CN111658772B

CN111658772B - Natural light induced controlled release medicine and preparation method and application thereof

Info

Publication number: CN111658772B
Application number: CN202010708743.XA
Authority: CN
Inventors: 夏栋林; 黄好; 陈超; 顾海鹰; 鲍鸿一; 王乡儿
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2022-06-03
Anticipated expiration: 2040-07-22
Also published as: CN111658772A

Abstract

The invention discloses a natural light induced controlled release medicament, which adopts an erythrocyte membrane to carry Endo and ICG; wherein, when the erythrocyte membrane is carried, the initial concentration of the Endo is 5.0-25.0 mg/mL, the initial concentration of the ICG is 1.0-7.0 mg/mL, and each milliliter of carrying solution contains 0.5mL of erythrocyte membrane extracted from whole blood. The invention relates the release amount of Endo to natural illumination, can effectively realize controlled release and maintain good blood concentration.

Description

Natural light induced controlled release medicine and preparation method and application thereof

Technical Field

The present invention relates to a novel controlled release drug of Endo.

Background

Indocyanine green (ICG) can generate a large amount of singlet oxygen under in-vitro natural light irradiation¹[O2]) And its metabolite hydrogen peroxide (H)₂O₂) The increase and accumulation of the drug can destroy the phospholipid bilayer of the erythrocyte membrane, open the pore channel of the erythrocyte membrane and release the entrapped drug from the pore channel, thereby completing the ICG responsive drug release; when the light irradiation is stopped, the release amount of singlet oxygen and the like is also reduced, the membrane pore is closed, and the drug release is stopped.

The growth of solid tumor and the generation of tumor blood vessel are closely related, and the relative hypoxia caused by the rapid proliferation of tumor cells enables tumor tissues to continuously generate angiogenesis promoting factors, destroys the original state of blood vessel balance, causes the tumor blood vessel to have obvious heterogeneity, makes the tumor microenvironment worsen, increases the malignancy degree of the tumor and reduces the treatment effect. Endo is a protein drug which can inhibit the proliferation and migration of vascular endothelial cells, can obviously inhibit the generation of new blood vessels, and can cause the growth cycle retardation and apoptosis of the vascular endothelial cells.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provide a medicament capable of effectively realizing the controlled release effect of the saturation.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a natural light induced controlled release medicine is prepared by encapsulating Endo and ICG with erythrocyte membrane (ERM); wherein, when the erythrocyte membrane is carried, the initial concentration of the Endo in the carrying solution is 5.0-25.0 mg/mL, the initial concentration of the ICG is 1.0-7.0 mg/mL, and each milliliter of the carrying solution contains 0.5mL of erythrocyte membrane extracted from whole blood.

When the erythrocyte membrane is entrapped, the preferred initial concentration of Endo is 20.0mg/mL, the initial concentration of ICG is 5.0mg/mL, and the solution pH is 7.4.

The invention also provides a preparation method of the natural light induced controlled release medicament, and the natural light induced controlled release medicament is obtained by extracting erythrocyte membranes and carrying ICG and Endo by adopting a physical ultrasonic method.

The preparation method comprises the following specific steps:

(1) extracting and preparing erythrocyte membrane liquid;

(2) ICG solution preparation: dissolving ICG in PBS solution;

(3) preparing an Endo solution: dissolving and diluting Endo in a PBS solution;

(4) preparing a mixed solution of ICG and Endo;

(5) ultrasonic package loading: the ICG and Endo mixed solution and the erythrocyte membrane solution are placed in a probe type ultrasonic instrument with the power of 100W for 6min of ultrasound at the temperature of 4 ℃, and then are placed and incubated for 30min at the temperature of 37 ℃.

Wherein, the extraction method of the erythrocyte membrane in the step (1) is as follows: taking 1mL of whole blood, adding 10mL of hypotonic solution, and cracking at 4 ℃ for 20-30 minutes; centrifuging at 2000rpm for 5min, washing with hypotonic solution for 3 times, and washing with isotonic PBS for 3 times to obtain erythrocyte membrane solution.

In the step (2), the concentration of ICG in the PBS solution is 2-14 mg/mL, and the pH value of the solution is adjusted to 7.4; in the step (3), the concentration of the Endo in the PBS solution is 10.0-50.0 mg/mL; mixing the ICG solution and the Endo solution in the step (4) in equal volume to prepare an ICG and Endo mixed solution; and (3) mixing the erythrocyte membrane liquid extracted from 1mL of whole blood, the ICG solution and the Endo solution in the step (5).

Preferably, the concentration of ICG in PBS solution in step (2) is 10.0mg/mL, and the concentration of Endo in PBS solution in step (3) is 40.0 mg/mL.

The invention also provides the application of the natural light induced controlled release medicament in the preparation of anti-tumor medicaments.

Compared with the prior art, the invention has the following advantages:

1. ICG-responsive controlled drug release: ICG responds by natural light irradiation to generate H₂O₂The opening of membrane pore channels, Endo release and ICG response membrane pore channel opening are the key points of successful research. The release amount of Endo is related to natural illumination, so that the controlled release is realized, and good blood concentration is maintained;

2. biocompatibility: ERM has good biocompatibility, and is different from the harm of other chemical synthesis carriers to animals;

3. the invention releases Endo through the response of ICG to natural light, effectively delays the release time, obviously improves the curative effect and fully exerts the treatment effect of inhibiting tumor vessels caused by slowly releasing Endo;

4. the early animal simulation experiment research result of the invention provides a foundation for the method for releasing Endo by ICG response to enter clinic early, and also provides a new idea for better solving the problems of tumor angiogenesis, tumor growth inhibition and the like.

Drawings

FIG. 1 is a flow chart of the preparation of the natural light induced controlled release drug (Endo-ICG-ERM) of the present invention;

FIG. 2 is a schematic view of the Endo-ICG-ERM natural light induced controlled release process of the present invention;

FIG. 3 is a SEM representation of the Endo-ICG-ERM of the present invention;

FIG. 4 is a graph showing the absorption spectrum of the Endo-ICG-ERM of the present invention with respect to natural light;

FIG. 5 is a graph of the Endo loading capacity under different ICG solution concentration conditions;

FIG. 6 is a graph showing the relationship of natural light response release time after entrapment under different ICG solution concentrations;

FIG. 7 shows the change in vivo release concentration of Endo encapsulated under different ICG solution concentrations;

FIG. 8 is an Endo-ICG-ERM constructed by erythrocyte membrane entrapment and an Endo-ICG-ER photoresponse release curve constructed by erythrocyte membrane entrapment;

FIG. 9 Effect of the time of exposure of the Endo-ICG-ERM of the present invention under natural light conditions on the amount of singlet oxygen production;

FIG. 10 is a graph of the effect of different natural light times on the production of malondialdehyde;

FIG. 11 shows the fluidity of the membrane of drug-loaded erythrocyte under different natural illumination time;

FIG. 12 is a graph of the change in the concentration of Endo in the serum of mice after administration;

FIG. 13 is a graph showing the resolution of tumor vessel normalization and the detection of hypoxic levels following Endo-ICG-ERM administration with CD31 and HIF in an efficacious example of the invention;

FIG. 14 is a graph of photoacoustic imaging (oxyhemoglobin) observed every 6 days after the Endo-ICG-ERM administration treatment in the effect examples of the present invention;

FIG. 15 is a graph showing the change in tumor size one month after administration in the present invention effect examples;

FIG. 16 is a graph of animal survival one and a half months after administration in the invention effect examples.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

The preparation process of the Endo-ICG-ERM comprises the following steps:

as shown in FIG. 1, first, the extraction of erythrocyte membrane is carried out, and 1mL of whole blood is added with 10mL of hypotonic solution (70mOsm/kg) and lysed at 4 ℃ for 20-30 minutes; centrifuging at 2000rpm for 5min, washing with hypotonic solution for 3 times, and washing with isotonic PBS solution for 3 times to obtain erythrocyte membrane solution (about 50 μ L); ICG was then dissolved in PBS and Endo was diluted in PBS, the concentration of ICG solution and Endo solution being 10mg eachmL, and the pH of the solution is adjusted to 7.4; carrying out erythrocyte membrane drug entrapment by ultrasound, mixing the prepared ICG solution and the Endo solution according to the volume ratio of 1: 1, mixing the mixed solution and erythrocyte membrane solution (mixing the erythrocyte membrane solution extracted from 1mL of whole blood, 1mL of ICG solution and 1mL of Endo solution according to the following proportion, namely the ICG and the Endo in the entrapment solution have initial concentrations of about 5 mg/mL) in a probe type ultrasonic instrument with the power of 100W, carrying out ultrasonic treatment for 6min at the temperature of 4 ℃, and standing and incubating for 30min at the temperature of 37 ℃ to obtain the Endo-ICG-ERM for standby preparation; injecting prepared drug-loaded erythrocyte membrane (Endo-ICG-ERM) into mouse via vein, exposing mouse to natural light, irradiating natural light to make ICG generate response, inducing cell to generate large amount of H₂O₂So that the erythrocyte membrane is broken and the Endo is released, thereby achieving the effect of treating the tumor (as shown in figure 2).

Characterization of the Endo-ICG-ERM prepared:

as shown in FIG. 3, which is a Scanning Electron Microscope (SEM) image of the Endo-ICG-ERM, and FIG. 4, which is an ultraviolet-visible spectrum image of the Endo-ICG-ERM, it can be seen that the ERM can successfully entrap both Endo and ICG.

Example 2

Effects of entrapping different concentrations of Endo solution on Endo in vivo release:

as can be seen from FIG. 5, the amount of Endo entrapped by the erythrocyte membrane is related to the initial concentration of Endo, and the greater the initial concentration of Endo, the greater the amount of Endo entrapped by the erythrocyte membrane. When the concentration is increased to 25.0mg/mL, the entrapment time (ultrasound) is over 6 minutes, and the entrapment efficiency is not obviously increased.

As can be seen from FIG. 6, the effective release time with Endo also increased with increasing initial concentration of Endo, and when the concentration increased to 20.0mg/mL, the release time exceeded 4 days; when the concentration was increased to 25.0mg/mL, the increase in release time was insignificant.

Therefore, in view of cost effectiveness, Endo is used at an initial concentration of 20.0mg/mL, with an entrapment time of 6 minutes being the optimum condition.

Example 3

Effect of loading ICG solutions of different concentrations on Endo release:

weighing a certain amount ofICG powder is dissolved in PBS solution to ensure that the concentration of the ICG solution is graded to carry out experiments, wherein the concentration is respectively 2mg/mL, 6mg/mL, 10mg/mL and 14mg/mL, and Na is used simultaneously₂HPO₄And KH₂PO₄Adjusting the pH to 7.4; endo was diluted to concentration (40.0mg/mL) with PBS solution and the pH was adjusted to 7.4; and (3) uniformly mixing the ICG solution with 1mL of the Endo solution and 1mL of ERM solution (erythrocyte membrane solution) extracted from whole blood respectively, carrying out ultrasonic treatment for 6 minutes, and standing at 37 ℃ for 30 minutes to prepare the Endo-ICG-ERM.

ICG solution is prepared according to a certain concentration gradient (namely the initial concentration of ICG in the entrapment solution is 1mg/mL, 3mg/mL, 5mg/mL and 7mg/mL respectively), the Endo-ICG-ERM medicament is prepared, the release amount of Endo is detected when the medicament is exposed to natural light for different time, and the result is shown in figure 7.

It can be seen from fig. 7 that the amount of change in Endo release is also related to the ICG concentration, with the greater the ICG concentration, the greater the amount of change in increase in Endo release, and the more release at the same time. After the Endo is exposed for 24 hours under natural light, when the ICG concentration is 1mg/mL or 3mg/mL, the release rate of the Endo is slow, the clearance rate in the Endo exceeds the release rate, and further the decrease of the concentration of the Endo serum occurs; when the ICG concentration is 5mg/mL, the Endo release rate is gentle, the sustained release is kept at a certain speed, and when the ICG concentration is 7mg/mL, the Endo still maintains the rapid release level, which exceeds the in vivo clearing efficiency, and the serum concentration continuously rises. Considering that the drug needs to be effectively and slowly released, the ICG initial concentration in the entrapment solution is preferably 5mg/mL for subsequent experiments.

Example 4

Comparing the influence of the erythrocyte membrane and erythrocyte on the Endo entrapment quantity and in vivo release when carrying out Endo and ICG entrapment:

erythrocyte membrane-carried Endoconment conditions for constructing Endo-ICG-ERM are as follows: in the entrapment solution, the initial concentration of Endo was 20.0mg/mL, the initial concentration of ICG was 5mg/mL, each mL of the solution contained 0.5mL of red cell membrane extracted from whole blood, and the solution was sonicated for 6 minutes and left at 37 ℃ for 30 minutes. The entrapment rate of Endo in the Endo-ICG-ERM was 27.34%.

The conditions of the erythrocyte entrapped Endo-ICG-ER are as follows: the initial concentration of Endo is 20.0mg/mL, the ICG concentration is 5mg/mL, each milliliter of solution contains 0.5mL of red blood cells extracted from whole blood, the solution is subjected to hypotonic dialysis for 12h, and the solution is subjected to hypertonic dialysis for 0.5h at 37 ℃ and finally isotonic for 2h to obtain the Endo concentration of 16.78mg/mL in the Endo-ICG-ER.

Release of Endo may be induced by exposure to natural light, which causes the encapsulated ICG to respond, as shown in fig. 9.

As can be seen from FIG. 8, the longer the exposure time to natural light, the greater the release amount of Endo, but the release rates of Endo-ICG-ERM and Endo-ICG-ER were significantly different. The release of Endo-ICG-ER is too little to carry out natural light controlled release to achieve the requirement of stabilizing the concentration of Endo serum.

Example 5

Exposure time under natural light is right¹[O2]Influence of production amount:

selecting Endo initial concentration of 20.0mg/mL, ICG concentration of 5mg/mL, treating with ultrasound for 6min, standing at 37 deg.C for 30min to obtain Endo-ICG-ERM, injecting into animal body via vein, exposing to natural light, and allowing ICG to respond and induce in vivo production¹[O₂]。¹[O₂]The amount of released (c) was correlated with the time of natural light irradiation, and the result is shown in fig. 9.

As can be seen from fig. 9, the longer the exposure time to natural light,¹[O₂]the more the amount of production. But after 12 hours of irradiation with natural light,¹[O₂]the production amount of (A) gradually becomes stable, and almost no more production is generated after 24 hours¹[O₂]. In summary, the natural light irradiation time is preferably 12 hours.

Meanwhile, the Endo-ICG-ERM drug prepared in this example was intravenously injected into the animals and irradiated with light for 12 hours each day, and the change in the in vivo release concentration of Endo is shown in FIG. 12. As can be seen from the figure, the Endo-ICG-ERM can keep the concentration of the Endo in the serum stable, which provides a basis for improving the action effect of the Endo.

Effects of the embodiment

1. Carrier erythrocyte membrane (ERM) peroxidation and fluidity study

As shown in fig. 10, the longer the exposure time is within 12 hours under natural light irradiation, the more the membrane lipid peroxidation product Malondialdehyde (MDA) is released, and the amount of MDA released after 12 hours tends to be stable, and less, hardly released. 12 hours is the optimum time for MDA release to reach higher amounts.

As shown in fig. 11, the fluidity of the drug-loaded erythrocyte membranes was monitored over 24 hours, and it was observed that the longer the exposure time to natural light, the worse the fluidity of the erythrocyte membranes. After 12 hours, the fluidity of the drug-loaded erythrocyte membrane tends to be stable, and the membrane fluidity hardly changes any more. According to the fluidity of the drug-loaded erythrocyte membrane, the optimal illumination time is 12 hours.

2. Immunohistochemical experimental study

Constructing a tumor-bearing mouse model: when the confluency of HeLa cells reaches 90%, the cells are collected and the concentration is adjusted to 3X 10⁷Each/mL, 100. mu.L (3X 10) were injected subcutaneously into BALB/c mice above the left leg using a 1mL syringe⁶Only). Around 2 weeks, tumor growth was visualized, body weight was recorded every three days and tumor volume was measured with a vernier caliper according to formula V ═ 0.5 × a × b²(a represents the longest diameter of the solid tumor and b represents the shortest diameter), the size of the solid tumor is calculated to be about 400-³Meanwhile, the tumor-bearing mice are modeled and can be used for in vivo experimental research.

Tumor-bearing mice were divided into 3 groups: a control group (tail vein injection with 0.1mL PBS), a Free Endo drug group (tail vein injection with 2.5mg/kg of Endo drug), and an Endo-ICG-ERM group (the Endo-ICG-ERM prepared in example 4 with 2.5mg/kg of dose) of the invention.

As shown in FIG. 12, the Endo-ICG-ERM of the present invention was administered intravenously 1 time every 3 days, and it was able to maintain the stability of the concentration of Endo in serum. In the Free Endo group, the administration was carried out by the caudal vein on a daily basis, considering that the half-life of Endo is only 10 hours, but the fluctuation of serum concentration was large. The effective action time of the Free Endo group is shorter than that of Endo-ICG-ERM, and the action effect is different, as shown in figures 13-16.

As shown in FIG. 13, the results of the normalization of tumor blood vessels using CD31 showed that Endo-ICG-ERM was able to normalize tumor blood vessels. The results of using HIF to detect hypoxia indicate that Endo-ICG-ERM can solve the problem of hypoxia in tumor tissues.

3. Photoacoustic imaging

As shown in fig. 14, in order to further prove whether Endo can solve the tumor hypoxia problem well, the oxyhemoglobin level of the tumor after drug injection is monitored by photoacoustic imaging, and research results show that Endo-ICG-ERM can effectively solve the hypoxia phenomenon in the tumor tissue.

4. Animal experiments

After the tumor-bearing mice are intravenously injected with the Endo-ICG-ERM solution and exposed for 12 hours under natural light, the ICG generates photoresponse and the Endo is released.

As shown in FIG. 15, after 28 days of treatment and observation, the tumor growth status after injection of Endo-ICG-ERM and combined radiotherapy (Endo-ICG-ERM + RT) was significantly inhibited compared to the Control group (Control), indicating that the tumor growth could be effectively inhibited.

As shown in FIG. 16, the survival rate of mice was observed continuously for 45 days, and at 45 days, the survival rate of the Endo-ICG-ERM group reached 80%, and the mice in the control group and the radiotherapy-only group (PBS + RT) did not survive.

Claims

1. The natural light-induced controlled release medicine is characterized in that the natural light-induced controlled release medicine adopts erythrocyte membranes to carry the Endottle and ICG; when the erythrocyte membrane is loaded, the initial concentration of the Endoticity in the loading solution is 5.0-25.0 mg/mL, the initial concentration of ICG is 5.0mg/mL, and each milliliter of the loading solution contains 0.5mL of erythrocyte membrane extracted from whole blood; wherein, the encapsulation of the eminence and the ICG of the erythrocyte membrane is carried out by the following methods: and (3) placing the mixed solution of ICG and the saturation and the erythrocyte membrane solution in a probe-type ultrasonic instrument with the power of 100W for 6min under the condition of 4 ℃, and then placing and incubating for 30min under the condition of 37 ℃.

2. The natural light induced controlled release drug of claim 1, wherein the initial concentration of the saturation in the encapsulating solution is 20.0mg/mL and the pH of the solution is 7.4 when the erythrocyte membrane is encapsulated.

3. The method for preparing a natural light induced controlled release drug according to claim 1 or 2, comprising the steps of:

(1) extracting and preparing erythrocyte membrane liquid;

(2) ICG solution preparation: dissolving ICG in PBS solution;

(3) preparing an Endu solution: dissolving and diluting the saturation in PBS (phosphate buffer solution);

(4) preparing a mixed solution of ICG and the saturation;

(5) ultrasonic entrapment: and (3) placing the mixed solution of ICG and the saturation and the erythrocyte membrane solution in a probe-type ultrasonic instrument with the power of 100W for 6min under the condition of 4 ℃, and then placing and incubating for 30min under the condition of 37 ℃.

4. The method for preparing a natural light-induced controlled-release drug according to claim 3, wherein the extraction method of the erythrocyte membrane in the step (1) is as follows: taking 1mL of whole blood, adding 10mL of hypotonic solution, and cracking at 4 ℃ for 20-30 minutes; centrifuging at 2000rpm for 5min, washing with hypotonic solution for 3 times, and washing with isotonic PBS for 3 times to obtain erythrocyte membrane solution.

5. The method for preparing a natural light-induced controlled-release drug according to claim 4, wherein the ICG solution in the step (2) has a pH of 7.4; the concentration of the concentration in the PBS solution in the step (3) is 10.0-50.0 mg/mL; mixing the ICG solution and the Endu solution in the step (4) in equal volume to prepare a mixed solution of the ICG and the Endu; in the step (5), the addition amount of the erythrocyte liquid is 1mL of erythrocyte membrane liquid extracted from whole blood, and the erythrocyte membrane liquid is mixed with 1mL of LICG solution and 1mL of saturation solution.

6. The method for preparing a natural light-induced controlled-release medicament according to claim 5, wherein the concentration of the saturation in the PBS solution in the step (3) is 40.0 mg/mL.

7. Use of the natural light induced controlled release drug of claim 1 or 2 for the preparation of an anti-tumor drug.