CN109432432B

CN109432432B - Construction and application of targeting to endoplasmic reticulum nano drug delivery system

Info

Publication number: CN109432432B
Application number: CN201811110809.4A
Authority: CN
Inventors: 游剑; 罗利华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-09-21
Filing date: 2018-09-21
Publication date: 2020-10-23
Anticipated expiration: 2038-09-21
Also published as: CN109432432A

Abstract

The invention provides construction of a target-to-cell endoplasmic reticulum nano drug delivery system, which is realized by modifying Pardaxin polypeptide on the surface of a liposome. The Pardaxin is a polypeptide which contains 33 amino acid residues and has a membrane penetrating effect in an amphiphilic cation alpha helical structure. The constructed nano drug-carrying system can be applied to preparing antiviral, antitumor and other drugs taking endoplasmic reticulum as a target. The nano drug-loading system can load water-soluble and fat-soluble drugs and transfer the drugs to endoplasmic reticulum parts, thereby enhancing the therapeutic effect of the drugs and reducing toxic and side effects. The nano-carrier is not limited to liposome, but also can be solid lipid nanoparticles, nano-emulsion, polymer micelle, inorganic nano-material and the like. The novel endoplasmic reticulum targeted nano-carrier constructed by the invention can obviously improve the concentration of the medicine taking the endoplasmic reticulum as a target at the action target part, and provides a new way for the curative effect exertion of the medicines.

Description

Construction and application of targeting to endoplasmic reticulum nano drug delivery system

Technical Field

The invention belongs to the field of medicines, and relates to a novel nano drug delivery system capable of targeting to an endoplasmic reticulum of a cell and application of the drug delivery system in preparation of antiviral, antitumor and other drugs taking the endoplasmic reticulum as a target.

Background

The drug can exert the drug effect only by reaching the action target, and the target of the drug is generally functional biological molecules such as protein, nucleic acid, enzyme, receptor and the like positioned in cells. Modern drug delivery systems require that the drug be transported to the target tissue, target cells, and even specific organelles. The nano carrier has the advantages of protecting the medicine, achieving slow release, small toxic and side effects and the like. The research on the tissue and cell targeted drug delivery system is mature at present, and the three-level targeting, namely organelle targeting, is a hotspot and difficulty in the research on the current drug delivery system. Tertiary targeting includes cytoplasmic targeting, organelle targeting, such as mitochondrial targeting, endoplasmic reticulum targeting, lysosomal targeting, etc., nuclear targeting, and the like.

Endoplasmic Reticulum (ER) is a closed reticulated duct system composed of inner membranes, which is the main site for intracellular protein synthesis, lipid synthesis, substance transport, carbohydrate metabolism, and detoxification, and its main function is to synthesize proteins and lipids. The endoplasmic reticulum contains multiple enzymes, the marker enzyme is glucose-6-phosphatase, which participates in glycogenosis, and the hydrolase in the endoplasmic reticulum cavity is used for metabolism and detoxification of various drugs. Cytochrome P450 on endoplasmic reticulum of liver and intestinal tract cells is a main enzyme for metabolizing drugs in human body and can catalyze the metabolism of various endogenous and exogenous substances (including most clinical drugs). Proteins and membrane proteins secreted by the body are transported into the endoplasmic reticulum during or after translation for modification, folding and correct assembly. When the number of unfolded or misfolded proteins in the endoplasmic reticulum is increased, a stress signal can be transmitted to the nucleus through the endoplasmic reticulum membrane, and then a series of specific target gene transcription up-regulation and protein translation level down-regulation are caused, so that the cells can continue to survive, and the reaction is Unfolded Protein Response (UPR), wherein the UPR has the functions of protecting the cells and cytotoxicity, and causes apoptosis, thereby causing a series of diseases, such as Alzheimer's disease, fibrocystic disease, diabetes and the like. Therefore, the endoplasmic reticulum can be used as a new target for treating the tumor, and some cytotoxic drugs can be adopted to induce the endoplasmic reticulum stress according to the pathway for inducing the apoptosis of the cells so as to accelerate the apoptosis of the cancer cells to treat the cancer. Drugs or cytokines may also be used to block disease-induced aberrant endoplasmic reticulum stress responses, reducing or even reversing apoptosis of cells.

Currently, targeted delivery of ER is not considered adequately. The relevant literature on which this has been studied is relatively rare. Costin et al entrapped the endoplasmic reticulum N-glycosylation inhibitor N-butyldeoxynojirimycin in pH sensitive liposomes (composed of DOPE and cholesterol hemisuccinate) successfully inhibited melanoma cell tyrosine activity in mice and significantly reduced the dose administered (Gertrude-E.Costin, Mihalea Trif, et al pH-sensitive liporeagents in animal healthcare carriers for endoplastic recombinant cells in mice cells [ J ], BBRC293(2002): 918).

Pardaxin (PA) is a 33 amino acid residue polypeptide (sequence: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH) isolated from Cynoglossus parvus Gunther and is the amphiphilic cation isolated from fish at the earliestThe alpha helical structure has the function of membrane penetration, and is an antibacterial active peptide with strong function. In addition, Pardaxin also has the effects of inhibiting proliferation and inducing apoptosis of human cancer cell lines. Its 33 amino acid structure contains many cationic and amphiphilic amino acids, making it easier to interact with anionic membranes, due to its special configuration, at concentrations below 10^-7Pardaxin at M can form a single channel with a pore diameter on a cell lipid bilayer membrane, and the concentration of the Pardaxin is 10^-7-10^-4M causes cell lysis (DoronRapaport, Yeast Shai. interaction of fluorescent labeled Pardaxin and Italiangues with labeled Bilayer]The Journal of Biological Chemistry 266(1991): 23769-23775；Peter I.Lelkes and Philip Lazarovici.Pardaxin inducesaggregation but not fusion of phosphatidylserine vesicles[J].FEBS LETTERS,230(1988):131-136； Bhunia A,Domadia PN,Torres J,et.al.NMR structure of pardaxin,a pore-forming antimicrobial peptide,in lipopolysaccharide micelles:mechanismof outer membrane permeabilization[J]Biol Chem,285(2010):3883-3895), low doses, Pardaxin entry, which can avoid mitochondria, golgi and lysosomes, and target the endoplasmic reticulum. The localization of Pardaxin after entry was studied by Chen-Hung pointing and found to accumulate on The endoplasmic reticulum after entry (Chen-Hung pointing, Han-Ning Huang, et al The mechanism by white partial, a natural antibiotic peptide, targets The endo plastic tissue and indemesc-FOS [ J ] J]. Biomaterials,35(2014):3627-3640)。

Disclosure of Invention

The invention aims to provide a construction of a targeting cell endoplasmic reticulum nano drug delivery system, which utilizes the specific tropism characteristic of water-soluble polypeptide Pardaxin to modify the water-soluble polypeptide Pardaxin on the surface of a nano carrier, thus promoting the internalization of nano carrier cells, having the specific targeting capability of the endoplasmic reticulum and realizing the specific accumulation of the endoplasmic reticulum sites of nano carriers and drugs in the carriers. The method is realized by the following scheme: modifying water-soluble polypeptide Pardaxin on the surface of the nano carrier in a chemical bonding or physical adsorption mode, wherein the Pardaxin is a polypeptide containing 33 amino acid residues, and the amino acid sequence is as follows: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH. .

The specific contents are as follows:

the Pardaxin modified nano-carrier is realized by the following chemical and/or physical methods:

(1) and directly carrying out chemical bonding with the nano-carrier by utilizing amino or carboxyl in the Pardaxin molecule to finish modification.

(2) Or the amino or carboxyl in the Pardaxin molecule is firstly chemically bonded with a compound (the connection effect of the compound), and then the compound with the connection effect is combined on the nano-carrier to complete the modification. Such linking compounds are, for example, polyethylene glycols, polyethyleneimines, etc., which have reactive groups.

(3) Or the modification of the Pardaxin polypeptide on the surface of the nanoparticle is realized through physical interaction such as charge adsorption and hydrogen bond interaction.

2. Constructing a Pardaxin modified nanoliposome carrier with endoplasmic reticulum targeting:

preparing liposome materials (phospholipid, cholesterol, DSPE-PEG-Pardaxin and the like; if the liposome materials are fat-soluble drugs, adding the drugs at the moment) by adopting a film dispersion method, an injection method and the like, mixing the materials according to a molar ratio (the molar ratio of the phospholipid to the cholesterol is 10-0.5: 1, and the DSPE-PEG-Pardaxin is 0.01-10% of the total lipid weight), adding an organic solvent for dissolving (such as chloroform, dichloromethane, ethanol and the like), carrying out 1) rotary evaporation to form a film, and adding an aqueous medium (aqueous solution containing salt and/or water-soluble drugs) for hydration to form liposome; or 2) directly into an aqueous medium to form liposomes. The liposome has a particle size of 1-1000 nm.

The invention also aims to provide application of the nano drug delivery system targeting to the endoplasmic reticulum of the cell in preparation of drugs targeting the endoplasmic reticulum. The carried medicine can be medicine with different physicochemical properties such as fat-soluble or water-soluble antitumor medicine, medicine in different treatment fields such as antitumor and anti-inflammatory medicine, or prodrug requiring further activation of enzyme on endoplasmic reticulum and therapeutic medicine for endoplasmic reticulum related diseases, and medicine or factor aiming at endoplasmic reticulum stress. The Pardaxin is exposed outside the nano-vector and serves as a target, the drug-loaded nano-liposome vector after entering cells can be specifically targeted to the endoplasmic reticulum of the cells, and drugs are released in the endoplasmic reticulum, or the drug-loaded nano-liposome vector can specifically cause the endoplasmic reticulum behavior stress reaction (such as excitation or inhibition of endoplasmic reticulum stress). Diseases involved include tumors, such as liver cancer; infectious inflammation, and diseases associated with endoplasmic reticulum stress, such as Alzheimer's disease, diabetes, etc.

In the application process, the preparation mode of the drug-loaded nanoparticles is different mainly according to the difference between the physicochemical properties (such as lipophilic hydrophilicity, electric property and the like) of the drug and the nano-carrier, and the lipid-soluble drug can be wrapped by adopting a similar intermiscibility method, and the drug and the lipid can be mixed, dissolved and formed into a film to complete loading. For water-soluble drugs, the loading of nano-drugs can be completed by utilizing the acid-base property or the charging property of the water-soluble drugs. Water soluble medicine such as cyclophosphamide and adriamycin, and fat soluble medicine such as dexamethasone acetate.

The invention relates to a method for binding water-soluble polypeptide Pardaxin to the surface of a nano carrier. The invention typically modifies Pardaxin on the surface of the liposome. Using DSPE-PEG-NH₂The amino group and the carboxyl group of the Pardaxin are subjected to dehydration condensation reaction to form a more stable amido bond. The modified DSPE-PEG-Pardaxin is formed, the modified substance is amphiphilic, the DSPE is a hydrophobic end, and the PEG-Pardaxin is a hydrophilic end. The hydrophobic end can be used as an anchor, the rivet is arranged on a bilayer membrane of a lipid carrier such as a liposome, and the hydrophilic end is exposed to the outside and used as a target head for targeting endoplasmic reticulum.

The endoplasmic reticulum targeted liposome constructed by the invention can be loaded with water-soluble and fat-soluble medicines, and can transfer the medicines to endoplasmic reticulum parts, thereby enhancing the curative effect of the medicines and reducing toxic and side effects. The nano-carrier is not limited to liposome, but also can be solid lipid nanoparticles, nano-emulsion, polymer micelle, inorganic nano-material and the like. The novel endoplasmic reticulum targeted nano-carrier constructed by the invention can obviously improve the concentration of the medicine taking the endoplasmic reticulum as a target at the action target part, and provides a new way for the curative effect exertion of the medicines.

Drawings

FIG. 1 is a NMR chart hydrogen spectrum of DSPE-PEG, Pardaxins, DSPE-PEG-Pardaxin.

FIG. 2 is the effect of the ratio of phospholipid to drug on the encapsulation efficiency of liposomes when prepared by thin film dispersion.

FIG. 3 is a graph showing the effect of phospholipid to drug ratio on liposome encapsulation efficiency when prepared using a calcium acetate gradient method.

Figure 4 is a map of endoplasmic reticulum co-localization of an endoplasmic reticulum-targeted cyclophosphamide liposome.

Figure 5 is a graph of the lysosomal escape of an endoplasmic reticulum-targeted cyclophosphamide liposome.

FIG. 6 is a cytotoxicity test of endoplasmic reticulum-targeted cyclophosphamide liposomes against SKOV-3, HepG-2.

Detailed Description

The invention is further explained by the accompanying drawings and examples.

Example 1

Synthesis of DSPE-PEG-Pardaxin: accurately weighing Pardaxin, dissolving in 3mL-10mL anhydrous DMF solvent, adding under dark and ice Bath (BOC)₂O reagent protects 4 free amino groups, (BOC) on the Pardaxin polypeptide₂O, Pardaxin in a molar ratio of 5.2:1, and reacting for 12 hours in a dark nitrogen gas seal mode. Warp (BOC)₂And after the protection of the O reagent, EDC and NHS are added to activate carboxyl on the Pardaxin polypeptide, wherein the molar ratio of EDC to Pardaxin is 10:1, and the molar ratio of NHS to Pardaxin is 5:1, and the activation reaction is carried out for 2 hours at normal temperature. After the activation is finished, DSPE-PEG-NH is added₂Reacting for 24 hours under magnetic stirring, DSPE-PEG-NH₂Pardaxin molar ratio is 1: 1. After the reaction is finished, stirring 1mL of 12M HCI to react for 2 hours to remove the BOC protection, then adjusting the pH value to be neutral by using 3M NaOH (1.2g is dissolved in 10mL of water), dialyzing and freeze-drying to obtain DSPE-PEG-Pardaxin, wherein the structural formula is as follows:

the structure of DSPE-PEG-Pardaxin is confirmed: DSPE-PEG-Pardaxin was performed by Nuclear Magnetic Resonance (NMR)¹H NMR and IR spectrum scans confirmed the structure (FIG. 1). In the hydrogen nuclear magnetic resonance spectrum of DSPE-PEG-PardaxinMoving 6.81-8.23ppm, a plurality of sharp signal peaks appear, and the peaks are classified as characteristic peaks of amido bonds; at a chemical shift of 4.46-4.65ppm, a distinct bimodal signal appears, which is assigned to the methine signal peak adjacent to the amide bond in the Pardaxin polypeptide; in addition, two carboxyl unimodal signals of the Pardaxin polypeptide at chemical shifts 12.15-12.76ppm have disappeared, indicating that the DSPE-PEG-Pardaxin has been successfully synthesized.

FIG. 1 is a NMR chart hydrogen spectrum of DSPE-PEG, Pardaxins, DSPE-PEG-Pardaxin. As shown in the figure: in a DSPE-PEG-Pardaxin nuclear magnetic resonance hydrogen spectrogram, a plurality of sharp signal peaks appear at chemical shift of 6.81-8.23ppm, and the peaks belong to characteristic peaks of amido bonds; at a chemical shift of 4.46-4.65ppm, a significant bimodal signal appears, which is attributed to the methine signal peak adjacent to the amide bond in the Pardaxin polypeptide; in addition, two carboxyl unimodal signals of the Pardaxin polypeptide at chemical shifts 12.15-12.76ppm have disappeared, indicating that the DSPE-PEG-Pardaxin has been successfully synthesized.

Example 2

Synthesis of Thiectionacid (TA) -PEG-Pardaxin: by NH₂-PEG-NH₂And lipoic acid (TA) to synthesize NH 2-PEG-TA. First, TA, DCC, NHS (molar ratio: 1:5:10) was dissolved in DMF and stirred at 60 ℃ for 2 hours to activate the carboxyl group on TA. Then, a certain amount of NH is added₂-PEG-NH₂(NH₂-PEG-NH₂TA 1:2, mol/mol) and stirring was continued for 24 hours. The crude product was dialyzed against distilled water for 48 hours and then lyophilized to give NH₂-PEG-TA。

Prior to synthesis of Pardaxin-PEG-TA, the amino group on the Pardaxin peptide was also replaced By (BOC) using the method described above₂And (4) protecting by O. Thereafter, EDC and NHS (Pardaxin: EDC: NHS ═ 1:5:10, mol/mol) were used to activate the carboxyl groups on FAL. Then, NH is added₂-PEG-TA(Pardaxin:NH₂PEG-TA ═ 1:1, mol/mol) and stirring was continued for 24 hours. At the end of the reaction, the protecting group was removed using HCl and the pH was adjusted by NaOH. After further dialysis and lyophilization, Pardaxin-PEG-TA was collected and stored at 4 ℃ prior to use.

Example 3

Synthesis of PCL-PEG-Pardaxin: adding under dark and ice Bath (BOC)₂O reagent for protecting free amino group (BOC) on Pardaxin₂O, Pardaxin in a molar ratio of 5.2:1, and reacting for 12 hours in a dark nitrogen gas seal mode. Warp (BOC)₂And after the protection of the O reagent, EDC and NHS are added to activate carboxyl on the Pardaxin polypeptide, wherein the molar ratio of EDC to Pardaxin is 10:1, and the molar ratio of NHS to Pardaxin is 5:1, and the activation reaction is carried out for 2 hours at normal temperature. After activation, adding polyethylene glycol-polycaprolactone (PCL-PEG-NH) with amino terminal₂) Reaction for 24 hours under magnetic stirring, PCL-PEG-NH₂Pardaxin molar ratio is 1: 1. After the reaction is finished, stirring 1mL of 12M HCI to react for 2 hours to remove the BOC protection, then adjusting the pH value to be neutral by using 3M NaOH (1.2g is dissolved in 10mL of water), dialyzing and freeze-drying to obtain the PCL-PEG-Pardaxin.

Example 4

Composition of endoplasmic reticulum targeted cyclophosphamide liposome:

the endoplasmic reticulum targeting effect and the drug effect result of the endoplasmic reticulum targeting liposome prepared by the invention are considered, and the cyclophosphamide endoplasmic reticulum targeting liposome in the example 4 is taken as an example for research.

Cyclophosphamide is the most common alkylating antineoplastic agent in clinic at present, and after entering the body, chloroethylphosphamide or phosphoramide mustard with strong alkylation effect is decomposed and released under the catalysis of liver microsome enzyme, so that the product has a cytotoxic effect on tumor cells. The traditional Chinese medicine composition is clinically used for malignant lymphoma, multiple myeloma, leukemia, breast cancer, ovarian cancer, cervical cancer, prostatic cancer, colon cancer, bronchial cancer, lung cancer and the like, and has certain curative effect. Can also be used for treating rheumatoid arthritis, children nephrotic syndrome and autoimmune diseases. The precursor drug cyclophosphamide has no antitumor activity in vitro, and can be converted into the aldehyde phosphoramidate by microsome functional oxidase on the endoplasmic reticulum of liver cells after entering the body, thereby exerting the drug effect.

The construction of the endoplasmic reticulum targeted cyclophosphamide can effectively improve the activation efficiency of the cyclophosphamide, reduce the administration dosage and reduce the toxic and side effects. Cyclophosphamide is a water-soluble drug, the pH value is between 5.6 and 6.5, and the water solution of the cyclophosphamide is weakly acidic, so that the invention firstly adopts a calcium acetate gradient method to construct an endoplasmic reticulum targeted cyclophosphamide liposome drug delivery system, and researches such as prescription investigation, cell uptake, cytotoxicity and the like are carried out.

1. Characterization of physicochemical Properties of endoplasmic reticulum-Targeted liposomes

Firstly, the prescription of cyclophosphamide liposome is researched, and the optimal preparation method is found to be a calcium acetate gradient method, phospholipid is hydrogenated soybean phospholipid, and the encapsulation efficiency of cyclophosphamide in the endoplasmic reticulum targeted liposome is measured by a High Performance Liquid Chromatography (HPLC), and is more than 40% (fig. 2 and 3). The particle size and potential were measured by Dynamic Light Scattering (DLS). The results are shown in Table 1. Table 1 shows physicochemical properties of the endoplasmic reticulum-targeted cyclophosphamide liposome, and the surface morphology of the endoplasmic reticulum-targeted liposome was observed using a Transmission Electron Microscope (TEM). As can be seen from Table 1, the distribution of the particle size of the endoplasmic reticulum-targeted liposomes was relatively uniform, and both the liposome encapsulation efficiency and drug loading were within acceptable ranges. The particle size of the liposome is less than 200nm and is uniform.

TABLE 1 endoplasmic reticulum-targeted cyclophosphamide liposomes physicochemical Properties

Drug loading (%)	Encapsulation efficiency (%)	Particle size	PDI
				1.89±0.07	48.99±2.405	184±10.75	0.206±0.045

FIG. 3 is a graph showing the effect of phospholipid to drug ratio on liposome encapsulation efficiency when prepared using a calcium acetate gradient method. HSPC effect was superior to S100 under the same prescription. And the liposome prepared by adopting the pH gradient method has higher encapsulation efficiency than that of a film dispersion method. The invention preferably adopts a calcium acetate gradient method to prepare cyclophosphamide liposome.

2. Endoplasmic reticulum targeting experiment verification (laser confocal)

Human ovarian cancer SKOV-3, human liver cancer HepG-2 and mouse colon cancer CT-26 cells are mixed according to the ratio of 5 multiplied by 10⁴One/well is inoculated to 10mm per well of a 24-well plate²On the slide of (a). After 24h of adherence, blank endoplasmic reticulum targeted liposome and blank non-targeted liposome which are fluorescently labeled by FITC and contain 500 mu g/mL of liposome are respectively added into each hole. After 12h incubation the wells were aspirated and each well was washed 3 times with PBS. Carrying out nuclear staining on cells by using hochest33342 dye, adding 100uL of nuclear dye with the concentration of 5ug/mL into each hole, incubating for 30min in a dark place at 37 ℃, sucking off staining solution, and washing for three times by using PBS; adding endoplasmic reticulum dye ER-tracker 100uL with the concentration of 5ug/mL, incubating in dark for 30min, marking the endoplasmic reticulum of the cell, sucking off the dye solution, washing with PBS for 2-3 times, adding 4% paraformaldehyde solution into each hole to fix the cell, taking out the slide after 20 min, sealing, and observing under a confocal laser microscope. The results are shown in FIG. 4, the coincidence of the endoplasmic reticulum fluorescence signal of the targeting group and the liposome fluorescence signal is better, and the coincidence degree of the non-targeting group and the endoplasmic reticulum is lower. Indicating that the endoplasmic reticulum targeting effect of Pardaxins is good (red fluorescence represents endoplasmic reticulum and green fluorescence represents liposome.)

3. Lysosome escape experiment (laser confocal)

The same method is carried out on human ovarian cancer SKOV-3, human liver cancer HepG-2 and mouse colon cancer CT-26 cells according to 5 multiplied by 10⁴Inoculating in glass-bottom culture dish. After 24h of adherence, blank endoplasmic reticulum targeted liposome and blank non-targeted liposome which are fluorescently labeled by FITC and contain 500 mu g/mL of liposome are respectively added into each dish. After 12h incubation the dish was aspirated and each well was washed 3 times with PBS. Carrying out nuclear staining on cells by using hochest33342 dye, adding 100uL of nuclear dye with the concentration of 5ug/mL into each hole, incubating for 30min in a dark place at 37 ℃, sucking off staining solution, and washing for three times by using PBS; adding lysosome Lys-tracker dye with the concentration of 5ug/mL for incubation for 30min in a dark place, labeling the lysosome of cells, sucking off dye liquor, washing for 2-3 times by PBS (phosphate buffer solution), adding 4% paraformaldehyde solution into each hole for fixing the cells, taking out a slide after 20 min, sealing the slide, and observing under a laser confocal microscope. The results are shown in FIG. 5, the overlap ratio of the lysosome fluorescence signal of the targeting group and the liposome fluorescence signal is lower, and the overlap ratio of the non-targeting group and the endoplasmic reticulum is higher. Indicating that Pardaxin has a role in aiding escape from lysosomes.

4. Cytotoxicity test

Human ovarian cancer SKOV-3, human liver cancer HepG-2 and mouse colon cancer CT-26 cells are mixed according to the ratio of 5 multiplied by 10⁴One, 200. mu.L/well was grafted onto a 96-well plate. After 24h of cell adherence, blank non-targeted liposome, drug-loaded liposome, blank targeted liposome and drug-loaded endoplasmic reticulum targeted liposome are added into the tumor cells according to the series concentration of the carrier (25ug/mL, 50ug/mL,100ug/mL,250 ug/mL). And (5) incubating for 72 h. Then 20. mu.L of MTT aqueous solution (5mg/mL) was added to each well, and after further incubation for 4 hours, the culture medium was discarded, 100. mu.L of DMSO was added to each well, and after shaking on a shaker for 20 minutes, the absorbance was measured at 570nm with a microplate reader. Blank liposome group and blank targeted liposome group were used as control groups, and free cyclophosphamide group was used as positive control. The result is shown in figure 6, the free cyclophosphamide is weak in vitro cytotoxicity due to water solubility and prodrug thereof, the ordinary cyclophosphamide liposome increases the cell entrance rate of the cyclophosphamide, the amount of the active cyclophosphamide in the system is increased, the in vitro cytotoxicity of the cyclophosphamide liposome is obviously higher than that of the free cyclophosphamide, and the liposome preparation can improve the uptake of the drug by cells and enhance the curative effect of the drug. The toxicity of cyclophosphamide on cells can be further increased after the liposome is modified by DSPE-PEG-Pardaxin, and the toxicity of cyclophosphamide on cells can be further increased along with the DSPE-PEG-PardaThe increase of the xin proportion has no obvious improvement on the cytotoxicity, which indicates that the identification of DSPE-PEG-Pardaxin and endoplasmic reticulum has saturation, the cytotoxicity of the drug in two different cell lines to HepG-2 is higher than that of SKOV-3, and the increase of the activation degree of cyclophosphamide under the targeting effect of the endoplasmic reticulum further indicates that the cytotoxicity is enhanced because the liver cancer endoplasmic reticulum has more cytochrome enzymes for activating the cyclophosphamide.

Example 5

Composition of endoplasmic reticulum-targeted doxorubicin liposomes:

prescription amounts of HSPC, Cholesterol, DSPE-PEG₂₀₀₀And DSPE-PEG-PAR, placing in a bottle shaped like a eggplant, adding organic solvent (chloroform) for dissolving, carrying out vacuum pressure reduction rotary evaporation in a water bath at 55 ℃ overnight, and forming a film on the wall of the bottle. Adding ammonium sulfate solution with pH of 5.4 into eggplant-shaped bottle, hydrating in water bath at 60 deg.C for 1 hr, and removing and dissolving the film to form multi-layer liposome solution. The above solution was sonicated to the desired particle size using probe sonication. The resulting liposome solution was passed through a Sephadex G50 column, eluted with PBS (pH 7.4), and the ammonium sulfate solution of the liposome external phase was removed. Adding 2mg/mL adriamycin solution into the endoplasmic reticulum targeted liposome to ensure that the mass ratio of the drug to the lipid is 1:20, and stirring and incubating for 30min in water bath at 50 ℃. And then removing free doxorubicin hydrochloride through a Sephadex G50 column, and finally collecting the doxorubicin-loaded endoplasmic reticulum targeted liposome.

Adriamycin is an antitumor antibiotic, can inhibit the synthesis of RNA and DNA, has the strongest inhibition effect on RNA, has wider antitumor spectrum, has the effect on various tumors, belongs to a periodic nonspecific medicament, and has the effect of killing tumor cells in various growth periods. Is mainly suitable for acute leukemia, and is effective for acute lymphocytic leukemia and granulocytic leukemia. The invention adds the Pardaxin in the classical prescription of the adriamycin liposome, the Pardaxin can help the adriamycin liposome to enter cells and target to endoplasmic reticulum membrane, and the adriamycin liposome can bypass lysosome so as to protect the medicine. The invention can reduce the administration dosage of the adriamycin and the toxic and side effects of the medicament.

Example 6

Composition of endoplasmic reticulum targeted dexamethasone acetate liposome:

prescription amounts of Dex-Ac, HSPC, Cholesterol, DSPE-PEG₂₀₀₀And DSPE-PEG-PAR, placing in a bottle shaped like a eggplant, adding organic solvent (chloroform) for dissolving, carrying out vacuum pressure reduction rotary evaporation in a water bath at 55 ℃ overnight, and forming a film on the wall of the bottle. PBS (pH 7.4) is added into the eggplant-shaped bottle, the mixture is hydrated in a water bath at 60 ℃ for 1 hour, and the film is peeled and dissolved to form a multi-layer liposome solution. The above solution was sonicated to the desired particle size using probe sonication. The resulting liposome solution was passed through a Sephadex G50 column, eluted with PBS (pH 7.4) and the free dexamethasone acetate drug in the liposomes was removed. Collecting the eluted liposome with opalescence, namely the endoplasmic reticulum targeted liposome carrying dexamethasone acetate.

Dexamethasone acetate is a corticoid drug. Has pharmacological effects of resisting inflammation, resisting endotoxin, suppressing immunity, resisting shock, enhancing stress reaction, etc., and can be widely used for treating various diseases such as autoimmune diseases, allergy, inflammation, asthma, dermatology, and ophthalmology. Dexamethasone acetate has more adverse reactions, is frequently generated when pharmacological dosage is applied, and has close relation with treatment course, dosage, medication types, usage, administration route and the like. The common adverse reactions include iatrogenic cushing syndrome facial appearance and body state, digestive tract ulcer, abnormal spirit, glucocorticoid withdrawal syndrome, diabetes, similar Cuxing syndrome, etc.

Dexamethasone acetate is metabolized into dexamethasone under the action of liver drug enzyme after administration, is a liver drug enzyme inducer, and can cause the activity of liver drug enzyme to be enhanced after long-term use, so that the content of cytochrome P450 is increased, and the endoplasmic reticulum hyperplasia of the liver and the synthesis of microsomal membrane-bound protein are promoted. Can be dissolved in ethanol or chloroform, so the dexamethasone acetate liposome can be prepared by adopting a film dispersion method. Dexamethasone acetate is prepared into liposome with endoplasmic reticulum targeting by utilizing a Pardaxin target head. The invention has three advantages: firstly, can improve the concentration of medicine at the target organ, reduce the distribution of medicine at other organs, secondly directly improve the activation efficiency of organism to dexamethasone acetate with the enzyme of medicine target to the usable endoplasmic reticulum of endoplasmic reticulum on, thirdly after giving tertiary target to the medicine, the dose of dosing of control medicine that can be better, it exerts the effect of liver medicine enzyme inducer and has controlled to it. Dexamethasone acetate has more adverse reactions, and the preparation of the dexamethasone acetate into the liposome preparation with endoplasmic reticulum targeting can effectively reduce the administration dosage, reduce the adverse reactions and control the induction effect of the dexamethasone acetate on liver drug enzymes.

Example 7

The composition of the endoplasmic reticulum targeted drug-loaded hemoglobin liposome is as follows:

prescription amounts of E80, Cholesterol, DSPE-PEG₂₀₀₀And DSPE-PEG-PAR, placing in a bottle shaped like a eggplant, adding organic solvent (chloroform) for dissolving, carrying out vacuum pressure reduction rotary evaporation in a water bath at 55 ℃ overnight, and forming a film on the wall of the bottle. Adding PBS containing Hb prescription amount into the eggplant-shaped bottle, hydrating in water bath at 60 ℃ for 1h, and stripping and dissolving the film to form a multi-layer liposome solution. The above solution was sonicated to the desired particle size using probe sonication. The resulting liposome solution was passed through a Sephadex G50 column, and eluted with PBS (pH 7.4) to remove free Hb in the liposome. Collecting the eluted liposome with opalescence, namely the endoplasmic reticulum targeted liposome carrying Hb. The liposome can combine oxygen molecules, and can directly deliver the oxygen molecules to the endoplasmic reticulum part of cells, so that the oxygen content of the part is improved, and the subsequent treatment aiming at the oxygen dependence of the endoplasmic reticulum part is facilitated.

Example 8

The composition of the solid lipid nanoparticle carrying paclitaxel in an endoplasmic reticulum targeting way is as follows:

paclitaxel 5mg

Glycerol monostearate 100mg

DSPE-PEG-Pardaxin 2mg

Paclitaxel, glyceryl monostearate and DSPE-PEG-Pardaxin in the prescription amount are added into absolute ethyl alcohol for dissolving, injected into PBS buffer solution, and then the required particle size is obtained by an ultrasonic or homogenizer. And (3) passing the obtained nanoparticle solution through a Sephadex G50 column, and eluting and purifying by PBS (pH 7.4) to obtain the endoplasmic reticulum targeted solid lipid nanoparticle loaded with paclitaxel.

Example 9

The composition of the endoplasmic reticulum targeted docetaxel-loaded nano micelle is as follows:

docetaxel 5mg

Polyethylene glycol-polycaprolactone (PEG-PCL) 100mg

PCL-PEG-Pardaxin 10mg

Adding docetaxel, polyethylene glycol-polycaprolactone and PCL-PEG-Pardaxin in a prescription amount into absolute ethyl alcohol for dissolving, injecting into a buffer solution of PBS, and obtaining the required particle size by an ultrasonic or homogenizer. And (3) passing the obtained nanoparticle solution through a Sephadex G50 column, and eluting and purifying by PBS (pH 7.4), thus obtaining the docetaxel-loaded endoplasmic reticulum targeted nano micelle.

Example 10

Synthesis of gold nanoparticles targeting endoplasmic reticulum: 2mg of Pardaxin-PEG-TA synthesized in the embodiment 2 is taken and incubated with 10mg of hollow gold nano material (HAuNS) (with the size of 40nm and a special absorption peak at 800 nm) in an aqueous medium for 24 hours at room temperature, and the hollow gold nano particle with the endoplasmic reticulum targeting function is obtained after centrifugation and purification. After entering cells, the nanoparticles are accumulated in endoplasmic reticulum, and further specific behaviors (photo-thermal stimulation and endoplasmic reticulum stress level enhancement) aiming at the endoplasmic reticulum can be realized by means of external stimulation (near infrared light).

Sequence listing

<110> Zhejiang university

Construction and application of nano drug delivery system targeting to endoplasmic reticulum of cell

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>33

<212>PRT

<213>Pardaxin

<400>1

Gly Phe Phe Ala Leu Ile Pro Lys Ile Ile Ser Ser Pro Leu Phe Lys

1 5 10 15

Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Ser Gly Gly Gln

20 25 30

Glu

Claims

1. A construction method of a targeting intracellular reticulum nano drug delivery system is characterized by comprising the following steps: modifying water-soluble polypeptide Pardaxin on the surface of the nano carrier in a chemical bonding or physical adsorption mode, wherein the Pardaxin is a polypeptide containing 33 amino acid residues, and the amino acid sequence is as follows: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH;

the specific construction method comprises the following steps:

(1) the Pardaxin modification is achieved by the following chemical and/or physical methods:

(a) directly chemically bonding amino or carboxyl in the Pardaxin molecule with a nano carrier to finish modification;

(b) or the amino or carboxyl in the Pardaxin molecule is firstly chemically bonded with a compound, and then the compound with the connecting function is combined on the nano-carrier to complete the modification, wherein the compound is selected from polyethylene glycol;

(c) or the modification of the Pardaxin polypeptide on the surface of the nanoparticle is realized through the physical interaction of charge adsorption and hydrogen bonds;

(2) constructing a Pardaxin modified nanoliposome carrier with endoplasmic reticulum targeting:

mixing liposome materials according to a molar ratio by adopting a film dispersion method or an injection method, dissolving the liposome materials by using an organic solvent, carrying out rotary evaporation to form a film, and adding an aqueous medium to hydrate to form the liposome or directly injecting the liposome into the aqueous medium to form the liposome.

2. The method for constructing the drug delivery system targeting to endoplasmic reticulum of cells according to claim 1, wherein step (1) comprises the step of first mixing Pardaxin with DSPE-PEG-NH₂By chemical reaction of carboxyl groups on Pardaxin with DSPE-PEG-NH₂The amino group is subjected to condensation reaction to form an amido bond; and then the distearoyl phosphatidyl ethanolamine is inserted into a liposome membrane or a lipid nanoparticle to finish the surface modification of the nano-liposome carrier by the Pardaxin.

3. The method for constructing the drug delivery system targeted to the endoplasmic reticulum of the cell according to claim 1, wherein the liposome material in the step (2) is selected from phospholipid, cholesterol and DSPE-PEG-Pardaxin, and the phospholipid and the cholesterol are mixed according to a molar ratio of 10-0.5: 1, and the DSPE-PEG-Pardaxin is 0.1-10% of the total lipid weight.

4. The method for constructing the drug delivery system targeting to endoplasmic reticulum of cells according to claim 1, wherein the drug delivery system is nanoliposome carrier, solid lipid nanoparticle, nanoemulsion, polymeric micelle or inorganic nanomaterial.

5. The application of the nano drug delivery system constructed by the method of claim 1 in preparing drugs targeting endoplasmic reticulum.

6. The use according to claim 5, wherein the drug is a lipid-soluble or water-soluble antitumor drug, an anti-inflammatory drug or a prodrug requiring further activation of an enzyme on the endoplasmic reticulum and a therapeutic drug for diseases associated with the endoplasmic reticulum.

7. The use according to claim 6, wherein the carried drugs are lipid soluble or water soluble antitumor drugs with different physicochemical properties, anti-inflammatory drugs or prodrugs requiring further activation of enzymes on the endoplasmic reticulum and therapeutic drugs for diseases related to the endoplasmic reticulum, and drugs or factors against endoplasmic reticulum stress.