METHOD FOR PRODUCING ALGINIC ACID-FOLIC ACID CONJUGATE, ALGINIC ACID-FOLIC ACID CONJUGATE PRODUCED THEREBY AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME

Abstract

The present invention relates to a method for producing an alginic acid-folic acid conjugate, an alginic acid-folic acid conjugate produced thereby, and a pharmaceutical composition containing the same. According to the method of producing an alginic acid-folic acid conjugate using a carboxy-protecting group and a leaving group, the hydroxyl group of alginic acid forms an ester group with the carboxyl group of folic acid. Thus, the alginic acid-folic acid conjugate may clearly distinguish cancer cells from normal tissue by more effectively targeting cancer cells than a conventional alginic acid-conjugated folic acid in which the amine group of folic acid is covalently bonded to the carboxyl group of alginic acid. Accordingly, the alginic acid-folic acid conjugate may be effectively used for precise diagnosis and efficient surgical resection of cancer lesions.

Claims

1. A method for producing an alginic acid-folic acid conjugate comprising steps of: a) introducing a protecting group to a carboxyl group of alginic acid; b) introducing a leaving group to a carboxyl group of folic acid; and c) obtaining a reaction product between the alginic acid to which the protecting group has been introduced in step a) and the folic acid to which the leaving group has been introduced in step b).

2. The method of claim 1, wherein the protecting group in step a) is an unsubstituted or substituted benzyl group; trimethylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl; an unsubstituted or substituted alkyl group having 1 to 4 carbon atoms; or tetrabutylammonium.

3. The method of claim 2, wherein the protecting group in step a) is tetrabutylammonium.

4. The method of claim 1, wherein the leaving group in step b) is a methanesulfonyl group, a p-toluenesulfonyl group, or a trifluoromethanesulfonyl group; an alkoxy group having 1 to 5 carbon atoms; a halogen; or imidazole.

5. The method of claim 4, wherein the leaving group in step b) is imidazole.

6. The method of claim 1, wherein the reaction product obtained in step c) is one in which a hydroxyl group of alginic acid and the carboxyl group of folic acid are bonded to each other via an ester bond.

7. The method of claim 1, wherein step a) comprises producing the alginic acid from alginic acid salt and introducing the protecting group to the carboxyl group of the alginic acid at a pH of 8 to 10.

8. An alginic acid-folic acid conjugate in which a carboxyl group of folic acid is linked to a hydroxyl group of alginic acid via an ester bond, or a pharmaceutically acceptable salt thereof.

9. The alginic acid-folic acid conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein an amine group bound to a dihydropteridine moiety remains unreacted.

10. The alginic acid-folic acid conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein the alginic acid-folic acid conjugate is represented by the following Formula 1: ##STR00008## wherein R is an alginic acid unit or an alginic acid polymer.

11. The alginic acid-folic acid conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein the salt of the alginic acid-folic acid conjugate is represented by the following Formula 1-1: ##STR00009## wherein R is an alginic acid unit or an alginic acid polymer, and M is Na, K, Mg, Ca, or Ba.

12. A pharmaceutical composition for diagnosing cancer containing the alginic acid-folic acid conjugate or pharmaceutically acceptable salt thereof according to claim 8.

13. The pharmaceutical composition of claim 12, further containing a fluorescence inducing substance.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1 illustrates that 5-ALA is delivered and released into cancer cells by a nanocarrier including an alginic acid-folic acid conjugate and 5-ALA according to one embodiment of the present invention.

[0065] FIG. 2 shows the .sup.1H NMR spectra of the alginic acid-folic acid conjugate according to one embodiment of the present invention, folic acid, and alginic acid.

[0066] FIGS. 3A and 3B show FT-IR spectra (FIG. 3A) and UV-vis spectroscopy (FIG. 3B) of the alginic acid-folic acid conjugate according to one embodiment of the present invention, folic acid, and alginic acid.

[0067] FIGS. 4A and 4B show the results of analyzing the physiochemical characteristics of the alginic acid-folic acid conjugate according to one embodiment of the present invention, and depicts a TEM image showing the morphology of NP4 (FIG. 4A), and a size distribution obtained by measuring the diameters of 230 NPs (FIG. 4B).

[0068] FIG. 5 shows the results of analyzing the physiochemical characteristics of the alginic acid-folic acid conjugate according to one embodiment of the present invention, and is a graph showing the stability of NP1 to NP4 over time.

[0069] FIG. 6 is a graph showing the intracellular cumulative 5-ALA release (%) of the alginic acid-folic acid conjugate according to one embodiment of the present invention.

[0070] FIGS. 7A, 7B, 7C and 7D show the results of evaluating the cytotoxicity of the alginic acid-folic acid conjugate according to one embodiment of the present invention. FIG. 7(A) is a fluorescence image of HFBs which are normal cells, and FIGS. 7(B) and 7(C) are fluorescence images of MCF-7, A549 and SKOV-3, respectively, which are cancer cell lines. FIG. 7D is a bar graph of cell viability versus concentration of Np.

[0071] FIG. 8 is a graph showing the results of quantitatively measuring the fluorescence of NP1 to NP4, each including the alginic acid-folic acid conjugate according to one embodiment of the present invention.

[0072] FIGS. 9A, 9B, 9C and 9D depict fluorescence images showing intracellular PpIX production by the alginic acid-folic acid conjugate according to one embodiment of the present invention. FIG. 9(A) is a fluorescence image of HFBs which are normal cells, and FIGS. 9(B), 9(C), and 9(D) are fluorescence images of A549, MCF-7 and SKOV-3, respectively, which are cancer cell lines. Cell nuclei are stained with DAPI and displayed in blue, and PpIX is displayed in red. Scale bar represents 50 μm.

[0073] FIG. 10 is a graph showing the results of quantitatively measuring fluorescence corresponding to intracellular PpIX production by the alginic acid-folic acid conjugate according to one embodiment of the present invention.

DETAILED DESCRIPTION

[0074] Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative examples.

Example 1. Production of Nanocarrier Including Alginic Acid-Folic Acid Conjugate

[0075] 1-1. Production of Alginic Acid-Folic Acid Conjugate

[0076] To produce an alginic acid-folic acid conjugate (AF), sodium alginate (Wako Pure Chemical Industries, 11 kDa, Japan) (1 g, 4.632 mmol) was added to 30 mL of a mixture of ethanol/0.6 M HCl and stirred for 18 hours at 4° C. Alginic acid was produced, filtered in vacuo using qualitative filter paper (Whatmanm), washed several times with alcohol and acetone, transferred to a vial, and dried under vacuum overnight. The dried alginic acid was dissolved in water (30 mL) and 4% TBAOH (Sigma-Aldrich, USA) was added to the solution with continuous stirring until the solution reached pH 9. The opaque solution was lyophilized under reduced pressure to obtain white TBA-alginate (see Reaction Scheme 1).

[0077] Folic acid (Sigma-Aldrich, USA) (0.408 g, 0.9264 mmol) was mixed with DMSO (10 mL) and 1,10-carbonyldiimidazole (CDI) (Sigma-Aldrich, USA) (0.1502 g, 0.9264 mmol) was added thereto. The produced compound was stirred under N.sub.2 gas at 25° C. in the dark for 24 hours (see Reaction Scheme 2).

[0078] Thereafter, TBA-alginate was dissolved in 50 mL of DMSO containing 1 wt % TBAF (tetrabutylammonium fluoride hydrate) (Sigma-Aldrich, USA). Under continuous stirring, the folic acid/CDI compound was added to the TBA-alginate solution and left to react overnight in the dark at 40° C. The product was precipitated in cold ethanol/methanol (1:1) containing 0.01 M HCl, filtered, and washed with alcohol. The product was neutralized by dissolving in a solution of sodium carbonate, and AF was obtained by lyophilization under reduced pressure (see Reaction Scheme 3 and Formula 1-2).

[0079] 1-2. Production of Nanocarriers Containing Alginic Acid-Folic Acid Conjugate

[0080] Nanocarriers (nanoparticles, NPs) containing AF were produced by referring to a conventional water-in-oil emulsion (W/O) preparation method (Jeong, Y. et al., Biomacromolecules 2019, 20, 1068-1076).

[0081] Briefly, soybean oil, a surfactant mixture (a mixture of Span80 and Tween80), and an aqueous phase component including AF and 5-ALA (98%) (Sigma-Aldrich, USA) were placed and mixed in a glass vial at a weight ratio of 7:2:1. For particle optimization, different nanocarriers (NPs) were prepared by adjusting the hydrophilic-lipophilic balance (HLB) values and AF concentration (0.5 to 1 wt %), while the concentration of 5-ALA was fixed at 1 wt %. The solution was mixed, followed by sonication without on-off pulse using a probe-type sonicator (VC-750, Sonics and Materials, USA) for 10 min at 40% amplitude. Then, the mixture was obtained as a yellowish opaque solution, which was redispersed in deionized (DI) water or phosphate-buffered saline (PBS) and centrifuged. The NPs collected in a liquid state were filtered using a syringe membrane filter (DISMIC-25, Advantec, Japan). Lastly, dialysis was performed for 24 hours using a dialysis membrane (Cellu-Sep MWCO 25 kDa) to remove impurities from the NP solution, thereby obtaining final NPs 1 to 4.

Comparative Example 1. Production of Nanocarrier Containing Alginic Acid

[0082] A nanocarrier containing an alginic acid was produced in the same manner as in Example 1, except that alginic acid was used instead of AF.

Experimental Example 1. Physicochemical Characterization of Alginic Acid-Folic Acid Conjugate and Nanocarriers Containing the Same

[0083] The structures of AF and NPs were analyzed by .sup.1H NMR (proton nuclear magnetic resonance), FT-IR (Fourier transform infrared) and UV-vis. .sup.1H NMR spectra (JNM-LA400, JEOL, Japan) were measured at 400 MHz; alginic acid and AF were measured at 80° C. and other compounds were measured at 25° C. (see FIG. 2). FT-IR spectra (ALPHA, Bruker, USA) were analyzed at a frequency range of 4,000 to 400 cm.sup.−1 to characterize AF (see FIG. 2A). UV-vis spectral analysis was performed using a Nanodrop 2000 spectrophotometer (Thermo Fisher, USA) (see FIG. 2B).

[0084] In addition, the characteristics of NP1 to NP4 produced to have different AF concentrations and surfactant mixture proportions were analyzed. The morphology of NPs was observed using transmission electron microscopy (TEM JEM-3010, JEOL, Japan). Furthermore, the stability of NPs was assessed by measuring the size thereof for 3 months during storage at 4° C.

[0085] As a result, as shown in FIG. 4, it was confirmed that the NPs had spherical morphology and were monodispersed without aggregation, and the average particle size of 230 NPs was about 25 nm. In addition, as shown in FIG. 5, it was confirmed that the NPs were stably maintained without a significant change in the size distribution for 3 months.

[0086] In addition, the average size, zeta (0 potential, 5-ALA loading capacity (LC) and encapsulation efficiency (EE) of the NPs were measured using a Zetasizer (Malvern, UK).

[0087] The average size of the NPs was measured using dynamic light scattering (DLS) at 25° C. at an angle of 173°.

[0088] To measure the concentration of 5-ALA contained in the NPs, NPs (1 mL) were dispersed in 1.5% hydrogen peroxide (1.5 mL) and sonicated in an ultrasonic water bath for 10 min at 37° C., and then stirred vigorously for 2 hours. Next, the NP decomposition product was centrifuged at 12,300×g using a Microsep device (MWCO 1 kDa), and the supernatant containing 5-ALA was collected and lyophilized under reduced pressure. Thereafter, 5-ALA was quantified using TNBSA (2,4,6-trinitrobenzene sulfonic acid) (5% w/v) (Thermo Fisher, USA) according to the manufacturer's instructions. The 5-ALA loading capacity (LC) and encapsulation efficiency (EE) were calculated according to the following equations:

LC (%)=5-ALA content in nanocarrier/nanocarrier weight×100

EE (%)=5-ALA content in nanocarrier/total 5-ALA content×100  [Equation 1]

[0089] As a result, as shown in Table 1 below, it was confirmed that NP4 was the smallest in size and the average particle size thereof was about 45 nm, which could efficiently penetrate cancer cells. Zeta potential is an indicator of the stability of the nanoparticles, and it was confirmed that all NPs had a negative average surface charge due to the carboxyl group of the alginic acid, suggesting that the NPs were stably formed in a suspended state. The 5-ALA loading capacity increased with increasing AF concentration, and the encapsulation efficiency was the highest at an AF concentration of 1 wt %. This was believed to be due to an increase in the number of ionic bonds between alginic acid and 5-ALA with amphoteric ions. Therefore, as the AF concentration increased, a higher concentration of 5-ALA was encapsulated, and thus the encapsulation efficiency also increased. Based on these results, in the subsequent experiment, 5-ALA release profile assessment, cytotoxicity assessment and PpIX quantification were performed using NP4.

TABLE-US-00001 TABLE 1 Alginic acid-folic Day 0 Zeta potential NP acid (wt %) HLB Size (nm) PDI (mV) LC % EE % NP1 0.5 7 117.9 ± 0.65 0.389 −27.4 ± 2.1 1.2% 6.33% NP2 0.5 8 53.56 ± 1.52 0.496 −23.3 ± 0.7 0.4% 8.8% NP3 1 7 83.45 ± 3.47 0.584 −22.8 ± 2.sup.  2.8% 27.14% NP4 1 8 45.89 ± 1.56 0.454 −29.3 ± 0.1 1.8% 31.6%

Experimental Example 2. 5-ALA Release Profile of Nanocarrier Containing Alginic Acid-Folic Acid Conjugate

[0090] The 5-ALA release profile was assessed under two different pH environments (pH 5.0 and pH 7.4) at 37° C. First, NP4 (2 mL) were placed in a dialysis membrane (MWCO 1 kDa) in 8 mL PBS at pH 5.0 or pH 7.4 and stirred at 37° C. After a fixed time, the release solution was taken out and fresh PBS was added thereto. The concentration of 5-ALA in the dialysis solution was analyzed with a microplate reader (Synergy H1, BioTek, USA) using TNBSA solution according to the manufacturer's instruction. The cumulative 5-ALA concentration released from NPs was calculated according to the following Equation 2:

[00001]$\begin{array}{cc}\mathrm{Cumulative}\mathrm{release}\left(\%\right)=\frac{V_0{C}_s+V_r.Math.C_{s-1}}{T_{\mathrm{ALA}}}\times 100& \left[\mathrm{Equation}2\right]\end{array}$

[0091] wherein T.sub.ALA indicates the total content of 5-ALA in the NPs, V.sub.0 indicates the total volume (10 mL) of the release solution, V.sub.r indicates the volume (1 mL) of the added PBS, and C.sub.s indicates the concentration of 5-ALA in the sample.

[0092] As a result, as shown in FIG. 6, it was confirmed that no more than 30% of 5-ALA was released over the experimental period at pH 7.4, indicating indicate that the 5-ALA contained in NPs remained stable under physiological conditions. However, more than 80% of 5-ALA was released after 80 hours at pH 5, while the 5-ALA was released from 12 to 24 hours. This release was due to the deprotonation and decomposition of the alginic acid carboxyl group at acidic pH. Thus, it can be seen that acid-catalyzed hydrolysis of alginic acid occurs in acidic environments, and NPs are deprotonated to release the drug in the carrier. Thus, these results suggest that a contrast agent containing NPs may be used for imaging gastrointestinal tract cancer.

Experimental Example 3. Measurement of Cytotoxicity of Nanocarrier Containing Alginic Acid-Folic Acid Conjugate

[0093] The human fibroblast cell line HFB was cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). MCF-7 (breast adenocarcinoma), A549 (lung cancer) and SKOV-3 (ovarian adenocarcinoma) cell lines were cultured in RPMI 1640 containing 10% FBS and 1% PS. All cell lines were procured from the Korea Cell Line Bank and cultured in a humidified 37° C. constant temperature incubator set to 5% CO.sub.2.

[0094] For cytotoxicity measurement, the HFB, MCF-7, A549 and SKOV-3 cell lines were used. First, cells were placed in in 96-well plates at a density of 5×10.sup.3 cells per well and preincubated for 24 hours at 37° C. Then, each well was treated with 12.5, 25, 50, 100 or 200 μg/mL of NP4 and incubated at 37° C. for 6, 12 or 24 hours. After the NP-containing medium was removed, the medium was treated with CCK-8 solution and allowed to react in an incubator at 37° C. for 2 hours. Absorbance was measured at 450 nm using a microplate reader.

[0095] Folic acid and 5-ALA are normally present in the body, and alginic acid is also a natural product and is a clinically approved substance. As shown in FIG. 7, it was confirmed that NPs had no cytotoxicity regardless of the concentration and incubation time thereof, suggesting that they had excellent biocompatibility.

Experimental Example 4. Quantification of PpIX by Nanocarrier Containing Alginic Acid-Folic Acid Conjugate

[0096] To quantify the intracellular accumulation of PpIX, cells were placed in 24-well plates at a density of 0.5×10.sup.6 cells per well. After 48 hours of culture, the medium was replaced with a fresh FBS-free medium containing NPs (0.1 mg/mL) and further incubated for 24 hours. After intracellular uptake of NPs and conversion of 5-ALA to PpIX, the culture medium containing NPs was removed and cells were washed with PBS. Then, 100 μL of cold RIPA lysis buffer solution was added to each well, mixed well, and incubated on ice for 30 min, followed by vortexing 4 to 6 times. The lysis solution was centrifuged at 14,000×g at 4° C. for 20 minutes. The supernatant was added to a black 96-well plate, and the fluorescence intensity at 635 nm emission wavelength (405 nm excitation wavelength) was measured with a microplate reader. BSA (bicinchoninic acid) (Thermo Fisher, USA) assay was used to obtain a quantitative fluorescence value according to the cell numbers by normalizing the fluorescence intensity to the total protein concentration of the cell lysate.

[0097] As a result, as shown in FIG. 8, it was confirmed that the fluorescence intensity was higher in the case in which the cancer cells were treated with the four types of NPs dispersed in PBS than the case in which the cells were treated with the NPs dispersed in DI water. In particular, NP4 showed the strongest fluorescence intensity, and was used in a subsequent cellular internalization test.

Experimental Example 5. Measurement of Cellular Internalization of Nanocarrier Containing Alginic Acid-Folic Acid Conjugate

[0098] To analyze the cellular uptake of NPs, a normal cell line and three cancer cell lines were placed in 48-well plates at a density of 0.05×10.sup.6 cells per well and preincubated at 37° C. for 48 hours. The medium was replaced with a fresh FBS-free medium containing NP4 (0.1 mg/mL) and incubated for 12 or 24 hours. Then, the cells were washed with PBS and 100 μL of 4% paraformaldehyde solution was added to each well, followed by fixing for 15 min. 4′,6-diamidino-2-phenylindole (DAPI) was used for cell nuclei staining and kept at room temperature for 20 min. After staining, the cells were washed using Dulbecco's PBS and fluorescence images were acquired using a confocal fluorescence microscope (Zeiss Z1 Axio Observer, Carl Zeiss, Germany). Fluorescence of PpIX was imaged under the conditions of AT560/40 nm excitation and 635/60 nm emission filters.

[0099] As a result, as shown in FIGS. 9 and 10, the nanocarrier (Alg) containing alginic acid did not display fluorescence in the cancer cells, whereas the nanocarrier (NP) containing the alginic acid-folic acid conjugate displayed fluorescence. In addition, while no fluorescence was observed in the normal cell line HFB, fluorescence was observed in the three cancer cell lines from 12 hours at which alginic acid started to be degraded after NP uptake. Thus, it could be seen that NPs were selectively uptaken into cancer cells. In addition, it was confirmed that the fluorescence intensity gradually increased up to 12 hours in cancer cells and rapidly increased up to 24 hours. The fluorescence intensities of the A549 and MCF-7 cell lines were similar up to 24 hours, and the SKOV3 cell line showed stronger fluorescence intensity than the MCF7 and A549 cell lines.

[0100] So far, the present invention has been described with reference to preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will appreciate that the present invention may be embodied in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present invention is defined by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.