CN115227838B - Preparation method and application of aptamer combined with nanobubble to construct nano assembly - Google Patents

Preparation method and application of aptamer combined with nanobubble to construct nano assembly Download PDF

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CN115227838B
CN115227838B CN202210519896.9A CN202210519896A CN115227838B CN 115227838 B CN115227838 B CN 115227838B CN 202210519896 A CN202210519896 A CN 202210519896A CN 115227838 B CN115227838 B CN 115227838B
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陈建福
陆永萍
梁还
孙月
杨寒凝
徐丽荣
王禹雪
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Kunming Medical University
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Abstract

The invention discloses a preparation method and application of a nano assembly constructed by aptamer combined with nano microbubbles, and the preparation method comprises the following steps: (1) preparing PLGA nano microvesicle; (2) nano microbubble lyophilized powder PLGANBs; (3) Au @ PLGANBs lyophilized powder; (4) nanometer assembly Apt @ Au @ PLGANBs freeze-dried powder. The nano assembly can be used as an efficient, stable and nontoxic ultrasonic contrast agent, can realize the development outside a blood pool, has higher diagnosis accuracy compared with the traditional micron-sized contrast agent, and achieves the effect of accurate positioning by the combination of the specific aptamer; meanwhile, different focuses (tumors, thrombi, plaques and the like) can be connected with corresponding therapeutic drugs or genes for precise treatment, so that the toxic and side effects of the drugs are greatly reduced.

Description

Preparation method and application of aptamer combined with nanobubble to construct nano assembly
Technical Field
The invention relates to the technical field of nano-drugs, in particular to a preparation method and application of a nano-assembly constructed by combining an aptamer and nano-microbubbles.
Background
Currently, targeting of nano drugs is mostly achieved by using protein antibodies as targeting guides, and the antibodies as guides have the limitations of large molecular weight, instability, inability of in vitro synthesis, high cost and the like. In order to overcome the limitation, the invention discovers a small molecular substance-aptamer serving as a targeting guide, and the aptamer has high affinity, strong specificity, specific recognition capability and better guiding effect compared with an antibody.
Aptamers are composed of single-stranded nucleotides (short RNA or single-stranded DNA sequences) obtained by exponential enrichment (SELEX) screening, which when folded into a unique three-dimensional conformation, have high specificity and affinity for specific binding to homologous targets. Although aptamers are functionally similar to protein antibodies, in biomedical and clinical applications, aptamers have the following advantages: (1) can be obtained by in vitro processes, and can bind ligands not recognized by antibodies, such as inorganic ions and small molecules; (2) can be artificially synthesized by Polymerase Chain Reaction (PCR), and has higher time efficiency and lower cost; (3) the chemical structure is simple, and the aptamer is easier to modify functional groups; (4) due to the non-immunogenicity characteristic, the protein is not easy to be decomposed by protease, and has high safety and strong stability.
Therefore, the selection of the nano material and the efficient and stable combination mode of the aptamer and the nanoparticle become key steps for breaking through the research field.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method and application of a green, efficient and novel aptamer combined nano microbubble constructed nano assembly.
In order to achieve the purpose, the invention adopts the following technical scheme: the invention relates to a preparation method of a nano assembly constructed by aptamer combined with nano microbubbles, which is characterized by comprising the following steps: (1) preparation of PLGA nanobubbles: preparing polylactic-co-glycolic acid (PLGA) nano microbubbles by adopting a single emulsification method;
(2) Nano microbubble PLGANBs freeze-dried powder: placing the nano microbubbles prepared in the step (1) in a freeze dryer to prepare freeze-dried powder, and then filling perfluoropropane C3F8 gas-filled vesicles under a vacuum condition to obtain PLGANBs freeze-dried powder;
(3) Au @ PLGANBs lyophilized powder: preparing nano gold Au particles by adopting a sodium borohydride reduction method; dissolving the freeze-dried powder prepared in the step (2) in a polyallylamine hydrochloride (PAH) solution, coating nanogold on the surface of PLGA micro-bubble by adopting an electrostatic adsorption method and a seed growth method to form a nanogold shell, and then carrying out freeze-drying to obtain Au @ PLGANBs freeze-dried powder;
(4) Nanometer assembly Apt @ Au @ PLGANBs freeze-dried powder: 3 of the aptamer ， Modifying sulfydryl-SH, dissolving a proper amount of the Au @ PLGANBs freeze-dried powder prepared in the step (3) in enzyme-free water, adding a proper amount of thiolated Aptamer Aptamer (Apt) into the solution, magnetically stirring the solution under an ice bath condition for reaction overnight, washing and centrifuging the reaction solution by PBS, removing redundant aptamers, and fully reacting nano-gold and sulfydryl to form a stable Au-S bond so that the aptamers are stably bonded on the surface of Au @ PLGA nano-microbubbles; freeze drying to obtain nanometer assembly Apt @ Au @ PLGANBs freeze dried powder.
Further, in step (1), PLGA nanobubbles were prepared: step1: preparing a basic solvent:
polyvinyl alcohol PVA solution: weighing 0.4g of PVA crystal, dissolving the PVA crystal in 20mL of ultrapure water, rapidly stirring the PVA crystal by a constant-temperature magnetic stirrer (at 80 ℃) until the PVA crystal is completely dissolved, and storing the PVA crystal in a refrigerator at 4 ℃ for later use;
polyallylamine hydrochloride PAH solution: weighing 100mg of PAH and 292.5mg of NaCl, and dissolving in 100mL of ultrapure water to obtain a required PAH solution;
step 2: weighing 80-100mg of PLGA and 8-10mg of camphor, dissolving in 3-4ml of dichloromethane, dropwise adding the solution into the PVA solution prepared in the step (1), and emulsifying by using 400W ultrasonic waves for 3-5min (On 4 s, off4 s) under the condition of ice-water bath to obtain white emulsion, thus obtaining the PLGA nano microbubble.
Further, in the step (2), adding 20ml of isopropanol solution (2%) into the emulsion prepared in the step (1) for stabilization, performing rotary evaporation for 4-6h to fully volatilize dichloromethane, centrifuging the solution (5 min, 2000rpm) to remove precipitates, taking supernate, centrifuging again (4 min, 15000rpm), washing for 3 times, and performing vacuum freeze drying on the precipitates for 18-24h;
and filling the obtained freeze-dried powder with C3F8 gas under a vacuum condition to prepare the perfluoropropane gas-encapsulated nano microbubble freeze-dried powder PLGANBS.
Further, in the step (3), 1-2mL of each of the chloroauric acid solution (1%) and the sodium citrate solution (1%) is added into 90-100mL of ultrapure water, stirred by a magnetic stirrer for 10min, then added with 1-2mL of sodium citrate solution containing 0.075% of sodium borohydride, and continuously stirred for 10-15min to prepare gold nano-solution Au NPs;
dissolving the prepared PLGANBs in 10mL of the PAH solution prepared in the step (1), stirring for 30-40min, centrifuging and washing for several times, and taking precipitates to obtain PAH-coated PLGANBs;
adding the prepared Au NPs solution into the obtained precipitate, stirring for 30min, centrifugally washing for 3 times, dissolving the precipitate in 2-3mL of 1% chloroauric acid solution, stirring for 30min, dropwise adding 300-400uL of hydroxylamine hydrochloride solution, stirring for 30min, centrifugally washing for 2-3 times, and freeze-drying the precipitate in vacuum for 18-24h to obtain Au @ PLGANBs freeze-dried powder.
Further, in the step (3), the aptamer takes the atherosclerotic macrophage-derived foam cell as a target, and the gene sequence of the aptamer is the nucleotide sequence shown in SEQ ID No. 1.
Further, in the step (4), a proper amount of the Au @ PLGANBs freeze-dried powder prepared in the step (3) is dissolved in enzyme-free water to prepare a 1mg/ml solution, a proper amount of thiolated Aptamer Aptamer and Apt are added into the solution, the solution is magnetically stirred and reacts overnight under the ice bath condition, and the reaction molar ratio of the nano-microbubbles to the Aptamer is 1:1-1.5; freeze drying to obtain final nanometer assembly Apt @ Au @ PLGANBs freeze dried powder, and using in Zeta granularity analysis, electron microscope detection, X-ray electron spectrum (XPS) and other characterization detection.
The aptamer combined with the nanobubble to construct the nano assembly is applied to the preparation of an ultrasonic contrast agent.
Further, the application in the preparation of in vitro diagnosis, in vivo imaging, targeted therapy, food safety and environmental monitoring.
Has the beneficial effects that: the nano assembly can be used as an efficient, stable and nontoxic ultrasonic contrast agent, can realize the development outside a blood pool, has higher diagnosis accuracy compared with the traditional micron-sized contrast agent, and achieves the effect of accurate positioning by the combination of the specific aptamer; meanwhile, different focuses (tumors, thrombi, plaques and the like) can be connected with corresponding therapeutic drugs or genes for precise treatment, so that the toxic and side effects of the drugs are greatly reduced.
Compared with the prior art, the invention has the following advantages: (1) The PLGA is a degradable functional polymer organic compound, has good biocompatibility, no toxicity and good performance of forming capsules and films, and adopts PLGA as a film material, stable C3F8 gas as a gas core, nanogold as a bridging tool and an aptamer as a targeting guide to prepare a nano assembly with the particle size of about 500-600 nm.
(2) The nano assembly combined by the aptamer and the nanoparticle is expected to be applied to a plurality of aspects such as in vitro diagnosis, in vivo imaging, targeted therapy, food safety, environmental monitoring and the like.
Drawings
FIG. 1 is a flow chart of the preparation of Apt @ Au @ PLGANBs according to the present invention. The microbubble takes PLGA as a membrane material and C3F8 as a core, nano-gold is coated on the surface of the PLGA microcapsule to form a PLGA @ Au nano microcapsule, and Apt nano Jin Lianyu PLGA NBs is coated on the surface to obtain the multifunctional nano assembly-Apt @ Au @ PLGA NBs.
FIG. 2 is a SAM plot of Apt @ Au @ PLGANBs according to the present invention (scale =2 μm; scale =500 nm).
FIG. 3 is a graph showing a particle size distribution of Apt @ Au @ PLGANBs according to the present invention.
FIG. 4 shows an XPS spectrum of Au-S bond of the present invention.
Detailed Description
The following specific examples are given to illustrate the technical solutions of the present invention, but the present invention is not limited in any way, and any modifications or substitutions based on the teaching of the present invention are included in the protection scope of the present invention.
Example 1
As shown in fig. 1, the method for preparing the nano assembly constructed by aptamer-conjugated nanobubbles comprises the following steps: (1) preparation of PLGA nanobubbles: preparing polylactic-co-glycolic acid (PLGA) nano microbubbles by adopting a single emulsification method; preparing PLGA nano microvesicles: step1: preparing a basic solvent:
polyvinyl alcohol PVA solution: weighing 0.4g of PVA crystal, dissolving the PVA crystal in 20mL of ultrapure water, rapidly stirring the PVA crystal by a constant-temperature magnetic stirrer (at 80 ℃) until the PVA crystal is completely dissolved, and storing the PVA crystal in a refrigerator at 4 ℃ for later use;
polyallylamine hydrochloride PAH solution: weighing 100mg of PAH and 292.5mg of NaCl, and dissolving in 100mL of ultrapure water to obtain a required PAH solution;
step 2: weighing 80mg PLGA and 9mg camphor, dissolving in 4ml dichloromethane, dropwise adding the solution into the PVA solution prepared in the step (1), and emulsifying with 400W ultrasonic waves for 5min (On 4 s, off4 s) under the condition of ice-water bath to obtain white emulsion, thus obtaining PLGA nano microvesicle.
(2) Nano microbubble PLGANBs freeze-dried powder: placing the nano microbubbles prepared in the step (1) in a freeze dryer to prepare freeze-dried powder, and then filling perfluoropropane C3F8 gas-filled vesicles under a vacuum condition to obtain PLGANBs freeze-dried powder;
adding 20ml of isopropanol solution (2%) into the emulsion prepared in the step (1) for stabilization, performing rotary evaporation for 6 hours to fully volatilize dichloromethane, centrifuging the solution (5 min, 2000rpm), discarding the precipitate, taking the supernatant, centrifuging again (4 min, 15000rpm), washing for 3 times, and performing vacuum freeze drying on the precipitate for 20 hours;
and filling the obtained freeze-dried powder with C3F8 gas under a vacuum condition to prepare the perfluoropropane gas-encapsulated nano microbubble freeze-dried powder PLGANBS.
(3) Au @ PLGANBs lyophilized powder: preparing nano gold Au particles by adopting a sodium borohydride reduction method; dissolving the freeze-dried powder prepared in the step (2) in a polyallylamine hydrochloride (PAH) solution, coating nanogold on the surface of PLGA micro-bubble by adopting an electrostatic adsorption method and a seed growth method to form a nanogold shell, and then carrying out freeze-drying to obtain Au @ PLGANBs freeze-dried powder;
adding 1mL of chloroauric acid solution (1%) and 1% of sodium citrate solution into 90mL of ultrapure water respectively, stirring for 10min by using a magnetic stirrer, adding 2mL of sodium citrate solution containing 0.075% of sodium borohydride, and continuing stirring for 15min to obtain gold nano solution AuNPs;
dissolving the prepared PLGANBs in 10mL of the PAH solution prepared in the step (1), stirring for 40min, centrifuging and washing for several times, and taking precipitates to obtain PAH-coated PLGANBs;
adding the prepared AuNPs solution into the obtained precipitate, stirring for 30min, centrifuging and washing for 3 times, dissolving the precipitate in 3mL of 1% chloroauric acid solution, stirring for 30min, dropwise adding 350uL of hydroxylamine hydrochloride solution, stirring for 30min, finally centrifuging and washing for 3 times, and carrying out vacuum freeze drying on the precipitate for 20h to obtain Au @ PLGANBs freeze-dried powder.
The aptamer takes an atherosclerosis macrophage derived foam cell as a target, and the gene sequence of the aptamer is a nucleotide sequence shown in SEQ ID No. 1.
(4) Nanometer assembly Apt @ Au @ PLGANBs freeze-dried powder: 3 of the aptamer ， Modifying sulfydryl-SH, dissolving a proper amount of the Au @ PLGANBs freeze-dried powder prepared in the step (3) in enzyme-free water, adding a proper amount of thiolated Aptamer Aptamer (Apt) into the solution, magnetically stirring the solution under an ice bath condition for reaction overnight, washing and centrifuging the reaction solution by PBS, removing redundant aptamers, and fully reacting nano-gold and sulfydryl to form a stable Au-S bond so that the aptamers are stably bonded on the surface of Au @ PLGA nano-microbubbles; freeze drying to obtain nanometer assembly Apt @ Au @ PLGANBs freeze dried powder.
Dissolving a proper amount of the Au @ PLGANBs freeze-dried powder prepared in the step (3) in enzyme-free water to prepare a 1mg/ml solution, adding a proper amount of thiolated Aptamer Aptamer and Apt into the solution, and reacting overnight under magnetic stirring under ice bath conditions, wherein the reaction molar ratio of the nano-microbubbles to the Aptamer is 1.5; and (3) freeze-drying to obtain the final nanometer assembly Apt @ Au @ PLGANBs freeze-dried powder, and performing characterization detection.
Apt @ Au @ PLGANBs freeze-dried powder is observed by a transmission electron microscope (SAM), as shown in figure 2, the nano assembly is observed to have good dispersibility and be in a sphere-like shape, and the rough nano gold shell formed by nano gold particles can be clearly seen on the surface of the nano microbubble. Apt @ Au @ PLGANBs particle size is measured by a Zeta particle size analyzer, as shown in FIG. 3, the average particle size is 508.4nm, the dispersion index is 0.765, the particle size is basically consistent with the observation result of SAM, the size is in unimodal distribution, and the nano-assembly is uniform in particle size and good in dispersibility. Electron microscopy and X-ray electron spectroscopy (XPS) and other characterization tests show that the Au-S bond of Apt @ Au @ PLGANBs can be detected, and as shown in FIG. 4, two peaks of the Au-S bond binding energy near 83.4eV and 87.1eV are respectively distributed to Au4F 7/2 and Au4F5/2, so that stable binding of the Au-S bond is demonstrated, and successful binding of the thiolated aptamer and the Au @ PLGA NBs is reflected.
The aptamer combined with the nanobubble to construct the nano assembly is applied to the preparation of an ultrasonic contrast agent. The application in the preparation of in vitro diagnosis, in vivo imaging, targeted therapy, food safety and environmental monitoring.
Example 2
Example 2 differs from example 1 in that:
the invention relates to a preparation method of a nano assembly constructed by aptamer combined with nano microvesicle, which comprises the following steps: (1) preparation of PLGA nanobubbles: preparing polylactic-co-glycolic acid (PLGA) nano microbubbles by adopting a single emulsification method; preparing PLGA nano microvesicles: step1: preparing a basic solvent:
polyvinyl alcohol PVA solution: weighing 0.4g of PVA crystal, dissolving the PVA crystal in 20mL of ultrapure water, rapidly stirring the PVA crystal by a constant-temperature magnetic stirrer (80 ℃) until the PVA crystal is completely dissolved, and storing the PVA crystal in a refrigerator at 4 ℃ for later use;
polyallylamine hydrochloride PAH solution: weighing 100mg of PAH and 292.5mg of NaCl, and dissolving in 100mL of ultrapure water to obtain a required PAH solution;
step 2: weighing 100mg of PLGA and 8mg of camphor, dissolving in 3ml of dichloromethane, dropwise adding the solution into the PVA solution prepared in the step (1), and emulsifying by using 400W ultrasonic waves for 4min (On 4 s, off4 s) under the condition of ice water bath to obtain white emulsion, thus obtaining the PLGA nano microbubble.
(2) Nano microbubble PLGANBs freeze-dried powder: placing the nano microbubbles prepared in the step (1) in a freeze dryer to prepare freeze-dried powder, and then filling perfluoropropane C3F8 gas-filled vesicles under a vacuum condition to obtain PLGANBs freeze-dried powder;
adding 20ml of isopropanol solution (2%) into the emulsion prepared in the step (1) for stabilization, performing rotary evaporation for 5 hours to fully volatilize dichloromethane, centrifuging the solution (5 min, 2000rpm), discarding the precipitate, taking the supernatant, centrifuging again (4 min, 15000rpm), washing for 3 times, and performing vacuum freeze drying on the precipitate for 24 hours;
and filling the obtained freeze-dried powder with C3F8 gas under a vacuum condition to prepare the perfluoropropane gas-encapsulated nano microbubble freeze-dried powder PLGANBS.
(3) Au @ PLGANBs lyophilized powder: preparing gold nanoparticles (Au) by a sodium borohydride reduction method; dissolving the freeze-dried powder prepared in the step (2) in a polyallylamine hydrochloride (PAH) solution, coating nanogold on the surface of PLGA micro-bubble by adopting an electrostatic adsorption method and a seed growth method to form a nanogold shell, and then carrying out freeze-drying to obtain Au @ PLGANBs freeze-dried powder;
adding 2mL of chloroauric acid solution (1%) and sodium citrate solution (1%) into 95mL of ultrapure water respectively, stirring for 10min by using a magnetic stirrer, adding 1.5mL of sodium citrate solution containing 0.075% of sodium borohydride, and continuing stirring for 10min to prepare gold nano solution AuNPs;
dissolving the prepared PLGANBs in 10mL of the PAH solution prepared in the step (1), stirring for 30min, centrifuging and washing for several times, and taking precipitates to obtain PAH-coated PLGANBs;
adding the prepared Au NPs solution into the obtained precipitate, stirring for 30min, centrifuging and washing for 3 times, dissolving the precipitate in 2mL 1% chloroauric acid solution, stirring for 30min, dropwise adding 300uL hydroxylamine hydrochloride solution, stirring for 30min, finally centrifuging and washing for 2 times, and carrying out vacuum freeze drying on the precipitate for 18h to obtain Au @ PLGANBs freeze-dried powder.
The aptamer takes an atherosclerosis macrophage derived foam cell as a target, and the gene sequence of the aptamer is a nucleotide sequence shown in SEQ ID No. 1.
(4) Nanometer assembly Apt @ Au @ PLGANBs freeze-dried powder: modifying sulfydryl-SH at the 3 end of an Aptamer, dissolving a proper amount of Au @ PLGANBs freeze-dried powder prepared in the step (3) in enzyme-free water, adding a proper amount of thiolated Aptamer Aptamer (Apt) into the solution, magnetically stirring the solution under an ice bath condition for reaction overnight, washing and centrifuging the reaction solution by PBS, removing the redundant Aptamer, and fully reacting nanogold and sulfydryl to form a stable Au-S bond so that the Aptamer is stably bonded on the surface of Au @ PLGA nano microbubble; freeze drying to obtain nanometer assembly Apt @ Au @ PLGANBs freeze dried powder.
Example 3
Example 3 differs from example 1 in that:
the invention relates to a preparation method of a nano assembly constructed by aptamer combined with nano microbubbles, which comprises the following steps: (1) preparation of PLGA nanobubbles: preparing polylactic-co-glycolic acid (PLGA) nano microbubbles by adopting a single emulsification method; preparing PLGA nano microvesicles: step1: preparing a basic solvent:
polyvinyl alcohol PVA solution: weighing 0.4g of PVA crystal, dissolving the PVA crystal in 20mL of ultrapure water, rapidly stirring the PVA crystal by a constant-temperature magnetic stirrer (at 80 ℃) until the PVA crystal is completely dissolved, and storing the PVA crystal in a refrigerator at 4 ℃ for later use;
polyallylamine hydrochloride PAH solution: weighing 100mg of PAH and 292.5mg of NaCl, and dissolving in 100mL of ultrapure water to obtain a required PAH solution;
step 2: weighing 90mg of PLGA and 10mg of camphor, dissolving in 3.5ml of dichloromethane, dropwise adding the solution into the PVA solution prepared in the step (1), and emulsifying by using 400W ultrasonic waves for 3min (On 4 s, off4 s) under the condition of ice-water bath to obtain white emulsion, thus obtaining the PLGA nano microbubble.
(2) Nano microbubble PLGANBs freeze-dried powder: placing the nano microbubbles prepared in the step (1) in a freeze dryer to prepare freeze-dried powder, and then filling perfluoropropane C3F8 gas-filled vesicles under a vacuum condition to obtain PLGANBs freeze-dried powder;
adding 20ml of isopropanol solution (2%) into the emulsion prepared in the step (1) for stabilization, performing rotary evaporation for 4 hours to fully volatilize dichloromethane, centrifuging the solution (5 min, 2000rpm), discarding the precipitate, taking the supernatant, centrifuging again (4 min, 15000rpm), washing for 3 times, and performing vacuum freeze drying on the precipitate for 18 hours;
and filling the obtained freeze-dried powder with C3F8 gas under a vacuum condition to prepare the perfluoropropane gas-encapsulated nano microbubble freeze-dried powder PLGANBS.
(3) Au @ PLGANBs lyophilized powder: preparing nano gold Au particles by adopting a sodium borohydride reduction method; dissolving the freeze-dried powder prepared in the step (2) in a polyallylamine hydrochloride (PAH) solution, coating nanogold on the surface of PLGA micro-bubble by adopting an electrostatic adsorption method and a seed growth method to form a nanogold shell, and then carrying out freeze-drying to obtain Au @ PLGANBs freeze-dried powder;
1.5mL of chloroauric acid solution (1%) and 1% of sodium citrate solution (1%) are added into 100mL of ultrapure water respectively, stirred by a magnetic stirrer for 10min, then added with 1mL of sodium citrate solution containing 0.075% of sodium borohydride, and stirred continuously for 12min to prepare gold nano solution AuNPs;
dissolving the prepared PLGANBs in 10mL of the PAH solution prepared in the step (1), stirring for 30-40min, centrifuging and washing for several times, and taking precipitates to obtain PLGANBs wrapped by PAH;
adding the prepared AuNPs solution into the obtained precipitate, stirring for 30min, centrifugally washing for 3 times, dissolving the precipitate in 2.5mL of 1% chloroauric acid solution, stirring for 30min, dropwise adding 400uL of hydroxylamine hydrochloride solution, stirring for 30min, centrifugally washing for 2.5 times, and freeze-drying the precipitate in vacuum for 24h to obtain Au @ PLGA NBs freeze-dried powder.
Test example 1
The freeze-dried powder of the nano-assembly in example 1 was used, XPS (X-ray photoelectron spectroscopy) was used to analyze the element ratios of Au @ PLGANBs and Apt @ Au @ PLGANBs, and the successful bonding of the nano-microbubbles and the aptamer was analyzed according to the surface elements. As shown in Table 1, nitrogen (N) and phosphorus (P) which are unique in aptamer and sulfur (S) contained in aptamer modified sulfydryl are detected in the aptamer-bonded nano assembly (Apt @ Au @ PLGA NBs), and the three elements are not detected in the aptamer-bonded nano microbubble (Au @ PLGANBs) without the aptamer, which indicates that the nano microbubble is successfully bonded with the aptamer. XPS analysis the elemental ratios of Au @ PLGA NBs and Apt @ Au @ PLGANBs are shown in Table 1, for example:
TABLE 1
Figure BDA0003641201890000091
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification and equivalents thereof.
Sequence listing
<110> university of Kunming medical science
<120> preparation method and application of nano assembly constructed by aptamer combined with nano microbubble
<130> 2022
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 81
<212> DNA
<213> Artificial sequence (aptamer)
<400> 1
cccctgcagg tgattttgct caagttgctg tggattggaa tcgctgtccg gacggccccc 60
agtatcgcta atcaggcgga t 81

Claims (5)

1. A preparation method for constructing a nano assembly by combining an aptamer and a nano microbubble is characterized by comprising the following steps: (1) preparing PLGA nano microvesicle: preparing polylactic-co-glycolic acid (PLGA) nano microbubbles by adopting a single emulsification method;
(2) Nanometer microbubble PLGA NBs freeze-dried powder: placing the nano microbubbles prepared in the step (1) in a freeze dryer to prepare freeze-dried powder, and then filling perfluoropropane C3F8 gas-filled vesicles under a vacuum condition to obtain PLGA NBs freeze-dried powder;
(3) Au @ PLGA NBs freeze-dried powder: preparing nano gold Au particles by adopting a sodium borohydride reduction method; dissolving the freeze-dried powder prepared in the step (2) in a polyallylamine hydrochloride PAH solution, coating nanogold on the surface of PLGA micro-bubble by adopting an electrostatic adsorption method and a seed growth method to form a nanogold shell, and then carrying out freeze drying to obtain Au @ PLGA NBs freeze-dried powder;
(4) Nanometer assembly Apt @ Au @ PLGA NBs freeze-dried powder: 3 of the aptamer ， Modifying sulfydryl-SH, dissolving a proper amount of Au @ PLGA NBs freeze-dried powder prepared in the step (3) in enzyme-free water, adding a proper amount of thiolated Aptamer Aptamer (Apt) into the solution, magnetically stirring the solution under an ice bath condition for reaction overnight, washing and centrifuging the reaction solution by PBS, removing redundant aptamers, and fully reacting nano-gold and sulfydryl to form a stable Au-S bond so that the aptamers are stably bonded on the surface of the Au @ PLGA nano microbubble; freeze dryingThen preparing Apt @ Au @ PLGA NBs freeze-dried powder of the nano assembly; the aptamer takes an atherosclerosis macrophage derived foam cell as a target, and the gene sequence of the aptamer is a nucleotide sequence shown in SEQ ID No. 1.
2. The method of claim 1 for preparing the aptamer-conjugated nanobubble for constructing a nano-assembly, wherein: in step (1), PLGA nanobubbles were prepared: step1: preparing a basic solvent:
polyvinyl alcohol PVA solution: weighing 0.4g of PVA crystal, dissolving the PVA crystal in 20mL of ultrapure water, rapidly stirring by a constant-temperature magnetic stirrer until the PVA crystal is completely dissolved, wherein the stirring temperature is 80 ℃, and storing the PVA crystal in a refrigerator at 4 ℃ for later use;
polyallylamine hydrochloride PAH solution: weighing 100mg of PAH and 292.5mg of NaCl, and dissolving in 100mL ultrapure water to obtain a required PAH solution;
step 2: weighing 80-100mg of PLGA and 8-10mg of camphor, dissolving in 3-4ml dichloromethane, dropwise adding the solution into the PVA solution prepared in the step (1), ultrasonically emulsifying by 400W for 3-5min under the condition of ice-water bath, ultrasonically switching on4 s and switching off4 s to obtain white emulsion, and preparing the PLGA nano microbubble.
3. The method of claim 1 for preparing the aptamer-conjugated nanobubble for constructing a nano-assembly, wherein: in the step (2), adding 20ml of 2% isopropanol solution into the emulsion prepared in the step (1) for stabilization, carrying out rotary evaporation for 4-6h to fully volatilize dichloromethane, centrifuging the solution and removing precipitates, wherein the centrifuging time is 5min, the centrifuging rotating speed is 2000rpm, taking supernate and carrying out centrifugal washing for 3 times again, the centrifuging time is 4min, the centrifuging rotating speed is 15000rpm, and carrying out vacuum freeze drying on the precipitates for 18-24h;
and filling the obtained freeze-dried powder with C3F8 gas under a vacuum condition to prepare the perfluoropropane gas-encapsulated nano microbubble freeze-dried PLGA NBs.
4. The method for preparing the aptamer-bound nanobubble-constructed nanoassembly according to claim 2, wherein: in the step (3), 1% chloroauric acid solution and 1% sodium citrate solution are taken and added into 90-100mL of ultrapure water respectively at the concentration of 1-2mL, stirred by a magnetic stirrer for 10min, then added into 1-2mL sodium citrate solution containing 0.075% sodium borohydride, and continuously stirred for 10-15min to prepare gold nano solution Au NPs;
dissolving the prepared PLGA NBs in the PAH solution 10mL prepared in the step (1), stirring for 30-40min, centrifuging and washing for times, and taking the precipitate to obtain the PLGA NBs coated by the PAH;
adding the prepared Au NPs solution into the obtained precipitate, stirring for 30min, centrifugally washing for 3 times, dissolving the precipitate in 2-3mL of 1% chloroauric acid solution, stirring for 30min, dropwise adding 300-400uL hydroxylamine hydrochloride solution, stirring for 30min, finally centrifugally washing for 2-3 times, and carrying out vacuum freeze drying on the precipitate for 18-24h to prepare Au @ PLGA NBs freeze-dried powder.
5. The method of claim 1 for preparing the aptamer-conjugated nanobubble for constructing a nano-assembly, wherein: in the step (4), a proper amount of Au @ PLGA NBs freeze-dried powder prepared in the step (3) is dissolved in enzyme-free water to prepare 1mg/ml solution, a proper amount of thiolated Aptamer is added into the solution, the solution is magnetically stirred and reacts overnight under the ice bath condition, and the reaction molar ratio of the nano-microbubbles to the Aptamer is 1:1-1.5; and (3) freeze-drying to obtain final nano assembly Apt @ Au @ PLGA NBs freeze-dried powder, and using the freeze-dried powder for Zeta particle size analysis, electron microscope detection and X-ray electron spectrum characterization detection.
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