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

Abstract

The present invention provides the construction of a nano-drug loading system targeting the endoplasmic reticulum of cells, which is achieved by modifying the Pardaxin polypeptide on the surface of the liposome. The Pardaxin is a polypeptide with 33 amino acid residues and an amphiphilic cationic α-helical structure that has a transmembrane effect. The constructed nano-drug loading system can be used in the preparation of anti-viral, anti-tumor and other drugs targeting endoplasmic reticulum. The nano-drug loading system can load water-soluble and lipid-soluble drugs, and deliver the drugs to the endoplasmic reticulum, which can enhance the therapeutic efficacy of drugs and reduce the toxic and side effects. The nanocarriers of the present invention are not limited to liposomes, but can also be solid lipid nanoparticles, nanoemulsions, polymer micelles, and inorganic nanomaterials. The novel endoplasmic reticulum-targeting nanocarrier constructed by the invention can significantly increase the concentration of drugs targeting endoplasmic reticulum in its target site, and provides a new way for the curative effect of these drugs to be exerted.

Description

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

technical field

The invention belongs to the field of medicine, and relates to a novel nano-drug-loading system that can be targeted to the endoplasmic reticulum of cells, and the application of the drug-carrying system in the preparation of endoplasmic reticulum-targeted antiviral and antitumor drugs.

Background technique

Drugs need to reach the target to exert their efficacy. The targets of drugs are generally functional biomolecules such as proteins, nucleic acids, enzymes and receptors located in cells. Modern drug delivery systems require drugs to be delivered to target tissues, target cells, and even specific organelles. Nanocarriers have the advantages of protecting drugs, achieving sustained release and having less toxic and side effects. At present, the research on tissue and cell-targeted drug delivery systems is relatively mature, and tertiary targeting, that is, organelle targeting, is the hotspot and difficulty in current drug delivery systems. Tertiary targeting includes cytoplasmic targeting, organelle targeting, such as mitochondrial targeting, endoplasmic reticulum targeting, lysosome targeting, etc., nuclear targeting, etc.

Endoplasmic reticulum (ER) is a closed reticulum system composed of inner membrane. It is the main site of intracellular protein synthesis, lipid synthesis, substance transport, carbohydrate metabolism and detoxification. Its main function is to synthesize protein. and lipids. The endoplasmic reticulum contains a variety of enzymes, and its marker enzyme is glucose-6-phosphatase, which is involved in the metabolism of glycogen. The hydrolase in the endoplasmic reticulum is used for various drug metabolism and detoxification. Cytochrome P450 in the endoplasmic reticulum of liver and intestinal cells is the main enzyme for drug metabolism in the human body, which can catalyze the metabolism of a variety of endogenous and exogenous substances (including most clinical drugs). The proteins and membrane proteins secreted by the body are transported to the endoplasmic reticulum lumen during or after translation for modification, folding and correct assembly. When unfolded or misfolded proteins in the endoplasmic reticulum increase, stress signals can be transmitted through the endoplasmic reticulum membrane to the nucleus, which in turn leads to up-regulation of transcription of a series of specific target genes and down-regulation of protein translation levels, so that cells can continue to survive , this response is the unfolded protein response (UPR), UPR has both protective and cytotoxic effects, causing cell apoptosis, which leads to a series of diseases, such as Alzheimer's disease, vesicular fibrosis disease occurrence, diabetes, etc. According to this, the endoplasmic reticulum can be used as a new target for tumor therapy. According to its apoptosis-inducing pathway, some cytotoxic drugs can be used to induce endoplasmic reticulum stress and accelerate the apoptosis of cancer cells to treat cancer. Some drugs or cytokines can also be used to block the abnormal endoplasmic reticulum stress response induced by disease, and reduce or even reverse cell apoptosis.

At present, the targeted drug delivery to the ER has not received enough attention. There are relatively few relevant literatures on its research. Costin et al. encapsulated the endoplasmic reticulum N-glycosylation inhibitor N-butyldeoxynojirimycin into pH-sensitive liposomes (composed of DOPE and cholesteryl hemisuccinate) and successfully inhibited mouse melanoma Cell tyrosine activity, and significantly reduce the dose (Gertrude-E. Costin, Mihaela Trif, et. al pH-sensitive liposomes are efficient carriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells [J], BBRC293 (2002 ): 918–923).

Pardaxin (PA) is a polypeptide containing 33 amino acid residues (sequence: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH) isolated from leopard sole. It is the first amphiphilic cationic α-helical structure isolated from fish and has a membrane-penetrating effect. It is a kind of antibacterial active peptide with strong effect. In addition, Pardaxin also inhibits proliferation and induces apoptosis in human cancer cell lines. Its 33-amino acid structure contains many cationic and amphiphilic amino acids, making it easier to interact with anionic membranes, and due to its special configuration, Pardaxin can bind to cellular lipid bilayers at concentrations below ^10-7 M. A single-channel pore size is formed on the membrane, which causes cell lysis at a concentration of 10 ^-7 -10 ^-4 M (Doron Rapaport, Yechiel Shai. Interaction of Fluorescently Labled Pardaxin and Its Analogues with Lipid Bilayer. [J] 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), at low doses, after Pardaxin enters cells It can avoid mitochondria, Golgi and lysosomes, and be directed to the endoplasmic reticulum. Chen-Hung Ting conducted a fluorescent tracing study on the localization of Pardaxin after entering cells, and found that Pardaxin accumulated in the endoplasmic reticulum after entering cells (Chen-Hung Ting, Han-Ning Huang, et al The mechanisms by which pardaxin, a naturalcationic antimicrobial antimicrobial peptide, targets the endoplasmic reticulum and inducesc-FOS[J]. Biomaterials, 35(2014):3627-3640).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a construction of a nano-drug-carrying system targeting the endoplasmic reticulum of cells. The present invention utilizes the water-soluble polypeptide Pardaxin to have endoplasmic reticulum-specific tropism, and modifies it on the surface of the nano-carrier, thus promoting the nano- After the carrier cells are internalized, they have endoplasmic reticulum-specific targeting ability, and realize the endoplasmic reticulum site-specific accumulation of nanocarriers and drugs in the carrier. It is achieved by the following scheme: the water-soluble polypeptide Pardaxin is modified on the surface of the nanocarrier by chemical bonding or physical adsorption. The Pardaxin is a polypeptide containing 33 amino acid residues, and its amino acid sequence is: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH. .

The details are as follows:

1. Pardaxin-modified nanocarriers are achieved by the following chemical and/or physical methods:

(1) Using the amino group or carboxyl group in the Pardaxin molecule to directly chemically bond with the nanocarrier to complete the modification.

(2) Or use the amino group or carboxyl group in the Pardaxin molecule to chemically bond with a compound (its linking function), and then combine the linking compound on the nanocarrier to complete the modification. Such linking compounds are such as polyethylene glycol, polyethylene imine, etc. with active reactive groups.

(3) The modification of the Pardaxin polypeptide on the surface of the nanoparticles can be realized by physical interactions such as charge adsorption and hydrogen bonding.

2. Construction of Pardaxin-modified nanoliposome carrier with endoplasmic reticulum targeting:

The liposome material (phospholipid, cholesterol, DSPE-PEG-Pardaxin, etc.; if it is a fat-soluble drug, add it at this time) will be prepared by the film dispersion method, injection method, etc., according to the molar ratio (phospholipid:cholesterol molar ratio is 10～0.5 : 1, DSPE-PEG-Pardaxin is 0.01%-10% of the total lipid weight) mix, add organic solvent to dissolve (such as chloroform, dichloromethane, ethanol, etc.), form a film by 1) rotary evaporation, add aqueous medium (Aqueous solution containing salt and/or water-soluble drug) hydration to form liposomes; or 2) direct injection into an aqueous medium to form liposomes. Liposomes range in size from 1-1000 nm.

Another object of the present invention is to provide the application of the nano-drug delivery system targeting the endoplasmic reticulum in the preparation of drugs targeting the endoplasmic reticulum. The contained drugs can be drugs with different physicochemical properties such as lipid-soluble or water-soluble anti-tumor drugs, drugs in different therapeutic areas such as anti-tumor, anti-inflammatory drugs, or prodrugs that require further activation of enzymes on the endoplasmic reticulum and endoplasmic reticulum-related diseases therapeutic drugs, and drugs or factors that can target endoplasmic reticulum stress. Pardaxin is exposed outside the nanocarrier and acts as a target, which can specifically target the drug-loaded nanoliposome carrier after entering the cell to the endoplasmic reticulum, and release the drug in the endoplasmic reticulum, or specifically cause the endoplasmic reticulum Behavioral stress responses (such as excitation or inhibition of endoplasmic reticulum stress, etc.). The diseases involved include tumors, such as liver cancer; infectious inflammation, and diseases related to endoplasmic reticulum stress, such as Alzheimer's disease, diabetes, etc.

In this application process, the preparation method of drug-loaded nanoparticles is mainly different according to the physicochemical properties of the drug (such as lipophilicity, hydrophilicity, electricity, etc.) and the nanocarrier. Encapsulation, the drug and lipid can be mixed and dissolved to form a film to complete the loading. For water-soluble drugs, their acid-base or charged properties can be used to complete the loading of nano-drugs. Water-soluble drugs such as cyclophosphamide, doxorubicin, fat-soluble drugs such as dexamethasone acetate, etc.

The present invention is to combine the water-soluble polypeptide Pardaxin to the surface of the nano-carrier. In the present invention, Pardaxin is typically modified on the surface of liposomes. The amino group of DSPE-PEG-NH ₂ and the carboxyl group of Pardaxin are used for dehydration condensation reaction to form a relatively stable amide bond. After modification, DSPE-PEG-Pardaxin is formed, and the modification is amphiphilic, DSPE is the hydrophobic end, and PEG-Pardaxin is the hydrophilic end. The hydrophobic end can be used as an "anchor", rivets on the bilayer membrane of lipid carriers such as liposomes, and the hydrophilic end is exposed as a "target head" targeting the endoplasmic reticulum.

The endoplasmic reticulum-targeted liposome constructed by the invention can load water-soluble and fat-soluble drugs, and deliver the drugs to the endoplasmic reticulum, so as to enhance the therapeutic efficacy of the drugs and reduce the toxic and side effects. The nanocarriers of the present invention are not limited to liposomes, but can also be solid lipid nanoparticles, nanoemulsions, polymer micelles, and inorganic nanomaterials. The novel endoplasmic reticulum-targeting nanocarrier constructed by the invention can significantly increase the concentration of drugs targeting endoplasmic reticulum in its target site, and provides a new way for the curative effect of these drugs to be exerted.

Description of drawings

Figure 1 is the NMR spectrum of DSPE-PEG, Pardaxins, DSPE-PEG-Pardaxin.

Figure 2 shows the effect of the ratio of phospholipids and drugs on the encapsulation efficiency of liposomes prepared by the film dispersion method.

Figure 3 shows the effect of the ratio of phospholipid to drug on the encapsulation efficiency of liposomes when prepared by the calcium acetate gradient method.

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

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

Figure 6 is the cytotoxicity test of endoplasmic reticulum-targeted cyclophosphamide liposome to SKOV-3 and HepG-2.

Detailed ways

The present invention will be further described with reference to the accompanying drawings and embodiments.

Example 1

Synthesis of DSPE-PEG-Pardaxin: Precisely weigh Pardaxin, dissolve it in 3mL-10mL anhydrous DMF solvent, add (BOC) ₂ O reagent to protect the 4 free amino groups on Pardaxin polypeptide in the dark and ice bath, (BOC ) ₂ O:Pardaxin molar ratio was 5.2:1, and the reaction was carried out for 12h in a nitrogen seal in the dark. After being protected by (BOC) ₂ O reagent, EDC and NHS were added to activate the carboxyl group on the Pardaxin polypeptide, the molar ratio of EDC:Pardaxin was 10:1, and the molar ratio of NHS:Pardaxin was 5:1, and the reaction was activated for 2 hours at room temperature. After the activation, DSPE-PEG-NH ₂ was added, and the reaction was carried out under magnetic stirring for 24 hours. The molar ratio of DSPE-PEG-NH ₂ : Pardaxin was 1:1. After the reaction was completed, the BOC protection was removed by stirring the reaction with 1ml 12M HCl for 2 hours, and then adjusted to neutrality with 3M NaOH (1.2g dissolved in 10mL water), dialyzed, and freeze-dried to obtain DSPE-PEG-Pardaxin. The structural formula is as follows :

DSPE-PEG-Pardaxin structure confirmation: Using nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR), DSPE-PEG-Pardaxin was confirmed by ¹ H NMR and infrared spectrum scanning (Fig. 1). In DSPE-PEG-Pardaxin 1H NMR spectrum, there are multiple sharp signal peaks at chemical shifts of 6.81-8.23ppm, which are attributed to the characteristic peaks of amide bonds; at chemical shifts of 4.46-4.65ppm, An obvious double-peak signal appeared, which was attributed to the methine signal peak adjacent to the amide bond in the Pardaxin polypeptide; in addition, the two carboxyl single-peak signals at the chemical shifts of 12.15-12.76 ppm in the Pardaxin polypeptide had disappeared, indicating that DSPE-PEG-Pardaxin has been successfully synthesized.

Figure 1 is the NMR spectrum of DSPE-PEG, Pardaxins, DSPE-PEG-Pardaxin. As shown in the figure: In DSPE-PEG-Pardaxin 1H NMR spectrum, there are multiple sharp signal peaks at chemical shifts of 6.81-8.23ppm, which are attributed to the characteristic peaks of amide bonds; at chemical shifts of 4.46 At -4.65ppm, there is an obvious double-peak signal, which is attributed to the methine signal peak adjacent to the amide bond in Pardaxin polypeptide; in addition, the two carboxyl mono groups of Pardaxin polypeptide at chemical shifts of 12.15-12.76ppm The peak signal has disappeared, indicating that DSPE-PEG-Pardaxin has been successfully synthesized.

Example 2

Synthesis of Thioctic Acid(TA)-PEG-Pardaxin: NH2-PEG-TA was synthesized by dehydration reaction between _NH2 -PEG- _NH2 and lipoic acid (ThiocticAcid, TA). First, TA, DCC, NHS (molar ratio: 1:5:10) were dissolved in DMF and stirred at 60 °C for 2 hours to activate the carboxyl groups on TA. Then, an amount of _NH2 -PEG- _NH2 ( _NH2 -PEG- _NH2 :TA=1:2, mol/mol) was added and stirring was continued for 24 hours. The crude product was dialyzed against distilled water for 48 hours and then lyophilized to yield _NH2 -PEG-TA.

The amino group on the pardaxin peptide was also protected with (BOC) ₂ O using the method described above prior to the synthesis of Pardaxin-PEG-TA. Afterwards, EDC and NHS (Pardaxin:EDC:NHS=1:5:10, mol/mol) were used to activate the carboxyl groups on FAL. Then, _NH2 -PEG-TA (Pardaxin: _NH2 -PEG-TA=1:1, mol/mol) was added and stirring was continued for 24 hours. At the end of the reaction, the protecting groups were removed using HCl and the pH was adjusted by NaOH. After further dialysis and lyophilization, Pardaxin-PEG-TA was collected and stored at 4°C until use.

Example 3

Synthesis of PCL-PEG-Pardaxin: (BOC) ₂ O reagent was added to protect free amino groups on Pardaxin in the dark and ice bath, the molar ratio of (BOC) ₂ O:Pardaxin was 5.2:1, and the reaction was carried out under nitrogen sealing in the dark for 12 h. After being protected by (BOC) ₂ O reagent, EDC and NHS were added to activate the carboxyl group on the Pardaxin polypeptide. The molar ratio of EDC:Pardaxin was 10:1, and the molar ratio of NHS:Pardaxin was 5:1. The reaction was activated at room temperature for 2 hours. After the activation, polyethylene glycol-polycaprolactone (PCL-PEG-NH ₂ ) with an amino terminal was added, and the reaction was carried out under magnetic stirring for 24 hours, and the molar ratio of PCL-PEG-NH ₂ : Pardaxin was 1:1. After the reaction, the reaction was stirred with 1 ml of 12M HCl for 2 hours to remove the BOC protection, and then adjusted to neutrality with 3M NaOH (1.2 g dissolved in 10 mL of water), dialyzed, and freeze-dried to obtain PCL-PEG-Pardaxin.

Example 4

Composition of endoplasmic reticulum-targeted cyclophosphamide liposomes:

To investigate the endoplasmic reticulum targeting effect and pharmacodynamic results of the endoplasmic reticulum targeting liposome prepared by the present invention, the present invention takes the cyclophosphamide endoplasmic reticulum targeting liposome of Example 4 as an example to conduct research.

Cyclophosphamide is currently the most commonly used alkylating agent antitumor drug in clinical practice. After entering the body, it is decomposed into chloroethyl phosphoramide or phosphoramide mustard with strong alkylating effect under the catalysis of liver microsomal enzymes. It has a cytotoxic effect on tumor cells, and this product also has a significant immunosuppressive effect. It is clinically used for malignant lymphoma, multiple myeloma, leukemia, breast cancer, ovarian cancer, cervical cancer, prostate cancer, colon cancer, bronchial cancer, lung cancer, etc., with certain curative effect. It can also be used in the treatment of rheumatoid arthritis, childhood nephrotic syndrome and autoimmune diseases. The prodrug, cyclophosphamide, has no antitumor activity in vitro, but it can be converted into aldophosphamide by microsomal functional oxidase on the endoplasmic reticulum of liver cells before it can exert its efficacy.

The construction of endoplasmic reticulum-targeted cyclophosphamide can effectively improve the activation efficiency of cyclophosphamide, reduce the dosage, and reduce the toxic and side effects. Cyclophosphamide is a water-soluble drug, and its pH value is between 5.6 and 6.5, and its aqueous solution is weakly acidic. Therefore, the present invention adopts the calcium acetate gradient method for the first time to carry out the endoplasmic reticulum targeting cyclophosphamide liposome drug delivery system. Constructed, and researched on formulation investigation, cellular uptake, cytotoxicity, etc.

1. Characterization of physicochemical properties of endoplasmic reticulum-targeted liposomes

Firstly, the formulation of cyclophosphamide liposome was studied, and it was found that the best preparation method was calcium acetate gradient method, and the phospholipid was hydrogenated soybean phospholipid. The encapsulation efficiency of the amide was found to be more than 40% (Figures 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 the physicochemical properties of endoplasmic reticulum-targeted cyclophosphamide liposomes. The surface morphology of endoplasmic reticulum-targeted liposomes was observed by transmission electron microscope (TEM). It can be seen from Table 1 that the particle size distribution of ER-targeted liposomes is relatively uniform, and the liposome encapsulation efficiency and drug loading are both within acceptable ranges. The particle size of liposomes is less than 200nm and relatively uniform.

Table 1 Physicochemical properties of endoplasmic reticulum-targeted cyclophosphamide liposomes

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

Figure 3 shows the effect of the ratio of phospholipid to drug on the encapsulation efficiency of liposomes when prepared by the calcium acetate gradient method. The effect of HSPC is better than that of S100 under the same prescription. And the encapsulation efficiency of liposomes prepared by pH gradient method is higher than that of film dispersion method. In the present invention, the calcium acetate gradient method is preferred to prepare cyclophosphamide liposomes.

2. Experimental verification of endoplasmic reticulum targeting (laser confocal)

Human ovarian cancer SKOV-3, human liver cancer HepG-2, and murine colon cancer CT-26 cells were seeded on a 10 mm ² glass slide in each well of a 24-well plate at 5×10 ⁴ cells/well. After 24 hours of adherence, blank endoplasmic reticulum-targeted liposomes and blank non-targeted liposomes containing 500 μg/mL liposomes labeled with FITC fluorescence were added to each well. After incubation for 12 h, the medium in the wells was aspirated, and each well was washed three times with PBS. The cells were nuclear-stained with hochest33342 dye, and 100uL of nuclear dye with a concentration of 5ug/mL was added to each well. After incubation at 37°C for 30min in the dark, the dye solution was aspirated and washed three times with PBS; Reticulum dye ER-tracker 100uL was incubated in the dark for 30min to label the endoplasmic reticulum of cells, aspirated the dye solution and washed 2-3 times with PBS, added 4% paraformaldehyde solution to each well to fix the cells, and removed the slides after 20 minutes The slides were mounted and observed under a laser confocal microscope. The results are shown in Figure 4. The fluorescence signal of the endoplasmic reticulum in the targeting group has a good coincidence with the fluorescence signal of the liposome, while the coincidence of the fluorescence signal in the non-targeting group with the endoplasmic reticulum is low. It shows that the endoplasmic reticulum targeting effect of Pardaxins is good (red fluorescence represents endoplasmic reticulum, green fluorescence represents liposomes.)

3. Lysosome escape experiment (laser confocal)

Human-derived ovarian cancer SKOV-3, human-derived liver cancer HepG-2, and mouse-derived colon cancer CT-26 cells were inoculated into glass-bottom petri dishes at 5×10 ⁴ cells/well in the same manner. After 24 hours of adherence, blank endoplasmic reticulum-targeted liposomes and blank non-targeted liposomes containing 500 μg/mL liposomes labeled with FITC fluorescence were added to each dish. After incubation for 12 h, the medium in the dish was aspirated, and each well was washed three times with PBS. The cells were nuclear-stained with hochest33342 dye, 100uL of nuclear dye with a concentration of 5ug/mL was added to each well, and after incubation at 37°C for 30min in the dark, the dye solution was aspirated and washed three times with PBS; lysozyme with a concentration of 5ug/mL was added. Incubate 100uL of Lys-tracker dye in the dark for 30min, label the cell lysosomes, remove the dye and wash 2-3 times with PBS, add 4% paraformaldehyde solution to each well to fix the cells, remove the slides after 20 minutes The slides were mounted and observed under a laser confocal microscope. The results are shown in Figure 5. The lysosomal fluorescence signal of the targeting group has a low degree of coincidence with the liposome fluorescence signal, and the non-targeting group has a high degree of coincidence with the endoplasmic reticulum. suggest that Pardaxin has a role in helping escape from the lysosome.

4. Cytotoxicity test

Human-derived ovarian cancer SKOV-3, human-derived liver cancer HepG-2, and mouse-derived colon cancer CT-26 cells were grafted on a 96-well plate in an amount of 5×10 ⁴ cells, 200 μL/well. After 24h of cell adhesion, the blank non-targeted liposomes, drug-loaded liposomes, blank targeted liposomes and drug-loaded endoplasmic reticulum-targeted liposomes were prepared according to the serial concentration of the carrier (25ug/mL). 50ug/mL, 100ug/mL, 250ug/mL) were added to tumor cells. Incubate for 72h. Then, 20 μL of MTT aqueous solution (5 mg/mL) was added to each well, and after further incubation for 4 h, the culture medium was discarded, and 100 μL of DMSO was added to each well. The blank liposome group and blank targeted liposome group were used as the control group, and the free cyclophosphamide group was used as the positive control. The results are shown in Figure 6. The free cyclophosphamide has weak cytotoxicity in vitro because it is water-soluble and a prodrug. Common cyclophosphamide liposomes increase the cell entry rate of cyclophosphamide and improve the active ring in the system. The amount of phosphoramide, its in vitro cytotoxicity was significantly larger than that of the free drug, indicating that the liposome preparation can improve the uptake of the drug by cells and enhance the efficacy of the drug. Modification of liposomes with DSPE-PEG-Pardaxin can further increase the toxicity of cyclophosphamide to cells, and the cytotoxicity of DSPE-PEG-Pardaxin does not increase significantly with the increase of the ratio of DSPE-PEG-Pardaxin, indicating that DSPE-PEG-Pardaxin may The recognition of Pardaxin and the endoplasmic reticulum is saturated. The two different cell lines are more cytotoxic to HepG-2 than SKOV-3, which may be because there are more cyclophosphamide-activating cytochromes in the endoplasmic reticulum of liver cancer cells , which further indicated that the activation of cyclophosphamide increased and the cytotoxic effect was enhanced under the effect of endoplasmic reticulum targeting.

Example 5

Composition of ER-targeted doxorubicin liposomes:

The recipe quantities of HSPC, Cholesterol, DSPE-PEG ₂₀₀₀ and DSPE-PEG-PAR were placed in an eggplant-shaped bottle, dissolved in an organic solvent (chloroform), and evaporated overnight in a 55°C water bath under vacuum and decompression, and the bottle wall formed a film. An ammonium sulfate solution with pH=5.4 was added to the eggplant-shaped bottle, and the solution was hydrated in a water bath at 60° C. for 1 hour, and the film fell off and dissolved to form a multilamellar liposome solution. The above solution was sonicated to the desired particle size using probe sonication. The obtained liposome solution was passed through a Sephadex G50 column, eluted with PBS (pH=7.4), and the ammonium sulfate solution in the outer phase of the liposome was removed. Add 2 mg/mL doxorubicin solution to endoplasmic reticulum-targeted liposomes to make the drug-lipid mass ratio 1:20, and incubate in a 50°C water bath with stirring for 30 min. Then go through a Sephadex G50 column to remove free doxorubicin hydrochloride, and finally collect doxorubicin-loaded endoplasmic reticulum-targeted liposomes.

Doxorubicin is an anti-tumor antibiotic that can inhibit the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA. It has a broad anti-tumor spectrum and has effects on a variety of tumors. Cyclic tumor cells have a killing effect. Mainly used for acute leukemia, effective for acute lymphoblastic leukemia and myeloid leukemia. In the present invention, Pardaxin is added to the classic prescription of doxorubicin liposome. Pardaxin can help doxorubicin liposome enter cells and target the endoplasmic reticulum membrane, and doxorubicin liposome can bypass lysosome to protect drug. The invention can reduce the dosage of doxorubicin and reduce the toxic and side effects of the drug.

Example 6

Composition of endoplasmic reticulum-targeted dexamethasone acetate liposomes:

Dex-Ac, HSPC, Cholesterol, DSPE-PEG ₂₀₀₀ and DSPE-PEG-PAR in recipe quantities were placed in an eggplant-shaped bottle, dissolved in an organic solvent (chloroform), and evaporated overnight in a water bath at 55°C under vacuum and reduced pressure. The walls form a thin film. PBS with pH=7.4 was added to the eggplant-shaped flask, hydrated in a water bath at 60°C for 1 h, the film fell off and dissolved and a multilamellar liposome solution was formed. The above solution was sonicated to the desired particle size using probe sonication. The obtained liposome solution was passed through a Sephadex G50 column and eluted with PBS (pH=7.4) to remove the free dexamethasone acetate drug in the liposome. The eluted liposomes with opalescence were collected, namely dexamethasone acetate-loaded endoplasmic reticulum-targeted liposomes.

Dexamethasone acetate is an adrenal cortical hormone drug. It has pharmacological effects such as anti-inflammatory, anti-endotoxin, inhibiting immunity, anti-shock and enhancing stress response. There are many adverse reactions of dexamethasone acetate, which mostly occur in the application of pharmacological doses, and are closely related to the course of treatment, dose, type of medication, usage and route of administration. Common adverse reactions include the following categories: iatrogenic Cushing's syndrome face and posture, peptic ulcer, mental disorder, glucocorticoid withdrawal syndrome, diabetes and Cushing-like syndrome, etc.

After administration of dexamethasone acetate, it is metabolized into dexamethasone under the action of liver drug enzymes. It is a liver drug enzyme inducer. Long-term use will lead to enhanced liver drug enzyme activity, resulting in an increase in cytochrome P450 content and promotion of liver endoplasmic reticulum. Proliferation and synthesis of microsomal membrane-bound proteins. It is soluble in ethanol or chloroform, so dexamethasone acetate liposomes can be prepared by thin film dispersion method. Dexamethasone acetate was prepared into liposomes with endoplasmic reticulum targeting using Pardaxin target head. The invention has three major advantages: first, it can increase the concentration of drugs in target organs and reduce the distribution of drugs in other organs; second, directly targeting drugs to the endoplasmic reticulum can use enzymes in the endoplasmic reticulum to improve the body's ability to respond to dexamethasone acetate The activation efficiency of metasone, thirdly, after giving the drug tertiary targeting, can better control the dose of the drug, and control its role as a liver drug enzyme inducer. Dexamethasone acetate has many adverse reactions, and preparing it into a liposome preparation with endoplasmic reticulum targeting can effectively reduce the dosage, reduce adverse reactions, and control its induction effect on liver drug enzymes.

Example 7

Composition of endoplasmic reticulum-targeted drug-loaded hemoglobin liposomes:

The recipe quantities of E80, Cholesterol, DSPE-PEG ₂₀₀₀ and DSPE-PEG-PAR were placed in an eggplant-shaped bottle, dissolved in an organic solvent (chloroform), and evaporated overnight in a 55°C water bath under vacuum and decompression, and the bottle wall formed a film. PBS containing the prescribed amount of Hb was added to the eggplant-shaped bottle, hydrated in a water bath at 60°C for 1 h, the film fell off and dissolved and a multilamellar liposome solution was formed. The above solution was sonicated to the desired particle size using probe sonication. The obtained liposome solution was passed through a Sephadex G50 column and eluted with PBS (pH=7.4) to remove free Hb in the liposomes. The eluted liposomes with opalescence were collected, which were Hb-loaded endoplasmic reticulum-targeted liposomes. The liposome can bind oxygen molecules, and can directly deliver the oxygen molecules to the endoplasmic reticulum part of the cell, so as to increase the oxygen content of the part, which is beneficial to the subsequent oxygen-dependent treatment of the endoplasmic reticulum part.

Example 8

Composition of ER-targeted paclitaxel-loaded solid lipid nanoparticles:

Paclitaxel 5mg

Glyceryl Monostearate 100mg

DSPE-PEG-Pardaxin 2mg

Paclitaxel, glycerol monostearate and DSPE-PEG-Pardaxin in prescription amounts are added to anhydrous ethanol to dissolve, injected into the buffer solution of PBS, and then ultrasonic or homogenizer is used to obtain the desired particle size. The obtained nanoparticle solution was passed through a Sephadex G50 column, eluted with PBS (pH=7.4) and purified, that is, the endoplasmic reticulum-targeted solid lipid nanoparticles loaded with paclitaxel.

Example 9

Composition of ER-targeted docetaxel-loaded nanomicelles:

Docetaxel 5mg

Polyethylene glycol-polycaprolactone (PEG-PCL) 100mg

PCL-PEG-Pardaxin 10mg

Docetaxel, polyethylene glycol-polycaprolactone and PCL-PEG-Pardaxin in the prescribed amount are dissolved in absolute ethanol, injected into the buffer solution of PBS, and then obtained by ultrasonic or homogenizer to obtain the desired particle size . The obtained nanoparticle solution was passed through a Sephadex G50 column, eluted with PBS (pH=7.4) and purified, that is, the endoplasmic reticulum-targeted nanomicelles loaded with docetaxel.

Example 10

Synthesis of endoplasmic reticulum-targeted gold nanoparticles: Take 2 mg Pardaxin-PEG-TA synthesized in Example 2, mix with 10 mg of hollow gold nanomaterials (HAuNS) (size 40 nm, specific at 800 nm) in an aqueous medium at room temperature. Type absorption peak), incubated for 24 hours, centrifuged and purified to obtain hollow gold nanoparticles with endoplasmic reticulum targeting function. After entering cells, the nanoparticles accumulate in the endoplasmic reticulum, and with the help of external stimulation (near-infrared light), specific behaviors against the endoplasmic reticulum can be realized (photothermal stimulation, enhancing the stress level of the endoplasmic reticulum).

sequence listing

<110> Zhejiang University

<120> Construction and application of nano-drug delivery system targeting the endoplasmic reticulum

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<170> SIPOSequenceListing 1.0

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<212> PRT

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

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Claims (7)

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.

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