CN110841065A

CN110841065A - Nano compound for pH/hypoxia dual-response drug release synergistic photodynamic therapy and preparation method thereof

Info

Publication number: CN110841065A
Application number: CN201911230654.2A
Authority: CN
Inventors: 高瑜; 李旭东; 陈海军; 王俊
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2020-02-28

Abstract

The invention discloses a pH/hypoxia dual-response drug release and photodynamic therapy nanocomposite and a preparation method thereof. The nano material is a Cs-NA nano material formed by modifying a hypoxia response group 2- (2-nitro-1H-imidazole-1-yl) acetic acid (NA) on the surface of chitosan, and is combined with a photosensitizer to form a nano compound. The nano-composite can respond to drug release in acidic and low-oxygen environments of tumors, has both molecular targeting effect and photodynamic curative effect, realizes the combination of photodynamic treatment and molecular targeting treatment, improves the accumulation of photosensitizer at tumor positions and reduces phototoxicity.

Description

Nano compound for pH/hypoxia dual-response drug release synergistic photodynamic therapy and preparation method thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a chitosan nano material for photodynamic therapy and a preparation method thereof.

Background

Photodynamic therapy (PDT) has been applied as a novel minimally invasive treatment for a variety of cancers. Photosensitizers (PSs) are excited under excitation of specific wavelengths of excitation light, transferring energy to the surrounding oxygen, generating highly toxic Reactive Oxygen Species (ROS), killing tumor cells (Robertson C A, Evans D H, Abrahamse H. Photodynamic therapy (PDT): A short review on cellular mechanisms and cancer applications for PDT [ J ]. Journal of Photochemistry and Photobiology B: Biology, 2009, 96(1): 1-8.). Compared with the traditional operations, chemotherapy and radiotherapy, the photodynamic therapy has the advantages of small wound, higher specificity and small damage to surrounding tissues, and needs specific laser excitation to reduce the toxic and side effects on normal tissues. However, photodynamic therapy has its own drawbacks, and patients receiving photodynamic therapy need to hide in the dark for a long time to avoid phototoxicity, and patient compliance is low.

Hypoxia is a hallmark feature of the tumor microenvironment resulting from aberrant angiogenesis, vascular damage and dysregulation of lymphatic drainage in solid tumors. The oxygen concentration in hypoxic tumor tissue is about (5 mm Hg), which is significantly lower than the hypoxic level in normal tissue (70 mm Hg).

The invention develops a novel chitosan nano material for high-efficiency treatment of lung cancer. Nitroimidazole acetate is coupled on chitosan chains through EDCI/NHS catalyzed amidation reaction to obtain the nano-composite with functions of hypoxia and acidic pH response release and photodynamic therapy.

Disclosure of Invention

The invention aims to provide a preparation method of a controlled-release nano composite medicine with low phototoxicity. The nitroimidazole group hypoxia response and the chitosan acidic pH response are utilized to actively target the tumor site for release, so that the accumulation of the drug on the tumor site is increased, the problem of high phototoxicity of the traditional photosensitizer is solved, and the curative effect of photodynamic therapy is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the nano-composite is obtained by taking chitosan Cs-NA modified by nitroimidazole acetate as a carrier and carrying a negative photosensitizer, wherein the chitosan Cs-NA modified by nitroimidazole acetate is obtained by modifying 2- (2-nitroimidazole-1-yl) acetic acid NA on amino of a chitosan Cs sugar chain, and the grafting rate of 2- (2-nitroimidazole-1-yl) acetic acid is 10% -30%.

The particle size of the nano-composite is 70-150 nm.

The molecular weight of the chitosan is 6-1000 kDa.

The negative photosensitizer comprises one of Rose Bengal (RB), Rose Bengal Derivative (RBD), indocyanine green (ICG), and Hematoporphyrin (HP). Wherein the rose bengal RBD is Rose Bengal Derivative (RBD) with side chain containing carboxyl, and the side chain has 8 carbon atoms.

The preparation method of the nano-composite comprises the following steps:

step (1): dissolving chitosan Cs in 0.1wt% acetic acid solution, sequentially adding NA, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI and N-hydroxysuccinimide NHS, stirring at room temperature for 12 hours, filling the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain a white flocculent Cs-NA carrier; the reaction steps are as follows:

step (2): dissolving a Cs-NA carrier in 0.1wt% acetic acid solution, placing the solution into a round-bottom flask, dropwise adding DMSO solution containing a photosensitizer with negative electricity at the dropping speed of 1-2 drops per second, stirring the solution at room temperature in a dark place for reaction for 12 hours, placing the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the nano-composite.

In the step (1), Cs: NA: EDCI: the molar ratio of NHS was 1: 1-3: 4: 4.

in the nano-composite, the mass ratio of the Cs-NA to the negative photosensitizer is 1: 1-1.5: 1.

the cut-off molecular weight of the dialysis bag in the steps (1) and (2) is 8000-14000 Da.

The nano-composite of the invention is used for the targeted therapy and photodynamic therapy of tumor cells.

The structural formulas of Cs-NA, RBD and RB in the invention are shown in the specification. The RBD can be prepared by a method described in the literature (Sugita N, Kawabata K I, Sasaki K, et al. Synthesis of Amphiphiic derivatives of Rose Bengal and thermal turbine administration [ J ]. bioconjugate ugatechemistry, 2007, 18(3): 866-873.).

First, the principle of the invention: -NH on Chitosan scaffold³⁺Has strong positive charge, can form a stable compound with a photosensitizer with negative charge, achieves the aim of transferring the photosensitizer, realizes the controlled release of the drug, and has better biocompatibility and great clinical application potential.

Secondly, the special pathological environment of tumor cell hypoxia increases the reduction stress, leads to the over-expression of nitroimidazole enzyme, azo reductase and quinone reductase, and utilizes nitroimidazole group to be reduced by nitroreductase in tumor tissue, so as to achieve the purpose of response and release.

Thirdly, the acidic pH value in the tumor tissue is utilized to promote the dissociation of the chitosan structure and promote the drug release.

Fourthly, the photosensitizers RB, RBD, ICG and HP can generate highly toxic reactive oxygen species ROS under the irradiation of exciting light with certain wavelength, and can effectively kill tumor cells.

Fifth, the present invention improves the water solubility of the photosensitizers RB, RBD, ICG, and HP, while reducing its phototoxicity.

The invention has the beneficial effects that:

firstly, the chitosan is used as a delivery carrier, passive targeting is realized by controlling the particle size of the nano-composite, and the medicine is delivered to a tumor part;

secondly, nitroimidazole as a hypoxia response group can be reduced in a hypoxic tumor environment, so that release of the entrapped drug is promoted, and the effects of accumulation of the photosensitizer at a tumor site and reduction of phototoxicity are achieved.

Drawings

FIG. 1 shows the preparation of Cs-NA carrier and raw material Cs in example 2¹H-NMR spectrum;

FIG. 2 is an infrared spectrum of the Cs-NA carrier and the raw Cs prepared in example 2;

FIG. 3 is a particle size peak plot of CBNs and CBDNs prepared in examples 5 and 6;

FIG. 4 is a UV spectrum of CBDNs prepared in example 8;

FIG. 5 is a graph showing the release characteristics of CBDNs of example 9;

FIG. 6 is an in vitro toxicity test of several nanoparticles on PC-9 cells in example 10.

The specific implementation method comprises the following steps:

the present invention is further described below in conjunction with specific examples to assist those of ordinary skill in the art in further understanding the present invention, but are not intended to limit the invention in any way.

Example 1

Rose Bengal (1 g) was dissolved in DMF (10 ml) and 8-bromooctanoic acid (0.68 g) was added. The reaction solution was heated at 80 ℃ and stirred for 7 hours. After completion of the reaction, DMF was removed by rotary evaporation, and the residue was stirred with ether overnight. The residue was filtered and washed thoroughly with diethyl ether and then stirred with water overnight. To remove excess rose bengal and impurities, after filtration, recrystallization from ethanol gives a purple-red powder RBD of the formula:

the hydrogen spectrum data are as follows

¹H NMR (DMSO-d ₆) δ (ppm): 0.89 (m, CH₂, 2H,J= 7.3 Hz), 1.03 (m, CH₂,2H,J= 7.7 Hz), 1.09 (m, CH₂, 4H,J= 6.8 Hz), 1.43 (m, CH₂, 2H,J= 7.5 Hz),2.18 (t, CH₂COOH, 2H,J= 7.8 Hz), 3.93 (t, OCH₂, 2H,J= 6.3 Hz), 7.48 (s,ArH, 2H), 8.17 (br.s, ArOH), 11.9 (br.s, COOH).

Example 2

Cs (20.00 mg) is weighed and dissolved in 2mL of 0.1wt% acetic acid solution, NHS (35.57 mg), EDCI (90.10 mg) and 2- (2-nitroimidazol-1-yl) acetic acid (18.58 mg) are sequentially added, after stirring reaction at room temperature for 12h, after the reaction is finished, the reaction solution is transferred into a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and dialyzed in secondary water for 3 days. Finally, the product (Cs-NA) was freeze-dried to give a white powder which was stored at-20 ℃. Using deuterated acetic acid as¹Test solvent of H-NMR spectrum, as can be seen from FIG. 1, the presence of imidazole group is confirmed by the absorption peaks of three aromatic rings appearing between 6.5 and 8. Infrared spectroscopy analysis was performed on Cs-NA and Cs, as shown in FIG. 2, with Cs-NA at 3000 cm^-1~3500 cm^-1The absorption peak is significantly enhanced, indicating that a strong aromatic ring is coupled, demonstrating the presence of an imidazole group at 1440 cm^-1The absorption peak is enhanced, the formation of amide is shown, and the combination of the nitroimidazole acetate and the chitosan is further proved. It is known that nitroimidazole acetate has been successfully coupled to chitosan. The nitroimidazole acetate grafting rate was 13.4% as determined by uv-vis spectroscopy.

Example 3

Cs (20.00 mg) is weighed and dissolved in 2mL of 0.1wt% acetic acid solution, NHS (35.57 mg), EDCI (90.10 mg) and 2- (2-nitroimidazol-1-yl) acetic acid (55.74 mg) are sequentially added, after stirring reaction for 12h at room temperature, after the reaction is finished, the reaction solution is transferred into a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and dialyzed in secondary water for 3 days. Finally, the product (Cs-NA) was freeze-dried to give a white powder which was stored at-20 ℃. The nitroimidazole acetate grafting rate was 29.6% as determined by uv-vis spectroscopy.

Example 4

Dissolving 1mg of Cs-NA carrier in 0.1wt% acetic acid solution, putting the solution into a round-bottom flask, dropwise adding 1ml of DMSO solution containing 1mg of indocyanine green (ICG), stirring the solution at room temperature in the dark for reaction for 12 hours, putting the reaction solution into a dialysis bag for dialysis, and freeze-drying the solution to obtain the Cs-NA/ICG nano-drugs (CINs). The grain diameter of the nanoparticles (CINs) is 89.6 +/-2.4 nm.

Example 5

Dissolving 1mg of Cs-NA carrier in 0.1wt% acetic acid solution, placing the solution into a round-bottom flask, dropwise adding 1ml of DMSO solution containing 1mg of C8 carboxyl side chain derivative (RBD), stirring the solution at room temperature in a dark place for reaction for 12 hours, filling the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the Cs-NA/rose bengal derivative nano-drug. As shown in FIG. 3, the nanoparticles (CBDNs) have a particle size of 115.4. + -. 1.9 nm.

Example 6

Dissolving 1mg of Cs-NA carrier in 0.1wt% acetic acid solution, putting the solution into a round-bottom flask, dropwise adding 1ml of DMSO solution containing 1mg of Rose Bengal (RB), stirring the solution at room temperature in a dark place for reaction for 12 hours, putting the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the Cs-NA/rose bengal nano-drug; as shown in FIG. 3, the Cs-NA/RB nanoparticles (CBNs) have a particle size of 86.89 + -1.63 nm.

Example 7

Dissolving 1mg of Cs carrier in 0.1wt% acetic acid solution, placing the solution into a round-bottom flask, dropwise adding 1ml of DMSO solution containing 1mg of C8 carboxyl side chain derivative (RBD), stirring the solution at room temperature in a dark place for reaction for 12 hours, filling the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the Cs/rose bengal derivative nano-drug; the particle size of the Cs/RBD nano-particles (CsDNs) is 128.7 +/-3.6 nm.

Example 8

10mg of Cs-NA was weighed out and dissolved in 2mL of 0.1wt% acetic acid solution, and RBD (8-carbon carboxy-side chain derivative of rose bengal) (10 mg, 2mL of DMSO) was added dropwise to the Cs-NA solution, and stirred at room temperature in the dark for 12 hours. The reaction solution was dialyzed against secondary water for 3 days, and then lyophilized to obtain CBDNs. As shown in FIG. 4, the fluorescence intensity of CBDNs was almost 0, demonstrating that the encapsulation efficiency of RBD in Cs-NA was close to 100%. The encapsulation and loading of CBDNs were 100% and 26.4% ± 0.03%, respectively.

Example 9

Sucking 2mL of CBDNs (rose bengal contains carboxyl side chain derivatives with 8 carbon atoms) solution, putting the CBDNs solution into a dialysis bag with the cut-off molecular weight of 8000-14000 Da, and putting the dialysis bag into 20 mL of PBS. Under the condition of stirring at 37 ℃, 1mL of the solution is taken for detection at a set time and supplemented at the same time1mL of the above buffer. The release characteristics of CBDNs are shown in FIG. 5, and the maximum release of RBD within 24h is 52% at pH 5.5, and pH 5.5+ Na₂S₂O₄The cumulative release of RBD in the group reached 58%. Because the nitro group in the nitroimidazole is easily reduced into amino under the reducing condition, the dissociation of the nano structure is promoted. In addition, the acidic pH causes hydrogen bonds in the chitosan molecule to be broken, thereby promoting the dissociation of the chitosan. The accumulated release of CBDNs is in a descending trend after 8h, and the phenomenon that the fluorescence intensity is increased and then reduced is caused because the RBD structure in the release medium is easy to damage. And no Na at pH =7.4₂S₂O₄In the group, the cumulative release was small, and the cumulative release at 24h was only 31%.

Example 10

Human lung cancer cell line PC-9 cells were used as the test cell line (cells purchased from cell resource center of Shanghai Life sciences institute of Chinese academy of sciences).

The cell culture method comprises the following steps: taking out the frozen cells from the liquid nitrogen tank, quickly placing the cells in a water bath at 37 ℃, continuously shaking the cells to quickly melt the cells, and centrifuging the cells at the room temperature of 1000 rpm; discarding the frozen stock solution, beating with 1ml culture solution to obtain cell suspension, transferring the cell suspension into culture flask, supplementing 3ml culture solution until the culture flask is placed at 37 deg.C and 5% CO₂After culturing for 24 hours in the incubator, the old culture solution is discarded, the new culture solution is replaced, and the culture is continued.

Cytotoxicity experiments: selecting PC-9 cells with good logarithmic phase growth and state, digesting with trypsin, and making into cell suspension (0.5-1 × 10)⁵one/mL). Inoculating the suspension into a 96-well plate according to the amount of 100 muL cell suspension per well, and placing the plate in a container containing 5% CO₂After incubation in an incubator at 37 ℃ for 24h, CBNs, CBDNs, CsDNs were added at different concentration gradients. After 4h incubation, the irradiated groups were given a laser (2.0W/cm)²10 min). After 24h of drug action, the wells were washed twice with PBS, 100ul of MTT solution (5 mg/ml, i.e., 0.5% MTT) was added to each well, and after further incubation for 4h, the incubation was terminated and the culture medium was carefully aspirated from the wells. Add 100ul DMSO into each well, shake for 10min at low speed on a shaker to dissolve the crystals sufficiently. Measuring each well at OD570 nm of an ELISA detectorAnd (4) light absorption value. And the survival rate of the cells was calculated as follows. Survival (%) = (experimental absorbance-solvent control absorbance)/(blank absorbance-solvent control absorbance).

The cytotoxicity results are shown in fig. 6. As can be seen from fig. 6, under the normoxic environment, almost none of the CBNs, CsDNs and CBDNs showed significant toxicity, whereas CBDNs showed some degree of cytotoxicity after light irradiation. As shown in fig. 6, under hypoxic environment, the cytotoxicity of CBNs and CBDNs was significantly increased, and after the light irradiation, the cytotoxicity was further increased. And the toxicity of CsDNs under the normal oxygen and low oxygen conditions is not obviously changed, so that the effect of the nano-composite under the low oxygen condition is illustrated, and the phototoxicity is properly reduced.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A pH/hypoxia double-response drug release synergistic photodynamic therapy nano-composite is characterized in that: the nano-composite is obtained by taking chitosan Cs-NA modified by nitroimidazole acetate as a carrier and carrying a photosensitizer with negative electricity, wherein the chitosan Cs-NA modified by nitroimidazole acetate is obtained by modifying amino of a chitosan Cs sugar chain with 2- (2-nitroimidazole-1-yl) acetic acid NA, and the grafting rate of 2- (2-nitroimidazole-1-yl) acetic acid is 10% -30%.

2. The nanocomposite of claim 1, wherein: the particle size of the nano-composite is 70-150 nm.

3. The nanocomposite of claim 1, wherein: the molecular weight of the chitosan is 6-1000 kDa.

4. The nanocomposite of claim 1, wherein: the negative photosensitizer comprises one of rose bengal, rose bengal derivatives, indocyanine green, and hematoporphyrin.

5. The nanocomposite as claimed in claim 4, wherein: the rose bengal derivative is a rose bengal derivative with a side chain containing carboxyl, and the carbon number of the side chain is 8.

6. A method of preparing the nanocomposite of claim 1, wherein: the method comprises the following steps:

step (1): dissolving chitosan Cs in 0.1wt% acetic acid solution, sequentially adding NA, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride EDCI and N-hydroxysuccinimide NHS, stirring at room temperature for 12 hours, filling the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain a white flocculent Cs-NA carrier;

7. The method of claim 6, wherein: in step 1), Cs: NA: EDCI: the molar ratio of NHS was 1: 1-3: 4: 4.

8. the method of claim 6, wherein: in the nano-composite, the mass ratio of the Cs-NA to the negative photosensitizer is 1: 1-1.5: 1.

9. the method of claim 6, wherein: the cut-off molecular weight of the dialysis bag in the steps (1) and (2) is 8000-14000 Da.