CN111529509B

CN111529509B - Nano-drug carrier and preparation method thereof, drug-loading system and preparation method and application thereof

Info

Publication number: CN111529509B
Application number: CN202010566247.5A
Authority: CN
Inventors: 王玉记; 李元明; 王彦明; 宋松林; 贾翌江; 桂琳
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2021-12-17
Anticipated expiration: 2040-06-19
Also published as: CN111529509A

Abstract

The invention provides a nano-drug carrier and a preparation method thereof, a drug-loading system and a preparation method and application thereof, belonging to the technical field of biological medicine. The nano-drug carrier provided by the invention is obtained by modifying gold nanoparticles by a trans-transcription activating factor, wherein the surfaces of the gold nanoparticles are negatively charged, and the particle size of the gold nanoparticles is 5-50 nm; the amino acid sequence of the trans-transcriptional activator is as follows: CYRGRKKRRQRRR are provided. The invention modifies the trans-transcription activator on the gold nanoparticle, and the PAD4 inhibitor 356 is delivered by taking the modified trans-transcription activator as a carrier, so that the delivery capability of 356 to the nucleus can be improved, and cancer cells can be killed.

Description

Nano-drug carrier and preparation method thereof, drug-loading system and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a nano-drug carrier and a preparation method thereof, a drug-loading system and a preparation method and application thereof.

Background

Peptidyl Arginine Deiminase (PAD) constitutes a family of calcium dependent enzymes, of which PAD4 is a post-transcriptional modification enzyme that catalyzes the deimination of positively charged arginine residues in the peptide chain to neutral citrulline, and citrullinated (Cit) proteins that cause DNA damage and are involved in human carcinogenesis. YW3-56 (chemical name is N- (1- (benzylamino) -5- (2-chlorooxalylamino) -1-oxypentan-2-yl) -6- (dimethylamino) -2-naphthamide, 356 for short) as a PAD4 inhibitor can effectively inhibit tumor growth in a mouse S-180 sarcoma xenograft model, but free 356 has poor water solubility, so that the delivery capability to cell nucleus is poor and the bioactivity is poor.

Disclosure of Invention

The invention aims to provide a nano-drug carrier and a preparation method thereof, a drug-loading system and a preparation method and application thereof, wherein trans-transcriptional activation factors are modified on gold nanoparticles, so that the gold nanoparticles are used as a carrier to deliver PAD4 inhibitor 356, the delivery capability of 356 to cell nucleus can be improved, cancer cells can be killed, and the nano-drug carrier is good in biological activity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a nano-drug carrier, which is obtained by modifying gold nanoparticles by a trans-transcription activating factor, wherein the surfaces of the gold nanoparticles are negatively charged, and the particle size of the gold nanoparticles is 5-50 nm; the amino acid sequence of the trans-transcriptional activator is as follows: CYRGRKKRRQRRR are provided.

Preferably, the content of the trans-transcription activator in the nano-drug carrier is 0.03-0.05 wt.%.

The invention provides a preparation method of the nano-drug carrier in the technical scheme, which comprises the following steps:

and mixing the gold nanoparticles, the trans-transcription activating factor and the first solvent, and performing coordination reaction to obtain the nano-drug carrier.

Preferably, the temperature of the coordination reaction is 15-35 ℃, and the time is 1.5-2.5 h.

The invention provides a drug-carrying system which comprises a nano-drug carrier and a PAD4 inhibitor loaded on the nano-drug carrier, wherein the nano-drug carrier is the nano-drug carrier prepared by the technical scheme or the preparation method of the technical scheme, and the PAD4 inhibitor is N- (1- (benzylamino) -5- (2-chlorooxalimido) -1-oxypentane-2-yl) -6- (dimethylamino) -2-naphthamide.

Preferably, the drug loading rate of the drug-loaded system is 60-90 wt.%.

The invention provides a preparation method of the medicine carrying system in the technical scheme, which comprises the following steps:

mixing a nano-drug carrier, a PAD4 inhibitor and a second solvent, and carrying out non-covalent combination on the nano-drug carrier and the PAD4 inhibitor to obtain a drug-carrying system.

Preferably, the temperature of the non-covalent bonding is 15-35 ℃, and the time is 50-70 min.

The invention provides application of the drug-loaded system in the technical scheme or the drug-loaded system prepared by the preparation method in the technical scheme in preparation of antitumor drugs.

Preferably, the tumor comprises colon cancer, lung cancer or breast cancer.

The invention provides a nano-drug carrier, which is obtained by modifying gold nanoparticles by a trans-transcription activating factor, wherein the surfaces of the gold nanoparticles are negatively charged, and the particle size of the gold nanoparticles is 5-50 nm; the amino acid sequence of the trans-transcriptional activator is as follows: CYRGRKKRRQRRR are provided. According to the invention, trans-transcription activating factors (TAT) are modified on gold nanoparticles (AuNPs), the obtained nano-drug carrier has excellent endocytosis performance, and PAD4 inhibitor 356 is delivered by using the nano-drug carrier, so that the delivery capability of 356 to cell nucleus can be improved, and cancer cells can be killed. Specifically, the sequence of the TAT is CYRGRKKRRQRRR, and the cell penetration capacity of AuNPs can be enhanced by bonding the sulfydryl of cysteine in the TAT with the AuNPs; meanwhile, the positive charges carried by the side chains in the 356 structure are electrostatically adsorbed with the negative charges on the surfaces of the AuNPs, so that the obtained drug-loaded nano system (namely 356-TAT-AuNPs) has higher cell uptake rate.

In the test example 1 of the invention, the in vitro activity of 356-TAT-AuNPs is studied, specifically, the cell activity of each preparation after treating tumor cells is determined by adopting an MTT colorimetric method, the distribution of gold nanoparticles and the change of cell autophagosomes are observed under an ultrastructure of cells by a transmission electron microscope, in order to more intuitively see the degree of H3 citrullination in the cells, the total amount change of H3 citrullinated protein is qualitatively detected by using immunofluorescence, and the change trend of intracellular protein is quantitatively determined by a Western Blot method (Western Blot). Among them, the in vitro MTT study showed that the IC50 values of 356-TAT-AuNPs on HCT-116 cells were increased by 4.5-fold (24h action) and 4.9-fold (48 h action) compared to free 356, indicating that TAT-modified AuNPs could enhance 356's anti-tumor proliferation activity with less time required for the effect.

In the test example 2, the in vivo anti-tumor activity of 356-TAT-AuNPs is evaluated, and specifically, the anti-tumor activity of a PD-1 antibody and 356-TAT-AuNPs in combination is investigated by adopting a nude mouse HCT-116 allograft tumor model. Experimental results show that when 356-TAT-AuNPs are fixed at a dose of 1 mu mol/kg, the antibody can be combined with PD-1 antibody (200 mu g/mouse) to remarkably inhibit tumor growth by tail vein injection administration.

Drawings

FIG. 1 is a schematic diagram of the preparation process and anti-tumor mode of 356-TAT-AuNPs of the present invention;

FIG. 2 is a transmission electron micrograph and a particle size distribution histogram of AuNPs, 356-AuNPs and 356-TAT-AuNPs, in which A1 and A2 are AuNPs (particle size 16.4nm), B1 and B2 are 356-AuNPs, and C1 and C2 are 356-TAT-AuNPs;

fig. 3 is an in vitro cytotoxicity profile of AuNPs against HCT-116 cells (n-9, dosing time 48 h);

figure 4 shows the 48h in vitro cytotoxicity of 356, 356-AuNPs and 356-TAT-AuNPs against HCT-116, MCF-7 and a549 (n-9, compound and nanosystems dosing time 48 h);

FIG. 5 is a transmission electron microscope image of HCT-116 cells, wherein A: 8 h; b: 24 h; c: (ii) 24h autophagy status; scale bar: 5 μm, low power (low magnetic); 1 μm, high power (high magnetic);

fig. 6 is a confocal laser image of HCT-116 cells incubated with drug for 12h, scale bar: 50 μm;

FIG. 7 is a graph of fluorescence intensity of drug uptake by HCT-116 single cells, wherein P < 0.05 compared to 356; p < 0.01 compared to group 356;

FIG. 8 is an expression diagram of the PADI4 protein and autophagy-related protein LC3, wherein A is 8h for drug to cell interaction and B is 24h for drug to cell interaction;

FIG. 9 is a graph showing the change in body weight of mice raised for 21 days (n-8);

fig. 10 is a graph of tumor volume change (n-8) in mice fed for 21 days, wherein a is the change in tumor volume; b is a tumor picture;

figure 11 is a graph of the effect of drug on tumor growth in HCT-116 xenograft mice (n-8);

FIG. 12 is a pathological section of HE-stained tumor, in which A is NS, B is AuNPs, C is PD-1, D is 356, E is 356-TAT-AuNPs, F is PD-1+356-TAT-AuNPs, scale bar: 100 μm.

Detailed Description

The invention provides a nano-drug carrier, which is obtained by modifying gold nanoparticles by a trans-transcription activating factor, wherein the surfaces of the gold nanoparticles are negatively charged, and the particle size of the gold nanoparticles is 5-50 nm; the amino acid sequence of the trans-transcriptional activator is as follows: CYRGRKKRRQRRR (the amino acid sequence is shown in SEQ ID No. 1).

In the present invention, the Trans-activating Transcription Activator (TAT) belongs to a cell-penetrating peptide (CPPs) derived from Human Immunodeficiency Virus (HIV) -I30, the sequence of which is CYRGRKKRRQRRR, and the arginine-rich basic domain RKKRRQRRR can penetrate cell membranes by adsorptive endocytosis. In the invention, the particle size of the gold nanoparticles (AuNPs) is 5-50 nm, preferably 6-35 nm, and further preferably 10-20 nm, and the average particle size can be 6.6nm, 16.4nm, 30.9nm or 47.1 nm. The AuNPs with the particle size within the range are adopted, and can be enriched in tumor tissues through the high Permeability and Retention (EPR) effect of solid tumors, so that the medicine can play a role in tumor parts. The invention realizes the modification of TAT on the surfaces of AuNPs through the coordination reaction between sulfydryl in TAT and AuNPs.

In the invention, the TAT content in the nano-drug carrier is preferably 0.03-0.05 wt.%, and more preferably 0.04 wt.%. The invention preferably controls the TAT content in the range, can modify all TAT on AuNPs, and is beneficial to enabling the AuNPs to efficiently penetrate cell membranes and enter cells.

In the invention, the gold nanoparticles, the trans-transcription activator and the first solvent are mixed, preferably, AuNPs colloidal solution (the solvent is preferably water) and TAT aqueous solution are mixed, and the first solvent is the solvent in the AuNPs colloidal solution and the water in the TAT aqueous solution; the concentration of AuNPs in the AuNPs colloidal solution is preferably 70-90 mug/mL, and more preferably 80-82 mug/mL; the concentration of the TAT aqueous solution is preferably 0.08-0.12 mmol/L, and more preferably 0.1 mmol/L; the volume ratio of the AuNPs colloidal solution to the TAT aqueous solution is preferably 190-210 mL: 38-42 μ L, more preferably 200 mL: 40 μ L.

The TAT is not particularly limited in source, and commercially available products well known to those skilled in the art can be used; in an embodiment of the present invention, the TAT is purchased from shanghai xin biotechnology limited.

The source of the AuNPs is not particularly limited in the present invention, and the AuNPs can be prepared by commercially available products or well-known methods well known to those skilled in the art; according to the invention, the AuNPs are preferably prepared by adopting a sodium borohydride reduction method or a sodium citrate reduction method, so that the surfaces of the prepared AuNPs are electronegative due to the existence of citrate ions, and can be bonded with electropositive compounds through electrostatic adsorption (for example, the AuNPs can be combined with positive charges carried by side chains in a 356 structure). In the invention, the AuNPs colloidal solution is prepared by adopting a sodium borohydride reduction method or a sodium citrate reduction method, and can be directly used subsequently.

After the AuNPs colloidal solution is obtained, the TAT aqueous solution is preferably added into the AuNPs colloidal solution dropwise under the stirring condition, and then the coordination reaction is carried out to obtain the nano-drug carrier. In the invention, the stirring speed is preferably 600-900 rpm, and more preferably 700 rpm; the dropping rate of the aqueous TAT solution is not particularly limited in the present invention, and a dropping rate known to those skilled in the art may be used.

In the invention, the temperature of the coordination reaction is preferably 15-35 ℃, more preferably 20-30 ℃, and the coordination reaction can be carried out at room temperature, i.e. no extra heating or cooling is needed; in the examples of the present invention, the coordination reaction is carried out specifically at 25 ℃. In the invention, the time of the coordination reaction is preferably 1.5-2.5 h, and more preferably 2 h; the coordination reaction time is counted by the completion of the dropping of the TAT aqueous solution. In the invention, in the process of the coordination reaction, sulfydryl in TAT is bonded with gold nanoparticles to obtain nano-drug carriers (TAT-AuNPs).

After the coordination reaction, the system obtained after the coordination reaction is preferably kept standing in a dark place under a sealed condition (the light-proof standing time is preferably 10-15 h, and more preferably 12h, so that the aggregation rate of AuNPs is reduced); and then carrying out post-treatment to obtain the nano-drug carrier. In the present invention, the post-treatment preferably comprises the steps of: and (3) carrying out first centrifugation on the system after being kept in a dark place, discarding the supernatant, carrying out second centrifugation after ultrasonically mixing the obtained sediment with ultrapure water, repeating the steps of discarding the supernatant, ultrasonically mixing and centrifuging for 2-3 times, and finally centrifuging and then pouring the supernatant back to remove the precipitate, wherein the obtained precipitate is the nano-drug carrier. In the invention, the rotation speed of each centrifugation in the post-treatment process is preferably 8000-12000 rpm independently, and more preferably 10000 rpm; the time of each centrifugation is independently preferably 8-12 min, and more preferably 10 min. In the present invention, the nano-drug carrier is preferably stored in a mixed solution of dimethyl sulfoxide (DMSO) and water, and specifically, the nano-drug carrier obtained by the last centrifugation may be resuspended in a mixed solution of dimethyl sulfoxide (DMSO) and water (the volume ratio of DMSO to water is preferably 1-2: 2-20, and more preferably 1: 5), so as to obtain a TAT-AuNPs colloidal solution. In the invention, the concentration of TAT-AuNPs in the TAT-AuNPs colloidal solution is preferably 500-900 mu g/mL, and more preferably 700 mu g/mL; the TAT-AuNPs colloidal solution is preferably used directly for drug loading.

The invention provides a drug-carrying system which comprises a nano-drug carrier and a PAD4 inhibitor loaded on the nano-drug carrier, wherein the nano-drug carrier is the nano-drug carrier prepared by the technical scheme or the preparation method of the technical scheme, and the PAD4 inhibitor is N- (1- (benzylamino) -5- (2-chlorooxalimido) -1-oxypentane-2-yl) -6- (dimethylamino) -2-naphthamide, namely YW3-56, which is 356 for short. In the invention, the drug loading rate (i.e. 356 content) of the drug-loaded system is preferably 60-90 wt.%, and more preferably 80-90 wt.%.

The source of the 356 is not particularly limited in the present invention, and the 356 can be prepared by methods known to those skilled in the art, and in the examples of the present invention, 356 can be specifically prepared by methods disclosed in US20120108562(Gong Chen, Yanming Wang, Pingxin Li, king Hu, Shu Wang, Yuji Wang.

In the present invention, the nano-drug carrier, the PAD4 inhibitor and the second solvent are mixed, preferably, a nano-drug carrier colloidal solution and a PAD4 inhibitor solution are mixed, and the second solvent is a solvent in the nano-drug carrier colloidal solution and a solvent in the PAD4 inhibitor solution. In the invention, the nano-drug carrier colloidal solution is the TAT-AuNPs colloidal solution prepared by the method; the concentration of the PAD4 inhibitor solution is preferably 0.8-1.2 mg/mL, more preferably 1mg/mL, and the solvent is preferably DMSO; the volume ratio of the nano-drug carrier colloidal solution to the PAD4 inhibitor solution is preferably 3.5-4.5: 1, and more preferably 4: 1.

According to the invention, preferably, the PAD4 inhibitor solution is dripped into the nano-drug carrier colloidal solution under the condition of stirring, and the nano-drug carrier and the PAD4 inhibitor are combined in a non-covalent manner to obtain a drug-loaded system. In the invention, the stirring speed is preferably 500-900 rpm, more preferably 700 rpm; the dropwise addition is preferably dropwise.

In the invention, the temperature of the non-covalent bonding is preferably 15-35 ℃, more preferably 20-30 ℃, and the non-covalent bonding can be carried out at room temperature. In the invention, the non-covalent binding time is preferably 50-70 min, and more preferably 1 h; the time for the non-covalent binding was counted after the addition of the PAD4 inhibitor solution was completed. According to the invention, through electrostatic adsorption of positive charges carried by a side chain in a 356 structure and negative charges on the surfaces of AuNPs, combination of a nano-drug carrier and a PAD4 inhibitor is realized, and a drug-loading system is obtained.

The invention provides application of the drug-loaded system in the technical scheme or the drug-loaded system prepared by the preparation method in the technical scheme in preparation of antitumor drugs. In the present invention, the tumor preferably includes colon cancer, lung cancer or breast cancer. In the invention, the anti-tumor medicine comprises the medicine carrying system and pharmaceutically acceptable auxiliary materials; the content of a drug-carrying system in the anti-tumor drug is preferably 80-95 wt.%, and the pharmaceutically acceptable auxiliary materials preferably comprise one or more of vitamin C, citric acid and arginine; the dosage form of the anti-tumor medicament preferably comprises nanoemulsion, liposome or freeze-dried powder.

The antitumor drug provided by the invention takes a drug-carrying system 356-TAT-AuNPs as an active ingredient, and the 356-TAT-AuNPs are used in cooperation with the PD-1 antibody, so that the antitumor activity effect is better, and particularly, the antitumor drug provided by the invention can be used in cooperation with a drug containing the PD-1 antibody, so that the antitumor activity effect can be further improved.

FIG. 1 is a schematic diagram of the preparation process and anti-tumor mode of 356-TAT-AuNPs of the present invention, specifically, TAT is bonded to AuNPs through Au-S to obtain a nano-drug carrier TAT-AuNPs that can be rapidly taken up by cells through endocytosis; then PAD4 inhibitor 356 with certain antitumor proliferation activity in vitro is loaded on TAT-AuNPs through electrostatic adsorption, so that 356 is rapidly accumulated in cells and then plays a role; meanwhile, due to the high permeability and retention effect (EPR effect) of the solid tumor, the nanoparticles tend to gather in tumor tissues in vivo, namely 356 is enriched in the tumor tissues through TAT-AuNPs, so that a better curative effect is obtained.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

AuNPs_6.6nmThe synthesis of (2): AuNPs with the average grain diameter of 6.6nm are prepared by a sodium borohydride reduction method. 3.75mg of NaBH was dissolved in 1mL of ultrapure water and the resulting NaBH solution was placed in an ice-water mixture for temporary storage. Accurately weigh 10.00mg 3H₂O·HAuCl₄Dissolving the AuNPs in 100mL of ultrapure water, adding 1mL of 1 wt.% sodium citrate aqueous solution under the ice-bath stirring condition, quickly adding the precooled NaBH aqueous solution after stirring for 1min, continuing to stir vigorously in the ice bath for 15min under the 800rpm condition, observing that the solution color is changed into red at the moment, indicating that the AuNPs are successfully synthesized at the moment, obtaining a colloidal solution of the AuNPs (the average particle size is 6.6nm), and sealing and keeping the colloidal solution away from light at 4 ℃.

Example 2

AuNPs with average particle sizes of 30.9nm and 47.1nm are prepared by a classical sodium citrate reduction method and are respectively marked as AuNPs_30.9nmAnd AuNPs_47.1nmThe method comprises the following specific steps:

49.63mL of a solution containing 3H₂O·HAuCl₄(14.65. mu. mol) of the aqueous solution was heated to boiling for 15min, 0.37mL of an aqueous sodium citrate solution (39.20 mM concentration) was added to the boiling HAuCl₄Reacting for 15min in the solution; after the mixture solution turns red, it is self-cleaningThen cooled to room temperature, and the prepared colloidal solution of AuNPs (average particle size of 30.9nm) is sealed and stored at 4 ℃ in a dark place.

49.26mL of a solution containing 3H₂O·HAuCl₄(14.65. mu. mol) of the aqueous solution was heated to boiling for 15min, 0.74mL of an aqueous sodium citrate solution (39.20 mM concentration) was added to the boiling HAuCl₄Reacting for 15min in the solution; after the mixture solution turns red, the mixture solution is naturally cooled to room temperature, and the prepared AuNPs (average particle size of 47.1nm) colloidal solution is sealed and stored at 4 ℃ in a dark place.

Example 3

AuNPs_16.4nmThe synthesis of (2): AuNPs with the average particle size of 16.4nm are prepared by a classical sodium citrate reduction method. Weighing 100.00mg of sodium citrate and dissolving the sodium citrate in 10.0mL of ultrapure water to obtain 1 wt.% of sodium citrate aqueous solution for later use; 17.39mg of 3H are weighed out₂O·HAuCl₄Adding 192mL of ultrapure water into a 250mL round-bottom flask (which is soaked in acid liquor in advance, washed with ultrapure water and naturally dried in the air), dissolving, heating to boiling through an oil bath device under the condition of reflux stirring, then quickly adding 10mL of the 1 wt.% sodium citrate aqueous solution into the round-bottom flask, and continuously boiling for 15 min; in the process, the color of the reaction solution is gradually changed from light yellow to wine red, which indicates that AuNPs are successfully synthesized; and naturally cooling the obtained wine red solution to room temperature to obtain AuNPs (average particle size of 16.4nm) colloidal solution, and sealing and keeping away from light for room temperature storage.

AuNPs prepared in example 3_16.4nmThe shape uniformity and sphericity are the best, and the invention utilizes the AuNPs_16.4nmSubsequent tests were performed.

Example 4

(1) Preparation of TAT-AuNPs: 200mL of AuNPs colloidal solution prepared in example 3 (the concentration of AuNPs is 82 μ g/mL) is transferred into a 500mL eggplant bottle, 40 μ L of TAT aqueous solution with the concentration of 0.1mmol/L is dropwise added under the condition of vigorous stirring at 700rpm, the color of the system is deepened from wine red at the moment, the stirring state is maintained at room temperature (25 ℃) for reaction for 2h after the dropwise addition is finished, the eggplant bottle is sealed after the reaction is finished, and the eggplant bottle is kept in the dark for 12 h; transferring the obtained colloidal solution into 50mL centrifuge tubes respectively, centrifuging for 10min at the rotation speed of 10000rpm, carefully absorbing and discarding supernatant liquid by using a suction tube after the centrifugation is finished, adding ultrapure water into the centrifuge tube, performing ultrasonic dispersion on the settled nanoparticles uniformly, continuing to perform centrifugal washing, and repeating twice; after the final centrifugal washing is finished, pouring out supernatant, resuspending the obtained sediment in a mixed solution of DMSO and water (the volume ratio of the DMSO to the water is 1: 5), combining the colloidal solution in each centrifuge tube, transferring the colloidal solution into a 10mL glass reaction bottle, and adding ultrapure water to fix the volume to 4mL to obtain TAT-AuNPs colloidal solution; the concentration of the TAT-AuNPs in the TAT-AuNPs colloidal solution is 700 mu g/mL.

(2) Preparation of 356-TAT-AuNPs: adding 1mL of 356 solution (the solvent is DMSO, the concentration of 356 is 1mg/mL, and 365 is prepared according to the method of the patent US 20120120108562) dropwise into 4mL of the TAT-AuNPs colloidal solution under the condition of vigorous stirring at 700rpm, maintaining the stirring state after the dropwise addition, reacting at room temperature for 1h to obtain 356-TAT-AuNPs colloidal solution (wherein the drug loading of 356-TAT-AuNPs is 90 wt.%) and storing at 4 ℃ in a sealed manner.

Comparative example

Preparation of 356-AuNPs: respectively transferring 200mL of AuNPs colloidal solution prepared in example 3 into 50mL centrifuge tubes, and centrifuging for 10min at the rotation speed of 10000 rpm; after the completion, carefully absorbing and discarding supernatant in each centrifugal tube by using a suction tube, adding ultrapure water into the centrifugal tube, performing ultrasonic oscillation on the settled nanoparticles to disperse the nanoparticles uniformly, continuing centrifugal washing, and repeating twice; after the last washing, the supernatant is poured off, the obtained sediment is resuspended in a mixed solution of DMSO and water (the volume ratio of the DMSO to the water is 1: 5), the colloidal solutions in all centrifuge tubes are combined, the mixed solution is transferred to a 10mL glass reaction bottle, ultrapure water is added to a constant volume of 4mL, a rotor is added, 0.1mL 356 solution (the 356 concentration is 10mg/mL, the solvent is DMSO) is added dropwise under the condition of vigorous stirring at 700rpm, the stirring state is maintained after the dropwise addition, the reaction is carried out for 1h at room temperature, and 356-AuNPs colloidal solution is obtained and stored at 4 ℃ in a sealed mode.

And (3) characterization:

(1) TEM characterization, sample: AuNPs, 356-TAT-AuNPs

Taking 1mL of sample colloidal solution, centrifuging (8000rpm, 10min), redissolving with absolute ethyl alcohol, dispersing uniformly by ultrasonic to obtain sample solution, spotting 10 microliter of sample solution on an electron microscope copper mesh, and drying at 37 ℃ with filter paper as a lower liner in an incubator. After the samples were completely dried, the particle size distribution of each group of samples was represented by a particle size distribution histogram by observing and photographing through a transmission electron microscope (220V). The results are shown in fig. 2, wherein a1 is a TEM image of AuNPs, and a2 is a particle size distribution histogram of AuNPs; b1 is a TEM image of 356-AuNPs, B2 is a particle size distribution histogram of 356-AuNPs; c1 is TEM image of 356-TAT-AuNPs, C2 is particle size distribution histogram of 356-TAT-AuNPs. As shown in FIG. 2, the prepared AuNPs, 356-AuNPs and 356-TAT-AuNPs have better sphericity and shape uniformity, wherein the grain diameter of the 356-loaded AuNPs is slightly increased.

(2) Zeta potential, sample: AuNPs, TAT-AuNPs, 356-TAT-AuNPs

Dispersing each sample colloidal solution in distilled water, absorbing 900 mu L of dispersion liquid after uniform ultrasonic dispersion, and measuring zeta potential by a laser nanometer particle size analyzer. Each sample was run in triplicate and the final results are expressed as mean plus minus standard deviation. The results are shown in Table 1.

TABLE 1 Zeta potential values

As can be seen from Table 1, the absolute value of the Zeta potential is slightly reduced after the TAT is modified on the surface of AuNPs, which indicates that the TAT rich in positive charges is successfully connected through Au-S bonds without influencing the stability of a nano system; and the Zeta potential absolute value is obviously reduced after 356 is loaded on the AuNPs surface, which shows that the stability of the nano system is relatively reduced due to the electrostatic adsorption of the citrate with negative charges on the AuNPs surface and 356 with positive charges.

Test example 1356-TAT-AuNPs Activity study in vitro

1.1 cell viability assay (MTT colorimetric method)

1.1.1 cell lines: HCT-116(human colon cancer cells) human colon cancer cells; a549(human lung cancer cell) human lung cancer cell; MCF-7(humanbreast cancer cell line) human breast cancer cells; LLC mice Lewis lung carcinoma cells. The cell lines used were provided by Nanjing Kaiky Biotechnology development, Inc.

1.1.2 Experimental methods

Experiment design:

1) experimental design grouping:

positive control group: free doxorubicin at various concentrations:

negative control group: a culture medium corresponding to the cultured cells;

blank dosage form group: 356 of AuNPs, TAT-AuNPs unloaded;

experimental groups: free 356, 356-AuNPs, 356-TAT-AuNPs.

2) Setting the concentration of the medicine: the experimental group concentration setting was based on the 356. mu.M loading concentration, and was set to 0.05. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M (obtained by dilution according to the desired concentration using the 356-TAT-AuNPs colloidal solution prepared in example 4 as a mother liquor); the free drug concentration was comparable to the experimental group.

3) Setting the cell concentration: 3000-5000 cells are inoculated to each hole of a 96-hole plate.

Preparation of reagents:

phosphate Buffer Solution (PBS): 8.20g NaCl, 0.20g KCl, 1.56g Na were weighed on an analytical balance₂HPO₄·12H₂O and 0.20g KH₂PO₄Fresh triple distilled water is added into a 500mL beaker, stirred vigorously to be dissolved completely, transferred to a 1L volumetric flask, and then the volume is determined by fresh triple distilled water, and the pH value is adjusted to 7.4. The solution was transferred to two 500mL reagent vials, autoclaved (15 lbs, 121 deg.C), sealed with Para film, and stored at 4 deg.C until needed.

MTT solution: 250mg of MTT is weighed by an analytical balance into a centrifuge tube, 50mL of sterile PBS solution (5mg/mL) is added, a Para film is sealed, and the centrifuge tube is wrapped by tinfoil paper and is subjected to ultrasonic oscillation in the dark to be fully dissolved. The bacteria were removed by filtration through a 0.22 μm filter, and the membrane was sealed and stored in a refrigerator at 4 ℃ in the dark.

DMEM high-glucose medium: after 50mLFBS is added into 450mL of incomplete DMEM high-sugar medium, 100U/mL of penicillin and 100 mu g/mL of streptomycin sulfate are continuously added, and the mixture is uniformly mixed to obtain the complete DMEM high-sugar medium containing 10% FBS. 5mL of the medium was taken out and placed in a 25cm rack²In a cell culture bottle with a square air-permeable cover and an inclined opening, 5 percent CO is added at 37 DEG C₂Culturing for four days under the condition, observing the generation of sterile spots under an inverted microscope, sealing by a Para film, and storing in a refrigerator at 4 ℃ for later use.

1640 medium: after adding 50mLFBS into 450mL of incomplete 1640 culture medium, adding 100U/mL of penicillin and 100 mu g/mL of streptomycin sulfate, and uniformly mixing to obtain complete 1640 culture medium containing 10% FBS. 5mL of the medium was taken out and placed in a 25cm rack²In a cell culture bottle with a square air-permeable cover and an inclined opening, 5 percent CO is added at 37 DEG C₂Culturing for four days under the condition, observing the generation of sterile spots under an inverted microscope, sealing by a Para film, and storing in a refrigerator at 4 ℃ for later use.

McCoy's 5A medium: 50mLFBS was added to 450mL of incomplete McCoy's 5A medium (containing the diabody) and mixed to give complete McCoy's 5A medium containing 10% FBS. 5mL of the medium was taken out and placed in a 25cm rack²In a cell culture bottle with a square air-permeable cover and an inclined opening, 5 percent CO is added at 37 DEG C₂Culturing for four days under the condition, observing the generation of sterile spots under an inverted microscope, sealing by a Para film, and storing in a refrigerator at 4 ℃ for later use.

Preparing the medicine:

free drug group: according to the concentration of the free drug, a proper amount of the free drug is weighed by an analytical balance, and after the trace amount of DMSO is dissolved, the blank culture medium is gradually diluted to obtain the free drug;

the dosage form comprises the following medicinal groups: based on 356 concentration, the culture medium is dispersed and diluted step by using a blank culture medium;

blank dosage form group: based on the content of Au, the Au-enriched culture medium is obtained by gradually dispersing and diluting the Au-enriched culture medium.

Cell culture:

1) culture of human colorectal cancer cell HCT-116

HCT-116 cells were adherent and appeared in irregular shapes (low density) and short fusiform, round shapes (high density) when observed under an inverted microscope. The frozen cells were quickly removed from the liquid nitrogen tank, gently shaken in a 37 ℃ water bath until the cells became fluid, quickly added with 10 volumes of McCoy's 5A medium, and centrifuged (800rpm, 10 min). After centrifugation, the supernatant was discarded, 3mL of fresh medium was added for resuspension, and the suspension was inoculated at 25cm²Cell culture flask, constant temperature at 37 ℃ and 5% CO₂Culturing in the incubator, and performing liquid change or passage according to the growth condition of the cells. HCT-116 cells grow slowly and are typically passaged once in 3-4 days. After the cell culture medium is transmitted to the third generation, the influence of the frozen stock solution on the cell growth is basically eliminated, and the cell which is in a good growth state and in a logarithmic growth phase is selected for the next experiment.

1mL of 0.25% trypsin-EDTA digest was added and the flask was gently shaken until the digest completely covered the bottom of the flask. And (3) incubating the cells in a cell incubator for 3-5 min until the cells are exfoliated and become round single cells, adding 5mL of culture medium to stop digestion, centrifuging (800rpm for 10min), and discarding the supernatant. Adding appropriate amount of culture medium to resuspend cells, blowing with pipette uniformly, counting, and making into the final product with concentration of 4 × 10⁴cells/mL of cell suspension. The cell suspension was seeded at 100. mu.L per well in 96-well cell culture plates (100. mu.L of LPBS buffer saline was added to the peripheral 36 wells for liquid seal), and the plates were placed in CO₂Culturing in an incubator, and observing under a microscope until the cells finish adherent administration.

2) Culture of human breast cancer cell MCF-7

MCF-7 cells are adherent cells, observed under an inverted microscope, and have a polygonal shape. 1640 culture medium, recovering, keeping constant temperature at 37 deg.C, 5% CO₂Culturing in a cell culture box, and performing liquid change or passage according to the growth condition of the cells. MCF-7 cells grow faster and are generally passaged once in 2-3 days. After the cell culture medium is transmitted to the third generation, the influence of the frozen stock solution on the cell growth is basically eliminated, and the cell which is in a good growth state and in a logarithmic growth phase is selected for the next experiment.

1mL of 0.25% trypsin-EDTA digest was added and the flask was gently shaken until the digest completely covered the bottom of the flask. And (3) incubating the cells in a cell incubator for 3-5 min until the cells are exfoliated and become round single cells, adding 5mL of culture medium to stop digestion, centrifuging (800rpm for 10min), and discarding the supernatant. Adding appropriate amount of culture medium to resuspend cells, blowing with pipette uniformly, counting, and making into the final product with concentration of 4 × 10⁴cells/mL of cell suspension. The cell suspension was seeded at 100. mu.L per well in 96-well cell culture plates (100. mu.L LPBS buffered saline added to the peripheral 36 wells)Liquid seal), place the plate in CO₂Culturing in an incubator, and observing under a microscope until the cells finish adherent administration.

3) Culture of human lung cancer cell A549

The A549 cells are adherent cells, observed under an inverted microscope, and have fusiform forms. After the F-12K culture medium is recovered, the temperature is kept constant at 37 ℃ and 5 percent CO₂Culturing in a cell culture box, and performing liquid change or passage according to the growth condition of the cells. A549 cells grow faster, typically once a passage of 2 days. After the cell culture medium is transmitted to the third generation, the influence of the frozen stock solution on the cell growth is basically eliminated, and the cell which is in a good growth state and in a logarithmic growth phase is selected for the next experiment.

4) Culture of mouse Lewis lung cancer cell LLC

LLC cells are adherent cells, observed under an inverted microscope, and are in spindle type. After the recovery of DMEM medium, keeping the temperature constant at 37 ℃ and 5% CO₂Culturing in a cell culture box, and performing liquid change or passage according to the growth condition of the cells. MCF-7 cells grow faster and are generally passaged once in 2-3 days. After the cell culture medium is transmitted to the third generation, the influence of the frozen stock solution on the cell growth is basically eliminated, and the cell which is in a good growth state and in a logarithmic growth phase is selected for the next experiment.

1mL of 0.25% trypsin-EDTA digest was added and the flask was gently shaken until the digest completely covered the bottom of the flask. Incubating the cells in a cell incubator for 3-5 min until the cells are exfoliated and become round single cellsDigestion was stopped by adding 5mL of medium, centrifugation (800rpm, 10min) and discarding the supernatant. Adding appropriate amount of culture medium to resuspend cells, blowing with pipette uniformly, counting, and making into the final product with concentration of 4 × 10⁴cells/mL of cell suspension. The cell suspension was seeded at 100. mu.L per well in 96-well cell culture plates (100. mu.L of LPBS buffer saline was added to the peripheral 36 wells for liquid seal), and the plates were placed in CO₂Culturing in an incubator, and observing under a microscope until the cells finish adherent administration.

Detecting the cell viability by an MTT method:

respectively sucking 25 μ L of each prepared solution, adding into 96-well plate, each group having 5 multiple wells, placing in CO₂Culturing in an incubator. After 24 and 48 hours of incubation, plates were removed and 25 μ of LMTT solution (5mg/mL) was added to each well and incubation continued in the incubator for 4 hours. After the completion of the reaction, the well plate was removed, the liquid in the well plate was carefully removed by a pipette, 100. mu.L of DMSO was added to each well, and the shaker was shaken for 10min to completely dissolve the formazan crystals. The microplate reader measures the absorbance (OD) value of the well plate at 490nm wavelength.

1.1.3 Experimental data processing and statistical methods

Calculating the vitality value of the tumor cells: cell viability (%) × (experimental OD value-blank OD value)/(control OD value-blank OD value); the cytostatic results were input to SPSS and the median Inhibitory Concentration (IC) was calculated₅₀): cell inhibition (%) ═ 1-cell survival; cell viability data, expressed as mean ± standard deviation (mean ± SD).

1.1.4 results of the experiment

TABLE 2 cell viability of drugs incubated with HCT-116 cells for 24h

Note: n is 9.

TABLE 3 cell viability of drugs incubated with HCT-116 cells for 48h

Note: n is 9.

TABLE 4 cell viability of drugs incubated with MCF-7 cells for 24h

Note: n is 9.

TABLE 5 cell viability of drugs incubated with MCF-7 cells for 48h

Note: n is 9.

TABLE 6 cell viability of drugs incubated with A549 cells for 24h

Note: n is 9.

TABLE 7 cell viability of drugs incubated with A549 cells for 48h

Note: n is 9.

TABLE 8 inhibition of LLC cell growth by drug for 24h

Note: n is 9.

TABLE 9 inhibition of LLC cell growth for 48h by the drug

Note: n is 9.

1.1.5 analytical discussion

The cytotoxicity results of the blank dosage form, the free drug combination and the drug-loaded dosage form after respectively acting on HCT-116, MCF-7, A549 and

LLC cell lines

24 and 48 hours are shown in the table 2-9, the trends of a plurality of cells are basically consistent, and the cytotoxicity effect tends to increase along with the prolonging of the acting time. And the 356-TAT-AuNPs group showed better inhibition at shorter action time (24h) than free 356. While the blank dosage form (as shown in figure 3) of AuNPs has high concentration of 100 mug/mL, the HCT-116 cell viability is still kept above 90%, and the AuNPs have no toxicity to the tumor cells.

The cell viability values of 48h of co-incubation with drug cells were plotted in a line graph, and the results are shown in FIG. 4. As shown by C in fig. 4, the drug showed low toxicity to a549 cells, but significantly reduced the activity of HCT-116 cells (a in fig. 4). In addition, 356-TAT-AuNPs have stronger inhibitory effect, which shows that TAT modified gold nanoparticles can enhance the in vitro anti-tumor activity of free 356. As shown in D in FIG. 4, the survival rate of HCT-116 cells decreased significantly with increasing concentration of 356-TAT-AuNPs, indicating that HCT-116 cells were more sensitive to 356-TAT-AuNPs. Based on the cell viability results, HCT-116 cell line was selected for subsequent experiments.

1.2 Transmission Electron Microscopy (TEM) Observation of intracellular distribution and autophagy of AuNPs

1.2.1 Experimental methods

Experiment design:

1) experimental design grouping:

AuNPs，TAT-AuNPs，356-AuNPs，356-TAT-AuNPs。

2) setting the concentration of the medicine: fixing the concentration of Au element of each group to be consistent, and the concentration of 356 medicine of the medicine carrying group to be 5 MuM;

3) setting the cell concentration: fine in growth stateInoculation of cell, six-well plate 1X 10⁵(one) of the steps.

Preparation of reagents:

Preparing the medicine:

dosage form drug groups (356-AuNPs, 356-TAT-AuNPs): based on 356 concentration, the culture medium is dispersed and diluted step by using a blank culture medium;

blank dosage form group (AuNPs, TAT-AuNPs): firstly, quantitatively determining the content of Au elements under the administration concentration of 356-AuNPs and 356-TAT-AuNPs by ICP-OES, and then diluting mother liquor of AuNPs and TAT-AuNPs to the same concentration by a blank culture medium.

Cell culture:

HCT-116 cells that grew well and were in logarithmic phase were selected, 1mL of 0.25% trypsin digest (without EDTA) was added, and the flask was gently shaken until the digest completely covered the bottom of the flask. And (3) incubating the cells in a cell incubator for 3-5 min until the cells are exfoliated and become round single cells, adding 5mL of culture medium to stop digestion, centrifuging (800rpm for 10min), and discarding the supernatant. Adding appropriate amount of culture medium to resuspend cells, blowing with pipette uniformly, counting, and making into 1 × 10⁵cells/mL of cell suspension. Six well plates were seeded with 1mL HCT-116 cell suspension at 37 ℃ in 5% CO₂₊Incubate under conditions for 24h until the cells are fully adhered to the bottom of the dish.

Removing the culture medium, washing the cells three times by using a proper amount of PBS, adding 1mL of a drug-containing culture medium into each component, and then continuously incubating for 8h and 24 h; after incubation, the drug-containing medium was removed, the cells were washed three times with PBS to remove residual nanoparticles and dead cells in the medium, 1mL of trypsin-EDTA digest was added to digest the cells, centrifugation was performed after completion, and the supernatant was discarded.

And (3) transmission electron microscope flaking:

fixing: and adding 5mL of 2.5% glutaraldehyde fixing solution special for an electron microscope into the obtained stem cell mass, and fixing for 2-4 h at 4 ℃. Centrifuging the cells to the tube bottom at low speed to obtain mung bean-sized cell mass, wrapping with 1% agarose, rinsing with PBS (pH 7.4) for 15min, and repeating twice;

post-fixing: 1% osmate was fixed at room temperature (20 ℃) for 2h, rinsed in PBS for 15min, and repeated twice;

and (3) dehydrating: sequentially adding 50% -70% -80% -90% -95% -100% -100% alcohol-100% acetone into the tissue, and dehydrating for 15min each time;

and (3) infiltration: the embedding medium 812 acetone acts for 2-4 h, the embedding medium 812 acetone acts for 1:2 overnight, the embedding medium 812 pure 812 acts for 7h, the embedding medium 812 pure 812 is poured onto an embedding plate, a sample is inserted into the embedding plate, and then the sample is placed in an oven at 37 ℃ overnight;

embedding: polymerizing for 48 hours in an oven at the temperature of 60 ℃;

slicing: and (5) slicing the thin slices by an ultrathin slicer to 60-80 nm to obtain the thin slices.

Dyeing: uranium lead double staining (2% uranium acetate saturated alcohol solution and lead citrate solution, each staining for 15min), slicing, standing at room temperature, and drying overnight.

And (5) observing under a transmission electron microscope, collecting images, photographing and analyzing.

1.2.2 discussion of results and analysis

HCT-116 cells are respectively incubated with AuNPs, TAT-AuNPs, 356-AuNPs and 356-TAT-AuNPs for 8h and 24h, and the distribution of the cells after the AuNPs are taken up and the influence of PAD inhibitor 356 on autophagy of the cells are observed by a transmission electron microscope.

As shown in a in fig. 5, AuNPs are widely distributed in cytoplasm, mitochondria (blue arrow), and sparingly distributed in lysosomes (purple arrow); both TAT-AuNPs and 356 loaded nanoparticles were found in the nucleus (red circles) suggesting that TAT-modified nanoparticles can penetrate the nuclear membrane into the nucleus. The number of nanoparticles in mitochondria and cytoplasm was significantly reduced at 24h (B in fig. 5) compared to 8h (a in fig. 5), and a large number of nanoparticles could be found aggregated in the lysosomal vesicular system (C in fig. 5), suggesting that particle efflux may occur before 24 h.

As shown by C in fig. 5, many large vesicles (red arrows) were observed in 356-treated cells, which contained phagocytosed and digested damaged organelles and the like, but such large vesicles were not observed in Control group (group without any treatment), indicating that 356 could indeed participate in apoptosis by affecting autophagy ability. Meanwhile, in the 356-TAT-AuNPs group, the vesicles show an increasing trend, which indicates that the vesicles are loaded on the TAT modified gold nanoparticles, and the efficiency of 356 entering cells can be obviously improved, so that a higher level of autophagy inhibition effect is generated. Meanwhile, the result is consistent with the fluorescence intensity increase result observed by a laser confocal microscope.

1.3H3 citrullination immunofluorescence assay

1.3.1 Experimental methods

Experiment design:

1) experimental design grouping:

control group: McCoy's 5A medium;

experimental groups: free 356, 356-AuNPs, 356-TAT-AuNPs.

2) Setting the concentration of the medicine: experimental group concentration settings were based on a loaded 356 concentration set at 10 μ M; the free drug concentration was comparable to the experimental group.

3) Setting the cell concentration: cells with good growth status were seeded on 20mm confocal dishes at 1X 10⁵(one) of the steps.

Preparation of reagents:

0.1% TritonX-100: 9.990mL of sterile PBS buffer was added to a 15mL centrifuge tube, 10. mu. LTritonX-100 was aspirated, transferred to the centrifuge tube, mixed and sealed for use.

1% BSA: weighing 20mg of BSA powder into a 4mL centrifuge tube, adding 2mL of PBS solution, and ultrasonically mixing uniformly for later use;

5% BSA: weighing 100mg BSA powder in a 15mL centrifuge tube, adding 10mL PBS solution, and ultrasonically mixing uniformly for later use;

1 × PhalloidinFITC Reagent working solution: accurately pipette 1. mu.L of 1000 × Phalloidin stock solution into 1mL of 1% BSA solution, gently blow the sample with a pipette, mix the sample and store the sample at-20 ℃.

Preparing the medicine:

cell culture:

HCT-116 cells that grew well and were in logarithmic phase were selected, 1mL of 0.25% trypsin digest (without EDTA) was added, and the flask was gently shaken until the digest completely covered the bottom of the flask. And (3) incubating the cells in a cell incubator for 3-5 min until the cells are exfoliated and become round single cells, adding 5mL of culture medium to stop digestion, centrifuging (800rpm for 10min), and discarding the supernatant. Adding appropriate amount of culture medium, resuspending cells, blowing with pipette uniformly, counting, and making into 1 × 10⁵cells/mL of cell suspension. A20 mm laser confocal dish was seeded with 1mL HCT-116 cell suspension at 37 deg.C in 5% CO₂₊Incubate under conditions for 24h until the cells are fully adhered to the bottom of the dish.

The culture medium is removed, the cells are washed three times by using an appropriate amount of PBS, 1mL of the drug-containing culture medium is added into each group, and then the incubation is continued for 12 h.

Immunofluorescence staining and Confocal observations H3 citrullination:

after incubation, removing the drug-containing culture medium, continuously washing the cells for three times by using PBS (phosphate buffer solution) to remove residual nanoparticles and dead cells, adding 4% paraformaldehyde fixing solution, and fixing the cells for 20min at room temperature; after completion, the cells were washed with PBS and repeated twice; adding 1mL of 0.1% TritonX-100 to treat cells for 5min to increase cell permeability; PBS washed cells 3 times; 1mL of 5% BSA solution acts for 2 hours at room temperature to block the nonspecific binding of the antibody; after completion, the cells were washed 3 times with PBS; adding 1mL of primary antibody (Anti-Histone H3, diluted 250 times with 1% BSA) into a confocal dish, placing the dish in an immunohistochemical light-proof wet box, and standing overnight at 4 ℃; after completion, the cells were washed 3 times with PBS; adding 1mL of secondary antibody (goat anti-rabbit IgG H & L, diluted 500 times with 1% BSA) into a confocal dish, placing the dish into an immunohistochemical light-resistant wet box, and acting for 1H at room temperature; after completion, the cells were washed 3 times with PBS; adding 1mL of 1 XPhalloidinFITC Reagent working solution, and processing at room temperature in the dark for 45min to stain cytoskeleton; after staining was complete, the cells were washed 3 times with PBS to remove excess dye; the cells were treated by adding 1mL of PBS solution and observed by confocal laser microscopy and photographed within 1 h.

All the above washing and reaction steps were performed on a shaker and the PBS washing operation lasted 10 min.

1.3.2 Experimental data processing and statistical methods

TABLE 10 excitation/emission wavelengths of the respective fluorescent substances

The fluorescence intensity in individual cells is expressed as Mean ± standard deviation (Mean ± SD) (n ═ 12). Statistical analysis was performed using SPSS, single sample t-test (one sample test) was performed for data analysis between the two groups, with P < 0.05 being defined as having statistical differences, denoted as x; p < 0.01 had significant statistical differences, expressed as x.

1.3.3 discussion of results and analysis

PAD enzyme can participate in the generation and development of tumors by citrullinating proteins including H3, so that the influence of a nano drug delivery system on H3 citrullination in HCT-116 cells can be evaluated by an immunofluorescence experiment to reflect the activity of PAD 4.

As shown in fig. 6, the red fluorescence channel of the confocal microscope indicates the citrullination degree of H3 protein, and is in positive correlation. The experimental result shows that the blank control group has the highest red fluorescence intensity, which indicates that the citrullination degree of the H3 protein in the cells is the highest; free 356 group H3 citrulline product has a decreasing trend, and 356-TAT-AuNPs group red fluorescence intensity is reduced more obviously, which visually shows that 356 has an inhibition effect on PAD4, and the drug-loaded nanoparticle group has an obvious inhibition effect.

The sets of red fluorescence channels were subjected to gray scale analysis and a histogram was plotted with gray scale values representing the fluorescence intensity values, as shown in fig. 7. The results show that the fluorescence intensity of the 3TA group is lowest and is statistically different from that of other groups, which suggests that the drug carrier can improve 356 inhibition of PAD4 protein.

1.4 Western analysis of intracellular protein content

1.4.1 Experimental methods

Experiment design:

1) experimental design grouping:

control group: McCoy's 5A medium, AuNPs;

experimental groups: free 356, 356-AuNPs, 356-TAT-AuNPs.

2) Setting the concentration of the medicine: experimental group concentration settings were based on a loaded 356 concentration set at 10 μ M;

3) setting the cell concentration: cells with good growth status were seeded on 35mm cell culture dishes at 5X 10⁵(one) of the steps.

Preparation of reagents:

Cracking mixed liquor: sucking 950 mu LRIPA, adding 40 mu LProtein kinases, adding 10 mu LPMSF continuously, and mixing for standby.

5% concentrated gum (10 mL): 8.075mL of distilled water was weighed into a 50mL beaker, 0.625mL of Buffer was added, 1.3mL of NaCl/Bis was further added, 0.05mL of Ammonium Sulfate (APs) was added, 0.005mL of TMEMD was added, and the mixture was mixed well.

12% separation gel (30 mL): 16.12mL of distilled water was added to a 50mL beaker, 1.88mL of Buffer (Tris-HCl: 10% SDS-25: 1) was added, 12mL of 30% Acr/Bis was added, 0.15mL of LAPs was added, 0.03mL of TMEMD was added, and the mixture was mixed well.

Preparing glue: aligning and clamping the glass plate into a frame, and adding water to detect leakage after the clamps on the two sides are tightly fixed; and (3) immediately pouring the prepared separation glue (which is used in the field) into a gap of the glass plate by using a liquid-transferring gun, pouring the glue to a position 1cm away from the short glass plate, and adding water to seal the glue. After the glue is solidified, the water layer is removed, the absorbent paper is sucked dry, then the concentrated glue (used as the preparation is carried out) is added, and a comb is inserted. After the colloid is solidified, carefully pulling out the comb for later use.

Preparing the medicine:

cell culture:

HCT-116 cells that grew well and were in logarithmic phase were selected, 1mL of 0.25% trypsin digest (without EDTA) was added, and the flask was gently shaken until the digest completely covered the bottom of the flask. And (3) incubating the cells in a cell incubator for 3-5 min until the cells are exfoliated and become round single cells, adding 5mL of culture medium to stop digestion, centrifuging (800rpm for 10min), and discarding the supernatant. Adding appropriate amount of culture medium to resuspend cells, blowing with pipette uniformly, counting, and making into 5 × 10⁵cells/mL of cell suspension. Inoculating 2mLHCT-116 cell suspension in 35mm cell culture dish at 37 deg.C in 5% CO₂₊Incubate under conditions for 24h until the cells are fully adhered to the bottom of the dish.

The medium was removed, the cells were washed three times with an appropriate amount of PBS, 2mL of drug-containing medium was added to each group, and incubation was continued for 8h and 24 h.

Immunoblot assay (Western blotting):

extraction of HCT-116 cell protein: the cell culture dish was decanted from the drug-containing medium, the cells were washed 2 times with pre-chilled PBS and then discarded, and the dish was placed on ice. Adding 500 μ L of the prepared lysis mixed solution into a culture dish, and gently shaking for lysis for 1 min; after completion, the pipette was gently pipetted, and the cell debris and lysate were aspirated and transferred to a 1.5mL centrifuge tube. The tube was centrifuged at 12000rpm for 15min at 4 ℃ and after centrifugation, the supernatant was carefully aspirated into a new 1.5mL centrifuge tube and 5 Xloadingbuffer was added (volume of supernatant: loadingbuffer: 4: 1). The protein was denatured by heating and shaking the centrifuge tube at 100 ℃ for 5min, and then temporarily stored in a refrigerator at-20 ℃.

SDS-PAGE gel electrophoresis: putting the prepared gel in an electrophoresis apparatus, and dropwise adding 80ug of the protein sample to be detected into the hole; electrophoresis conditions: the concentrated gel has a constant pressure of 80V for about 20 min; the separation gel was kept at a constant pressure of 120V, and electrophoresis was stopped until bromophenol blue reached near the bottom of the gel.

Wet film transfer: soaking the PVDF membrane in methanol for 30s, transferring the PVDF membrane into a WB transfer buffer, completely soaking thick filter paper with the same size as the PVDF membrane in the WB transfer buffer, taking out the filter paper, putting the filter paper, gel, the PVDF membrane and the filter paper into a sponge pad interlayer in sequence, forcibly pressing the sponge pad to drive off bubbles, and putting the sponge pad into a Bio-Rad standard wet type membrane transfer device. Film transferring conditions: the membrane is rotated at a constant current of 300mA for 30min and 2 h.

WesternBlot detects the protein of interest: (1) and (3) sealing: after the film transfer is finished, soaking the PVDF film in 5 percent skim milk powder, and gently shaking for 1h on a shaking table at room temperature; (2) primary antibody incubation: diluting the primary antibody by 500 times by using 1% BSA/5% skimmed milk powder, and adding the diluted primary antibody into a hybridization bag; putting the sealed PVDF membrane in the same way, sealing the membrane by a membrane sealing machine, and incubating for 1h at room temperature on a side swing table; (3) washing the membrane: rinsing the PVDF membrane for 5min by TBST, and repeating twice; (4) and (3) secondary antibody incubation: diluting the secondary antibody 500 times with 5% skimmed milk, and adding into a hybridization bag; putting the PVDF membrane in the device, sealing the opening of the device by a membrane sealing machine, and incubating the device for 1h at room temperature on a side swing table; (5) washing the membrane: rinsing the PVDF membrane for 5min by TBST, and repeating for three times; (6) and (3) placing the PVDF film in a dark room, dropwise adding 1mL of chemiluminescent agent into a liquid-transferring gun, waiting for 1min, and then exposing, developing and fixing.

All the washing and reaction steps are carried out on a side shaking bed.

1.4.2 Experimental data processing and statistical methods

The band gray values were read using Quantity One v.4.6.2 software.

1.4.3 discussion of results and analysis

Phage and autophagosome formation is associated with lipidation of LC3-I by phosphatidylethanolamine, resulting in the production of membrane-associated LC3-II protein. To investigate whether treatment with 356-TAT-AuNPs could modulate autophagy of HCT-116 cells, the levels of LC3-I and LC3-II were analyzed by Western blotting 8h after treatment with 356-TAT-AuNPs. Consistent with the accumulation of autophagic vesicles, LC3-II accumulated after treatment with 356, 356-AuNP, and 356-TAT-AuNPs (B in FIG. 8). LC3-II accumulated significantly more in 356-TAT-AuNPs treated cells compared to 356 and 356-AuNPs treated cells, indicating that 356-TAT-AuNPs modulate autophagy flux by inhibiting lysosomal autophagic vesicle breakdown.

Test example 2356-TAT-AuNPs evaluation of in vivo antitumor Activity

2.1 cell lines and laboratory animals

Cell line: HCT-116(human colon cancer cells) human colon cancer cells.

Experimental animals: male BALB/cNude mice, weighing 16-18 g, were purchased from Experimental animals, Inc., Wei Tong Li Hua, Beijing.

2.2 Experimental methods

2.2.1 grouping and dosing

Negative control group: physiological saline; IgG: 200 mu g/20 g;

a free drug group: 356: 10 mu mol/kg; PD-1 antibody: each dose is 200 mug;

vector group: AuNPs: 1 mg/kg; 356-TAT-AuNPs: 1 mu mol/kg;

combination group: PD-1+ 356-TAT-AuNPs: 200. mu.g/20 g + 1. mu. mol/kg.

2.2.2 modes and times of administration

All the medicines in each group are administrated by tail vein injection; the treatment was completed by continuing the feeding for 21 days after the first day of administration.

Wherein IgG, PD-1 antibodies are administered once a week; AuNPs, NS, 356-TAT-AuNPs were administered daily for the first week and weekly for the next two weeks.

2.2.3 animal feeding

Male BALB/cNude mice, weighing 16-18 g, were housed in the Central barrier System of laboratory animals of the university of capital medical sciences, 5 mice per cage. All mice had free water and diet, and the light 12/12 was switched on and off alternately, and the bedding was changed every day.

2.2.4 extraction of human lymphocytes

27mL of human peripheral whole blood was taken at 10: 1, adding 3mL of 4% sodium citrate solution to obtain anticoagulated whole blood; the anticoagulated whole blood was diluted with 30mL of PBS and mixed well.

5mL of human peripheral blood lymphocyte separation solution is sucked into a 15mL centrifuge tube, an equal volume of diluted blood is slowly dripped by using a disposable Pasteur pipette, the diluted blood is tiled above the liquid level of the separation solution, and the interface between the two liquid levels is kept clear (because of the density difference between the two liquid levels, an obvious layered interface is formed). This procedure was repeated with the remaining diluted blood.

The liquid obtained after centrifugation (800 g of horizontal rotor at room temperature, 25min) was clearly stratified: the uppermost layer is diluted plasma layer, the middle transparent layer is separating liquid, and a white membrane layer is arranged between the plasma and the separating liquid and is the required lymphocyte layer.

Carefully sucking the white membrane layer between the plasma and the separation liquid into a 15mL clean centrifuge tube, adding 10mL cell washing liquid to wash the white membrane layer cells, and centrifuging (250g for 10 min); the supernatant was discarded, 5mL of cell wash was added to resuspend the cells and centrifuged (250g, 10 min). After repeating this operation twice, PBS resuspended the final stem cell pellet for use.

2.2.5 establishment of animal tumor model simulating human immune Environment

Cell culture

HCT-116 cells were revived and cultured in complete McCoy's 5A medium. Subculturing the cells to the third generation, basically eliminating the influence of the frozen stock solution on the cell growth, then selecting the cells with good growth state and logarithmic phase for digestion, and centrifuging (800rpm, 10min) for collection. Cell viability>95% of the cells were present in the culture,resuspend and count with 4 ℃ precooled PBS and dilute it to 1X 10⁷The cells/0.1 mL were placed in 0.4mL centrifuge tubes and stored temporarily in ice bath boxes for later use.

Establishment of HCT-116 cell allotransplantation tumor model

After fixing the mouse, the armpit is disinfected by 75% alcohol, the right hand holds a syringe which is sucked with prepared tumor cell suspension, the needle is inserted at the position of the armpit at an oblique angle of 45 degrees, 0.1mL of tumor cells are injected into the underarm subcutaneous part, the right hand slightly presses the needle hole for 1min after the needle is withdrawn quickly, and the tumor liquid is prevented from leaking. The mice were returned to their cages and observed for abnormal reactions. 5 mice were inoculated in this order as primary mice.

Observing abnormal reaction and tumor growth condition of mouse every day, removing neck of primary mouse in barrier to kill it by eighth day, separating tumor tissue, placing it in sterile surface dish, washing with PBS for 3 times, removing blood vessel on tumor surface, shearing tumor tissue into size smaller than 1mm with sterile ophthalmic scissors³The volume of the small block, the tumor small block obtained by PBS washing, is put on the tip of the tumor-planting needle for standby.

The mouse is fixed by the left hand, the right side of the abdomen is disinfected by 75% alcohol, the ophthalmology scissors cut a small incision of about 0.5cm at the disinfection position of the right side of the abdomen, the subcutaneous fascia and the skin are separated by a bent forceps, the tumor seeding needle is inserted from the small incision and runs along the subcutaneous part until the deep part of the armpit, the tumor seeding needle is slightly rotated, the tip end is marked out a small cavity at the armpit, at the moment, the tumor cut block is carefully ejected out, the needle is withdrawn by fast rotation, the collodion is smeared at the incision of the abdomen, the mouse is put back to the mouse cage after waiting for 1min, and the abnormal reaction is observed. 10 mice were sequentially inoculated as second generation xenograft tumor mice according to this method.

Observing whether the mice have abnormal reaction and tumor growth condition every day, selecting the mice with good tumor growth till the tenth day, and inoculating according to the tumor-seeding step of passage to obtain 90 third generation allograft tumor mice.

Establishment of xenotransplantation tumor model for simulating human body immune environment

The mice were observed daily for abnormal reactions and tumor growth, and 10 days after inoculationThe tumor volume is uniform and is all 1mm³At this time, 200. mu.L of human lymphocytes (about 5X 10 in cell number) isolated from mice were administered by tail vein injection⁵One), a nude mouse model of HCT-116 cell xenograft presenting human lymphocyte circulation was obtained, rested for one day, and awaited for further processing.

2.2.6 animal treatment methods

One day after completion of T lymphocyte injection, mice were randomly divided into 7 groups of 8 mice each. In order to ensure that the weight between each group has no significant difference, all data are recorded into the SPSS for verification, and adjustment is performed according to the result.

2.3 evaluation index

2.3.1 body weight and general State

Weighing and recording every two days for a fixed time;

the posture, gait, spirit, fur, feces and tail injury of the mice were observed.

2.3.2 tumor weight and tumor volume

After the experiment, the mice were killed by cervical dislocation and were isolated by blunt force to obtain intact tumor tissues. After the tumor tissue is washed by PBS buffer solution, filter paper is dipped to be dry, the tumor weight is weighed and recorded by an analytical balance, and the data is recorded into SPSS for statistical analysis.

Fixing the time, measuring the long diameter and the short diameter of the tumor by using a vernier caliper every two days, recording, and observing the growth condition of the tumor; tumor volume was calculated as follows: tumor volume (tumor major diameter × tumor minor diameter)²)/2。

2.3.3 pathological section of tumor tissue

Material taking: after tumor tissue was removed, the cells were soaked in 4% paraformaldehyde solution and fixed overnight at room temperature. Taking out the tumor tissue from the fixing solution, flattening the tumor tissue in a fume hood by using a scalpel, cutting the tumor tissue into small samples with the thickness of 2mm, and placing the small samples and the labels in a dehydration box correspondingly to wait for dehydration.

And (3) dehydrating: and (4) putting the dehydration box into a dehydration machine, and dehydrating by using gradient alcohol solution in sequence. Dehydrating with 75% alcohol for 4h, dehydrating with 85% alcohol for 2h, dehydrating with 90% alcohol for 22h, dehydrating with 95% alcohol for 1h, dehydrating with absolute ethanol (I) for 30min, dehydrating with absolute ethanol (II) for 30min, treating with xylene (I) for 10min, treating with xylene (II) for 10min, treating with wax (I) for 1h, treating with wax (II) for 1h, and treating with wax (III) for 1 h.

Paraffin section dewaxing to water: placing the slices in xylene (I) for 15min, xylene (II) for 15min, anhydrous ethanol (I) for 10min, anhydrous ethanol (II) for 10min, 95% ethanol for 5min, 90% ethanol for 5min, 80% ethanol for 5min, 70% ethanol for 5min, and distilled water for 5 min.

Hematoxylin staining of cell nucleus: the sections were stained with hematoxylin for 5min, washed with running water for 3s, 1% alcohol with hydrochloric acid for 3s, washed with running water for 30s, rewetted with 0.6% ammonia water, and washed with running water.

Eosin staining of cytoplasm: the sections were immersed in eosin stain for 3min and rinsed with distilled water for 2 s.

Dewatering and sealing: placing the slices in 95% alcohol (I) for 5min, 95% alcohol (II) for 5min, anhydrous alcohol (I) for 5min, anhydrous alcohol (II) for 5min, xylene (I) for 5min, and xylene (II) for 5min, and dehydrating to obtain transparent product. The slices are taken and dried, and the neutral gum is sealed.

Observing by a microscope, and taking a picture and storing by an image acquisition and analysis system.

2.4 statistical analysis method

The measured data is expressed by mean plus or minus standard deviation (mean plus or minus SD), statistical analysis is carried out by applying SPSS software, one-way ANOVA (one-way ANOVA) is adopted among a plurality of groups, and the P < 0.05 is defined as having statistical difference and is expressed as x; p < 0.01 had significant statistical differences, expressed as x.

2.5 Experimental results and discussion

2.5.1 general status of mice

The mice in each group have no death, and the fur of the mice is smooth and can move freely, and the abnormal phenomenon of defecation does not occur. Some mice have bited tails, but the distribution is random, and the mice are not concentrated on fixed groups, and presumably the bites are caused by the discomfort of the tails of the mice due to the more times of tail veins.

The body weight change of the mice is one of indexes reflecting the toxicity of the antitumor drugs. As can be seen from FIG. 9, the body weight of the normal group mice increased normally and steadily during the feeding period. The body weight of the tumor-bearing mice is reduced to a certain degree, and the change among the groups has no significant difference. The weight of the NS group and the mice in the administration group decreased by about 13% within 21 days, which indicates negligible systemic toxicity of the nanoparticles, considering that the weight loss may be due to the effect of tumor-bearing mice on feeding.

2.5.2 tumor weight and tumor volume

Tumor volume was monitored in real time every two days after receiving treatment. As shown in a in fig. 10, during the course of 21 days of treatment, the AuNPs group and the IgG group did not detect any inhibition of tumor growth, while the 3TA group and the PD-1 antibody combined with 3TA group were effective in inhibiting the increase in tumor volume.

As can be seen from fig. 11, the average tumor weight of the mice in the combination group of 3TA and PD-1 antibody was significantly different from that in the control group.

2.5.3 pathological results

Sections obtained by hematoxylin-eosin staining (HE) were stained. The hematoxylin staining solution is alkaline, and can stain basophilic part in cell to be bluish purple, such as chromatin in cell nucleus and nucleic acid in cytoplasm; eosin staining solution is acidic, can stain eosinophilic part in cells to be red pink, and mainly stains cytoplasm and components in extracellular matrix.

FIG. 12 is a histopathological section of tumor. The pathophysiology of tumor cells is characterized by relatively large nuclear volume, clear nuclear envelope, and significant nuclear division as observed in HE staining results due to vigorous proliferation. Compared with the NS group, the treatment group has the advantages that the number of the cells with obvious nuclear division is reduced, and part of the cells has histological damage, which indicates that part of the tumors have apoptosis; the reduction in the number of mitotic cells in the PD-1+3TA combination group was evident, indicating that limited therapeutic benefit was indeed achieved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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<120> nano-drug carrier and preparation method thereof, drug-loading system and preparation method and application thereof

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<170> SIPOSequenceListing 1.0

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Claims

1. A drug loading system comprises a nano drug carrier and a PAD4 inhibitor loaded on the nano drug carrier, wherein the nano drug carrier is obtained by modifying gold nanoparticles through a trans-transcription activating factor, the surface of the gold nanoparticles is negatively charged, and the particle size of the gold nanoparticles is 5-50 nm; the amino acid sequence of the trans-transcriptional activator is as follows: CYRGRKKRRQRRR, respectively; the content of the trans-transcription activating factor in the nano-drug carrier is 0.03-0.05 wt.%;

the PAD4 inhibitor is N- (1- (benzylamino) -5- (2-chlorooxalimido) -1-oxypentan-2-yl) -6- (dimethylamino) -2-naphthamide; the drug loading rate of the drug loading system is 60-90 wt.%.

2. The drug delivery system of claim 1, wherein the preparation method of the nano-drug carrier comprises the following steps:

3. The drug delivery system of claim 2, wherein the temperature of the coordination reaction is 15-35 ℃ and the time is 1.5-2.5 h.

4. The preparation method of the drug-carrying system of any one of claims 1 to 3, which comprises the following steps:

5. The method according to claim 4, wherein the temperature of the non-covalent bonding is 15 to 35 ℃ and the time is 50 to 70 min.

6. The application of the drug-carrying system of any one of claims 1 to 3 or the drug-carrying system prepared by the preparation method of any one of claims 4 to 5 in preparing an antitumor drug.

7. The use of claim 6, wherein the tumor comprises colon, lung or breast cancer.