CN102641495A - Liver targeting interleukin-12/chitosan nano drug feeding system for intravenous injection and preparation method thereof - Google Patents

Abstract

The invention discloses a liver-targeted interleukin-12/chitosan nano drug delivery system for intravenous injection and a preparation method thereof. The drug delivery system is made of the liver-targeting compound sodium polyphosphate-chitosan as a carrier material, cross-linked and embedded with interleukin-12, the particle size of the nanosphere is 200-500nm, and the drug-loading rate is 2-25%. ; The degree of deacetylation of the chitosan ≥ 80%. Nanoparticles are prepared by cross-linking sodium tripolyphosphate and chitosan to encapsulate interleukin-12 to obtain the drug delivery system. The invention has good liver targeting; the drug release rate of the obtained drug delivery system is >60% after 16 hours under the condition of pH6.5.

Description

A liver-targeted interleukin-12/chitosan nano drug delivery system for intravenous injection and its preparation method

technical field

The invention relates to a targeted drug delivery system, in particular to a liver-targeted interleukin-12/chitosan nano drug delivery system for intravenous injection and a preparation method thereof.

Background technique

Interleukin-12 (Interleukin-12, IL-12) has been widely valued because of its powerful anti-tumor activity and anti-metastasis effect. However, since 1997, two fatal cases of intravenous injection of IL-12 in phase II clinical trials were reported (Leonard JP, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gammaproduction. Blood.1997;90:2541-8) The clinical application of IL-12 has so far stopped. Over the years, scientists from various countries have been looking for effective ways to reduce its toxic and side effects. Chitosan (CS), as a biocompatible, degradable, low-toxic polycation natural polymer material, and modifiable in physical and chemical properties, has become a research hotspot for biotherapeutic carriers in recent years. Recently, some scholars have improved the bioavailability of IL-12 by preparing IL-12/chitosan nanoparticle injection, and successfully inhibited the growth of tumors in the mouse bladder (Zaharoff DA, et al. Intravesicalimmunotherapy of superficial bladder cancer with chitosan/interleukin-12. Cancer Res. 2009;69:6192-9.). However, this method of intratumoral injection is limited to the local treatment of some palpable tumors, and systemic administration still faces the risk of poor targeting and severe side effects.

my country is a high-incidence area for liver diseases, so the development of a new liver-targeted drug delivery system can reduce drug dosage and reduce side effects. The liver-targeted IL-12 carrier system can improve the immune microenvironment in the liver by releasing IL-12 in the liver, which can not only improve the curative effect of primary liver cancer, but also effectively prevent liver metastasis of colorectal cancer. Liver-targeted drug delivery systems can be divided into passive targeting and active targeting according to the guiding mechanism. The former refers to the uptake of drug-loaded particles by monocyte-macrophages (especially Kuffer cells in the liver) and transported to the liver through normal physiological processes. Liver therapy; the latter refers to drug carriers modified with "targeting molecules", such as asialoglycoprotein receptor-mediated targeting drugs. Since sugar molecules can increase the affinity between drugs and tumor cells, and facilitate the passage of drugs through tumor cells, chitosan, as a polysaccharide cation, is one of the most suitable carriers for tumor drugs.

Chinese patent CN101254308A discloses a preparation method for modifying chitosan nanoparticles with glycyrrhetinic acid-polyethylene glycol. This invention makes full use of the high liver targeting of glycyrrhetinic acid, and prepares it through hydrophilic modification at different sites. The macromolecular complex achieved liver targeting, but no detailed information on the intrahepatic drug release efficiency of the complex drug was provided. Chinese patent CN101766820A discloses cross-linking chitosan nanoparticles with tripolyphosphate negative ions and glutaraldehyde, and then connecting folic acid to the carrier. Chinese patent CN102225051A utilizes the pH sensitivity of carboxymethyl chitosan and the tumor targeting of folic acid to prepare folic acid-carboxymethyl chitosan modified pH-sensitive nano-doxorubicin (Doxorubicin) nano-liposomes. These composite carriers all take advantage of the pH-sensitive property of carboxymethyl chitosan, and realize the purpose of tumor targeting by folic acid targeting molecules, but do not have the effect of liver targeting.

Contents of the invention

The object of the present invention is to provide a liver-targeting interleukin-12/chitosan nano drug delivery system for intravenous injection. The drug-loading system screens the preparation conditions of nanospheres with strong liver targeting according to the passive liver targeting mechanism; The pH-sensitive drug-loading system was prepared by using the ampholyte properties of the modified nanospheres.

In order to achieve the above-mentioned purpose of the invention, the technical solution adopted in the present invention is: a liver-targeting interleukin-12/chitosan nano drug delivery system for intravenous injection, which is based on the liver-targeting compound sodium polyphosphate-chitosan The carrier material is made of cross-linking and embedding interleukin-12 (IL-12), the particle size of the nanosphere is 200-500nm, and the drug loading rate is 2-25%; the deacetylation degree of the chitosan is more than or equal to 80%.

The preparation method of the liver-targeted interleukin-12/chitosan nano drug delivery system for intravenous injection comprises the following steps:

(1) Preparation of sodium polyphosphate-chitosan modified interleukin-12 nanospheres:

Prepare interleukin-12 aqueous solution with a concentration of 1-5 μg/mL and chitosan-acetic acid solution with a concentration of 1-5 mg/mL respectively. The deacetylation degree of chitosan is ≥80%. ~ 1:6 mixing, under the condition of magnetic stirring 200 ~ 400r/min, slowly add sodium tripolyphosphate aqueous solution with a concentration of 0.5 ~ 1.6mg/mL, the total volume of sodium tripolyphosphate solution added is interleukin-12 0.5 to 4 times the volume of the aqueous solution to obtain a suspension of interleukin-12 nanospheres;

(2) Add the suspension obtained in step (1) into acetic acid-sodium acetate buffer and mix well, the volume ratio of the suspension and acetic acid-sodium acetate buffer is 1:1～1:3, adjust with glacial acetic acid When the pH value is between 5.5 and 6.5, it is uniformly cross-linked and then filtered through a microporous membrane to prepare a suspension of interleukin-12 chitosan nanospheres chemically modified by sodium polyphosphate.

Preferred technical scheme, in described step (1), the concentration of interleukin-12 aqueous solution is 2 μ g/mL, the concentration of chitosan acetic acid solution is 2 mg/mL, the volume ratio of above-mentioned two solutions is 1: 3, tripolyphosphoric acid The concentration of the sodium aqueous solution was 0.84 mg/mL, and the total volume of the added sodium tripolyphosphate solution was 1.2 times the volume of the interleukin-12 aqueous solution.

Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

1. The present invention uses sodium polyphosphate to modify the surface of chitosan, which reduces the positive charges on the surface of nanoparticles, prevents the neutralization of opsonins rich in negative charges in blood circulation, and delays the action of mononuclear macrophages. Systemic clearance increases the opportunity for nanoparticles to enter the liver through blood circulation, and has good liver targeting;

2. The present invention rationally selects the concentration of each component of the nanosphere and the proportional relationship between them, and the particle size of the nanosphere. Experiments show that the drug release rate of the obtained drug delivery system is >60% after 16 hours under the condition of pH6.5 .

Description of drawings

Fig. 1 is the figure of influence of the ratio between chitosan and sodium tripolyphosphate on the particle size of nanospheres in embodiment 2.

FIG. 2 is a graph showing the influence of IL-12 concentration on the particle size of nanospheres in Example 3.

Fig. 3 is the in vivo distribution of FITC-labeled IL-12 nanospheres with different particle sizes in Example 4 after 24 hours of tail vein injection in mice.

Fig. 4 is the drug release rate of the pH-sensitive CS/TGF-β1 siRNA nano-drug sustained-release system in Example 5 under different pH conditions.

Figure 5 is the effect of IL-12 nanospheres in Example 7 on the liver metastasis of CRC in mice.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Embodiment 1: Preparation of sodium polyphosphate-chitosan modified IL-12 nanospheres

Prepare 1 mL of IL-12 aqueous solution with a concentration of 2 μg/mL and 3 mL of chitosan (deacetylation degree ≥ 80%) acetic acid solution with a concentration of 2 mg/mL. Under certain conditions, 1.2 mL of sodium tripolyphosphate with a concentration of 0.84 mg/mL was slowly added dropwise, uniformly cross-linked and then filtered through a microporous membrane to prepare a suspension of IL-12 chitosan nanospheres chemically modified by sodium polyphosphate.

Embodiment 2: Effect of the ratio between different chitosan and sodium tripolyphosphate on the particle size of nanospheres

Prepare 1 mL of IL-12 aqueous solution with a concentration of 2 μg/mL and chitosan acetic acid solution with a concentration of 1 to 5 mg/mL. The degree of deacetylation of chitosan is ≥80%. Under the condition of ～400r/min, slowly add sodium tripolyphosphate aqueous solution with a concentration of 0.5～1.6mg/mL dropwise, filter through a microporous membrane after uniform crosslinking, and detect the difference between different chitosans and sodium tripolyphosphate with a particle size analyzer. The impact of the ratio on the particle size of the nanosphere is shown in Figure 1, wherein the abscissa is the mass ratio of chitosan to sodium tripolyphosphate.

Example 3: Effects of different IL-12 concentrations on the particle size of nanospheres

Prepare 1 mL of IL-12 aqueous solution with a concentration of 0.5-10 μg/mL and 3 mL of chitosan (deacetylation degree ≥ 80%) acetic acid solution with a concentration of 2 mg/mL, mix the above two solutions, and stir in a magnetic field for 200-400 r Under the condition of 0.84mg/mL sodium tripolyphosphate 1.2mL slowly dropwise, uniformly cross-linked and filtered through a microporous membrane, the particle size analyzer detects the influence of different IL-12 concentrations on the particle size of nanospheres, see the attached Figure 2 shows.

Example 4: Sodium polyphosphate modified chitosan nanosphere liver targeting detection

①Preparation of different particles by FITC-sodium polyphosphate-chitosan nanoparticles: Weigh 10mg of fluorescein isothiocyanate (FITC), dissolve it in 10mL of absolute ethanol, and add it dropwise to 20mL of more than 20mL under the condition of magnetic stirring. Sodium polyphosphate-chitosan acetic acid solution (chitosan: sodium polyphosphate are 2; 4; 8; 16 respectively), dark reaction to make carbon atoms on FITC react with amino groups on chitosan for labeling. The nanoparticle size was detected with a nanometer particle size analyzer. ②Animal experiment: 0.2ml of nanosuspension was injected into the tail vein of mice, and the tissue distribution and liver targeting of nanoparticles were detected at different times with a live imaging instrument (KODAK In-Vivo Imaging System FX Pro). ③ Fluorescence assay was used to detect the tissue distribution of nanoparticles. At 1, 12 and 24 hours after the administration, the mice were killed, the liver, kidney, spleen and blood were quantitatively weighed, and 9 times the volume of 1M HCI solution was added to centrifuge, and the supernatant was taken with a fluorescence spectrometer (F- Model 4000, HITACHI, Japan) was used to measure the fluorescence intensity FI, which was corrected with tissue recovery ratio R (Recovery ratio) and tissue weight. Fsa, Fbl and Fst represent the fluorescence intensity of the sample, blank control and standard control, respectively; Cst and Csa represent the concentration of the standard control and sample, respectively.

$\mathrm{<span\; class="notranslate">\; R</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; Fsa</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; <figure-callout\; id="1"\; label="Fb"\; filenames="1205071700162.png"\; state="\{\{state\}\}">Fb</figure-callout></span>}\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Fst</span>}}<span\; class="notranslate">\; \&times;</span>\frac{\mathrm{<span\; class="notranslate">\; Cst</span>}}{\mathrm{<span\; class="notranslate">\; Csa</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span>$

The results showed that the recovery rates of chitosan nanospheres modified by sodium polyphosphate were >70% in the liver, exceeding the recovery rates in kidney, spleen and blood. And the intrahepatic recoveries of nanocarrier systems with particle sizes of 200nm, 300nm and 500nm were 75%, 88% and 80%, respectively, suggesting that the IL-12 nanospheres modified by sodium polyphosphate had a liver target within the range of 200-500nm. The tropism was significantly increased (see Figure 3).

Example 5: Preparation of pH Sensitive Sodium Polyphosphate-Chitosan Modified IL-12 Nanospheres

① The preparation materials are the same as above. ② Use glacial acetic acid to prepare sodium polyphosphate-chitosan solutions with different pH values (4.5-8.5) with acetic acid-sodium acetate buffer solution, and use sodium polyphosphate-chitosan solutions with different pH values to prepare particle sizes of 200-500nm IL-12 nanospheres (method refer to Example 1). ③ IL-12 encapsulation rate: Centrifuge the IL-12 nanosphere solutions prepared from the above sodium polyphosphate-chitosan solutions with different pH values, detect the concentration and content of IL-12 in the supernatant with a UV spectrophotometer, and calculate the encapsulation rate. efficiency. Encapsulation efficiency (%) = 100% × (total amount of IL-12 added - amount of unencapsulated IL-12)/total amount of IL-12 added. ④ In vitro detection of IL-12 release rate: Take the IL-12 nanosphere suspension prepared above, dilute it with acetate buffer with different pH values, and then use the above method to detect the change of the encapsulation efficiency, so as to evaluate the drug release of IL-12 nanospheres in vitro pH sensitivity. To screen the optimal preparation conditions for the construction of pH-sensitive IL-12 nano-delivery system. See Figure 4.

The data showed that the prepared pH-sensitive IL-12 nano-drug sustained-release system had a drug release rate of >60% after 16 hours at pH 6.5.

Example 6: Detection of the Toxic Effect of IL-12 Nanospheres on Mice

① Intraperitoneal orthotopic inoculation of spleen to establish a mouse model of colorectal cancer liver metastasis: SPF Balb/c mice were inoculated, injected intraperitoneally with chloral hydrate (3ml/10g), fixed in supine position after anesthesia, and cut open the mouse The spleen was exposed on the left side of the abdomen, and 0.2 mL of CT26 cell suspension was slowly injected into the spleen with a 1 mL syringe. After killing the exposed tumor cells with 75% alcohol cotton balls and pressing to stop bleeding, the spleen was removed and the incision was sutured. Some mice were sacrificed at different time points, and the number and volume of colorectal cancer liver metastases in mice were detected. ② Toxic and side effects detection of IL-12 nanospheres: construct IL-12 nanospheres modified by sodium polyphosphate and non-modified respectively, the methods refer to the second part. Sixty SPF-grade Balb/c mice were randomly divided into two groups, and each mouse was injected with the above-mentioned nano-medicine (both containing 0.2 μg IL-12) through the tail vein every 2 days, and then sacrificed after five weeks of continuous injection, and detected by ELISA Serum IFN-γ, ALT, and AST levels in mice; histopathological techniques were used to detect pathological changes in the lung, kidney, liver, and brain of mice in each group.

The results showed that the serum levels of ALT and AST were 1800 μmol/LS and 2800 μmol/LS respectively after 2 weeks in the IL-12 treatment group without nanosphere coating. Infiltration, partial hepatic cell water denaturation; while the levels of ALT and AST in the serum of mice in the IL-12 treatment group modified by sodium polyphosphate modified chitosan were 1000 μmol/LS and 1500 μmol/LS after 2 weeks, and histological detection The results showed that only some inflammatory cells infiltrated in the portal area in the liver. This result indicated that the chitosan nanospheres modified by sodium polyphosphate greatly reduced the toxic effect of IL-12.

Example 7: Effect of IL-12 Nanospheres on Liver Metastasis of Colorectal Cancer in Mice

①Establishment of mouse liver metastasis model: CRC liver metastasis model was established in 60 SPF Balb/c mice, the method was the same as above. ② Preparation of chitosan IL-12 nanosphere suspension: refer to the above-mentioned detection indicators for preparation conditions. ③Tail vein administration: After one week of modeling, the mice were randomly divided into three groups, and each mouse was injected with 0.2ml of the above-mentioned nano-drug suspension (containing 0.1μg IL-12 low-dose treatment group and Containing 0.2 μg IL-12 high-dose treatment group). ④ Separation of tumor microenvironment tissue: liver tissue within 0.5 cm around liver metastases was defined as tumor microenvironment tissue. Some fresh tumor microenvironment tissues were taken for in vitro tissue culture; some of the remaining tissues were stored in liquid nitrogen; some were fixed with formaldehyde for histopathological detection and immunohistochemical experiments. ⑤ Normal liver tissues, liver metastases tissues and liver metastases tissues in the treatment group were weighed respectively, and after homogenization, the supernatant was taken to detect the gene and protein expression levels of TGF-β1 by RT-PCR and Western-blot. ③ Observation indicators: observe the growth of mice, sacrifice some mice at different time points, and detect the number and volume of liver metastases in each group. The results showed that the number of intrahepatic metastatic tumors in the treatment group decreased, and the total tumor volume was significantly reduced compared with the control group (see Figure 5).

Claims (3)

1. A liver-targeted interleukin-12/chitosan nano drug delivery system for intravenous injection, characterized in that: it uses the liver-targeted compound sodium polyphosphate-chitosan as a carrier material, cross-linked and embedded interleukin -12, the particle size of nanospheres is 200-500nm, and the drug loading rate is 2-25%; the degree of deacetylation of the chitosan is ≥80%. 2. the preparation method of liver targeting interleukin-12/chitosan nano drug delivery system for intravenous injection described in claim 1, is characterized in that comprising the steps: (1) Preparation of sodium polyphosphate-chitosan modified interleukin-12 nanospheres: Prepare interleukin-12 aqueous solution with a concentration of 1-5 μg/mL and chitosan-acetic acid solution with a concentration of 1-5 mg/mL respectively. The deacetylation degree of chitosan is ≥80%. ~ 1:6 mixing, under the condition of magnetic stirring 200 ~ 400r/min, slowly add sodium tripolyphosphate aqueous solution with a concentration of 0.5 ~ 1.6mg/mL, the total volume of sodium tripolyphosphate solution added is interleukin-12 0.5 to 4 times the volume of the aqueous solution to obtain a suspension of interleukin-12 nanospheres; (2) Add the suspension obtained in step (1) into acetic acid-sodium acetate buffer and mix well, the volume ratio of the suspension and acetic acid-sodium acetate buffer is 1:1～1:3, adjust with glacial acetic acid When the pH value is between 5.5 and 6.5, it is uniformly cross-linked and then filtered through a microporous membrane to prepare a suspension of interleukin-12 chitosan nanospheres chemically modified by sodium polyphosphate. 3. preparation method according to claim 2, is characterized in that: in step (1), the concentration of interleukin-12 aqueous solution is 2 μ g/mL, and the concentration of chitosan acetic acid solution is 2mg/mL, the volume of above-mentioned two solutions The ratio is 1:3, the concentration of the sodium tripolyphosphate aqueous solution is 0.84 mg/mL, and the total volume of the added sodium tripolyphosphate solution is 1.2 times the volume of the interleukin-12 aqueous solution.

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