CN112603896A

CN112603896A - Preparation method of RGD cyclopeptide/R8 peptide modified ERG combined Afa double-drug-loaded liposome freeze-dried powder

Info

Publication number: CN112603896A
Application number: CN202011526863.4A
Authority: CN
Inventors: 黄挺; 黄绳武
Original assignee: Hangzhou Red Cross Hospital
Current assignee: Hangzhou Red Cross Hospital
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-04-06
Anticipated expiration: 2040-12-22
Also published as: CN112603896B

Abstract

The invention relates to a preparation method of an ergosterol and afatinib combined liposome, and belongs to the technical field of pharmaceutical preparations. The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome freeze-dried powder comprises the following steps: adding the freeze-drying protective agent into a pre-prepared RGD/R8-ERG/Afa-LIP liposome suspension in an external addition mode; finally, freeze-drying to prepare the compound liposome freeze-dried powder; the RGD/R8-ERG/Afa-LIP liposome suspension is prepared by the following method: ERG/Afa-LIP is prepared first and then by the post-insertion method. The invention selects proper auxiliary materials and a process method to prepare the ERG/Afa-LIP into a stable liposome freeze-dried powder preparation, and the bioavailability of the medicament can be correspondingly improved. And then the liposome in liquid form is prepared into a freeze-dried preparation by utilizing a freeze-drying technology, so that the storage time is prolonged. And carrying out quality evaluation and stability research on the obtained liposome, and analyzing to obtain conditions suitable for a drug storage environment and factors influencing stability.

Description

Preparation method of RGD cyclopeptide/R8 peptide modified ERG combined Afa double-drug-loaded liposome freeze-dried powder

Technical Field

The invention relates to a preparation method of a liposome, in particular to a preparation method of an ergosterol and afatinib combined liposome, and belongs to the technical field of pharmaceutical preparations.

Background

Malignant tumors of the lung are the leading cause of non-benign tumor-related deaths in the world today and pose a serious threat to human health. The global cancer incidence and mortality estimates in 2018 report that 1810 new cancer cases and 960 ten cancer death cases are expected in 2018, wherein non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer, but 75% of patients are found to be in the middle and advanced stage of cancer and miss the optimal period of surgical treatment.

The cisplatin and the taxol are put into clinical application for a long time, but the cisplatin and the taxol do not improve the survival benefit of patients, have drug resistance and toxic and side effects, and even harm the health and the life of the patients. The molecular targeted drug therapy is more and more widely applied due to the characteristics of strong pertinence and less side effect. In 2004, NSCLC was found to be associated with Epidermal Growth Factor Receptor (EGFR) mutations, since treatment of lung cancer entered the stage of molecular targeted studies.

Gefitinib (Gefitinib) is a first generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI), and has better curative effect on EGFR mutation patients than chemotherapeutic drugs, but the TKI resistance phenomenon appears in patients 10-14 months after the drugs are taken, and finally the curative effect of the drugs is reduced. Relevant researches show that the TKI drug resistance is mainly caused by mutation of threonine-790 of an EGFR exon into methionine, namely T790M mutation, c-Met gene amplification and the like. In the previous experimental study, the laboratory adopts the in vitro mechanism study on PC-9 cells sensitive to the first generation of molecular targeted drug gefitinib and A549 cells resistant to the gefitinib. The research finds that the inhibition rate of the combined administration group to A549 and PC-9 cells is improved to different degrees compared with that of the two single-use groups of ergosterol and gefitinib at different administration time, which indicates that the combination of ergosterol and gefitinib obviously enhances the sensitivity of GEF to A549 and PC-9 cells.

Afatinib (Afatinib) is a second-generation irreversible EGFR tyrosine kinase inhibitor, is an EGFR and HER2 dual multi-target drug developed by the company briglingmine, germany, namely is developed aiming at TKI secondary drug resistance development, carries a reactive acrylamide group, is an ATP competitive anilino derivative, and can prevent EGFR, HER2 and HER4 kinases from forming covalent bonds and irreversible valence bonds. However, the second generation of targeting drug afatinib can generate drug resistance after about 9-13 months of use, lose the anti-tumor effect and seriously affect the treatment effect.

Currently, afatinib which is commercially available is a tablet, the oral administration bioavailability is low, the adverse reactions are more, the most common adverse reactions are gastrointestinal adverse reactions, and people who do not have serious adverse reactions need to take measures of interrupting the administration or reducing the dosage. In order to improve the life quality of patients, reduce the occurrence of drug resistance and better exert the drug effect, the development of a novel afatinib preparation with targeting property, high bioavailability and less adverse reaction is imperative.

Simple independent administration does not achieve the desired effect, taking into account the different physicochemical and pharmacokinetic properties of the drugs, such as solubility, half-life, etc. Therefore, a drug co-delivery system is needed to address these issues.

Disclosure of Invention

The invention aims to solve the technical problems, and provides a preparation method of RGD cyclopeptide/R8 peptide modified ERG combined Afa double-drug-loaded liposome freeze-dried powder. According to the invention, DSPE-PEG3400-c (RGDFk) and DSPE-PEG1000-R8 are embedded into an ERG/Afa-LIP lipid membrane layer by adopting a post-insertion method to prepare the ERG/Afa-LIP (RGD/R8-ERG/Afa-LIP) modified by RGD cyclopeptide/R8 peptide, and considering that the liposome is easy to aggregate, precipitate, oxidize and the like after being placed for a long time, the liposome is prepared into a freeze-dried powder formulation, and the optimal freeze-dried powder preparation method is obtained by examining a prescription process and a freeze-drying process of the liposome. Finally, the apoptosis effect of the RGD/R8-ERG/Afa-LIP freeze-dried powder on PC-9 cells is examined by a flow cell apoptosis technology.

The technical scheme for solving the problems is as follows:

the preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome freeze-dried powder comprises the following steps: adding the freeze-drying protective agent into a pre-prepared RGD/R8-ERG/Afa-LIP liposome suspension in an external addition mode; finally, freeze-drying to prepare the compound liposome freeze-dried powder; the RGD/R8-ERG/Afa-LIP liposome suspension is prepared by the following method: ERG/Afa-LIP is prepared first and then by the post-insertion method.

Preferably, the freeze-drying method is a rapid freezing method, in which the temperature of a cold trap part in the apparatus is reduced to a minimum temperature in advance, and then the sample is placed in the cold trap.

Preferably, the prefreezing time of the freeze-drying method is 4 hours.

Preferably, the freeze-drying time of the freeze-drying method is 35 hours.

Preferably, the lyoprotectant is formulated as: each 5mL of liposome freeze-dried powder contains 147mg of sucrose and 147mg of mannitol, the glycolipid ratio is 3:1, and the mass ratio of the sucrose to the mannitol is 1: 1.

Preferably, a slow freezing method is adopted, and after the equipment is placed at a cold trap for 4 hours, the equipment is transferred to the upper layer to be subjected to freeze-drying for 35 hours; freeze-drying procedure: -30 to-20 ℃: 350 min; -20 to-10 ℃: 350 min; -10 to-0 ℃: 350 min; 0-10 ℃: 350 min; 10-20 ℃: 350 min; 20-30 ℃: and (5) 350 min.

The invention also aims to provide the liposome freeze-dried powder prepared by the method.

The invention aims to provide the application of the liposome freeze-dried powder in preparing targeted drugs for inhibiting tumor cell growth and/or inducing tumor cell apoptosis.

The invention has the following beneficial effects:

1. the invention selects proper auxiliary materials and a process method to prepare the ERG/Afa-LIP into a stable liposome freeze-dried powder preparation, and the bioavailability of the medicament can be correspondingly improved. And then the liposome in liquid form is prepared into a freeze-dried preparation by utilizing a freeze-drying technology, so that the storage time is prolonged. And carrying out quality evaluation and stability research on the obtained liposome, and analyzing to obtain conditions suitable for a drug storage environment and factors influencing stability;

2. the invention successfully prepares RGD/R8-ERG/Afa-LIP freeze-dried powder. Using ERG/Afa-LIP to carry out investigation of a freeze-drying process and optimization experiments of a prescription, screening out an optimal prescription process, and applying the optimal prescription process to RGD/R8-ERG/Afa-LIP for verification; the optimal freeze-drying and prescription process is as follows: and (3) placing the mixture in a cold trap of equipment for 4 hours by adopting a slow freezing method, transferring the mixture to the upper layer, and freeze-drying the mixture for 35 hours (the freeze-drying procedure is carried out for 350 minutes at-30 to-20 ℃, 350 minutes at-20 to-10 ℃, 350 minutes at-10 to-0 ℃, 350 minutes at 0 to 10 ℃, 350 minutes at 10 to 20 ℃, 350 minutes at 20 to 30 ℃ and 350 minutes). The prescription of the freeze-drying protective agent comprises: each liposome lyophilized powder (5 mL) contained 147mg of sucrose and 147mg of mannitol (glycolipid ratio of 3:1, sucrose to mannitol mass ratio of 1: 1). After the RGD/R8-ERG/Afa-LIP freeze-dried powder is redissolved, the particle size, the potential, the encapsulation rate and the serum stability are good, and the apoptosis of PC-9 cells can be obviously promoted by detecting the freeze-dried powder through flow type apoptosis.

Drawings

FIG. 1 is a DSPE-PEG1000-RRRRRR Maldi-Tof map;

FIG. 2 is a DSPE-PEG1000-RRRRRR Maldi-Tof map;

FIG. 3 is a DSPE-PEG3400-c (RGDFK) Maldi-Tof map;

FIG. 4 is a DSPE-PEG3400-c (RGDFK) Maldi-Tof map;

FIG. 5 is a graph of the temperature drop of the sample and the spacer;

FIG. 6 is a transmission electron micrograph of individual liposomes;

FIG. 7 is the particle size distribution of each liposome;

FIG. 8 is the particle size distribution of each liposome;

FIG. 9 is Zeta potential for each liposome;

FIG. 10 is Zeta potential for each liposome;

FIG. 11 shows the cumulative release of AFA and RGD/R8-ERG/AFA-LIP lyophilized powders in different pH release media;

FIG. 12 is a flow chart of apoptosis of PC-9 cells.

Detailed Description

The invention is further explained below with reference to the drawings.

This detailed description is to be construed as illustrative only and is not limiting, since modifications will occur to those skilled in the art upon reading the preceding specification, and it is intended to be protected by the following claims.

First, experimental material

(I) experimental drugs and reagents

Afatinib bulk drug (Aiblin biotechnology limited, batch number: abs 47020632)

Ergosterol bulk drug (more than or equal to 95%, Sigma company, batch number: BCBN 4049V)

Lecithin (Soybean, >98%, Shanghai Aladdin Biotechnology GmbH, batch number: G1813018)

High purity cholesterol (injection grade, Shanghai ai Wei special medical science Co., Ltd., batch number: B01221)

Ammonium sulfate (Shanghai Lingfeng Chemicals Co., Ltd., batch No. 20120313)

Citric acid (Shanghai Lingfeng chemical reagent Co., Ltd., batch No. 20110725)

Trichloromethane (Shanghai Lingfeng Chemicals Co., Ltd., batch No. 20171017)

Methanol (chromatographic grade, Tiandi limited USA, batch number MS 1922-801)

Polycarbonate track-etched film (type: 0.8, 0.4, 0.2, 0.1 μm, Whatman Co., UK)

Glucose (national drug group chemical reagent limited, F20090921)

Sucrose (Nanning sugar industry, Inc., Clear sugar Mill, lot number 171801)

Mannitol (Qingdao Mingyue seaweed group Co., Ltd., batch No. 20180314)

Trehalose (Yuan Ye Biotechnology Co., Ltd., batch No. S11052-25 g)

Resin: 2-Chlorotrityl Chloride Resin with a degree of substitution of 1.03mmol/g (Nankai Synthesis technology Co., Ltd., Tianjin)

Amino acids: Fmoc-Arg (pbf) -OH (> 99%) Fmoc-Gly-OH (Chen Nuo, > 99%)

DMF (Korea of origin)

DCM (Korea of origin)

MEOH (origin Japan)

DIEA (New German chemical industry, 99%)

HBTU (Hao San Biotechnology, 99%)

Piperidine (Shanghai chemical reagent company, national drug group, 99%)

95% cutting fluid: TFA (J.T.Baker, 99%)

TIS (Shanghai Darui fine chemical industry, 98%)

EDT (Shanghai Darui fine chemical industry, 98%)

(II) experimental equipment

TS-1 horizontal shaking table (Qinlinbel apparatus manufacturing Co., Ltd, Haimen city)

JA203H analytical electronic balance (Changzhou city lucky electronic devices Co., Ltd.)

XS105DU analytical balance (Mettler-Torledo Switzerland)

DK-450B electric heating constant temperature water tank (Shanghai Senxin experiment instrument Co., Ltd.)

Scientz-IID ultrasonic cell disruption instrument (Ningbo Xinzhi Biotech Co., Ltd.)

RE-52A rotary evaporator (Shanghai Yangrong biochemical instrument factory)

WH-861 vortex mixer (Taicang Hualida experimental equipment Co., Ltd.)

KQ5200DE model digital control ultrasonic cleaner (Kunshan ultrasonic Instrument Co., Ltd.)

Agilent 1260 high performance liquid chromatograph (Agilent technologies, Inc. of America)

HOMEEX-25 high pressure film extruder (Shanghai Hermeishi electromechanical science Co., Ltd.)

Zetasizer Nano ZS90 laser particle analyzer/Zeta potentiometer (British Marvin instruments Co., Ltd.)

H-7650 Transmission scanning Electron microscope (HITACHI, Japan)

Twelve-channel semi-automatic polypeptide instrument (Shanghai Qiao Yao biological science and technology Co., Ltd.)

VirTis freeze dryer (Shanghai Si Gaylor Biotechnology Co., Ltd.)

DHS20-A moisture meter (Shanghai precision scientific instruments Co., Ltd.)

Eppendorf 5427R desk-top high speed refrigerated centrifuge (Eppendorf Co., Germany)

Eppendorf 5702 Table centrifuge (Eppendorf Co., Germany)

HH-2 digital display constant temperature water bath (Changzhou Guohua electric appliance Co., Ltd.)

Refrigerator-freezers (Qingdao Haier GmbH)

Thermo 905 ultra-low temperature refrigerator (American Saimer Feishale Co.)

Micro-pipette (Eppendorf Co, Germany)

Synergy H1MFD multifunctional enzyme-labeling instrument (BioTek company, USA)

Twelve-channel semi-automatic polypeptide synthesizer (semi-automatic polypeptide synthesizer of Shanghai Qiang biological science and technology Limited, patent No. 201020226529.2)

SHIMADZU high performance liquid chromatograph (model: preparative, analytical, software: Class-VP. visual System, manufacturer: SHIMADZU)

Second, method and results

(I) DSPE-PEG₁₀₀₀Synthesis of-RRRRRRRR polypeptide

1 Experimental procedure

1.1 swelling of the resin

2-Chlorotrityl Chloride Resin was placed in a reaction tube, and DCM (15 ml. g) was added^-1) Shaking for 30 min.

1.2 grafting with the first amino acid

The solvent was filtered off by suction through a sand core, 3 times molar excess of Fmoc-Arg (pbf) -OH amino acid was added, DMF was added for dissolution, 10 times molar excess of DIEA was added, and shaking was carried out for 60 min. Blocking with methanol.

1.3 deprotection

DMF was removed and 20% piperidine DMF solution (15 ml. g) was added^-1) 5 min, remove and add 20% piperidine DMF solution (15 ml. g)^-1)，15 min。

1.4 detection

And (3) pumping out the piperidine solution, taking dozens of particles of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5 min, and turning dark blue to be a positive reaction.

1.5 washing

DMF(10 ml·g^-1) Twice, DCM (10 ml. g)^-1) Twice, DMF (10 ml. g)^-1) Twice.

1.6 condensation

And (3) dissolving protected amino acid and HBTU in triple excess by using DMF as little as possible, adding the solution into a reaction tube, immediately adding DIEA in ten-fold excess, and reacting for 30 min.

1.7 detection

Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5 min, and taking colorless negative reaction.

1.8 washing

DMF(10 ml·g^-1) Once, DCM (10 ml. g)^-1) Twice, DMF (10 ml. g)^-1) Twice.

1.9 linking amino acids

Repeating the three-six steps, and connecting the amino acids in the sequence from right to left. Until the straight sequence is completed. 1.10 draining and washing the resin

DMF(10 ml·g^-1) Twice, methanol (10 ml. g)^-1) Twice, DMF (10 ml. g)^-1) Twice, DCM (10 ml. g)^-1) Twice, and then is dried for 10 min.

1.11 cleavage of the polypeptide

Preparation of cutting fluid (10 ml. g)^-1) TFA 95%; 1% of water; 2% of EDT; TIS 2%, cutting time: and (4) 120 min.

1.12 drying and washing

The lysate is blown dry as much as possible with nitrogen, washed six times with ether and then evaporated to dryness at normal temperature.

1.13 ESI-MS identification

Straight-line sequences were identified by ESI-MS as our pre-linkage-modification spectra.

1.14 reaction

Adding DMF to swell for 15 min, adding 1.5 mmol of excessive DSPE-PEG₁₀₀₀-COOH, HATU in triple excess, dissolved in as little DMF as possible, was added to the reaction tube and DIEA was added immediately in ten-fold excess. The reaction was carried out for 45 min.

1.15 crude product

And (4) performing the polypeptide according to the tenth, eleventh and twelfth steps to obtain a colloidal polypeptide crude product.

1.16 Freeze drying

Adding the polypeptide into water and acetonitrile solution, putting into a freeze dryer for concentration, and freeze-drying to obtain the final product.

2 results of the experiment

As shown in FIG. 1, the raw material DSPE-PEG₁₀₀₀-COOH, MW:567+ polymeric moiety ≈ 1700, (normal distribution in the figure can clearly see each peak difference of 44), the final product DSPE-PEG₁₀₀₀-RRRRRRRR MW: 1700+1267≈2900。

As shown in fig. 2, the final polymer portion 2800 may be slightly smaller than 2900 and may have a slightly inferior spectrum.

(di) DSPE-PEG₃₄₀₀-c Synthesis of (RGDFK) Polypeptides

1 Experimental procedure

1.1 swelling of the resin

1.2 grafting with the first amino acid

The solvent was filtered off by suction through a sand core, 3-fold molar excess of Fmoc-Asp (Oall) -OH amino acid was added, DMF was added for dissolution, 10-fold molar excess of DIEA was added and shaking was carried out for 60 min. Blocking with methanol.

1.3 deprotection

DMF was removed and 20% piperidine DMF solution (15 ml. g) was added^-1)，5minAfter removal of the solvent, 20% piperidine DMF solution (15 ml. g) was added^-1)，15min。

1.4 detection

1.5 washing

DMF(10 ml·g^-1) Twice, DCM (10 ml. g)^-1) Twice, DMF (10 ml. g)^-1) Twice.

1.6 condensation

1.7 detection

1.8 washing

DMF(10 ml·g^-1) Once, DCM (10 ml. g)^-1) Twice, DMF (10 ml. g)^-1) Twice.

1.9 linking amino acids

Repeating the three-six steps, and sequentially connecting amino acids Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-Lys (Dde) -OH in the sequence from right to left. Cyclization, removing OALL protecting group, dissolving HBTU with DMF as little as possible, adding into a reaction tube, adding DIEA for ten times of excessive amount, reacting for 60 min, and removing side chain Dde protecting group.

1.10 Wash resin

1.11 cleavage of the polypeptide

Preparation of cutting fluid (10 ml. g)^-1) TFA 95%; 1% of water; 2% of EDT; and (3) TIS 2%. Cutting time: 120 min

1.12 drying and washing

1.13 ESI-MS identification

The straight-line cyclic peptide sequence is identified by ESI-MS to be correct for the mass spectrum before linkage modification.

1.14 reaction

Adding DMF to swell for 15 min, adding 1.5 mmol of excessive DSPE-PEG₃₄₀₀Three times of excess of-COOH and HATU, dissolving with DMF as little as possible, adding into a reaction tube, immediately adding DIEA ten times of excess, and reacting for 45 min.

1.15 crude product

1.16 Freeze drying

2 results of the experiment

The MALDI-tof spectrum of the PEG glycol polymer is similar to normal distribution, and each peak in the normal distribution strictly speaking should be about 44 different. Wherein, DSPE-PEG phospholipid polyethylene glycol raw material partially falls off when MALDI-tof is detected, accurate molecular weight is two additions, and polymer part has deviation of about 10% due to polymerization. The end product may fall off the DSPE for sequence reasons and may not.

As shown in FIG. 3, the raw material DSPE-PEG₃₄₀₀-COOH, MW 607+ polymeric moiety ≈ 4100, with slightly poorer pattern of the polymeric moiety. End product DSPE-PEG₃₄₀₀-c (rgdfk) molecular weight: feed 4100+ line 607 ≈ 4700.

As shown in FIG. 4, the tip falls off, and 701+ the polymerized portions 3800-3900 are slightly smaller than 4700. Since the polymer is used, it is possible that the error is within 10%.

(III) research on prescription process of ERG/Afa-LIP freeze-dried powder

The storage form of the common liposome is liquid, but the liposome is quite unstable in the liquid form, which is easy to cause the phenomena of drug leakage, phospholipid hydrolysis, oxidation, precipitation and related substance increase, thereby causing the instability of the liposome. In the experiment, the ERG/Afa-LIP freeze-dried liposome is developed by adopting a freeze-drying method, so that the stability of the ERG/Afa-LIP freeze-dried liposome is improved, and the storage time is prolonged. The liposome is easy to phase separate in the freeze drying process, and the generated ice crystals can destroy the structure of liposome phospholipid bilayer, so that the particle size of the liposome is increased after redissolution, and insoluble precipitate is generated. Thus, this can be solved by adding a suitable lyoprotectant. The freeze-drying protective agent has four characteristics: poor water absorption, high glass transition temperature, no reducing group and low crystallization rate.

The experiment selects proper auxiliary materials and a process method to prepare the ERG/Afa-LIP into a stable liposome freeze-dried powder preparation, and the bioavailability of the medicament can be correspondingly improved. And then the liposome in liquid form is prepared into a freeze-dried preparation by utilizing a freeze-drying technology, so that the storage time is prolonged. And carrying out quality evaluation and stability research on the obtained liposome, and analyzing to obtain conditions suitable for a drug storage environment and factors influencing stability.

1 preparation of lyophilized liposomes

The ice crystals generated in the freeze-drying process can damage the liposome structure and cause the phenomena of sample non-shaping, shrinkage and the like. In order to reduce the damage of freeze-drying to the liposome and a certain aesthetic degree, so that the liposome does not collapse and collapse, a freeze-drying protective agent is generally required to be added. The adding mode of the freeze-drying protective agent is divided into an internal addition method and an external addition method. The external addition method is to add the protective agent directly into the prepared liposome suspension, and the internal addition method is to add the protective agent into the hydration medium (194.63 mM ammonium sulfate solution) of the liposome for co-hydration. In the experiment, 5% of sucrose is added as a freeze-drying protective agent for experiment. The results in the table below show that the particle size of the liposomes obtained by the internal addition after lyophilization is significantly higher than that obtained by the external addition method (A)P<0.01) and the appearance of the lyophilized liposomes was concave and wrinkled. In summary, the experiment selects the addition method as the addition mode of the liposome freeze-drying protective agent.

2 optimization of the Freeze-drying Process

2.1 determination of Co-melting Point of lyophilized powder

The temperature of the material cannot be higher than the eutectic point during sublimation drying, otherwise the material can be melted to cause boiling of the material, and the dried liposome is easy to generate bubbles, inflate and expand, deepen color, dry shrinkage, difficult dissolution and the like^[62]Thereby affecting the quality of the lyophilized formulation. And (3) placing the prepared freeze-dried sample on a partition plate, recording the temperature of the partition plate and the change value of the temperature of the sample along with time, and drawing a cooling curve, wherein the cooling curve is as shown in figure 5.

As can be seen from FIG. 5, the temperature of the sample decreases with time in 0-13 min, and a small temperature rise process occurs in 13-14 min, wherein the temperature rises from 1.7 ℃ to 2.8 ℃, which indicates that the lowest eutectic point of the sample is between 1-3 ℃, so the temperature rise temperature in the first stage (sublimation drying) should not exceed 1 ℃. The freeze dryer can meet the production requirement.

2.2 investigation of prefreezing Rate

The prefreezing rate is one of the important parameters of the lyophilization process. The pre-freezing rate is divided into a fast freezing method and a slow freezing method. The quick freezing method is characterized by that firstly, the cooling procedure of freeze-drying machine is started, the temperature of cold trap is reduced to-40 deg.C, then the sample is placed in the cold trap, and said method can quickly cool the sample, and the sample can be formed into compact network structure. The slow freezing method is to put the freeze-dried sample into the cold trap and then start the cooling function of the equipment, and the method is characterized in that the temperature of the sample is slowly reduced, the formed ice crystals are coarse, but the drying efficiency can be improved. The different prefreezing rates may cause osmotic pressure to develop between the frozen outer and molecular layers of the liposomes, resulting in drug leakage from the liposomes. Therefore, the experiment is carried out by adopting a slow freezing method and a quick freezing method respectively. The results are shown in the table below, and the slow and fast freezing methods had little effect on the Afa and ERG encapsulation efficiencies. However, the slow freezing method is smaller in particle size than the quick freezing method, and the appearance is full, loose and free of depression, so that the slow freezing method is selected for pre-freezing the freeze-dried sample in the experiment.

Effect of prefreezing Rate on Liposome Freeze-drying Effect

2.3 investigation of prefreezing time

The length of the prefreezing time determines whether the sample is completely frozen, which is a key factor affecting the quality of the lyophilized product. In the experiment, the quality of freeze-dried samples after 1, 2, 3, 4, 6 and 8 hours of pre-freezing in a cold trap by a slow freezing method is respectively considered. Carrying out sublimation drying according to the program. Whether the appearance of the sample is full, the particle size change, the encapsulation efficiency of ERG and Afa and the re-solubility are used as evaluation indexes, the scoring method is shown in the table, and the scores are better. The results show that the composite score obtained by pre-freezing for 4h is highest, so the pre-freezing time is selected to be 4 h.

2.4 examination of Freeze-drying time

The storage temperature of the freeze-dried powder is required to be lower than the glass transition temperature of the medicine, otherwise, the medicine has the adverse symptoms of dent, surface atrophy, hardening and discoloration, caking and the like. The water content in the freeze-dried powder is an important factor influencing the glass transition temperature. The higher the water content in the freeze-dried powder is, the lower the glass transition temperature of the freeze-dried powder is, and the worse the stability of the freeze-dried powder is, so that the water content is also an important factor for controlling the quality of freeze-dried products. The experiment respectively considers the water content under the freeze-drying time of 20, 24, 35, 48 and 72 h to determine the optimal freeze-drying time. As a result, as shown in the following table, the moisture content gradually decreased with the lapse of the lyophilization time. In consideration of cost and time, lyophilization is finally selected for 35h as the lyophilization time.

2.5 verification of Freeze-drying prescription Process

Preparing three batches of ERG/Afa-LIP liposomes according to the optimized prescription and preparation process, adding a protective agent, subpackaging the liposomes containing the protective agent into penicillin bottles with 5mL of each bottle, and respectively inspecting the appearance, re-solubility, particle size distribution, entrapment rate and the like of the lyophilized liposomes.

3 screening index of freeze-drying protective agent

3.1 evaluation of appearance

The volume of the ERG/Afa-LIP after freeze-drying is the same as that of the ERG/Afa-LIP before freeze-drying, no collapse is generated, and the ERG/Afa-LIP is compact and full when observed by naked eyes and a transmission electron microscope. The liposome has round microscopic shape and uniform size.

3.2 evaluation of reconstitution Capacity

Adding the freeze-dried ERG/Afa-LIP into the original volume of normal saline for injection, and shaking slightly by hand without other equipment to obtain clear liposome redissolution. And observing the redissolution time and after redissolution, the color is clear and transparent, and the color is milky white and has no precipitate.

3.3 evaluation of particle diameter

Adding the lyophilized ERG/Afa-LIP into normal saline for injection, and manually dispersing. The reconstituted particle size after lyophilization should be within specification or not significantly altered from that before lyophilization.

3.4 evaluation of encapsulation efficiency

Adding the ERG/Afa-LIP after freeze-drying into normal saline for injection, and shaking by hand to disperse. The encapsulation efficiency of ERG/Afa-LIP freeze-dried powder of each prescription is measured, and the change of the encapsulation efficiency before and after freeze-drying is small.

3.5 screening of lyoprotectants

The freeze-drying protective agent has the following characteristics: a. poor water absorption; b. the crystallization rate is low; c. the glass transition temperature is high; d. the product is tasteless and does not react with protein, liposome and the like; e. the price is cheap and easy to obtain. In the experiment, the single use and the combined use of the freeze-drying protective agent are respectively inspected, and the proper and optimal prescription process is screened out.

The protective effect of different types and concentrations of protective agents on liposomes in the freeze-drying process is different, and the effect of mannitol, glucose, sucrose and trehalose on freeze-dried samples is respectively considered, and the results are shown in the following table. The result shows that mannitol has strong supporting capacity due to self-crystallization and is easy to obtain full appearance, but the formation of the crystallization can damage the liposome structure, so that the entrapment rate of the liposome is remarkably reduced and the particle size of the liposome is remarkably increased. Sucrose has poor appearance and collapse, but has small particle size, short redissolution time, good dispersibility and high ERG encapsulation efficiency and Afa encapsulation efficiency. Glucose and trehalose are used as protective agents, and the particle size PDI value after redissolution is higher and the dispersibility is poor. Thus considering the use of mannitol as a proppant to give a good appearance to the freeze-dried product, it was further screened for co-use with sucrose.

3.6 determination of the amount and the proportions of the lyoprotectant

This experiment was carried out by varying the glycolipid ratio and the ratio between sucrose and mannitol to design 9 sets of experiments, the specific experiments being shown in the table below. The protective effect of the liposome on the freeze-dried product is examined by taking the appearance, redissolution time, mean particle size, ERG and Afa entrapment rate of the liposome before and after freeze-drying as selection indexes. The results of the tests are shown in the following table. The results show that prescription 4 scored the highest. Therefore, the finally determined freeze-dried powder prescription process comprises the following steps: glycolipid ratio =3:1, sucrose: mannitol =1:1 (147 mg:147 mg).

3.7 validation test

RGD/R8-ERG/Afa-LIP samples were prepared and tested for validation using formula 4 in the above table, with the results shown in the table below. The results show that the appearance of each group is completely loose, uniform and full. The redissolving time of three batches of freeze-dried powder is less than 30s, the average particle size is 100.2 +/-0.43 nm, the PDI is 0.210 +/-0.007, the ERG encapsulation rate is 92.87 +/-1.32%, and the Afa encapsulation rate is 92.60 +/-0.77%. In summary, from the above lyophilization process and formulation review, the final lyophilization process was determined as follows: and (3) placing the mixture in a cold trap of equipment for 4 hours by adopting a slow freezing method, transferring the mixture to the upper layer, and freeze-drying the mixture for 35 hours (the freeze-drying procedure is carried out for 350 minutes at-30 to-20 ℃, 350 minutes at-20 to-10 ℃, 350 minutes at-10 to-0 ℃, 350 minutes at 0 to 10 ℃, 350 minutes at 10 to 20 ℃, 350 minutes at 20 to 30 ℃ and 350 minutes). The prescription of the freeze-drying protective agent comprises: each liposome lyophilized powder (5 mL) contained 147mg of sucrose and 147mg of mannitol (glycolipid ratio of 3:1, sucrose to mannitol mass ratio of 1: 1).

4. Quality evaluation of RGD/R8-ERG/Afa-LIP liposome freeze-dried powder

4.1 morphological Observation

4.1.1 appearance

RGD-ERG/Afa-LIP solution, R8-ERG Afa-LIP solution, RGD/R8-ERG/Afa-LIP solution and RGD/R8-ERG/Afa-LIP freeze-dried powder are milky, uniform in color and luster, transparent and bright, and free of precipitate after redissolving. The RGD/R8-ERG/Afa-LIP freeze-dried powder is complete and loose in appearance, uniform and full and free of pits.

4.1.2 microscopic morphology

The liposomes were observed by transmission electron microscopy. The samples were prepared by negative staining. At room temperature, taking RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP freeze-dried powder samples, dripping the RGD/R8-ERG/Afa-LIP freeze-dried powder samples onto a copper net special for an electron microscope, sucking excess sample solution with filter paper, standing for 1min, carrying out negative dyeing with 1% phosphotungstic acid, standing for 30s, sucking excess dye solution of the copper net with the filter paper, naturally volatilizing, observing with the electron microscope and taking a picture, wherein the result is shown in figure 6. The transmission electron microscope result shows that each liposome has round shape and uniform particle size distribution, and the particle size distribution is about 120 nm.

4.2 particle size and distribution thereof

At room temperature, taking ERG/Afa-LIP, RGD-ERG/Afa-LIP, R8-ERG/Afa-LIP, RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP freeze-dried powder samples, pouring 1mL of the samples into a sample cell, and measuring the average particle size and the distribution thereof by using a laser particle sizer. The results are shown in FIGS. 7 and 8. The results showed that the average particle size of ERG/Afa-LIP was 107.9. + -. 0.19 nm, the polydispersity number PDI was 0.235. + -. 0.013, less than 0.3, the average particle size of RGD-ERG/Afa-LIP was 125.2. + -. 1.76 nm, PDI was 0.249. + -. 0.016, less than 0.3, the average particle size of R8-ERG/Afa-LIP was 112.0. + -. 0.77 nm, PDI was 0.252. + -. 0.013, less than 0.3, the average particle size of RGD/R8-ERG/Afa-LIP after reconstitution was 107.2. + -. 0.287 nm, PDI was 0.293. + -. 0.007, less than 0.3, the average particle size of RGD/R8-ERG/Afa-LIP after reconstitution was 107.2. + -. 0.294 nm, the particle size of PDI was 0.294, less than 0.3, and the distribution of liposomes was more concentrated.

4.3 Zeta potential measurement

At room temperature, ERG/Afa-LIP, RGD-ERG/Afa-LIP, R8-ERG/Afa-LIP, RGD/R8-ERG/Afa-LIP, RGD/R8-ERG/Afa-LIP freeze-dried powder samples are taken, injected into a sample cell and measured for potential by a Zeta potentiometer, and the result is shown in figure 9 and figure 10. The results show that the Zeta potential of ERG/Afa-LIP is-1.15 + -0.31 mV, the Zeta potential of RGD-ERG/Afa-LIP is-11.27 + -0.73 mV, the Zeta potential of R8-ERG/Afa-LIP is-8.00 + -0.66 mV, the Zeta potential of RGD/R8-ERG/Afa-LIP is-8.61 + -0.15 mV, and the Zeta potential of RGD/R8-ERG/Afa-LIP freeze-dried powder after redissolving is-2.49 + -0.18 mV.

4.4 encapsulation efficiency and drug content determination

3 RGD/R8-ERG/Afa-LIP freeze-dried powder samples, wherein the average entrapment rate of Afa is 92.45 +/-0.61%. The average encapsulation efficiency of ERG was 92.87 ± 1.32%.

4.5 degree of Release

This experiment examined the in vitro release profile of the Afa drug at different pH conditions. Precisely sucking 4mL of RGD/R8-ERG/Afa-LIP lyophilized powder solution and 4mL of Afa citric acid solution, placing in a dialysis bag, clamping with a dialysis clamp, adding into 100 mL of 40% methanol-containing phosphate buffer solutions with pH of 7.4 and 6.5, respectively, placing in a constant temperature water bath oscillator (shaking rate of 100 rpm min)^-1Release temperature 37 ℃), 1mL of dialysate was aspirated at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 24h and immediately supplemented with 1mL of fresh dialysis medium. Filtering the sample with 0.45 μm microporous filter membrane, detecting by HPLC, introducing 20 μ L of sample to determine peak area, substituting into linear regression equation to calculate out drug release concentration of Afa at each sampling point, and calculating to obtain c₁The total dose of Afa is recorded as M₀. The calculation is performed according to the following formula:

wherein, c₁Is the concentration of Afa released at each sampling point, V₀To release the volume of medium, V is the sampling volume, M₀The total amount of Afa is shown.

The results in FIG. 11 show that in phosphate buffer containing 40% methanol at pH7.4, the cumulative release percentage of Afa drug substance in 24h is 94.92%, and the cumulative release percentage of RGD/R8-ERG/Afa-LIP lyophilized powder is 95.25%. Under the condition of pH6.5, in phosphate buffer containing 40% of methanol, the cumulative release percentage of the Afa raw material drug in 24h is 98.21%, almost completely released, and the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder is 96.95%. In phosphate buffer solution containing 40% of methanol and at the pH value of 7.4, the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder in 0.5 h is 21.26% <40%, and the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder in 6 h is 82.64% > 80%; the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder is 95.25 percent after 24 hours. In phosphate buffer solution containing 40% of methanol and with the pH value of 6.5, the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder in 0.5 h is 22.47% <40%, the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder in 6 h is 87.09% >80%, and the cumulative release percentage of RGD/R8-ERG/Afa-LIP freeze-dried powder in 24h is 96.95%. The 2015 edition of Chinese pharmacopoeia relates to the requirements of liposome burst release effect: the initial release amount of 0.5 h should be less than or equal to 40%, and the cumulative release percentage of 24h should be more than 80%. RGD/R8-ERG/Afa-LIP lyophilized powder meets the requirements under the condition that the release medium is phosphate buffer solution with pH6.5 and pH7.4 and containing 40% methanol.

4.6 serological stability Studies

RGD/R8-ERG/Afa-LIP and 1mL of freeze-dried powder thereof are taken and mixed with fetal calf serum filtered by a 0.22 mu m microporous membrane in equal volume, 100 mu L of the mixture is taken for 0, 0.5, 1, 3, 5, 8 and 24 hours respectively, diluted by 10 times with ultrapure water, and the particle size is measured by adopting a Malvern particle size analyzer. The result is shown in the table below, the particle size of the RGD/R8-ERG/Afa-LIP freeze-dried powder is stabilized at about 110 nm within 24h, and the serum stability is good.

4.7 flow apoptosis assay

PC-9 cells are cultured in vitro, and ERG/Afa-LIP, RGD-ERG/Afa-LIP, R8-ERG/Afa-LIP, RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP freeze-dried powder groups are given for stimulation for 48 h, and the apoptosis condition of each group is detected by Annexin V-FITC/PI double-staining flow cytometry. Taking the number of PC-9 cells in logarithmic growth phase as 1.5X 10⁵each.mL^-1，800 rpm·min^-1Centrifuging for 5 min, blowing and mixing with dropper to obtain single cell suspension, adding 2 mL of complete culture medium into each well, setting 3 multiple wells for each concentration, and performing parallel assay for 3 times. At 37 ℃ with 5% CO₂Culturing in incubator until cell fusion is 80%, adding medicine, treating for 48 hr, collecting supernatant and cell at 1.0 × 10⁶Washed 2 times with precooled PBS at 800 rpm min^-1Centrifuging for 5 min, discarding the supernatant, adding 500 μ L of Apoptosis Positive Control Solution for resuspension, incubating on ice for 30 min, adding appropriate amount of precooled 1 × Binding Buffer for resuspension, adding the same amount of living cells called treated, mixing with the living cells, adding pre-cooled PBS for centrifugal washing, discarding the supernatant, supplementing to 1.5 mL with precooled 1 × Binding Buffer, and adding into each single staining tube5 μ L Annexin V-FITC or 10 μ L PI were added and light irradiation was avoided for 5 minutes. On the flow cytometer, the voltage of the FSC, SSC and fluorescence channels is adjusted by using a blank tube, and under the condition of the voltage, the compensation of the screen light channel is adjusted by using a single dyeing tube. And finally, after passing through a 400-mesh screen, detecting the apoptosis rate by adopting a flow cytometer within 1 hour. The experiment was repeated 3 times.

The test results are shown in FIG. 12 and the following table. The results show that the apoptosis rate of each administration group is very higher than that of a blank control group: (P<0.01). There was no significant difference in the apoptosis rate between the RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP lyophilized powder groups ((P>0.05), indicating that the preparation of RGD/R8-ERG/Afa-LIP into lyophilized powder does not reduce the apoptosis effect on PC-9 cells. Wherein, the apoptosis rates of the RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP freeze-dried powder groups are very different with respect to that of the single target head modified liposome (the apoptosis rates of the RGD/R8-ERG/Afa-LIP and the RGD/R8-ERG/Afa-LIP freeze-dried powder groups are all very significantP<0.01). RGD-ERG/Afa-LIP and R8-ERG/Afa-LIP have no significant difference compared to ERG/Afa-LIP (R) (A)P>0.05). The double-target modification effect is better than that of the single-target modification effect.

Stability study of 5 RGD/R8-ERG/Afa-LIP liposome freeze-dried powder

During long-term storage, the drug can generate degradation products, so that the curative effect of the drug is reduced, and toxic and side effects are generated, thereby affecting the quality, safety and effectiveness of the drug. The stability of the medicine is set according to the contained components or the preparation characteristics and quality requirements so as to objectively and comprehensively evaluate the stability of the medicine. The experiment mainly carries out stability investigation on the liposome from two aspects of physical stability and chemical stability.

5.1 physical stability

The liposome belongs to a colloid dispersion system, and is a thermodynamically unstable system. The physical stability of the ERG/Afa-LIP lyophilized liposome mainly includes appearance, reconstitution time, particle size after reconstitution, and entrapment rate of ERG and Afa of the ERG/Afa-LIP lyophilized liposome. The ERG/Afa-LIP liposome freeze-dried powder is placed in a dark and closed manner at the temperature of 4 ℃ and the temperature of 25 ℃ respectively, the physical stability of the ERG/Afa-LIP liposome freeze-dried powder is investigated by using the appearance, the redissolution time, the particle size distribution after redissolution and the entrapment rate of ERG and Afa as evaluation indexes, and the results are shown in the table below.

As can be seen from the above table, after standing at 4 ℃ and 25 ℃ for 10 days, the redissolution time of the freeze-dried powder at 4 ℃ is shorter than that of the freeze-dried powder at 25 ℃; the particle size is not changed greatly and is stabilized at about 120 nm; however, the Afa encapsulation efficiency and the ERG encapsulation efficiency at 4 ℃ are slightly higher than those at 25 ℃. Therefore, the freeze-dried powder has better physical stability at 4 ℃ than at 25 ℃.

5.2 chemical stability

The chemical stability of liposomes has two aspects, firstly the stability of the drug encapsulated in the liposomes, which can be examined by examining the content of the drug. On the other hand, since the membrane material component of liposomes contains phospholipids, which are unstable and easily undergo oxidative hydrolysis due to unsaturated bonds contained in the structure, it is necessary to examine the index of the substance. The ERG/Afa-LIP freeze-dried liposome is respectively placed at 4 ℃ and 25 ℃, the chemical stability of the ERG/Afa-LIP freeze-dried liposome is examined by using the contents of ERG and Afa and related substances as indexes, and the results are shown in the following table.

As can be seen from the table above, after standing at 4 ℃ and 25 ℃ for 10 days, the Afa content and the ERG content of the freeze-dried powder both have slight reduction tendency, but the reduction content is not much different; but the impurities produced at 4 c were slightly less than the impurity level at 25 c. Therefore, the freeze-dried powder has better chemical stability at 4 ℃ than at 25 ℃.

5.3 influential factor test

The influential factor test is conducted under severe conditions in order to understand the factors affecting the stability of the drug, thereby providing scientific reference to the packaging material, the storage conditions, and the expiration date of the drug. Meanwhile, theoretical basis is provided for conditions such as temperature and humidity and the like adopted in accelerated tests and long-term tests. Three batches of ERG/Afa-LIP liposome freeze-dried powder are respectively subjected to high-temperature, high-humidity and illumination tests according to the specification of 2015 version Chinese pharmacopoeia and the 'pharmaceutical preparation stability guiding principle'.

5.3.1 high temperature test

The ERG/Afa-LIP freeze-dried lipid preparation is sealed and protected from light, and is placed in a constant-temperature oven at 60 ℃ to sample for detection at 0, 5 and 10 days respectively, and the results are shown in the following table.

From the above table it follows that: after being placed at 60 ℃ for 5 days and 10 days, the appearance is slightly concave, the redissolution time is obviously prolonged, the particle size is obviously increased, the content changes of ERG and Afa are slightly small, but the ERG encapsulation efficiency and the Afa encapsulation efficiency are obviously reduced. The high temperature has obvious influence on the freeze-dried powder liposome, so the freeze-dried powder liposome needs to be stored at low temperature.

5.3.2 high humidity test

And (3) taking three batches of liposome freeze-dried powder to be opened, keeping out of the sun, placing the three batches of liposome freeze-dried powder in a constant-humidity closed container, sampling at 0, 5 and 10 days respectively under the environment of RH 90% +/-5% at 25 ℃, and detecting by HPLC (high performance liquid chromatography), wherein the results are shown in the following table.

From the above table, it can be seen that, after being placed at 25 ℃ and RH 90% +/-5% for 5d and 10 d, the appearance is obviously concave, the redissolution time is obviously increased, the particle size is obviously increased, the content changes of ERG and Afa are slightly smaller, but the ERG encapsulation rate and the Afa encapsulation rate are obviously reduced. The result shows that the liposome freeze-dried powder is obviously affected by high humidity, so the liposome freeze-dried powder is required to be placed under a dry and closed condition during storage.

5.3.3 light test

Three batches of liposome freeze-dried powder are taken for sealing, and the three batches of liposome freeze-dried powder are respectively sampled and detected at 0, 5 and 10 days under the illumination condition of 4500Lx +/-500 Lx, and the results are shown in the following table.

As can be seen from the above table, the product has a loose, uniform and full appearance and a short redissolution time after being placed for 10 days under the illumination condition of 4500Lx +/-500 Lx. The particle size is not greatly changed; the ERG content is slightly changed, and the encapsulation efficiency is almost unchanged; the Afa content is not changed greatly, the encapsulation efficiency is obviously reduced, which shows that strong light has obvious influence on the stability of the liposome freeze-dried powder, so that attention needs to be paid to avoiding light during storage.

Third, analysis and discussion

In this part of the experiment, DSPE-PEG was inserted after₃₄₀₀C (RGDFk) and DSPE-PEG₁₀₀₀R8 is embedded into lipid membrane layer of ERG/Afa-LIP to prepare RGD cyclic peptide and R8 peptide modified ERG/Afa-LIP (RGD/R8-ERG/Afa-LIP). RGD consists of an arginine-glycine-aspartic acid tripeptide, which can be targeted for recognition and binding to a variety of integrin receptors. R8 belongs to a cell penetrating peptide, is a cationic oligomeric cell penetrating peptide consisting of 8 arginines, is called R8 for short, and has the characteristics of good penetrating effect, low cytotoxicity and the like. The RGD cyclopeptide and R8 peptide are connected to the DSPE through PEG, and the DSPE-PEG is used₃₄₀₀C (RGDFk) and DSPE-PEG₁₀₀₀-R8 as phospholipid material to directly modify liposomes to avoid detachment from liposome surface during blood circulation and to improve stability of co-modified liposomes. Considering the problem of instability of the liposome, the liposome is prepared into a freeze-dried powder, the form, the particle size distribution, the Zeta potential, the serological stability, the in-vitro release, the stability and related substances of the liposome are preliminarily inspected, and the in-vitro efficacy is inspected through an in-vitro flow cytometry detection apoptosis experiment. The stability of the RGD/R8-ERG/Afa-LIP lyophilized powder was also examined.

The experiment was first investigated for the lyophilization process. The RGD/R8-ERG/Afa-LIP temperature can not be higher than the eutectic point during sublimation drying, otherwise, the temperature can be melted to cause boiling phenomenon, and the dried liposome is easy to generate bubbles, inflate and expand, deepen color, shrink by dryness, difficult to dissolve and the like, thereby affecting the quality of the freeze-dried product. The temperature rise in the first stage (sublimation drying) is not more than 1 ℃ through inspection. The freeze dryer can meet the production requirement.

The pre-freezing rate is one of the important parameters of the freeze-drying process, and directly influences the freeze-drying effect of the freeze-dried sample. The pre-freezing rate is divided into a fast freezing method and a slow freezing method. The quick freezing method is that the temperature lowering program of freeze drier is started to lower the temperature of cold trap to-40 deg.c before setting the sample in the cold trap. The slow freezing method is to put the freeze-dried sample into the cold trap and then start the equipment to cool, and the method is characterized in that the temperature of the sample is slowly reduced, the formed ice crystals are coarse, but the drying efficiency can be improved. The different prefreezing rates may cause osmotic pressure to develop between the frozen outer and molecular layers of the liposomes, resulting in drug leakage from the liposomes. Therefore, the experiment is carried out by adopting a slow freezing method and a quick freezing method respectively. The pre-freezing rate observed in this experiment was a slow freezing method.

The length of the prefreezing time determines whether the sample is completely frozen, which is a key factor affecting the quality of the lyophilized product. In the experiment, the quality of freeze-dried samples after 1, 2, 3, 4, 6 and 8 hours of pre-freezing in a cold trap by a slow freezing method is respectively considered. Carrying out sublimation drying according to the program. And (4) comprehensively scoring by taking whether the appearance of the sample is full, the particle size change, the encapsulation efficiency of ERG and Afa and the re-solubility as evaluation indexes, and finally, investigating that the pre-freezing time is 4 h.

The storage temperature of the freeze-dried powder is required to be lower than the glass transition temperature of the medicine, otherwise, the medicine has the adverse phenomena of dent, surface atrophy, hardening, discoloration, caking and the like. The water content in the freeze-dried powder is an important factor influencing the glass transition temperature. The higher the water content in the freeze-dried powder is, the lower the glass transition temperature of the freeze-dried powder is, and the worse the stability of the freeze-dried powder is, so that the water content is also an important factor for controlling the quality of freeze-dried products. In order to save time, the experiment adopts drying for 35 hours as drying time on the basis that the water content meets the specification.

Ice crystals produced during the lyophilization process can damage the liposome structure and cause the sample to be unformed and produce shrinkage. In order to reduce the damage to the liposome and to ensure a certain aesthetic degree, the liposome does not collapse and collapse, and a freeze-drying protective agent is generally required to be added. The freeze-drying protective agent examined in the text is mannitol, glucose, sucrose and trehalose, and the screening is carried out by comprehensively scoring by taking appearance, redissolution time, particle size and encapsulation rate as evaluation indexes. Mannitol easily obtains a full appearance due to the supporting capacity of self-crystallization, but the formation of crystallization can cause mechanical damage to liposome membranes, so that the entrapment rate of the liposome is remarkably reduced and the particle size is remarkably increased. Sucrose has poor appearance, but smaller particle size, shorter redissolution time, better dispersibility, higher ERG encapsulation efficiency and Afa encapsulation efficiency. Glucose and trehalose are used as protective agents, and the particle size PDI value after redissolution is higher and the dispersibility is poor. Thus considering the use of mannitol as a proppant to give a good appearance to the freeze-dried product, it was further screened for co-use with sucrose. The freeze-dried powder prescription finally determined by comprehensive grading comprises the following steps: glycolipid ratio =3:1, sucrose: mannitol =1:1 (147 mg:147 mg)

The finally determined freeze-drying process and prescription process are as follows: and (3) placing the mixture in a cold trap of equipment for 4 hours by adopting a slow freezing method, transferring the mixture to the upper layer, and freeze-drying the mixture for 35 hours (the freeze-drying procedure is carried out for 350 minutes at-30 to-20 ℃, 350 minutes at-20 to-10 ℃, 350 minutes at-10 to-0 ℃, 350 minutes at 0 to 10 ℃, 350 minutes at 10 to 20 ℃, 350 minutes at 20 to 30 ℃ and 350 minutes). The prescription of the freeze-drying protective agent comprises: each liposome lyophilized powder (5 mL) contained 147mg of sucrose and 147mg of mannitol (glycolipid ratio of 3:1, sucrose to mannitol mass ratio of 1: 1), and the quality thereof was evaluated. The result shows that the RGD/R8-ERG/Afa-LIP freeze-dried powder has uniform, full, complete and smooth appearance, uniform and milky color after redissolution, small particle size, concentrated distribution, stability of about 100 nm and high encapsulation efficiency, the average encapsulation efficiency of Afa is 92.45 +/-0.61 percent, and the average encapsulation efficiency of ERG is 92.87 +/-1.32 percent. The 2015 edition of Chinese pharmacopoeia relates to the requirements of liposome burst release effect: the initial release should be less than or equal to 40% at 0.5 h and the cumulative release percentage should exceed 80% at 24 h. RGD/R8-ERG/Afa-LIP lyophilized powder meets this requirement in a phosphate buffer containing 40% methanol with a release medium of pH6.5 and pH 7.4. The serum stability is good. Stability tests show that the RGD/R8-ERG/Afa-LIP freeze-dried powder needs to be stored under the conditions of low temperature, light protection and drying.

The apoptosis result shows that the apoptosis rate of each administration group is obviously higher than that of a blank control group. The apoptosis rates of the RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP freeze-dried powder groups have no significant difference, which indicates that the apoptosis effect of the RGD/R8-ERG/Afa-LIP prepared into freeze-dried powder on PC-9 cells is not weakened. Wherein, the apoptosis rates of the RGD/R8-ERG/Afa-LIP and RGD/R8-ERG/Afa-LIP freeze-dried powder groups are very different compared with that of the single target head modified liposome. RGD-ERG/Afa-LIP and R8-ERG/Afa-LIP have no significant difference compared to ERG/Afa-LIP. The double-target modification effect is better than that of the single-target modification effect.

Four, small knot

RGD/R8-ERG/Afa-LIP freeze-dried powder is successfully prepared in the experiment. In order to save the experiment cost and reduce the experiment cost, the ERG/Afa-LIP is used for the investigation of the freeze-drying process and the optimization experiment of the prescription, and the optimal prescription process is screened out and then applied to RGD/R8-ERG/Afa-LIP for verification. The optimal freeze-drying and prescription process is as follows: and (3) placing the mixture in a cold trap of equipment for 4 hours by adopting a slow freezing method, transferring the mixture to the upper layer, and freeze-drying the mixture for 35 hours (the freeze-drying procedure is carried out for 350 minutes at-30 to-20 ℃, 350 minutes at-20 to-10 ℃, 350 minutes at-10 to-0 ℃, 350 minutes at 0 to 10 ℃, 350 minutes at 10 to 20 ℃, 350 minutes at 20 to 30 ℃ and 350 minutes). The prescription of the freeze-drying protective agent comprises: each liposome lyophilized powder (5 mL) contained 147mg of sucrose and 147mg of mannitol (glycolipid ratio of 3:1, sucrose to mannitol mass ratio of 1: 1). After the RGD/R8-ERG/Afa-LIP freeze-dried powder is redissolved, the particle size, the potential, the encapsulation rate and the serum stability are good, and the apoptosis of PC-9 cells can be obviously promoted by detecting the freeze-dried powder through flow type apoptosis. The storage conditions are as follows: drying at low temperature and keeping out of the sun.

Claims

The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome freeze-dried powder comprises the following steps: adding the freeze-drying protective agent into a pre-prepared RGD/R8-ERG/Afa-LIP liposome suspension in an external addition mode; finally, freeze-drying to prepare the compound liposome freeze-dried powder; the RGD/R8-ERG/Afa-LIP liposome suspension is prepared by the following method: ERG/Afa-LIP is prepared first and then by the post-insertion method.
2. The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome lyophilized powder as claimed in claim 1, which is characterized in that: the freeze-drying method is specifically a quick freezing method, and specifically comprises the steps of reducing the temperature of a cold trap part in equipment to the lowest temperature in advance, and then placing a sample in the cold trap.
3. The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome lyophilized powder as claimed in claim 2, which is characterized in that: the prefreezing time of the freeze-drying method was 4 hours.
4. The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome lyophilized powder as claimed in claim 2, which is characterized in that: the freeze-drying time of the freeze-drying method was 35 h.
5. The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome lyophilized powder as claimed in claim 1, which is characterized in that: the prescription of the freeze-drying protective agent comprises: each 5mL of liposome freeze-dried powder contains 147mg of sucrose and 147mg of mannitol, the glycolipid ratio is 3:1, and the mass ratio of the sucrose to the mannitol is 1: 1.
6. The preparation method of the RGD cyclopeptide/R8 peptide modified ERG combined Afa double drug-loaded liposome lyophilized powder as claimed in claim 1, which is characterized in that: placing the mixture at a cold trap of equipment for 4 hours by adopting a slow freezing method, transferring the mixture to the upper layer, and carrying out freeze-drying for 35 hours; freeze-drying procedure: -30 to-20 ℃: 350 min; -20 to-10 ℃: 350 min; -10 to-0 ℃: 350 min; 0-10 ℃: 350 min; 10-20 ℃: 350 min; 20-30 ℃: and (5) 350 min.
7. A lyophilized liposome powder prepared according to the method of any one of claims 1-6.
8. The use of the lyophilized liposome powder of claim 7 in the preparation of a targeted drug for inhibiting tumor cell growth and/or inducing tumor cell apoptosis.