AU2021104340A4

AU2021104340A4 - Preparation and Application of Matrix Metalloproteinase-9-Responsive Surface Charge-Reversible Nanocarrier

Info

Publication number: AU2021104340A4
Application number: AU2021104340A
Authority: AU
Inventors: Luyuan Li; Qiangzhe Zhang
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2021-09-16
Anticipated expiration: 2029-07-20

Abstract

The invention discloses a surface charge-reversible nanocarrier for drug delivery based on a novel charge reversible amphiphilic molecule, which can be applied to specifically detect and high- effectively treat of MMP9-expressing tumors. The functional amphiphilic molecule consists of three parts: oleic acid is the hydrophobic region of the molecule, the middle connecting part is a MMP9 responsive cleavage polypeptide (-GPLGLPG-), and the other part is a segment of poly-glutamic acid residues (-GEEEEEG), which constitutes a negatively charged amphiphilic molecule (named as OMPE). The nanocarrier (0-NP) consists of cationic lipid core and OMPE. The cationic lipid core is composed of 1,2-dioleoyl-3 trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Soy L-a-phosphatidylcholine (SPC), Cholesterol (CHO), and 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[poly(ethylene glycol)2000] (DSPEPEG2000). After modified by OMPE, the surface charge is reversed to negative charge. Cell and animal experiments show that the nanocarrier can effectively deliver detection probes and anti-tumor drugs, which improve the sensitivity of diagnosis agents and the antitumor effects of drugs in vivo by overcoming their low solubility and/or nonspecific enrichment. So this nanocarrier can be a promising delivery platform for cancer diagnosis and therapy..

Description

Preparation and Application of Matrix Metalloproteinase-9-Responsive

Surface Charge-Reversible Nanocarrier

TECHNICAL FIELD

The invention belongs to the field of nano drugs, and particularly relates to a

surface charge-reversible nanocarrier for drug delivery based on a novel charge

reversible amphiphilic molecule

BACKGROUND

Nanotechnology has attracted increasing attention in the diagnosis and

therapeutics of cancer, largely due to its unique properties, such as improving drug

delivery efficiency, achieving long-term circulation in the body and reducing toxicity.

A large number of nano-drug carriers, such as liposomes, polymer micelles,

dendrimers and other nanotechnology models, have been used in clinical research and

even approved for cancer therapeutics. Due to the leakage characteristics of tumor

blood vessels, nanoparticles of appropriate size can be preferentially enriched in

tumor lesions to effectively provide chemotherapeutic drugs or imaging probes.

However, there are multiple complex biological steps in the effective delivery of the

payload to the tumor site, such as nanocarrier/protein interaction, blood circulation,

extravasation, tumor penetration, and cell uptake. In addition, the properties of

nanocarriers (e.g., surface charge, size, geometry, porosity, and targeting ligands)

have been shown to influence or interact with these biological processes. Compared

with anions, PEGylated cationic nanoparticles showed better inhibition of tumor

growth, but poorer effects on blood circulation time and tumor enrichment.

Nonetheless, smart nanomaterials with sensitive biological responses remain

attractive for cancer diagnosis and therapeutics.

Bio-specific stimulation-responsive nanomaterials are typically triggered by that

microenvironment of the lesion. Bioresponsive nanomaterials generally contain

functional molecules that have specific sensitivity to tumor microenvironment

features, such as factors that respond to specific functions such as enzyme, pH,

hypoxia, redox state, and glucose. Since enzymes are of great significance in many

biological processes, tumor-related enzymes have become new targets for drug

therapy.

Matrix metalloproteinases (MMPs), as a kind of proteolytic enzyme, play a vital

role in all stages of tumor development by degrading the extracellular matrix. MMPs

can regulate apoptosis, angiogenesis, tumor growth, metastasis and many other

aspects, making them become specific biological candidate molecules for malignant

tumors. MMP9 in MMPs can be released and activated as secreted type IV

collagenase/gelatinase in locally inflamed tissues. The expression of MMP9 in tumor

tissues is related to the malignant degree of various cancers including breast cancer,

colon cancer and gastric cancer. At present, the drug delivery systems developed

based on MMPs response are mainly of the following types, such as binding ligands,

enzyme-responsive cleavage prodrugs, MMPs-related effect targets and matrix

degradation. Over-expression of MMPs in various solid tumors makes MMPs

sensitive antibodies or polypeptides become popular target components. Polypeptides,

as targeted functional ligands, are more favoured by people because of their simple preparation method, low cost, low immunogenicity and low opsonin function. In addition, peptides modified by long circulating motifs can overcome the obvious defect of rapid metallization.

At present, it is still difficult to detect tumors with non-invasive, repeatability

and high accuracy during or after clinical therapeutics of tumors, so specific image

detection technology is urgently needed to guide tumor therapeutics. Molecular

imaging plays an increasingly important role in immunotherapy and personalized

medicine. The preparation of molecular probes is the key to molecular imaging. Only

after the molecular probes with high sensitivity and specificity are introduced into the

body, can they specifically bind to specific target molecules in cells and generate

certain signals, which are then collected in vitro by specific imaging devices, such as

positron emission computed tomography (PET-CT), single-photon emission

computed tomography (SPECT), magnetic resonance imaging (MRI), and

chemiluminescent devices, so as to achieve the purpose of specific diagnosis.

Therefore, it is an effective way to solve the above-mentioned problems by adopting

non-invasive methods, rationally designing molecules responding to cancer cells

according to tumor markers, and then developing them into diagnostic reagents and

therapeutic drugs for tumors. In a word, the research and development of nano-drug

delivery carrier with specific targeting/responsiveness for tumors with high

expression of MMP9 will have great breakthrough significance in the diagnosis and

therapeutics of various cancers.

SUMMARY

The technical problem to be solved by the invention is how to provide a surface

charge-reversible nanocarrier for drug delivery based on a novel charge reversible

amphiphilic molecule, which is particularly suitable for microenvironment of MMP9

expressing tumors, such as colorectal cancer, gastrointestinal cancer, bladder cancer,

brain cancer and the like, which can promote the charge reversion of the invention.

According to the technical scheme of the invention, a functional amphiphilic

molecule OMPE is formed by connecting oleic acid and a polypeptide segment,

wherein the amino acid sequence of the polypeptide segment is GPLGLPGGEEEEEG

(SEQ ID No.1), and the structure is as follows: OH 0 H

HO HN

0 0 00 HN 0 HN NH C N NH O 0 OH o o 0 N ~ HHN O H

_NH OH 0 0

The functional amphiphilic molecule consists of three parts: oleic acid is the

hydrophobic region of the molecule, and the intermediate connecting part is a MMP9

responsive cleavage polypeptide, as well as a glutamic acid-rich polypeptide segment,

which constitute negatively charged amphiphilic molecules.

A tumor microenvironment responsive charge-reversible nanocarrier is prepared

from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero

3-phosphocholine (DOPC), Soy L-a-phosphatidylcholine (SPC), Cholesterol (CHO),

distearyl phosphatidyl ethanolamine-polyethylene glycol 2000 with the mass ratio of

m : 4: m : 1: 1 (m =2-4) , the foundation frame with positive charge was formed, and the nano-carrier with negative charge on the surface was formed by OMPE modification.

The preparation method of the tumor microenvironment responsive charge

reversible nanocarrier comprises the following steps:

1) Weighing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2

dioleoyl-sn-glycero-3-phosphocholine (DOPC), Soy L-a-phosphatidylcholine (SPC),

Cholesterol (CHO), distearyl phosphatidyl ethanolamine-polyethylene glycol 2000

and the OMPE in a mass ratio of m: 4: m: 1: 1/2 (m = 2-4), and dissolving in

trichloromethane/methanol solution;

2) After completely dissolving, rotating and evaporating the solvent at 39 deg°C

to form a uniform film, and removing the residual organic solvent by inert gas;

3) Adding PBS, heating and hydrating in water bath at 60°C for 15 min, and

ultrasonicating in water bath for 15 min to obtain clear suspension; Filtering the

suspension to obtain the tumor microenvironment responsive charge-reversible nano

carrier.

The delivery system is composed of the tumor microenvironment responsive

charge-reversible nanocarrier and the loaded active ingredient described above.

Further, the active ingredient is any one of a chemical drug, a biological drug, a

nano drug, a radiopharmaceutical, a photothermal therapy, a photodynamic therapy

drug or a carrier wrapping these drugs capable of killing cancer cells.

Further, the active ingredient is any one of an alkylating agent, an antimetabolite,

an anti-tumor natural drug, an anti-tumor antibiotic, a hormone, a metal complex or a

tumor radiotargeted marker.

The functional amphiphilic macromolecule derived from the functional

amphiphilic molecule OMPE of the invention is responsively cut off by other matrix

metalloenzymes and the like to generate charge reversion.

The tumor microenvironment responsive charge-reversible nanocarrier of the

present invention derivatizes a functional nanodrug/probe delivery carrier.

Compared with that prior art, the invention has the following beneficial effects:

(1) The invention can be widely applied to diagnosis and therapeutics of tumors

with high expression of MMP9. It can be used for targeted therapy or diagnosis of

various tumors by being conjugated or mixed with a preparation or a detection probe

capable of killing cancer cells.

(2) The amphiphilic functional molecules in the invention can be cut off by the

specific response to reverse the surface charge of the nanocarrier and realize the

efficient delivery of drugs.

(3) The nanocarrier loads the diagnostic reagent or the killer drug to realize high

efficiency tumor diagnosis and therapeutic effect.

(4) The invention has simple preparation method, low economic cost and wide

application prospect. It can be used to monitor the curative effect of drugs and the

corresponding patient's condition in real time. The invention provides a simple, rapid,

economical and accurate detection means for monitoring the recurrence and metastasis of tumor after therapeutics in real time, so as to adjust the treatment scheme in time, carry out clinical intervention as soon as possible, prevent the disease from progressing and improve the prognosis of patients.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a structure diagram of OMPE;

Figure 2 is an O-NP composition diagram and a characterization diagram;

Figure 3 is a cell uptake diagram of O-NP;

Figure 4 is a drug delivery diagram of O-NP in vivo. Nanocarrier (0-NP)

enhanced specific enrichment of H2S probe in LS180 xenografts. A) Distribution of

Probe 1 encapsulated in the indicated nanocarriers in whole body post 48 h injection.

B) NIR images of retrieved tumors post 48 h treatment with Probe 1 encapsulated in

the indicated nanocarriers. C) Quantification of relative fluorescence intensities of the

tumors (data are mean SD, n = 3; **p <0.01; Student's t-test with two tails)

Figure 5 : Therapeutic outcomes of mice with LS180 xenografts. A)

Photographs of tumors retrieved from experimental animals treated with PBS,

Nanocarrier (0-NP), free DOX, or Nanocarrier (0-NP)-DOX. The animals were

treated seven times, intravenously with the indicated nanoparticles every other day,

and the tumors were retrieved on day 15. B) Growth curves of the LS180 tumors with

the indicated treatments (data are mean SD, n = 5 biologically independent samples;

**p <0.01, ***p <0.001; two-way ANOVA with Dunnett's multiple comparisons

test). C) Typical images of tumor infiltration of DOX (green) with various treatments

via frozen sections. Nuclei were indicated by DAPI (blue). Scale bar, 25 im. D)

Typical images of H&E and Ki67 immunohistochemical staining of the tumors with

various treatments. Scale bar, 100 im.

DESCRIPTION OF THE INVENTION

The technical scheme of the present invention will be further explained by

specific embodiments below. It should be clear to those skilled in the art that the

described embodiments are only to support the present invention, and should not be

regarded as a specific limitation of the present invention.

The methods used in the following examples are all conventional methods unless

otherwise specified.

Materials and reagents used in the following examples are commercially

available unless otherwise specified.

Example 1: Synthesis of amphiphilic molecule OMPE according to the present

invention

(1) Materials

N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA),

dichloromethane (DCM), ninhydrin, vitamin C, phenol, benzotriazole -N,N,N',N'

tetramethylurea hexafluorophosphate (HBTU), diisopropylethylamine (DIPEA),

triisopropylsilane (TIS), N,N- dimethylformamide (DMF), anhydrous ether, Wang

resin, methanol, various Fmoc-protected amino acids, oleic acid, polypeptide

synthesis tube, shaker, vacuum water pump, rotary evaporator. The above reagents

and materials are commercially available.

(2) Preparation of solvent

The reaction solution (act)-n-methylmorpholine: n, n dimethylformamide (1: 24)

Deprotection solvent-piperidine: N, N dimethylformamide (1: 4)

Lysates-trifluoroacetic acid (95%), triisopropylsilane (2.5%) and ultrapure water

(2.5%).

Ninhydrin detection solution-5% ninhydrin: 5% vitamin C: 4g/mL phenol (1: 1:

1).

(3) Synthesis of OMPE

The Fmoc solid-phase peptide synthesis method is adopted to synthesize OMPE,

and the specific method is as follows:

(A) Fmoc-Gly-Wang resin was weighed, placed into a polypeptide synthesis

tube, and 8 mL DMF DMF was added for swelling for 30 min.

(B) Deprotection: adding a deprotection solvent, and performing deprotection at

room temperature for 10 min; ; Washing: DMF and DCM were washed alternately

for 3 times.

(C) Coupling: sequentially weighing the corresponding amino acids with 5 times

of redundancy amount from theC end to the N end according to the sequence,

mixing with HBTU with an equal molar ratio, adding 6 mL of activation reaction

reagent (ACT) for dissolution, adding into the polypeptide synthesis tube, placing on

a shaking table, and reacting at room temperature for 40 min-I h; The N end is

connected with oleic acid for reaction, namely, 10 times of redundant amount of

corresponding amino acid is mixed with HBTU with equal molar ratio, 6 mL of

activation reaction reagent containing 1% DIPEA is added for dissolution, and then the mixture is added into the polypeptide synthesis tube and placed on a shaking bed for reaction overnight at room temperature;

(D) Washing: DMF and DCM are washed alternately for 3 times (the same as the

washing process mentioned above). Deprotection: The deprotection reagent is added

and the mixture is deprotected at room temperature for 10 min; . Washing:DMFand

DCM are washed alternately 3 times each.

At each step of coupling and deprotection, the free amino group is detected by

ninhydrin detection reagent to determine whether the deprotection and coupling are

complete. Deprotection may only be carried out after the test coupling is complete;

Similarly, the coupling reaction of the next amino acid residue cannot proceed until

the deprotection is complete.

Cyclic reaction: according to the above deprotection-washing-coupling-washing

deprotection cycle until all the amino acids are coupled. In each step, the free amino

group can be detected by ninhydrin detection reagent to determine whether the

deprotection and coupling are complete.

Resin shrinkage: the resin is washed after synthesis of the target sequence.

Adding methanol into that polypeptide synthesis tube for washing, contracting and

draining.

Transfer the prepared OMPE- resin to a 10 mL brown bottle, add 5 mL of lysis

buffer, place on ice, and lyse under mild stirring in the dark for 3 h. Subsequently, that

polypeptide lysate is evaporate under the condition of vacuum rotation at 42C until

the lysate is basically volatilize; Precipitate the crude product in a flask with 10 mL of pre-cooled ether, which was centrifuged several times. Vacuum dry the crude product of OMPE, and store the dried crude product at -20 °C for standby.

Figure 1 is a structure diagram of OMPE. The structural sequences are as

follows: Oleic acid-gplglp-ggeeeeeeg; The results of high performance liquid

chromatography and time-of-flight mass spectrometry confirme that the synthesized

OMPE could be used for subsequent experiments.

Example 2 construction of functional nanocarrier O-NP

(1) Materials

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero

3-phosphocholine (DOPC), Soy L-a-phosphatidylcholine (SPC), Cholesterol (CHO),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)2000]

(DSPEPEG2000),Methanol, Trichloromethane, Phosphate Buffer Salt Solution (PBS).

(2) Preparation of O-NP

The nanocarrier O-NP is prepared by a film dispersion method, and that specific

steps are as follow:

Accurately weigh 1mg of DOTAP, 2 mg of DOPC, 1 mg of SPC, 0.5 mg of

DSPE-PEG 2000, 0.5 mg of CHO, and 0.5 mg of OMPE in 5 mL of solvent

(trichloromethane/methanol, 2: 1 by volume) and place in a round-bottomed flask

after complete dissolution. At 39C. The rotary evaporator is slowly and uniformly

rotated to volatilize the organic solvent, and a uniform film is formed and apply to the

flask wall. Remove the residual organic solvent from the flask with inert gas. Then 5

mL PBS is added, heated and hydrated in a 60°Cwater bath for 15 min, following by water-bath ultrasound for 15 min to obtain a clear suspension. The prepared samples are filtered (0.224m) three times to obtain sample O-NP (1 mg/mL) and stored at 4C for subsequent use.

Placed 1 mL of nanoparticle suspension O-NP (1 mg/mL) in a Dynamic Light

Scattering (DLS) sample cell, and the size of O-NP and its surface potential (Q) can be

measured by a DLS granulometer. The test is repeated three times at the test

temperature of 25C, laser wavelength of 633 nm, probe angle of 173, and sample

equilibration time of 2 min. Experimental data are processed and analysed by

software Zetasizer Software.

The morphology of nanoparticles is observed by electron microscope (TEM).

Take 8 L of O-NP (1 mg/mL) and drop it on the copper mesh supported by common

carbon membrane. After 2 min, absorb the excess sample with filter paper. Then drop

6 L of 1% phosphotungstic acid for negative dyeing for 5 min. Absorb the excess

liquid with filter paper. Finally, wash it with water once and put it in a drying oven for

later use. Under the voltage of 200 kV, it is observed by biological transmission

electronmicroscope TalosTF200C (TEM, FEI, USA)

Example 3 Uptake of O-NP by cells

(1) Preparation of imaging agent (0-NP-Cy2): According to the preparation

method of O-NP, 1 mg DOTAP, 2 mg DOPC, 1 mg SPC, 0.5 mg DSPE-PEG2000, 0.5

mg CHO, 0.5 mg OMPE and 0.5 mg Cy2 are accurately weighed and dissolved in 5

mL solvent (chloroform/methanol, volume ratio 2:1). At the temperature of 39C, the

rotary evaporator rotates slowly and evenly. After the organic solvent volatilizes, a uniform film is formed and attached to the flask wall. The residual organic solvent in the flask Is removed with inert gas. Adding 5 mL of PBS, heating and hydrating in 60

°C water bath for 15 min, and ultrasonic treatment in water bath for 15 min, a clear

suspension is obtained. The prepared sample is filtered (0.22 m) for three times to

obtain sample O-NP (1 mg/mL), which is stored at 4C for later use.

(2) Cell culture: Human colorectal cancer cell LS180 and human breast cancer

cell Hela are cultured in a 5% carbon dioxide incubator at 37Cwith high glucose

DMEM containing 10% fetal bovine serum and 1% double antibodies (100 U/mL

penicillin and 100 g/mL streptomycin).

(3) O-NP-Cy2 interacts with cells and cell uptake is observed.

Hela cells are incubated at a cell concentration of 1x105/mL, with 1 mL of cell

suspension in a circular petri dish (35 mm) at 37C in a 5% C02 cell incubator. After

12 h, discarding the culture solution, then the petri dishes are incubated with different

concentrations of exogenous MP9 (0, 1, or 10 ng/mL) for 1 h, following by 0-NP

Cy2(30 [g/mL, 1 mL) for 2 h. Then washed twice with pre-cooled 1xPBS. The cells

are incubated with Hoechst 33342 containing 10 g/mL for 10 min at 37 C, and

washed twice with pre-cooled 1xPBS. The fluorescence distribution in the cells is

detected by a laser scanning confocal microscope (ZEISS TCS SP8).

As shown in Part A of Figure 3, the addition of exogenous MP9 promoted the

entry of O-NP into cells, and in particular, 10 ng/mL MMP9 significantly promoted

the entry of O-NP into Hela cells.

Hela and LS180 are incubated 1 mL of the cell suspension at a cell concentration

of 1x105/mL in a round petri dish (35 mm) at 37C in a 5% C02 cell incubator for 12

h, discarding the culture medium and incubating with the MMP9 inhibitor 3B-3CT (0

or 20 g/mL) for 30 min following by 0-NP-Cy2 (30 [g/mL, 1 mL) for 2 h. Then

washed twice with pre-cooled 1xPBS. The cells are incubated with Hoechst 33342

containing 10 g/mL for 10 min at 37C, and washed twice with pre-cooled 1xPBS.

The fluorescence distribution in the cells is detected by a laser scanning confocal

microscope (ZEISS TCS SP8).

As shown in part B of Figure 3, the high expression of MMP9 in tumor cell line

LS180 results in higher uptake of O-NP than in Hela cells. The addition of inhibitor

3B-3CT could significantly inhibit the uptake of O-NP by cells.

Example 4 Enrichment of O-NP in solid tumor

(1) The experimental animals are female BALB/c nude mice with the age of 6-8

weeks, which were purchased from Unilever. The animal experiments are conducted

in an environment free of specific pathogenic microorganisms. All animal

experiments are conducted in accordance with the Guidefor LaboratoryAnimal Care

and Use issued by Tianjin Science and Technology Commission.

(2) Preparing O-NP-Cy7. Referring to the preparation method of O-NP-Cy2, 0.5

mg of Cy2 was replaced by an equal mass of Cy7 to obtain sample O-NP-Cy7 (1

mg/mL) which was stored at 4C for later use.

(3) Establishing animal model of tumor in vivo.

1x108/mL (100 L) of Hela cells are subcutaneously inoculated in the left thigh

of mice, and 5x107/mL(100 L) of LS180 cells are subcutaneously inoculated in the

right thigh. O-NP-Cy7(1 mg/mL, 200 L) is given by tail vein when the tumor size

was about 150 mm3, corresponding to 200 ng Cy7. Six hours after administration,

IVIS Lumina II (Xenogen/Perkin Elmer); The 710 nm excitation is observed by IVIS

Lumina II (Xenogen/Perkin Elmer), and ICG transmitted and filtered to receive

signals.

Figure 4 shows that the LS180 tumor had a higher residual uptake of O-NP than

the Hela tumor tissue.

In summary, from experimental examples 1to 4, it can be concluded that the

nanocarrier of the present invention has the characteristic of specific charge reversion

to increase the drug uptake efficiency by cell. In practical application, the nanocarrier

of the present invention can be conjugated or mixed with a preparation capable of

killing or indicating cancer cells as a functional delivery system for targeted therapy

or imaging of tumors.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The functional amphiphilic molecule OMPE is formed by connecting oleic

acid and a polypeptide segment, the amino acid sequence of the polypeptide segment

is GPLGLPGGEEEEEG, and the structure is as follow: OH

HOO

HO I NH

0 0 0 0 HN

N NH OH H

NHN O 0 N NH 0 OH

00

2. The tumor microenvironment responsive charge-reversible nanocarrier

consists of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn

glycero-3-phosphocholine (DOPC), Soy L-a-phosphatidylcholine (SPC), Cholesterol

(CHO), and distearyl phosphatidyl ethanolamine-polyethylene glycol 2000 in a mass

ratio of m: 4: m: 1: 1, m = 2-4, and forms a basic frame with positive charge, and the

nanocarrier with negative surface charge is formed after modification by OMPE as

claimed in claim 1.

3. The preparation method of the tumor microenvironment responsive charge

reversible nanocarrier according to claim 2, which is characterized in that the steps are

as follows:

1) Weighing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2

dioleoyl-sn-glycero-3-phosphocholine (DOPC), Soy L-a-phosphatidylcholine (SPC),

Cholesterol (CHO), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[poly(ethylene glycol)2000] (DSPEPEG2ooo) and OMPE of claim 1in a mass ratio of

m: 4: m: 1: 1/2, m= 2-4, and dissolving in trichloromethane/methanol solution;

2) After complete dissolution, rotary evaporation is carried out at 39C to form a

uniform film, and residual organic solvent is removed by inert gas;

3) PBS is added, and the mixture is hydrated by heating in a 60°C water bath for

min, following by water-bath ultrasound for 15 min to obtain a clear suspension;

Filter that suspension agent by a filter membrane to obtain the tumor

microenvironment responsive charge-reversible nanocarrier.

4. A delivery system comprising the tumor microenvironment responsive

charge-reversible nanocarrier and a loaded active ingredient according to claim 2.

5. The delivery system according to claim 4, which is characterized in that the

active ingredient is any one of a chemical drug, a biological drug, a nano drug, a

radiopharmaceutical, a photothermal therapy, a photodynamic therapy drug, or a

carrier encapsulating these drugs that can kill cancer cells.

6. The delivery system according to claim 4, which is characterized in that the

active ingredient is any one of an alkylating agent, an antimetabolite, an antineoplastic

natural drug, an antineoplastic antibiotic, a hormone, a metal complex or a tumor

radiotargeted marker.