontrollable release. Summary of the Invention

[0007] The purpose of this invention is to provide a deoxyribonucleic acid-poly-L-lysine composite hydrogel and its preparation method, which can achieve efficient and specific exosome separation.

[0008] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0009] A deoxyribonucleic acid-poly-L-lysine composite hydrogel includes a DNA backbone and poly-L-lysine residues encapsulating the DNA backbone, wherein the DNA backbone contains nucleic acid aptamers that target exosome surface proteins.

[0010] In one or more embodiments of the present invention, the exosome surface protein is CD63 protein, and the nucleic acid aptamer is CD63 DNA aptamer.

[0011] In one or more embodiments of the present invention, the CD63 DNA aptamer sequence is shown in SEQ ID NO.5.

[0012] Another specific embodiment of the present invention provides the following technical solution:

[0013] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel, the method comprising the following steps:

[0014] Rolling circle amplification was performed using a circular DNA template containing the antisense sequence of the CD63 DNA aptamer to obtain the RCA product; then the RCA product was inactivated.

[0015] Poly-L-lysine was dissolved in water to obtain a poly-L-lysine solution;

[0016] The inactivated RCA product was mixed with a poly-L-lysine solution and incubated to obtain a deoxyribonucleic acid-poly-L-lysine composite hydrogel.

[0017] In one or more embodiments of the present invention, the circular DNA template containing the CD63 DNA aptamer antisense sequence is prepared as follows:

[0018] Design and synthesize 5' phosphorylated modified ssDNA-1, wherein the ssDNA-1 contains the CD63 DNA aptamer complementary sequence;

[0019] Design primer one for ssDNA-1, with the 5' and 3' ends of ssDNA-1 being complementary to the 3' and 5' ends of primer one, respectively.

[0020] Mix ssDNA-1 and primer 1, add sodium chloride, and use sterile water to make up to the appropriate volume. Synthesize circular DNA-1 with a notch at the end by heating and annealing.

[0021] Circular DNA-1, T4 DNA ligase, and buffer were mixed and reacted at 4 ℃-16 ℃ for 6 h-12 h to obtain a circular DNA template containing the CD63 DNA aptamer antisense sequence.

[0022] In one or more embodiments of the present invention, the ssDNA-1 and primer 1 are mixed in a molar ratio of 1:1 to 1:1.2, and NaCl with a final concentration of 80 mmol / L is added, and the volume is brought up to 20 μL with sterile water.

[0023] In one or more embodiments of the present invention, the concentration of the poly-L-lysine solution is 20 μmol / L to 160 μmol / L.

[0024] In one or more embodiments of the present invention, the rolling circle amplification operation is as follows: 5×10 -3 nmol-1×10 -2 The following solutions were prepared: a circular DNA template (nmol), phi29 DNA polymerase (final concentration 0.2 U / µL-0.5 U / µL), phi29 DNA polymerase buffer (final concentration 1×phi29 DNA polymerase), BSA (final concentration 0.2 mg / mL), dNTPs (final concentration 0.5 mmol / L-1 mmol / L), and NaCl (final concentration 80 mmol / L). The mixture was then brought to a final volume of 100 µL with sterile water. The mixture was incubated at 350 rpm-450 rpm at 35 ℃-37 ℃ for 4 h-16 h.

[0025] In one or more embodiments of the present invention, the inactivation conditions of the RCA product are: 65 ℃-75 ℃, for 8 min-12 min.

[0026] In one or more embodiments of the present invention, the inactivated RCA product and the poly-L-lysine solution are mixed and shaken at 350 rpm-450 rpm for 4 h-16 h.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. Regarding molecular recognition mechanisms, traditional methods often employ direct modification with short-chain DNA aptamers, which suffers from limited recognition sites and weak binding affinity. In contrast, this invention utilizes rolling circle amplification technology to construct ultra-long DNA chains, where repeating CD63 aptamer units form a multivalent binding interface, increasing exosome separation efficiency to over 85%. This three-dimensional network structure not only enhances separation capability but also significantly improves recognition specificity.

[0029] 2. Regarding stability, existing DNA materials generally face the challenge of easy degradation by nucleases. This invention creatively introduces poly-L-lysine (PLL) as a protective layer, which encapsulates the DNA backbone through electrostatic interactions, forming a unique "molecular armor" structure. Experiments have confirmed that this design extends the half-life of DNA in a serum environment to over 24 hours, fully meeting the needs of clinical applications. Compared to existing exosome separation technologies, this invention offers superior overall performance: high separation efficiency, significantly improved ease of operation, and complete avoidance of chemical cross-linking agents.

[0030] 3. This invention has advantages in clinical translation. Currently, in vitro separation systems require complex operating equipment. This invention has no toxic residues (no chemical cross-linking agent required) and is compatible with image monitoring (fluorescent agents can be incorporated to achieve visualization). Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 Electrophoresis images of the synthesized circular DNA templates used in Examples 2 and 5 of this invention;

[0033] Figure 2 This is a photograph of the deoxyribonucleic acid-poly-L-lysine composite hydrogel in Example 11 of the present invention;

[0034] Figure 3 This is a DNA-PLL hydrogel cytotoxicity diagram from one embodiment of the present invention;

[0035] Figure 4 This is a confocal fluorescence image of exosomes on a DNA-PLL hydrogel in one embodiment of the present invention;

[0036] Figure 5 This is a nuclease resistance diagram of a DNA-PLL hydrogel in one embodiment of the present invention;

[0037] Figure 6 This is a diagram showing the efficiency of DNA-PLL hydrogel separation of exosomes and electrostatic adsorption in one embodiment of the present invention. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0039] One specific embodiment of the present invention provides a deoxyribonucleic acid-poly-L-lysine composite hydrogel, which includes a DNA backbone and poly-L-lysine encapsulating the DNA backbone, wherein the DNA backbone contains nucleic acid aptamers that target exosome surface proteins.

[0040] Specifically, firstly, the DNA backbone is repeatedly arranged with nucleic acid aptamers that target exosome surface proteins, forming multivalent binding interfaces and increasing exosome separation efficiency to over 85%, enhancing not only separation capability but also recognition specificity. Secondly, poly-L-lysine (PLL) acts as a protective layer, encapsulating the DNA backbone through electrostatic interactions to form a unique "molecular armor" structure, extending the DNA's half-life in serum to over 24 hours, meeting clinical application requirements.

[0041] Furthermore, the exosome surface protein is CD63 protein, and the nucleic acid aptamer is CD63 DNA aptamer. The CD63 DNA aptamer sequence is shown in SEQ ID NO.5, specifically CACCCCACCTCGCTCCCGTGACACTAATGCTA. Of course, in other embodiments, different nucleic acid aptamers can be selected according to the type of exosome surface protein.

[0042] Another specific embodiment of the present invention provides a method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel, which specifically includes the following steps:

[0043] Step 1: Design the antisense sequence of the CD63 DNA aptamer onto the circular DNA template.

[0044] Specifically, 5' phosphorylated modified ssDNA-1 was designed and synthesized, and primer one for ssDNA-1 was designed. The 5' and 3' ends of ssDNA-1 were complementary to the 3' and 5' ends of primer one, respectively.

[0045] Phosphorylated ssDNA-1 at the 5' end and primer 1 were mixed in a molar ratio of 1:1 to 1:1.2. NaCl was added to a final concentration of 80 mmol / L, and sterile water was added to bring the volume to 20 μL. Circular DNA-1 with a nick at the end and a final concentration of 10 mmol / L was synthesized according to the designed heating annealing program. When ssDNA-1 and primer 1 were mixed in a 1:1 ratio, the concentration of ssDNA-1 in the system was 100 mmol / L, and the concentration of primer 1 was 100 mmol / L.

[0046] Add 1 U of T4 DNA ligase to 100 ng of circular DNA-1, and react at 4℃-16℃ for 6 h-12 h in a final concentration of 1 × T4 buffer to obtain the circular DNA template product.

[0047] Step 2: Use a circular DNA template containing the antisense sequence of the CD63 DNA aptamer for rolling circle amplification to obtain the RCA product.

[0048] Specifically, 5×10 -3 -1×10 -2 A circular DNA template of nmol, phi29 DNA polymerase at a final concentration of 0.2 U / µL-0.5 U / µL, phi29 DNA polymerase buffer at a final concentration of 1×phi29 DNA polymerase, BSA at a final concentration of 0.2 mg / mL, dNTPs at a final concentration of 0.5 mmol / L-1 mmol / L, and NaCl at a final concentration of 80 mmol / L are mixed together and then brought to a final volume of 100 µL with sterile water. The mixture is then shaken at 350 rpm-450 rpm at 35 ℃-37 ℃ for 4 h-16 h to obtain the RCA product.

[0049] Step 3: Inactivate RCA products.

[0050] Specifically, the inactivation conditions are: 65 ℃-75 ℃, time 8 min-12 min, preferably inactivation at 65 ℃ for 10 min.

[0051] Step 4: Mix the inactivated RCA product with the poly-L-lysine solution and incubate to obtain a deoxyribonucleic acid-poly-L-lysine composite hydrogel.

[0052] Specifically, poly-L-lysine was dissolved in water to obtain a poly-L-lysine solution with a concentration of 20 μmol / L–160 μmol / L. The inactivated RCA product and the poly-L-lysine solution were mixed and shaken at 350 rpm–450 rpm for 4–16 h.

[0053] The present invention will be further described in detail below with reference to specific embodiments.

[0054] In this invention, RCA is short for rolling circle amplification, BSA is short for bovine serum albumin, and ssDNA is short for linear single-stranded DNA.

[0055] Example 1

[0056] Preparation method of circular DNA template:

[0057] The sequences of 5' phosphorylated ssDNA-1, primer 1, 5' phosphorylated ssDNA-2, primer 2, and CD63 aptamer are shown in Table 1.

[0058] Table 1: Deoxynucleotide sequences

[0059]

[0060] Preparation of circular DNA template:

[0061] We designed and synthesized ssDNA-1 with phosphorylation modification at the 5' end. The ssDNA-1 has 128 bases. We designed primer 1 for ssDNA-1 with 20 bases. The 3' and 5' ends of primer 1 are complementary to the 5' and 3' ends of ssDNA-1, respectively.

[0062] ssDNA-1 contains a complementary sequence to the aptamer AptCD63.

[0063] The nucleotide sequence of ssDNA-1 is shown in SEQ ID NO.1, as shown in Table 1.

[0064] The nucleotide sequence of primer 1 is shown in SEQ ID NO.3, as shown in Table 1.

[0065] The nucleotide sequence of aptamer AptCD63 is shown in SEQ ID NO.5, as shown in Table 1.

[0066] Phosphorylated ssDNA-1 at the 5' end and primer 1 were mixed in a 1:1 molar ratio. NaCl was added to a final concentration of 80 mmol / L, and the volume was adjusted to 20 µL with sterile water. The concentrations of ssDNA-1 and primer 1 were both 100 mmol / L. Circular DNA-1 with a notched end was synthesized according to the following heating annealing procedure.

[0067] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0068] Step 1: 95 ℃ for 2 min;

[0069] Step 2: 65 ℃ for 2 min;

[0070] Step 3: 60 ℃ for 5 min 30 s;

[0071] Step 4: 20 ℃ for 30 s;

[0072] Step 5: 4 ℃ for 10 min.

[0073] Add 2 U of T4 DNA ligase to 200 ng of the product to a final concentration of 1 × T4 buffer, and incubate at 22 °C for 8 hours to obtain circular DNA template 1, which is then stored at 4 °C.

[0074] Example 2

[0075] DNA template preparation method:

[0076] The sequences of ssDNA-1 and primer 1 with 5' phosphorylation modification and ssDNA-2 and primer 2 with 5' phosphorylation modification are shown in Table 2:

[0077] Table 2: Deoxynucleotide sequences

[0078]

[0079] Preparation of circular DNA template 1:

[0080] a. Design and synthesize ssDNA-1 with phosphorylation modification at the 5' end. The number of bases in ssDNA-1 is 92. Design primer 1 for ssDNA-1 with 24 bases. The 3' and 5' ends of primer 1 are complementary to the 5' and 3' ends of ssDNA-1, respectively.

[0081] ssDNA-1 contains a complementary sequence to the aptamer AptCD63.

[0082] The nucleotide sequence of ssDNA-1 is shown in SEQ ID NO.6, as shown in Table 2.

[0083] The nucleotide sequence of primer 1 is shown in Table 2 as SEQ ID NO. 8.

[0084] b. Mix 5' phosphorylated ssDNA-1 and primer 1 at a molar ratio of 1:1, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of ssDNA-1 and primer 1 are both 100 mmol / L. Synthesize nicked circular DNA-1 using the following heating annealing procedure.

[0085] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0086] Step 1: 95 ℃ for 2 min;

[0087] Step 2: 65 ℃ for 2 min;

[0088] Step 3: 60 ℃ for 5 min 30 s;

[0089] Step 4: 20 ℃ for 30 s;

[0090] Step 5: 4 ℃ for 10 min.

[0091] c. Add 2 U of T4 DNA ligase to 200 ng of the product to a final concentration of 1 × T4 buffer. Incubate at 22 °C for 8 hours to obtain circular DNA template 1, and store at 4 °C.

[0092] In this embodiment, the electrophoresis diagram of the synthesized circular DNA template is shown below. Figure 1 As shown in the figure, Template 1 refers to ssDNA-1, and Circ-DNA 1 refers to circular DNA template one. As can be seen from the figure, ssDNA-1 was successfully circularized into circular DNA.

[0093] Example 3

[0094] DNA template preparation method:

[0095] The sequences of ssDNA-1 and primer 1 with 5' phosphorylation modification and ssDNA-2 and primer 2 with 5' phosphorylation modification are shown in Table 3:

[0096] Table 3: Deoxynucleotide sequences

[0097]

[0098] Preparation of circular DNA template 1:

[0099] a. Design and synthesize ssDNA-1 with phosphorylation modification at the 5' end. The number of bases in ssDNA-1 is 174. Design primer 1 for ssDNA-1 with 27 bases. The 3' and 5' ends of primer 1 are complementary to the 5' and 3' ends of ssDNA-1, respectively.

[0100] ssDNA-1 contains a complementary sequence to the aptamer AptCD63.

[0101] The nucleotide sequence of ssDNA-1 is shown in SEQ ID NO.10, as shown in Table 3.

[0102] The nucleotide sequence of primer 1 is shown in Table 3 as SEQ ID NO.12.

[0103] b. Mix 5' phosphorylated sDNA-1 and primer 1 in a 1:1 molar ratio, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of ssDNA-1 and primer 1 are both 100 mmol / L. Synthesize nicked circular DNA-1 using the following heating annealing procedure.

[0104] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0105] Step 1: 95 ℃ for 2 min;

[0106] Step 2: 65 ℃ for 2 min;

[0107] Step 3: 60 ℃ for 5 min 30 s;

[0108] Step 4: 20 ℃ for 30 s;

[0109] Step 5: 4 ℃ for 10 min.

[0110] c. Add 2 U of T4 DNA ligase to 200 ng of the product to a final concentration of 1 × T4 buffer. Incubate at 22 °C for 8 hours to obtain circular DNA template 1, and store at 4 °C.

[0111] Example 4

[0112] Method for preparing DNA template II:

[0113] The circular DNA template 2 was prepared using the following method:

[0114] a. Design and synthesize ssDNA-2 with phosphorylation modification at the 5' end. The number of bases in ssDNA-2 is 128. Design primer 2 for ssDNA-2 with 20 bases. The 5' end and 3' end of ssDNA-2 are complementary to the 3' end and 5' end of primer 2, respectively.

[0115] The nucleotide sequence of ssDNA-2 is shown in SEQ ID NO.2, as shown in Table 1.

[0116] The nucleotide sequence of primer 2 is shown in SEQ ID NO.4, as shown in Table 1.

[0117] Phosphorylated ssDNA-2 at the 5' end and primer 2 were mixed in a 1:1 molar ratio. NaCl was added to a final concentration of 80 mmol / L, and the volume was adjusted to 20 µL with sterile water. The concentrations of both ssDNA-2 and primer 2 were 100 mmol / L. Circular DNA-2 with a notched end was synthesized according to the following heating annealing procedure.

[0118] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0119] Step 1: 95 ℃ for 2 min;

[0120] Step 2: 65 ℃ for 2 min;

[0121] Step 3: 60 ℃ for 5 min 30 s;

[0122] Step 4: 20 ℃ for 30 s;

[0123] Step 5: 4 ℃ for 10 min.

[0124] c. Add 2 U of T4 DNA ligase to 200 ng of the product obtained in step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 5 hours to obtain circular DNA template II, which is then stored at 4 °C.

[0125] Example 5

[0126] Method for preparing DNA template II:

[0127] The circular DNA template 2 was prepared using the following method:

[0128] a. Design and synthesize ssDNA-2 with phosphorylation modification at the 5' end. The number of bases in ssDNA-2 is 92. Design primer 2 for ssDNA-2 with 24 bases. The 5' end and 3' end of ssDNA-2 are complementary to the 3' end and 5' end of primer 2, respectively.

[0129] The nucleotide sequence of ssDNA-2 is shown in SEQ ID NO.7, as shown in Table 2.

[0130] The nucleotide sequence of primer 2 is shown in SEQ ID NO.9, as shown in Table 2.

[0131] b. Mix 5' phosphorylated ssDNA-2 and primer 2 at a molar ratio of 1:1, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of both ssDNA-2 and primer 2 are 100 mmol / L. Synthesize terminally nicked circular DNA-2 according to the following heating annealing procedure.

[0132] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0133] Step 1: 95 ℃ for 2 min;

[0134] Step 2: 65 ℃ for 2 min;

[0135] Step 3: 60 ℃ for 5 min 30 s;

[0136] Step 4: 20 ℃ for 30 s;

[0137] Step 5: 4 ℃ for 10 min.

[0138] c. Add 2 U of T4 DNA ligase to 200 ng of the product obtained in step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 5 hours to obtain circular DNA template II, which is then stored at 4 °C.

[0139] In this embodiment, the electrophoresis diagram of the synthesized circular DNA template is shown below. Figure 1 As shown in the figure, Template 2 refers to ssDNA-2, and Circ-DNA 2 refers to circular DNA template 2. As can be seen from the figure, ssDNA-2 was successfully circularized into circular DNA.

[0140] Example 6

[0141] Method for preparing DNA template II:

[0142] The circular DNA template 2 was prepared using the following method:

[0143] a. Design and synthesize ssDNA-2 with phosphorylation modification at the 5' end. The number of bases in ssDNA-2 is 174. Design primer 2 for ssDNA-2 with 27 bases. The 5' end and 3' end of ssDNA-2 are complementary to the 3' end and 5' end of primer 2, respectively.

[0144] The nucleotide sequence of ssDNA-2 is shown in SEQ ID NO.11, as shown in Table 3.

[0145] The nucleotide sequence of primer 2 is shown in SEQ ID NO.13, as shown in Table 3.

[0146] Phosphorylated ssDNA-2 at the 5' end and primer 2 were mixed in a 1:1 molar ratio. NaCl was added to a final concentration of 80 mmol / L, and the volume was adjusted to 20 µL with sterile water. The concentrations of both ssDNA-2 and primer 2 were 100 mmol / L. Circular DNA-2 with a notched end was synthesized according to the following heating annealing procedure.

[0147] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0148] Step 1: 95 ℃ for 2 min;

[0149] Step 2: 65 ℃ for 2 min;

[0150] Step 3: 60 ℃ for 5 min 30 s;

[0151] Step 4: 20 ℃ for 30 s;

[0152] Step 5: 4 ℃ for 10 min.

[0153] c. Add 2 U of T4 DNA ligase to 200 ng of the product obtained in step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 5 hours to obtain circular DNA template II, which is then stored at 4 °C.

[0154] Example 7

[0155] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0156] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0157] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 20 µmol / L).

[0158] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P20, which is used to separate exosomes.

[0159] Implementing Column 8

[0160] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0161] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0162] b. Add 2 µL of PLL solution (concentration of 20 µmol / L) to the above RCA product mixture system.

[0163] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P20 (without AptCD63 aptamer), thus obtaining the nD-P20 hydrogel for capturing exosomes.

[0164] Example 9

[0165] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0166] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0167] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 40 µmol / L).

[0168] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P40, which is used to separate exosomes.

[0169] Implementation of Column 10

[0170] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel:

[0171] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0172] b. Add 2 µL of PLL solution (concentration of 40 µmol / L) to the above RCA product mixture system.

[0173] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P40 (without AptCD63 aptamer), thus obtaining an nD-P40 hydrogel for capturing exosomes.

[0174] Example 11

[0175] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0176] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0177] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 80 µmol / L).

[0178] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled D-P80, thus obtaining the D-P80 hydrogel for exosome separation, as detailed below. Figure 2 As shown.

[0179] Implementation of Column 12

[0180] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0181] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0182] b. Add 2 µL of PLL solution (concentration of 80 µmol / L) to the above RCA product mixture.

[0183] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P80 (without AptCD63 aptamer), thus obtaining the nD-P80 hydrogel for capturing exosomes.

[0184] Example 13

[0185] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel:

[0186] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0187] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 160 µmol / L).

[0188] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P160, thus obtaining the D-P160 hydrogel for separating exosomes.

[0189] Implementation List 14

[0190] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0191] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0192] b. Add 2 µL of PLL solution (concentration of 160 µmol / L) to the above RCA product mixture.

[0193] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P160 (without AptCD63 aptamer), thus obtaining the nD-P160 hydrogel for capturing exosomes.

[0194] Example 15

[0195] A cytotoxicity assessment assay for DNA-PLL hydrogels used to isolate exosomes includes the following steps:

[0196] a. Immerse 22 mg of DNA-PLL hydrogel used for exosome isolation into 1 mL of complete culture medium to obtain the hydrogel extract. Add 5 × 10⁻⁶ mg of the extract to each well of a 96-well plate. 3 H9C2 cells were added to a 96-well plate with an extraction solution and cell culture medium at a volume ratio of 1:9. The plate was then incubated at 37 °C for 24 hours in a CO2 incubator.

[0197] b. Aspirate the supernatant, add 90 µL of cell culture medium, then add 10 µL of MTT solution, and incubate at 37°C for 4 hours in a CO2 incubator. Then, measure the absorbance at 490 nm using a microplate reader and calculate the relative cell viability of each sample well. Experimental results are shown below. Figure 3 A.

[0198] The experimental results showed that the cell viability of H9C2 cells treated with different concentrations of DNA-PLL hydrogel extract was not significantly different from that of the control group (unincubated with extract).

[0199] Example 16

[0200] A cytotoxicity assessment assay for DNA-PLL hydrogels used to isolate exosomes includes the following steps:

[0201] a. Treat 22 mg of DNA-PLL hydrogel used for exosome isolation with 1 U DNase I to obtain hydrogel degradation products. Add 5 × 10⁻⁶ mg of the degradation product to each well of a 96-well plate. 3 H9C2 cells were added to a 96-well plate at a volume ratio of 1:9, and the plate was incubated at 37 °C for 24 hours in a CO2 incubator.

[0202] b. Aspirate the supernatant, add 90 µL of cell culture medium, then add 10 µL of MTT solution, and incubate at 37°C for 4 hours in a CO2 incubator. Then, measure the absorbance at 490 nm using a microplate reader and calculate the relative cell viability of each sample well. Experimental results are shown below. Figure 3 B.

[0203] Experimental results showed that the cell viability of H9C2 cells treated with degradation products of different DNA-PLL hydrogels catalyzed by DNase I was not significantly different from that of the control group (incubated with an equal volume of PBS). Both cytotoxicity assays indicated that the DNA-PLL hydrogels used for exosome isolation were non-cytotoxic.

[0204] Example 17

[0205] Validation of DNA-PLL hydrogel capture of exosomes includes the following steps:

[0206] a. The RCA products obtained in Examples 7-14 were incubated with SYBR Green II at 37 °C and 450 rpm for 30 minutes.

[0207] b. Incubate the exosomes with CM-DiI at 37 ℃ and 450 rpm for 30 minutes.

[0208] c. Incubate the RCA products obtained in steps a and b with exosomes at 37 °C and 450 rpm for 30 minutes.

[0209] d. Add PLL solutions of the corresponding concentrations according to Examples 7-14, and incubate at 37 °C and 450 rpm for 30 minutes.

[0210] e. Observe and photograph using an inverted fluorescence microscope; results are shown below. Figure 4 .

[0211] The results showed that, compared with the nD-P20, nD-P40, nD-P80, and nD-P160 groups, the exosome-specific fluorescence signals of the D-P20, D-P40, D-P80, and D-P160 groups were stronger, confirming the key role of AptCD63 in mediating exosome-specific recognition and enrichment.

[0212] Example 18

[0213] A DNA-PLL hydrogel nucleic acid resistance assay for exosome isolation includes the following steps:

[0214] a. The concentration of DNA in a 200 µL ssDNA strand solution was measured using a micro-spectrophotometer (C0). After forming a DNA-PLL hydrogel, the concentration of DNA in the supernatant was measured (C1).

[0215] b. Then place the DNA-PLL hydrogel in DMEM-H medium to a total volume of 200 µL and measure the initial DNA concentration in the supernatant.

[0216] c. Incubate the entire system at 37°C for 30 days, measuring the DNA concentration (C) in the supernatant every 24 hours. The degradation rate is calculated using the formula: The experimental results are shown in [the table]. Figure 5 , Figure 5 D-PO in Example 2 refers to the RCA product without the addition of PLL solution.

[0217] Experimental results showed that D-P20, D-P40, D-P80, and D-P160 were completely dissolved at 2, 5, 7, and 11 days, respectively. These results confirm that the polylysine protective coating effectively prevents nucleases from digesting DNA molecules in the hydrogel, supporting the feasibility of expanding in vivo applications.

[0218] Example 19

[0219] A quantitative analysis of the exosome separation amount and electrostatic adsorption ratio using a DNA-PLL hydrogel for exosome separation includes the following steps:

[0220] a. After exosome capture, collect CD63 aptamer (AptCD63) functionalized DNA-PLL hydrogels (DP) and non-functionalized control hydrogels (nD-P). Immerse these exosome capture hydrogels and blank hydrogels in 1×PBS at 4 °C for 2 hours, remove the supernatant, and repeat the washing step twice.

[0221] b. All hydrogels were physically disintegrated by ultrasonic disruption and then centrifuged at 12,000×g for 30 minutes at 4 °C to obtain the supernatant;

[0222] c. Collect the supernatant and determine the total protein concentration using the BCA method; establish a standard curve using BSA standards, calculate the total amount of exosome-related proteins, and calculate the exosome capture efficiency based on the protein content of exosomes per 1 mg of hydrogel, and use the difference to calculate electrostatic adsorption. Experimental results are shown in […]. Figure 6 , Figure 6 In Example 2, nD-PO refers to the RCA product (without PLL solution) and nD-PO refers to the RCA product (without PLL solution) in Example 4.

[0223] The results showed that the exosome capture efficiency gradually increased from D-P20 (2.5 µg / mg) to D-P80 (11.2 µg / mg), indicating that a higher cross-linking density can better preserve the accessibility and recognition ability of aptamers. Electrostatic adsorption accounted for 94.7%, 22.8%, 14.3%, and 31.0% of the total exosome capture in D-P20, D-P40, D-P80, and D-P160, respectively, highlighting the dominant role of aptamer-mediated specific recognition in the hydrogel network's capture of exosomes. This demonstrates that the DNA-PLL hydrogel achieves efficient separation of exosomes through the electrostatic interaction of PLLs and the specific recognition of the DNA aptamer (AptCD63).

[0224] In summary, this invention optimizes cross-linking strategies (such as physical entanglement or chemical covalent bonds) to regulate gel pore size and stability, and introduces DNA aptamers that can target and recognize exosome surface proteins. This invention is expected to achieve efficient and specific exosome separation, solving key problems such as low efficiency, poor specificity, and insufficient stability in existing exosome separation technologies. It has broad application prospects in liquid biopsy, targeted therapy, and other fields, and provides a revolutionary technical platform for the clinical application of exosomes.

[0225] Compared with existing technologies, the main differences of this invention include: a. Material innovation: The DNA-PLL dynamic network combines targeting (CD63 aptamer) and protection functions, with a separation efficiency >85% and aptamer stability >48 h. b. Intelligent release: Nuclease / microenvironment triggers precise controlled release, superior to traditional passive diffusion or uncontrolled degradation. c. Gentle injection: Biocompatibility >95%, avoiding toxic cross-linking agents.

[0226] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0227] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A DNA-poly-L-lysine complex hydrogel, characterized by, The DNA backbone contains a nucleic acid aptamer targeting an exosome surface protein, and the poly-L-lysine wraps the DNA backbone.

2. The DNA-poly-L-lysine complex hydrogel according to claim 1, wherein The exosome surface protein is a CD63 protein, and the nucleic acid aptamer is a CD63 DNA aptamer.

3. The DNA-poly-L-lysine complex hydrogel according to claim 2, wherein The sequence of the CD63 DNA aptamer is shown in SEQ ID NO.

5.

4. A method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 1, characterized in that, The preparation method comprises the following steps: Rolling circle amplification is performed using a circular DNA template containing a CD63 DNA aptamer antisense sequence to obtain an RCA product; and the RCA product is inactivated; Poly-L-lysine is dissolved in water to obtain a poly-L-lysine solution; The inactivated RCA product and the poly-L-lysine solution are mixed and incubated to obtain a deoxyribonucleic acid-poly-L-lysine composite hydrogel.

5. The method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 4, characterized in that, The circular DNA template containing the CD63 DNA aptamer antisense sequence is prepared as follows: A 5'-end phosphorylation modified ssDNA-1 containing a CD63 DNA aptamer complementary sequence is designed and synthesized; A primer one of the ssDNA-1 is designed, and the 5' end and the 3' end of the ssDNA-1 are complementary to the 3' end and the 5' end of the primer one, respectively; The ssDNA-1 and the primer one are mixed, sodium chloride is added, sterile water is added to the appropriate volume, and a heating annealing program is used to synthesize a circular DNA-1 with a notched end; The circular DNA-1, T4 DNA ligase and buffer are mixed, and the reaction is carried out at 4-16 ℃ for 6-12 h to obtain a circular DNA template containing a CD63 DNA aptamer antisense sequence.

6. The method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 5, characterized in that, The ssDNA-1 and the primer one are mixed at a molar ratio of 1:1-1:1.2, NaCl is added at a final concentration of 80 mmol / L, and sterile water is added to 20 μL.

7. The method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 4, characterized in that, The concentration of the poly-L-lysine solution is 20-160 μmol / L.

8. The method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 4, characterized in that, The rolling circle amplification operation is: mixing 5×10 -3 nmol-1×10 -2 nmol of circular DNA template, a final concentration of 0.2 U / µL-0.5 U / µL of phi29 DNA polymerase, a final concentration of 1×phi29 DNA polymerase buffer, a final concentration of 0.2 mg / mL of BSA, a final concentration of 0.5 mmol / L-1 mmol / L of dNTPs, a final concentration of 80 mmol / L of NaCl, and using sterile water to make up to 100 µL; at a speed of 350 rpm-450 rpm, 35 ℃-37 ℃ shaking for 4 h-16 h.

9. The method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 4, characterized in that, The inactivation conditions of the RCA product are 65-75 ℃ for 8-12 min.

10. The method for preparing the deoxyribonucleic acid-poly-L-lysine composite hydrogel according to claim 4, characterized in that, After the inactivated RCA product and the poly-L-lysine solution are mixed, they are shaken at 350-450 rpm for 4-16 h.

Images