PH SENSITIVE METAL AND NANOPARTICLE AND PREPARATION METHOD

Abstract

The present invention relates to a pH sensitive particle, a method of preparation thereof, and a use thereof. More particularly, the invention provides a pH sensitive metal nanoparticle and its use for medical treatment utilizing cell necrosis during photothermal therapy. The pH sensitive metal nanoparticle based on this invention consists of a pH sensitive ligand compound whose charge changes depending on the pH of the metal nanoparticle. The particle can be collected in cells, such as cancer cells which present an abnormal pH environment. The pH sensitive metal nanoparticle based on this invention can induce cell death through a photothermal procedure after aggregation. Therefore, the invention enables medical treatment using cell necrosis for e.g. cancer treatment.

Claims

1-14. (canceled)

15. A method for destroying abnormal cells, comprising: administering pH-sensitive metal nanoparticles to aggregate; and irradiating the aggregated metal nanoparticles with light, thereby destroying the abnormal cells.

16. The method according to claim 15, wherein the abnormal cells are cells representing an acidic pH.

17. The method according to claim 15, wherein the abnormal cells are cancer cells.

18. The method according to claim 15, wherein the metal nanoparticles are introduced into the cells and form aggregations in the cells.

19. The method according to claim 15, wherein the metal nanoparticles are gold particles or gold-coated particles.

20. The method according to claim 15, wherein the charge of at least a portion of the compounds changes under an acidic pH environment.

21. The method according to claim 15, wherein the charge of the compound changes upon hydrolysis.

22. The method according to claim 21, wherein the charge of the compound changes from negative to positive after the compound is hydrolyzed under an acidic pH environment.

23. The method according to claim 15, wherein the compound is represented by the following chemical formula I: ##STR00011##

24. The method according to claim 15, wherein the metal nanoparticles have an average diameter of 20 nm or less.

25. The method according to claim 15, wherein the light is a red or infrared light.

26. The method according to claim 25, wherein the light is a laser.

27.-41. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0047] FIG. 1 is of transmission electron microphotographs showing the pH-sensitive gold nanoparticles of the present invention which are dispersed in an aqueous solution at pH 7.3 and at pH 5.5 with the lapse of 10 min, 30 min, 120 min, 90 min and 60 min, respectively (from the left upper panel in a clockwise direction) (scale bars represent 50 nm for pH 7.3 and 500 nm for pH 5.5).

[0048] FIG. 2 shows light absorption spectra of the pH-sensitive gold nanoparticles of the present invention according to pH and time. Absorbance was measured 24 hrs after the pH-sensitive gold nanoparticles were dispersed at pH 7.3 (black) and 10 min, 30 min and 90 min after the pH-sensitive gold nanoparticles were dispersed in an acetate buffer solution at pH 5.5 (blue, green and red, respectively).

[0049] FIG. 3 is a dark-field microphotograph showing the pH-sensitive gold nanoparticles of the present invention captured by uterine cervical cancer cells. The gold nanoparticles which aggregate within the cells are visualized red.

[0050] FIG. 4 is of optical photographs showing the cells treated with (left row) and without the pH-sensitive gold nanoparticles of the present invention (right row) after the cells were exposed for 10 min to a laser of 660 nm wavelength at 140 mW (top panels), 85 mW (middle panels) and 55 mW (bottom panels) and stained with trypan blue. The cells killed by the photothermal effect of the pH-sensitive gold nanoparticles are visualized in blue.

BEST MODEL

EXAMPLES

Synthesis of pH-Sensitive Ligand

[0051] A solution of lipoic acid (1) in anhydrous chloroform, as illustrated in the following reaction scheme, were first mixed at room temperature for 5 min with 1.3 equivalents of carbonyldiimidazole under a vacuum condition with stirring, followed by separating the reaction solution layer from the remaining carbonyldiimidazole. Ethylenediamine was dissolved in an amount corresponding to 5 equivalents of the lipoic acid in anhydrous chloroform under a nitrogen atmosphere, cooled in an ice bath, and mixed for 1 hr with the separated reaction solution by stirring. The resulting reaction solution containing the product (2) was extracted three times with 10% NaCl and once with deionized water and mixed at room temperature for 24 hrs with citraconic anhydride to form a solid substance (3). After filtration, the solid substance was dissolved in an aqueous solution which was adjusted to a pH of 9 with NaOH. The resulting solution was stirred at room temperature for 4 hrs along with 1 equivalent of NaBH.sub.4 to afford a pH-sensitive ligand (4) which was used without further purification.

##STR00007##

[0052] Under an acidic condition, the product (4) is hydrolyzed at its amide bond into a primary amine and citraconic acid, as illustrated in the following reaction scheme. The primary amine is positively charged at an acidic pH.

##STR00008##

Synthesis of Gold Nanoparticles Stabilized with Citrate

[0053] A solution of the gold precursor HAuCl.sub.4 in distilled water was heated at 120 C. for 30 min with stirring, and then for an additional 2 hrs along with trisodium citrate with stirring. In this regard, while the trisodium citrate acted as a reducing agent and a surface ligand, the solution turned from yellow to red, indicating the construction of gold nanoparticles. Afterwards, the solution was cooled at room temperature with stirring. (Ind. Eng. Chem. Res. 2007, 46, 3128-3136)

##STR00009##

Synthesis of pH-Sensitive Gold Nanoparticles

[0054] To an aqueous solution containing an excess of the synthetic pH-sensitive ligand were added the citrate-stabilized gold nanoparticles, followed by stirring at room temperature for 8 hrs. Because the dithiol of the pH-sensitive ligand binds more strongly to the surface of the gold nanoparticles than does the carboxylic acid of citrate, the pH-sensitive ligand is exchanged for the citrate. Excess ligands were removed by dialysis.

##STR00010##

Aggregation Characteristics of pH-Sensitive Gold Nanoparticles

[0055] The pH-sensitive gold nanoparticles were dispersed in aqueous solutions at pH 7.3 and pH 5.5, respectively. 10 min, 30 min, 120 min, 90 min, and 60 min after the dispersion, the pH-sensitive gold nanoparticles were observed through transmission electron microimages. The results are given in FIG. 1. As shown in the TEM of FIG. 1, the pH-sensitive nanoparticles were observed to aggregate at pH 5.5. In contrast, the pH-sensitive gold nanoparticles were well dispersed at an average size of 15 nm under a pH 7.3 condition irrespective of the lapse of time. When the condition was changed to a pH of 5.5, the particles formed aggregates which were observed to gradually increase in size to ones of microns with time.

Light Absorption Properties According to Aggregation of Gold Nanoparticles

[0056] After being exposed to a pH of 7.3 and 5.5 similar respectively to the surrounding conditions of normal cells and cancer cells, the light absorption of the synthetic pH-sensitive gold nanoparticles was measured over time. Light absorption spectra of the synthetic pH-sensitive gold nanoparticles observed 24 hrs after dispersion under a pH 7.3 condition and 10 min, 30 min and 90 min after dispersion under a pH 5.5 condition are depicted in FIG. 2.

[0057] At pH 7.3 corresponding to a normal biological condition, the pH-sensitive gold nanoparticles were found to intensively absorb only a band of visible light less than 600 nm, but when the condition was adjusted to pH 5.5, their absorption wavelength was observed to shift toward longer wavelengths with time and finally to the red-near infrared ranges. This is attributed to the fact that the pH-sensitive gold nanoparticles aggregate to each other thanks to the electrostatic attraction generated while the charge of the surface ligand of the particles changes from positive to negative when hydrolyzed.

[0058] The pH-sensitive gold nanoparticles around normal cells which form a neutral environment of pH 7.3-7.4 absorb only light in a visible range of less than 600 nm. In contrast, when around cancer cells which form an acidic condition of pH 5.5, the pH-sensitive gold nanoparticles form aggregates which absorb light in the red-infrared range. In addition to the selective photothermal therapeutic potential of the pH-sensitive gold nanoparticles for cancer, the red-infrared light exhibits the advantage of increasing the photothermal effect of metal nanoparticles because it is only slightly absorbed or scattered by bio-substances such as the skin, blood, etc.

Observation with Dark-Field Microscope

[0059] Uterine cervical cancer cells were treated with the pH-sensitive gold nanoparticles before observation with a dark-field microscope. The image photographed by the dark-field microscope is given in FIG. 3. As the gold nanoparticles aggregated, their absorption wavelengths shifted toward longer wavelengths. Thus, because they absorbed red-near infrared light, a red image was observed with a dark-field microscope.

[0060] After they are introduced into cells through endocytosis by endosomes, the pH-sensitive gold nanoparticles are exposed to an acidic condition during the fusion of the endosomes with lysosomes and thus form aggregates.

Test for Photothermal Therapy

[0061] Tests for photothermal therapy were conducted in vitro with the pH-sensitive gold nanoparticles. Uterine cervical cancer cells were incubated in combination with pH-sensitive gold nanoparticles to introduce the nanoparticles into the cells (experimental group). For a control, the cells were incubated alone. The experimental group and the control were exposed to a laser of 660 nm for 10 min at a power of 140 mW, 85 mW and 55 mW. Thereafter, the cells were stained with trypan blue before observation under an optical microscope. The images are given in FIG. 4. Irradiated portions are indicated by circles.

[0062] Trypan blue selectively stains dead cells. No dead cells were detected in both the experimental group and the control when they were exposed to a laser at a power of 55 mW less than a threshold, indicating that the pH-sensitive gold nanoparticles exhibit no cytotoxicity under a light condition less than the threshold. That is, the pH-sensitive gold nanoparticles of the present invention meet the requirement of therapeutic photosensitizers that are non-toxic under a condition of darkness. When irradiated with a laser with a power of 85 mW, only cells in the experimental group were selectively killed. At a power of 140 mW, the cells of the experimental group were also selectively killed. More cells were killed at a power of 140 mW than at a power of 85 mW, indicating that photothermal therapy induces cell death in a dose-dependent manner. Consequently, the pH-sensitive gold nanoparticles of the present invention can be useful in the photothermal therapy of cancer.