KR20160081052A - Hyperbranched polyglycerol having disulfide bonds - Google Patents

Abstract

The present invention relates to a highly branched polyglycerol having disulfide bonds. More specifically, the present invention relates to a highly branched polyglycerol and a use thereof. The highly branched polyglycerol includes a glycidol-based compound having a disulfide bond as a monomer or includes a glycidol or a glycidol-based compound having the disulfide bond as a comonomer. According to the present invention, the highly branched polyglycerol is biocompatible and includes disulfide bonds in a main chain and a branched chain, making the polyglycerol undegradable under a reducing condition. Therefore, the highly branched polyglycerol can be useful as a drug delivering object.

Description

[0001] HYPERBRANCHED POLYGLYCEROL HAVING DISULFIDE BONDS WITH DISSULFIDE BINDING [0002]

The present invention relates to a high molecular weight polyglycerol having a disulfide bond, and more particularly to a high molecular weight polyglycerol having a disulfide bond in a main chain or a branch chain and its use as a drug carrier.

The hyperbranched polyglycerol (PG) is a spherical highly branched polymer composed of a polyether skeleton having a large number of hydroxyl groups. PG is synthesized by a ring opening multiband polymerization reaction of glycidol of a wide molecular weight having a relatively narrow polydispersity as a monomer. Similar to polyethylene glycol (PEG), a polyether analogue, PG exhibits excellent biocompatibility, immunogenicity, and low toxicity. PG also has great interest as a promising material in biomedical fields such as drug delivery systems, polymer therapeutics, proteomics, and human serum albumin substitutes due to ease of synthesis, ease of access to various structures, and ease of attachment of various functional groups .

PG has a long and controllable in vivo circulating half-life, and is excellent in therapeutic efficacy. For example, PGs with molecular weights of 100 kg / mol and 500 kg / mol have a cyclic half-life of 32 hours and 57 hours, respectively. However, according to recent studies, approximately 500% of the injected dose of PG accumulates in organs such as the liver, and this accumulation is higher with higher molecular weight of the polymer. It is therefore necessary to develop polymers that are biocompatible and degradable under physiological conditions.

To this end, PGs with acid-degradable moieties have recently been introduced. One group has developed two types of pH-responsive, highly branched PGs by introducing acetal bonds, respectively, into monomers or initiators (Dingels, C. et al., Biomacromolecules 2013, 14 , 448-459; Tonhauser, C. et al., ACS Macro Letters 2012, 1 , 1094-1097). In addition, other groups have controlled the degradation kinetics of the hyperbranched PG by introducing structurally different ketone linkages into the polymer backbone (Shenoi, RA et al., Biomaterials 2013, 34 , 6068-6081; Shenoi, RA et al., J. Am . Chem . Soc . 2012, 134 , 14945-14957). Nonetheless, these pH-reactive degradation approaches are still incomplete because the polymer can be decomposed in aqueous solutions at neutral pH conditions.

In biological systems, disulfide bonds are degraded into thiols in response to a reduction potential, which is predominantly produced by the high concentration of glutathione (2-10 mM) in the cytoplasm versus the glutathione concentration in the extracellular fluid (<10 μM). In contrast, disulfide is stable in an extracellular oxidative environment, because the concentration of cystine is higher than that of cysteine and reduced glutathione. A large concentration gradient between the intracellular environment and the extracellular environment provides a new means for the development of polymers that degrade specifically after cell uptake. Techniques have been reported for using disulfide bonds in the degradation of PEG- and PG-containing materials (Ko, NR et al., Biomacromolecules 2014, 15 , 3180-3189; Chen, W. et al., Macromolecules 2013, 46 , 699-707; And Lee, Y. et al., Biomacromolecules 2005, 6 , 24-26), which are limited to crosslinking agents for conjugation linkers or nanogels between polymers and drugs, and using disulfide bonds in the PG backbone Not reported.

 Dingels, C. et al., Biomacromolecules 2013, 14, 448-459;  Tonhauser, C. et al., ACS Macro Letters 2012, 1, 1094-1097  Shenoi, R. A. et al., Biomaterials 2013, 34, 6068-6081  Shenoi, R. A. et al., J. Am. Chem. Soc. 2012, 134, 14945-14957  Ko, N. R. et al., Biomacromolecules 2014, 15, 3180-3189  Chen, W. et al., Macromolecules 2013, 46, 699-707; And Lee, Y. et al., Biomacromolecules 2005, 6, 24-26

Accordingly, it is an object of the present invention to provide a biocompatible and redox-degradable hyperbranched polyglycerol.

Another object of the present invention is to provide the use of the above-mentioned highly branched polyglycerol as a drug carrier.

In order to achieve the above object, the present invention provides a high molecular weight polyglycerol having a disulfide bond in the main chain or the branch chain.

To achieve these and other objects, the present invention provides a drug carrier comprising the above-described highly branched polyglycerol.

The hyperbranched polyglycerol according to the present invention is biocompatible and can be decomposed under reducing conditions by presence of a disulfide bond in the main chain or the branch chain, and thus can be usefully used as a drug carrier.

1a shows a reaction scheme for preparing 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (SSG) as a monomer (repeating unit) (P (G-co-SSG)) which is a homopolymer of the above monomers and a high molecular weight polyglycerol (PSSG) which is a copolymer of the monomer and glycidol.
FIG. 2A shows a ¹ H NMR spectrum of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (SSG) as a monomer (repeating unit) ¹ &lt; / RTI &gt; NMR spectrum of the homopolymer (polymer 2), and Fig. 2C shows the ¹ H NMR spectrum of the copolymer (polymer 7).
FIG. 3A shows a ¹ H NMR spectrum of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (SSG) as a monomer (repeating unit) ^13C NMR spectrum of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (SSG) as a monomer (repeating unit).
Figure 4a shows the MALDI-ToF spectrum of the homopolymer (polymer 1), and Figure 4b shows the MALDI-ToF spectrum of the copolymer (polymer 7). In addition, the illustration in Figure 4b shows the detailed molar mass and placement of the copolymer in the range of 2600 to 2700 g / mol.
FIG. 5 shows a ¹³ C NMR spectrum of in situ copolymerization kinetics of G and SSG (monomer ratio 1: 1). The left side shows the overlay of the spectrum collected at the designated time point, and the right side shows G Showing a zoom-in spectrum showing methine carbon in epoxides at 53 and 55 ppm, corresponding to SSG monomers.
Figure 6 shows monomer conversion percent versus total conversion for copolymerization of G (blue square) and SSG (red circle) measured from ¹³ C NMR kinetics at 50 占 폚 in bulk.
FIG. 7A shows the decomposition diagram of the PSSG chain treated with DTT, and FIG. 7B shows the change in ¹ H- and ¹³ C NMR spectra of the PSSG homopolymer (polymer 2) after DTT treatment (10 min).
Figure 8a shows the GPC traces of P (G ₁₆₅ -co-SSG ₅₅ ) (polymer 4) before DTT treatment (black curve) and after (red curve), Figure 8b shows an illustration of the degradation products, 8c shows possible mechanisms for the origin of three peaks in the GPC trace after DTT treatment.
Figure 9a shows in vitro cell survival analysis of PG ₁₅₀ (black), P (G ₆₀ -co-SSG ₃₀ ) (gray) and PSSG ₂₀ polymer (white) in normal cells WI-38 as measured by MTT assay FIG. 9B shows the in vitro cell survival assay in cancer cell HeLa as measured by MTT assay, and FIG. 9C shows the in vitro cell survival assay in normal cell WI-38 as measured by CCK assay , And Figure 9d shows the in vitro cell survival assay in cancer cell HeLa as measured by CCK assay.

The present invention provides hyperbranched polyglycerols having disulfide bonds in the backbone or branched chain.

Conventionally, 'hyperbranched polyglycerol' is a polymer composed of a polyether main chain, a polyether branch chain and a plurality of hydroxyl groups, whereas the hyperbranched polyglycerol according to the present invention has a disulfide bond in addition to a main chain or a branch chain .

In one embodiment, the high molecular weight polyglycerol having a disulfide bond in the main chain or branching chain according to the present invention may be a polymer comprising a glycidol-based compound having a disulfide bond as a repeating unit (monomer) Preferably a homopolymer polymerized from a glycidol-based compound having a disulfide bond as a repeating unit (monomer).

The 'glycidol-based compound having a disulfide bond' can be prepared by reacting 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan- ).

&Lt; Formula 1 >

The high molecular weight polyglycerol prepared by polymerizing the compound of Formula 1 in the form of a repeating unit is referred to as' poly (2- ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan- Quot; PSSG &quot;). An exemplary structure of the PSSG is shown in FIG.

The hyperbranched polyglycerol according to the present invention can be produced by anionic ring-opening multibranching polymerization of the glycidol-based compound having the disulfide bond. For example, a hyperbranched polyglycerol according to one embodiment of the present invention may be produced by the scheme shown in FIG. 1B.

Preferably, the hyperbranched polyglycerol according to one embodiment of the present invention is a glycidol-based compound having disulfide bonds under an argon atmosphere, 2 - ((2- (oxiran-2-ylmethoxy) ethyl) Yl) ethan-1-ol (SSG) to an initiator.

Examples of the polymerization initiator include an alcohol-containing compound such as trimethylolpropane (TMP) or methoxyethanol.

The polymerization reaction may be carried out using potassium alkoxide (e.g., potassium methoxide) or cesium hydroxide monohydrate (CeOH.H ₂ O) as a catalyst.

The polymerization reaction is carried out in an initiation step of reacting under an argon atmosphere at room temperature for 0.5 to 3 hours, preferably 1 hour, and for 2 to 4 hours, preferably 5 hours to 24 hours, preferably 12 hours, At a temperature of 70 to 120 DEG C, preferably 90 DEG C for 8 hours, preferably 5 hours.

The hyperbranched polyglycerol according to one embodiment of the present invention may contain 10 to 50 glycidol-based compounds having disulfide bonds. For example, the highly branched polyglycerol may be PSSG ₁₀ , PSSG ₂₀ , PSSG ₃₀ , PSSG ₄₀ Or PSSG ₅₀ .

Highly branched in accordance with an aspect of the present invention polyglycerol may have a molecular weight of from 2000 to 11000 g / mol, it may have a M _w / M _n of 1.10 to 1.15. The molecular weight of the hyperbranched polyglycerol can be controlled by varying the monomer to initiator ratio. The characteristics of the exemplary hyperbranched polyglycerol can be seen in Table 1.

In another embodiment, the high molecular weight polyglycerols having disulfide bonds in the main chain or branching chain according to the present invention may be polymers which additionally contain glycidol as repeating units, preferably disulfide bonds as comonomers And a copolymer copolymerized with glycidol and a glycidol.

The 'glycidol-based compound having a disulfide bond' can be prepared by reacting 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan- ).

&Lt; Formula 1 >

The high molecular weight polyglycerol produced by the copolymerization reaction of the compound of formula (1) and glycidol is referred to as' poly (glycerol-co-2- (2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) -1-ol) '(referred to as' P (G-co-SSG)'). Examples of the P (G-co-SSG) is _{P (G 165 -co-SSG 55} ), P (G 60 -co-SSG 30), P (G 20 -co-SSG 20) and P (G ₁₀ - co-SSG ₂₀ ). &lt; / RTI &gt; An exemplary structure of P (G-co-SSG) is shown in FIG.

The hyperbranched polyglycerol (copolymer) according to another aspect of the present invention may be produced by the scheme shown in FIG. 1B. Preferably, the high molecular weight polyglycerol (copolymer) according to another aspect of the present invention is a 2 - ((2- (oxiran-2-ylmethoxy) ethyl) glycidol compound having a disulfide bond under an argon atmosphere, Disulfanyl) ethan-1-ol (SSG) and glycidol (G) to an initiator.

Examples of the polymerization initiator include an alcohol-containing compound such as 1,1,1-tris (hydroxymethyl) propane (TMP) or methoxyethanol.

The polymerization reaction may be carried out using potassium alkoxide (e.g., potassium methoxide) or cesium hydroxide monohydrate (CeOH.H ₂ O) as a catalyst.

The polymerization reaction is carried out in an initiation step of reacting under an argon atmosphere at room temperature for 0.5 to 3 hours, preferably 1 hour, and for 2 to 4 hours, preferably 5 hours to 24 hours, preferably 12 hours, At a temperature of 70 to 120 DEG C, preferably 90 DEG C for 8 hours, preferably 5 hours.

The hyperbranched polyglycerol according to another embodiment of the present invention may have a molecular weight of 5000 to 24000 g / mol and may have an M _w / M _n of 1.23 to 1.38. The molecular weight of the hyperbranched polyglycerol can be controlled by varying the monomer to initiator ratio. For example, the molecular weight of the high molecular weight polyglycerol according to another aspect of the present invention can be achieved by adding 25 to 75 mole% of SSG. The characteristics of the exemplary hyperbranched polyglycerol can be seen in Table 1.

The high molecular weight polyglycerol according to the present invention can be decomposed under reducing conditions due to the disulfide groups present in the main chain or the branch chain and is biocompatible so that it is safer than conventional hyperbranched polyglycerols without disulfide bonds in the main chain or branch chain Do.

On the other hand, the present invention provides a drug carrier comprising the aforementioned hyperbranched polyglycerol. The hyperbranched polyglycerol according to the present invention may be useful as a polymer therapeutic agent, proteomics, and human serum albumin substitute in addition to a drug carrier.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited to the Examples.

Manufacturing example  1: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) Ethan-1-ol ( SSG , Monomer)

For reducing degradation and oxidation it is to produce a functional adult mounds polyglycerol of the terrain type, as shown in Figure 1a, the redox instability ₂ AB-type monomer, 1 to 2 of the formula - ((2- (oxiran -2 -Ylmethoxy) ethyl) disulfanyl) ethan-1-ol (SSG) was synthesized (Fig.

&Lt; Formula 1 >

Specifically, a solution of 2-hydroxyethyl disulfide (13.88 g, 0.09 mol) in t-butanol (150 mL) was added to potassium-t-butoxide (7.41 g, 0.09 mol) in t- Under stirring at room temperature for 15 minutes while stirring, and then continued stirring for 15 minutes. Subsequently, excess epichlorohydrin (54.6 g, 0.6 mol) was added dropwise using a syringe for 30 minutes and then stirred at room temperature for 15 hours. The salt formed was filtered off and the filtrate was evaporated on a rotary evaporator to give a mixture of product, by-product (diepoxide) and unreacted diol. The crude product was dissolved in 150 mL of methylene chloride and washed three times with water (30 mL each time). The organic layer was evaporated using a rotary evaporator and dried in a vacuum oven. The mixture was further purified by silica gel column chromatography using ethyl acetate / hexane (2: 1 v / v) to give 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl ) Ethan-1-ol (SSG) (6.2 g, 33%).

^{1 H NMR (600 MHz, CDCl} 3): δ ppm 3.90 (dd, 2H, J = 12.1, 6.0 Hz), 3.85 3.73 (m, 3H), 3.39 (dd, 1H, J = 11.7, 6.1 Hz), 3.19 - 3.15 (m, 1H), 2.90 (dt, 4H, J = 22.5, 6.1 Hz), 2.82 - 2.90 (m, 1H), 2.63 (dd, 1H, J = 5.0, 2.7 Hz) 2.20 (t, 1H, J = 6.3 Hz). ¹³ C NMR (150 MHz, CDCl ₃ ):? Ppm 70.91, 68.84, 59.66, 50.19, 43.50, 40.56, and 37.8.

Example  1: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) &Lt; / RTI &gt; ethan-1-ol ( PSSG ₁₀ ; Synthesis of polymer 1)

As shown in FIG. 1 b, a potassium alkoxide initiator made by the reaction of trimethylol propane (TMP) with a potassium methoxide solution Poly (2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol, a homopolymer, was prepared as follows by anionic ring- opening multibranched polymerization. In this example, a slow monomer addition reaction was used to synthesize the polymer in a controlled manner. The molecular weight of the polymer was controlled by the monomer to initiator ratio.

Specifically, trimethylol propane (TMP) (45 mg, 0.3352 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 37.5 μL, 0.127 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was then purged with argon and heated to 90 &lt; 0 &gt; C. Then, 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethane-1-ol monomer obtained in Production Example 1 (0.70 g, 3.32 mmol) . After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 占 폚 for 1 day to obtain a homopolymer of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (Polymer 1).

Example  2: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) &Lt; / RTI &gt; ethan-1-ol ( PSSG ₂₀ ; Synthesis of polymer 2)

Trimethylolpropane (TMP) (24 mg, 0.1788 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 20 μL, 0.0678 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was then purged with argon and heated to 90 &lt; 0 &gt; C. Then, 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethane-1-ol monomer obtained in Production Example 1 (0.70 g, 3.32 mmol) . After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 占 폚 for 1 day to obtain a homopolymer of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (Polymer 2).

Example  3: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) &Lt; / RTI &gt; ethan-1-ol ( PSSG ₅₀ ; Synthesis of Polymer 3)

Trimethylolpropane (TMP) (9 mg, 0.06705 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 7.5 μL, 0.0254 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was then purged with argon and heated to 90 &lt; 0 &gt; C. Then, 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethane-1-ol monomer obtained in Production Example 1 (0.70 g, 3.32 mmol) . After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 占 폚 for 1 day to obtain a homopolymer of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (Polymer 3).

Example  4: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) Ethan-1-ol and Glycidol  Copolymer (P ( G ₁₆₅ - co - SSG ₅₅ ); Synthesis of Polymer 4)

(Glycerol-co-2 - ((2- (oxiran-2-yl) -1H-pyrazol-3-yl) propionic acid as a copolymer was obtained by copolymerization of SSG prepared in Production Example 1 and non-decomposable glycidol Yl) methoxy) ethyl) disulfanyl) ethan-1-ol) (P (G-co-SSG)).

Specifically, trimethylol propane (TMP) (24 mg, 0.1788 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 20 μL, 0.0678 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was purged with argon and the flask was heated to 90 &lt; 0 &gt; C. A mixture of SSG (2.1 g, 10 mmol) and glycidol (G) (2.22 g, 30 mmol) was added dropwise using a syringe pump for 12 hours. After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 캜 for 1 day to obtain a copolymer of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan- &Lt; / RTI &gt;

Example  5: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) Ethan-1-ol and Glycidol  Copolymer (P ( G ₆₀ - co - SSG ₃₀ ); Synthesis of Polymer 5)

Trimethylolpropane (TMP) (12 mg, 0.0894 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 10 μL, 0.0339 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was purged with argon and the flask was heated to 90 &lt; 0 &gt; C. A mixture of SSG (0.568 g, 2.7 mmol) and glycidol (G) (0.4 g, 5.4 mmol) was added dropwise using a syringe pump for 12 h. After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 캜 for 1 day to obtain a copolymer (Polymer 5) of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan- &Lt; / RTI &gt;

Example  6: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) Ethan-1-ol and Glycidol  Copolymer (P ( G ₂₀ - co - SSG ₂₀ ); Synthesis of Polymer 6)

Trimethylolpropane (TMP) (24 mg, 0.1788 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 20 μL, 0.0678 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was purged with argon and the flask was heated to 90 &lt; 0 &gt; C. A mixture of SSG (0.757 g, 3.6 mmol) and glycidol (G) (0.266 g, 3.6 mmol) was added dropwise using a syringe pump for 12 hours. After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 占 폚 for 1 day to obtain a copolymer of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol and glycidol &Lt; / RTI &gt;

Example  7: 2 - ((2- ( Oxirane -2- Ylmethoxy )ethyl) Disulfanyl ) Ethan-1-ol and Glycidol  Copolymer (P ( G ₁₀ - co - SSG ₂₀ ); Synthesis of Polymer 7)

Trimethylolpropane (TMP) (24 mg, 0.1788 mmol) was placed in a 2-neck round bottom flask. Potassium methoxide (25 wt%, 20 μL, 0.0678 mmol) in methanol was diluted with 0.70 mL of methanol and the solution was added to the flask and stirred at room temperature under an argon atmosphere for 30 min. Excess methanol was removed using a rotary evaporator and dried in a vacuum oven (90 &lt; 0 &gt; C, 3 h) to give a white salt initiator. The flask was purged with argon and the flask was heated to 90 &lt; 0 &gt; C. A mixture of SSG (0.757 g, 3.6 mmol) and glycidol (G) (0.133 g, 1.8 mmol) was added dropwise using a syringe pump for 12 hours. After completion of monomer addition, the reaction was continued for an additional 5 hours. The resulting polymer was dissolved in 1.0 mL of methanol, precipitated with excess diethyl ether, and washed twice with diethyl ether. The product was dried under vacuum at 90 占 폚 for 1 day to obtain a copolymer of 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol and glycidol &Lt; / RTI &gt;

Test Example  1: Characterization of Monomers and Polymers

Successful synthesis of the monomer (SSG) synthesized in Preparation Example 1 was confirmed by ¹ H-NMR and ¹³ C-NMR spectroscopy (FIGS. 2A, 3 A and 3 B). As shown in Figures 2a, 3a and 3b, in the case of 2- ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan-1-ol (SSG, monomer) And a hydroxyl group.

On the other hand, in the case of the polymer prepared from the monomer, the molecular weight was controlled to the monomer-to-initiator ratio and it was confirmed by GPC and ¹ H NMR spectroscopy. The ¹ H NMR spectra of polymer 2 and polymer 8 are shown in FIGS. 2B and 2C, respectively.

The molecular weight of polymer 2 was calculated to be 3709 g / mol, as obtained from the NMR data of Fig. 2b using the following formula:

Number of repeating units ( n ) = 22 Integral value x 3 Proton number of TMP (methyl, 3H) / 4 Disulfide of SSG (4H) Proton number of neighboring protons = 17;

M _n = 210.31 < molecular weight of SSG monomer > x 17 + 134.17 < molecular weight of TMP initiator > = 3709.44 g / mol.

The molecular weight of Polymer 7 was also calculated to be 4424 g / mol, as obtained from the NMR data of FIG. 2C using the following formula:

Number of repeating units (SSG) = 17.47 <integral value> x3 <number of protons of TMP (methyl, 3H)> / 4 <number of disulfide proton adjacent to the proton of SSG (4H)> = 13;

The number of repeating units (G) = number of protones of [75.52 <integral value> x3 <TMP (methyl, 3H)> x {(13 <number of repeating units of SSG> x9 <proton of SSG except disulfide neighboring proton The number of protons in the monomer (5H) > = 21; the number of protons in the monomer;

M _n = 74.08 (molecular weight of G monomer) x 21 + 210.31 (molecular weight of SSG monomer) x 13 + 134.17 (molecular weight of TMP) = 4423.88 g / mol.

Considering the branched structure of the product and the PS (polystyrene) reference material used for the homopolymers and copolymers prepared in Examples 1 to 7, the controlled molecular weight (homopolymer: 2200 - 10650 g / mol; : 5000 - 24000 g / mol) and narrow molecular weight distribution (homopolymer M _w / M _n <1.15; Copolymer M _w / M _n <1.38). Interestingly, the homopolymer showed complete conversion (90-100%) of the SSG monomer, but the addition of glycidol as comonomer slightly reduced the SSG conversion (60-85%). For example, by varying the supply amount of SSG (25-75 mol%) in the polymerization reaction of the copolymer, copolymers of various compositions containing SSG in a proportion of 28 to 55 mol% were obtained. This result shows that the two monomers have different reactivities.

All of the homopolymers (PSSG) and copolymers (P (G-co-SSG) were soluble in organic polar solvents such as methanol, DMSO and DMF in addition to water. In Figure 2b, ¹ H of the homopolymer (polymer 2) NMR spectra were determined for each of the disulfide neighboring carbons (d, e at 2.8-3.1 ppm), the methyl and methylene groups of the initiator (0.8 ppm, respectively), with the polyether backbone (polyether backbone of PSSG at 3.4-4.2 ppm and c) And a characteristic proton peak of b at 1.3 ppm.) Dispersed at 2.8-3.1 ppm The average molecular weight M _n was calculated by measuring the integral of the initiator signal at 0.8 ppm for the signal of the neighboring carbon Together, the target molecular weight and the data obtained by NMR spectroscopy were consistent.

In addition, the ¹ H NMR spectrum of the copolymer (polymer 7) shown in Figure 2C shows a chemical shift similar to that of the homopolymer. However, in the case of the copolymer, a much stronger signal (3.4-4.2 ppm) was observed in the polyether backbone signal due to the additional polyether backbone present in the PG as compared to the homopolymer. The coverage and molecular weight of SSG were calculated by integrating peaks of proton from the polyether skeleton at 3.4-4.2 ppm with disulfide adjacent carbons at 2.8-3.1 ppm (SSG) for initiator signals at 0.8 ppm. Detailed analysis of ¹ H and inverted-gated ¹³ C NMR also confirmed the branched structure of the PSSG polymer.

The GPC results show the unipolar distribution and the controlled molecular weight values (Table 1). However, mismatches in molecular weights as measured by NMR and GPC have been found and these results can be explained by the presence of branched structures and multiple hydroxyl functionalities. These spherical algebraic structures do not contribute to the overall hydrodynamic radius of the polymer, and the fact that GPC has been corrected with PS may be another source of variation.

The characteristic analysis data of all the polymers synthesized in the present invention are shown in Table 1 below.

Polymer composition
(Target) Polymer Composition (NMR) ^a Mn
(Target) % SSG
(Target) Mn
(NMR) ^a % SSG
(NMR) ^a Mn
(GPC) ^b Mw / Mnb
(GPC) ^b Reference PG ₁₅₀ PG ₁₃₉ 11200 0 10400 0 14300 1.50 Polymer 1 PSSG ₁₀ PSSG ₉ 2240 100 2030 100 3780 1.10 Polymer 2 PSSG ₂₀ PSSG ₁₉ 4340 100 4130 100 3600 1.12 Polymer 3 PSSG ₅₀ PSSG ₅₃ 10650 100 11280 100 8500 1.15 Polymer 4 P (G ₁₆₅ -co-SSG ₅₅ ) P (G ₁₁₄ -co-SSG ₄₈ ) 23920 25.00 18670 29.63 15800 1.28 Polymer 5 P (G ₆₀ -co-SSG ₃₀ ) P (G ₄₆ -co-SSG ₁₈ ) 10890 33.33 7460 28.12 5370 1.38 Polymer 6 P (G ₂₀ -co-SSG ₂₀ ) P (G ₂₁ -co-SSG ₁₃ ) 5820 50.00 4420 38.23 5000 1.25 Polymer 7 P (G ₁₀ -co-SSG ₂₀ ) P (G ₁₀ -co-SSG ₁₂ ) 5080 66.67 3530 54.54 4620 1.23

^a &lt; ¹ &gt; H NMR spectroscopy.

^b Measured using GPC-RI in DMF with polystyrene reference material.

PG ₁₅₀ is a polymer synthesized for comparison with PSSG.

The MALDI-ToF spectra of the homopolymer (polymer 1) and the copolymer (polymer 7) according to the present invention are shown in FIGS. 4A and 4B, respectively. The illustration in Figure 4b shows the detailed molar mass and placement of the copolymer in the range of 2600 to 2700 g / mol. The signal spacing corresponds to the mass of the linear combination of each monomer in the homopolymer and the copolymer (G: 74.08 g / mol, SSG: 210.31 g / mol).

The presence of each fragment of the TMP initiator and the functional monomer in the homopolymer and the copolymer was clearly confirmed by the MALDI-ToF measurement method. As shown in Figure 4a, two distribution modes were observed due to different ions such as H ⁺ and K ⁺ with fragmented species. For example, the major molecular weight distribution at 1224.82 corresponds to the molecular weight (TMP (134.17) + SSG (210.31) x 5 + K ⁺ (39.1)) of PSSG with potassium as counterion and the sub- Corresponds to the molecular weight (TMP (134.17) + SSG (210.31) x 5 + H ⁺ (1)) of PSSG having H ⁺ as an ion. In addition, the spacing between the signals (210.31 g / mol) matches well with the functional SSG units contained in the PSSG, demonstrating the presence of SSG monomers. Unlike the results of the PSSG homopolymer, the MALDI-ToF results of the P (G-co-SSG) copolymer show the presence of two modes of distribution. As shown in Figure 4b, the presence of TMP-initiated copolymers of G and SSG is clearly demonstrated. In particular, peaks corresponding to the variously combined copolymers of G and SSG are separated from the mass spectrum (the illustration in FIG. 4B). For example, the mass peak at 2670.63 corresponds to a copolymer having TMP as initiator, G in eleven units, SSG in eight units, and K ⁺ as counterion (TMP (134.17) + G (74.08) x11 + SSG (210.31) x 8 + K ⁺ (39.1)). It should be emphasized that the spacing of the signals corresponds to the mass of linear combinations of various monomers in the copolymer (G: 74.08 g / mol, SSG: 210.31 g / mol) Prove the reaction clearly.

Test Example  2: In-situ ( in situ Copolymerization reaction kinetics

In order to analyze the distribution of comonomers during the copolymerization reaction, the copolymerization reaction behavior between the SSG monomers of the present invention and other monomers was studied. Online ¹ H NMR spectroscopy has been used to observe the dynamics of epoxide monomers because it is a quantitative method that allows easy access between the two spectra in a fraction of a second at shortest intervals of minimum and consumption of comonomers. However, when ¹ H NMR is used in the present invention, it is limited to monitoring the monomer consumption since the ¹ H NMR signals of the two monomers and polymer overlap. Therefore, we used quantitative in situ monitoring of the two monomers for bulk copolymerization reactions based on ¹³ C NMR spectroscopy. Specifically, the copolymerization reaction of G and SSG was carried out in a conventional NMR tube at 50 DEG C without using any solvent. In the bulk polymerization, a quantitative ¹³ C NMR spectrum can be observed in a few minutes due to the natural abundance of the ¹³ C isotope. In this way, the microstructure of the polymer growing at any time over the reaction can be observed.

Specifically, methoxyethanol (0.5 g, 6.57 mmol) and cesium hydroxide monohydrate (0.378 g, 2.25 mmol) were reacted in a round bottom flask at 60 ° C under an argon atmosphere to prepare an initiator. The initiator solution and comonomers (G and SSG) were placed in 4.0 mL vials and stirred thoroughly in an ice bath. The mixture was transferred to a conventional NMR tube under an argon atmosphere and then sealed with a septum on an ice bath. Kinetic measurements using ¹³ C NMR were recorded on a 600 MHz VNMRS system with a 5 mm PFG AutoX DB probe and measured without solvent. Reference Dynamics ¹³ C NMR experiments required 64 transient signals, a spectral width of 1894 Hz, and a recirculation delay time of 10 s for one experiment to be achieved with 13.7 μs 90 ° pulses and 70 experiments Was used with a flip angle of 45 [deg.] And an inverse gated decoupling experiment over a period of 13 hours.

FIG. 5 shows ¹³ C NMR spectra of in-situ copolymerization kinetics of G and SSG (monomer ratio 1: 1). The left side shows the overlay of the spectra collected at the designated time and the right side shows the zoom-in spectrum showing the methine carbon in the epoxide at 53 and 55 ppm, corresponding to G and SSG monomers, respectively 150 MHz, 323 K).

Figure 5 shows a typical series of ¹³ C NMR spectra collected for the copolymerization of G with SSG, which shows the simultaneous appearance of the polyether skeleton of the polymer at 70-78 ppm with two monomers (labeled G and red highlighted in blue The SSG). In order to measure the monomer consumption, the resonance (55 ppm for G and 53 ppm for SSG) of representative methine groups in the two epoxide monomers was compared during the course of the reaction. As shown in Fig. 5, the time required for complete monomer consumption of G in the copolymerization reaction was shorter than that of SSG. For example, signals for each monomer could not be detected after 165 and 440 minutes, respectively, indicating different reactivity of each monomer. This difference is because the structure of the disulfide spacer in the SSG interferes with access from the other SSG monomers, thereby reducing the reactivity during the polymerization reaction.

On the other hand, the inventors have chosen 50 ° C as the polymerization temperature in the kinetic studies to prevent the possibility of explosion in the NMR tube due to the low flash point of the glycidol monomer (66 ° C).

Figure 6 shows the monomer conversion percent versus total conversion for copolymerization of G (blue square) and SSG (red circle) measured from ¹³ C NMR kinetics quantitatively at 50 占 폚 in bulk.

It should be noted that in the first ¹³ C NMR spectra the monomer conversion is set to 0% and the conversion ratio is calculated using the measured integral for the signal of the two neighboring disulfides, It remained constant. As shown in Figure 6, the molar ratio of SSG units in the polymer chain was lower than the monomer feed in the initial stage and was rapidly increased upon consumption of the G monomer near the final stage of the reaction, for example, a total conversion of 51% , The conversion rates of G and SSG are 80% and 22%, respectively (G is 3.6 times more reactive than SSG). Thus, there is a gradient of monomer inclusion during the copolymerization of P (G-co-SSG). Nevertheless, unlike the one-pot reactions performed in bulk during NMR measurements, the present inventors used slow monomer addition methods to synthesize copolymers in solvents. Therefore, it was assumed that the difference in reactivity between the two monomers should be suppressed in order to obtain a more homogeneous distribution of the two monomers.

Test Example  3: Polymer degradation

Decomposition of the homopolymer (PSSG) through disulfide reduction was studied using NMR and GPC.

a) NMR analysis: To study the D ₂ O disulfide die thio tray toll -d ₁₀ 2 equivalents of a solution _(d-10 DTT) solution treatment before and after the chemical shift value of the redox-dependent degradation of the polymer as compared to the. For the NMR study, about 15 mg of PSSG polymer was dissolved in 0.60 mL of DTT solution and disulfide reduction and degradation of the polymer were observed by ¹ H and ¹³ C NMR spectroscopy, respectively.

b) GPC analysis: DTT (2 equivalents of disulfide) was added to a solution of P (G-co-SSG) in DMF and samples were analyzed using GPC-MALLS in DMF containing 10 mM lithium bromide. The molecular weight ( M _n And M _w ) and PDI were measured and compared before and after DTT treatment.

The degradation diagram of the PSSG chain treated with DTT is shown in FIG. 7A, and the change in the ¹ H- and ¹³ C NMR spectra of the PSSG homopolymer (polymer 2) after DTT treatment (10 min) is shown in FIG.

The disulfide groups present in the monomer and polymer chains degrade the chains under reducing conditions. Dithiothreitol (DTT) has a high disulfide reduction efficiency due to (1) a structural tendency to form a stable six-membered ring having an internal disulfide bond and (2) a variety of organic solvents capable of dissolving the PG- , Dithiothreitol was used as a reducing agent. To study DTT-induced degradation of PSSG (Fig. 7a), reduction of PSSG was observed by dissolving PSSG in a solution of 2 equivalents of DTT-d _{10 in} D ₂ O and ¹ H and ¹³ C NMR before and after DTT treatment The spectra were compared. As shown in the ¹ H and ¹³ C NMR results, disulfide neighboring protons and carbon signals disappeared with simultaneous appearance of carbon signal with thiol adjacent protons that could be monitored during DTT treatment (Fig. 7B). It was also confirmed that the disulfide bond of the polymer chain in the aqueous solution was rapidly degraded within 10 minutes, because the solubility of PSSG and DTT in water is high so that DTT is easy to approach the polymer chain.

On the other hand, the decomposition products of the copolymer were further investigated by GPC analysis. GPC traces of P (G ₁₆₅ -co-SSG ₅₅ ) (polymer 4) before DTT treatment (black curve) and after red curve (polymer 4) are shown in FIG. 8A and the diagrams of decomposition products are shown in FIG. A possible mechanism for the origin of the three peaks in the post-treatment GPC trace is shown in Figure 8c.

Figure 8 shows how the molecular weight of the P (G ₁₆₅ -co-SSG ₅₅ ) chain changes after DTT-induced degradation in DMF for 1 hour. As expected, the molecular weight peak located at 18000 g / mol shifted to the left, indicating that the small polymer P (G ₁₆₅ -co-SSG ₅₅ ) corresponds to three different peaks at 2600, 4600 and 12900 g / mol, respectively Indicating that the fragment was successfully degraded. It is interesting that each of the peaks exhibits different intensities. Considering that the non-degradable G monomers are more reactive than the redox-degradable SSG monomers, the core block inside the copolymer contains more of the non-degradable G fraction while the outer periphery contains more degradable SSG There will be many. This gradient in the distribution of the monomers in the highly branched copolymer of P (G ₁₆₅ -co-SSG ₅₅ ) results in a higher intensity of the lower molecular weight fraction than the higher molecular weight (Figure 8b). We have proposed a possible mechanical insight into the degradation of the polymer from differences during polymerization (Figure 8c). Specifically, the incoming monomers may react with either a 2 ° hydroxyl group (blue, -OH group) or a 1 ° hydroxyl group (green, -OH group). When one reacts through a 2 ° hydroxyl group, the new polymer chain remains after DTT treatment. Conversely, when the subsequent monomer reacts with a 1 DEG hydroxyl group, the new polymer chain can be decomposed from the parent polymer chain. For this reason, the structure of the degradation product is divided into small sections and large sections, where the difference in molecular weight is almost double. On the other hand, the presence of a large core slice is due to the TMP initiator comprising three hydroxyl groups. Theoretically, a TMP initiator can have three large segments, so a large core segment can have a molecular weight three times the molecular weight of a large segment. In summary, the structure of the degradation products can be divided into three different structures by two different reactive sites of initiator and monomer.

Test Example  4: Biocompatibility analysis (cytotoxicity analysis)

The cytotoxicity of PG ₁₅₀ , P (G ₆₀ -co-SSG ₃₀ ) and PSSG ₂₀ was evaluated to examine their potential as drug carriers.

Specifically, human epithelial carcinoma cells (HeLa) and human diploid cells (WI-38) were purchased from Korean Cell Line Bank (Seoul, Korea). Cytotoxicity assays were performed using conventional MTT assays. Cells were seeded in 96-well plates at a density of 7 x 10 ³ cells per well and cultured at 37 ° C in 5% CO ₂ for 24 hours. HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. WI-38 cells were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies) supplemented with 10% FBS, 25 mM sodium bicarbonate, and 1% penicillin-streptomycin. After removing the culture medium, the wells were washed with PBS. Next, 90 μL of fresh medium, 10 μL of various concentrations of PG ₁₅₀ , P (G ₆₀ -co-SSG ₃₀ ) and PSSG ₂₀ solution were replenished in each well and incubated for an additional 24 hours. For MTT analysis, each well was washed with PBS. Then, 10 μL of thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) stock solution (5 mg / mL) and 90 μL of fresh medium were added. After incubation for 4 hours, 100 μL of the DMSO solution was replaced with the polymer solution to dissolve the MTT-formazan product, and the plate was gently stirred at room temperature for 15 minutes. Absorbance of the solution was read at 540 nm using 620 nm as reference. For CCK-8 analysis, each well was washed with PBS. Then, 10 μL of a water-soluble tetrazolium salt (CCK-8, Dojindo Molecular Technologies) and 90 μL of fresh medium were added. After incubation for 4 hours, the plate was gently agitated for 15 minutes at room temperature and the absorbance of the solution read at 450 nm.

The results are shown in Fig. Figure 9a shows in vitro cell survival analysis of PG ₁₅₀ (black), P (G ₆₀ -co-SSG ₃₀ ) (gray) and PSSG ₂₀ polymer (white) in normal cells WI-38 as measured by MTT assay FIG. 9B shows the in vitro cell survival assay in cancer cell HeLa as measured by MTT assay, and FIG. 9C shows the in vitro cell survival assay in normal cell WI-38 as measured by CCK assay , And Figure 9d shows the in vitro cell survival assay in cancer cell HeLa as measured by CCK assay.

MTT assay was based on mitochondrial dehydrogenase activity, while CCK-8 assay was performed using an electron mediator, 1-methoxyphenazinium methyl (H), to detect the activity of intracellular dehydrogenases NAD (H) and NADP Sulfate (1-methoxy PMS) is used. Thus, CCK-8 is more sensitive than the MTT assay, which can reflect overall cell viability, whereas the MTT assay shows mitochondrial activity. As shown in Figure 9, the cell viability of each cell line treated with various concentrations of the polymer solution exceeded 90% to a concentration of 100 μg / mL in both analyzes. PG1 showed good cell viability at all tested concentration ranges, but PSSG ₂₀ showed relatively lower cell viability at higher concentrations of 1000 μg / mL than the other two samples. This is because degradation of the disulfide bond under the intracellular redox condition affects the intracellular redox mechanism and weakens the cytotoxicity. Generally, a copolymer of P (G ₆₀ -co-SSG ₃₀ ) with 30 mol% disulphide bonds exhibits a higher cell survival rate than the homopolymer PSSG ₂₀ at 1000 μg / mL. Taken together, these results demonstrate that the amount of degradable linkage affects the overall cell viability. It should also be noted that the content of degradable linkages in P (G ₆₀ -co-SSG ₃₀ ) is much higher than that of conventionally-known degradable polymers (generally containing 6 to 15 mol% of degradable linkages). Considering that the preferred concentration for the biologic application of polymers is generally less than 1000 μg / mL, homopolymers and copolymers with novel redox-degradable residues of SSG have no side effects on mitochondrial activity and cell viability.

Claims (12)

Hyperbranched polyglycerols having disulfide bonds in the backbone or branched chain.
The hyperbranched polyglycerol according to claim 1, wherein the hyperbranched polyglycerol comprises a glycidol-based compound having a disulfide bond as a repeating unit.
3. The method according to claim 2, wherein the glycidol compound having a disulfide bond is 2 - ((2- (oxiran-2-ylmethoxy) ethyl) disulfanyl) ethan- &Lt; RTI ID = 0.0 > polyglycerol:
&Lt; Formula 1 &gt;

3. The process according to claim 2, wherein the hyperbranched polyglycerol is produced by anionic ring-opening multibranching polymerization of the glycidol-based compound having the disulfide bond. Glycerol.
The hyperbranched polyglycerol according to claim 2, wherein the hyperbranched polyglycerol comprises 10 to 50 glycidol-based compounds having the disulfide bond.
The high molecular weight polyglycerol according to claim 2, wherein the high molecular weight polyglycerol has a molecular weight of 2000 to 11000 g / mol.
The highly branched polyglycerol according to claim 2, wherein the highly branched polyglycerol has an M _w / M _n of 1.10 to 1.15.
The highly branched polyglycerol according to claim 2, wherein the highly branched polyglycerol further comprises glycidol as a repeating unit.
9. The high molecular weight polyglycerol according to claim 8, wherein the highly branched polyglycerol is produced by a copolymerization reaction of a glycidol-based compound having the disulfide bond and glycidol.
The highly branched polyglycerol according to claim 8, characterized in that said highly branched polyglycerol has a molecular weight of 5000 to 24000 g / mol.
The highly branched polyglycerol according to claim 8, characterized in that said highly branched polyglycerol has an M _w / M _{n of} from 1.23 to 1.38.
12. A drug carrier comprising a high molecular weight polyglycerol according to any one of claims 1 to 11.

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