1. Introduction

The aqueous solubility of drugs is a vital feature for their bioavailability which governs their dissolution and transfer process and is thus critical for drug delivery and drug development ^[1]. A number of newly discovered drugs have failed in commercialization due to their poor water solubility and cell membrane permeability ^[2]. To overcome this issue,various solubility-enhancing techniques are developed,and many enhancing auxiliary techniques are investigated to improve the dissolution of the drugs exhibiting a low aqueous solubility ^[3]. Among these auxiliary solubilizing enhancers,dendrimers are ideal candidates due to their distinctive and interesting dendritic architecture which make them admirably feasible in versatile biological applications ^{[4, 5]}. In particular,polyamidoamine (PAMAM) dendrimer is one of the most popular species and is extensively investigated as an enhancer to improve the dissolution of hydrophobic drugs,such as ketoprofen ^[6],ibuprofen ^[7], aceclofenan ^[8],and riboflavin ^[9]. PAMAM dendrimers present primary amines on the surface which may electrostatically interact with the negatively charged moieties in the drug molecules. Besides,with varying dimensions the internal cavities of the molecules provide a hydrophobic environment that can encapsulate hydrophobic drugs in water. In addition,the tertiary amines of the dendrimeric structure may interact with certain functional groups of the drug molecules via hydrogen bond formation. Altogether,these characteristic properties make dendrimers suitable for drug solubilization.

Folic acid (Fig. 1a) is a well known member of the vitamin B family as well as a tumor-targeting molecule thanks to its high affinity toward the folate receptor (FR) ^[10]. It is essential for a variety of bodily functions,such as promoting rapid cell division and growth during infancy and pregnancy,and acting as coenzyme in the regeneration of methionine from homocysteine ^[11]. A folic acid deficiency can result in many health problems and symptoms, including neural tube defects,diarrhea,macrocytic anemia, pregnancy complications,and mental confusion,as examples ^[12]. The solubility of folic acid is thus crucial for its applications, nevertheless it is practically insoluble in water with a solubility reported as 1.6 mg/L at 25 ℃ ^[13]. Hence,we have interests in solubility enhancement of folic acid which is rarely reported in the literatures. Considering the costly preparation and purification of high generational molecules,dendrimer with a lower generation is of special interest for us to start the primary study. In this work, G0-G2 PAMAM dendrimers were synthesized and investigated for improving the solubility of folic acid. For the purpose of comparison,ethylenediamine (EDA,Fig. 1a) was chosen as a small-molecule reference. Although it is widely believed that the dendritic architecture accounts for the enhancing capability of dendrimers,few publications have made direct comparison between dendrimers and small molecules in terms of solubility performance. In order to reveal the possible ‘‘dendritic effect’’ ^{[14, 15]} in the dissolution of folic acid,the enhancement of PAMAM dendrimers and EDA on folic acid solubility was evaluated,both at pH 5 and pH 11. In addition,we believe that discovery of the solubilization pattern of the drug molecules under acidic/basic conditions will help us improve drug dissolution and drug delivery.

Fig. 1

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Fig. 1.Molecular structure of folic acid,ethylenediamine and G1 PAMAM dendrimer.

Folic acid,methyl acrylate and ethylenediamine were purchased from Shanghai Sinopharm Chemical Reagent Co.,Ltd. (Shanghai,China). Methyl acrylate and ethylenediamine were distilled before use. Double-distilled water was used to prepare all the solutions for solubility studies. 2.2. Synthesis of PAMAM dendrimers

PAMAM dendrimers were synthesized using the classic divergent method developed by Tomalia et al. ^[16]. In brief, ethylenediamine (0.45 g,7.49 mmol) in MeOH (10 mL) was added to methyl acrylate (5.15 g,59.8 mmol) and the solution was stirred at 25 ℃ for 24 h under nitrogen. The solvent and excess methyl acrylate were removed by rotary evaporation and then under vacuum to give an intermediate product bearing four terminal methyl ester groups (2.98 g,98.6%). Subsequently,ethylenediamine (4.93 g,82.2 mmol) was added to a methanol solution (10 mL) of that product (1.0 g,2.47 mmol). The reaction solution was stirred at 25 ℃ for 24 h under nitrogen,and then the solvent and excess ethylenediamine were removed. The residue was washed with ether and concentrated under vacuum to give a product bearing four terminal amino groups,defined as G0 PAMAM dendrimer (1.23 g,96.3%). By repeating the Michael addition and amidation reaction mentioned above,G1 (yield: 96.7%,Fig. 1a) and G2 (yield: 97.3%) PAMAM dendrimers were synthesized,and their structures confirmed by 1H NMR and IR spectra (see Supporting information). The characteristics of these PAMAM dendrimers are listed in Table 1.

Table 1

Table 1 Characteristic data of the G0/G1/G2 PAMAM dendrimers.

Table 1 Characteristic data of the G0/G1/G2 PAMAM dendrimers.

The solubility of folic acid in the presence of PAMAM dendrimers was determined at pH 5 and pH 11. These two pH values were chosen to provide typical acidic/basic conditions because we believed the ionization status of folic acid significantly affects its solubilization process. The concentration of dendrimers is in the range from 1 mmol/L to 10 mmol/L. Sodium hydroxide and hydrochloric acid were used to adjust the pH value of the sample solutions. The sample preparation is similar for each system. In brief,folic acid was added to the solution containing dendrimer,or ethylenediamine,and the mixture was incubated in a vibrating water bath at 25 ℃ for 24 h. The solution was then centrifuged and the supernatant was diluted to the proper concentration for UV characterization. The absorbance of the solution at the characteristic wavelength of 280 nm was measured using a UV-visible spectrophotometer (TU-1900) and 10 mm path-length quartz cells. For the calculation of solubility,0.05 mg/mL folic acid in 0.1 mol/L acetate buffer (pH 5.8) was prepared and diluted to several sample solutions of certain concentration (0.001-0.03 mg/ mL). The absorbance of the sample solutions at 280 nm was measured and plotted to make the calibration curve of folic acid. 3. Results and discussion 3.1. PAMAM dendrimers enhance the solubility of folic acid at pH 11

The solubility of folic acid under the basic condition was first investigated in the presence of G0-G2 PAMAM dendrimers. Because of the low pKa values (4.65 and 6.75) ^[17],the two carboxylic acid groups in folic acid are entirely deprotonated and negatively charged at pH 11. The solubility of folic acid without any enhancer compound was determined to be ~2 mg/mL,indicating that the ionization of folic acid plays an important role in its solubilization process. When PAMAM dendrimers are introduced, the dissolution of folic acid in water is significantly enhanced (Fig. 2a). For example,the solubility increases to ~20 mg/mL in the presence of 1 mmol/L G0 PAMAM dendrimer and to ~135 mg/mL with 10 mmol/L G2 dendrimer. The solubility of folic acid increases with the concentration of dendrimer in all three generations, which is consistent with the literature ^{[6, 7, 8, 9]}. At the higher concentration,there are more dendrimer molecules in the solution that can interact with folic acid,help the solvation of folic acid and in turn enhance its solubility. Meanwhile,the solubility of folic acid also increases with the generation of the dendrimer. At the same concentration,the higher generation of dendrimer possesses more functional groups on the surface and more internal cavities, providing more ‘‘interaction sites’’ to improve the solubility of folic acid. For the same reason,the G2 dendrimer displays the best enhancing effect in terms of the dendrimer concentration,while the G0 dendrimer showing the worst (Fig. 2a).

Fig. 2

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Fig. 2.Solubility of folic acid at pH 11 in the presence of (a) varied concentration of G0/G1/G2 PAMAM dendrimers,(b) G0/G1/G2 PAMAM dendrimers with the same concentration (32 mmol/L) of primary amine groups,and (c) ethylenediamine/G2 PAMAM dendrimer with the same concentration (20 mmol/L or 80 mmol/L) of primary amine groups.

The dendritic structure is a fundamental feature of dendrimers and always plays an important role in dendrimer applications ^{[14, 15]}. The term ‘‘dendritic effect’’ is widely used to describe the unique physico-chemical property observed for dendrimers in terms of their generation level,generally implying that higher generation exhibits better capability. However,this property is rarely examined in solubility enhancement studies ^[18]. We are thus interested in discovering the dendritic effect in the dendrimer-related solubilization process. This can be carried out by comparing the folic acid solubility in the presence of different generational dendrimers with the same concentrations of primary amines. The solubility of folic acid in 8 mmol/L G0,4 mmol/L G1 and 2 mmol/L G2 PAMAM dendrimer solutions are shown in Fig. 2b. The concentration of primary amine group is 32 mmol/L for each sample based on the number of the end groups (Table 1). All three samples give similar result,with G0 showing a slightly higher enhancing capability than G1 and G2. This suggests that at pH 11, the solubility of folic acid is simply related to the total availability of primary amines,and G2 does not display the advantage of dendritic effect over G0 and G1 dendrimers.

To clarify this conclusion,the solubilization of folic acid was further investigated with a small molecule,i.e.,ethylenediamine, which has a very simple molecular structure consisting of two primary amines. As shown in Fig. 2c,ethylenediamine displayed an enhancing effect on the dissolution of folic acid similar to G2 PAMAM dendrimer in the presence of 20 mmol/L of primary amines. In the case of higher concentration (80 mmol/L primary amines),the ethylenediamine solution dissolved even more folic acid than G2 dendrimer. This result further proves that under the basic condition of pH 11,the ionization of folic acid and the number of primary amine groups are the key factors for the dendrimerrelated dissolution of folic acid. The electrostatic interaction between ionized folic acid and the primary amine of the dendrimer is the major entity responsible for the solubility enhancement. This also explains why G2 dendrimer shows a lower enhancing level than G0 and G1. In the G2 PAMAM dendrimer,more primary amine groups are fixed on one molecule,hence,the interaction between folic acid and the amine groups is hindered because of the steric effect. In addition,since there is no tertiary amine group in ethylenediamine,the possible interaction between folic acid and the tertiary amine of the dendrimer,if any,is insignificant. 3.2. PAMAM dendrimers enhance the solubility of folic acid at pH 5

In order to identify the interaction between folic acid and dendrimer molecules under the acidic condition,we investigated the dissolution of folic acid in the presence of G0-G2 PAMAM dendrimers at pH 5. The solubilization assay shows a similar tendency as at pH 11,i.e.,the solubility of folic acid increases with the concentration of dendrimer for all three generations,and with the dendrimer generation at a fixed concentration,showing an identical order of the increasing rate G2 > G1 > G0 (Fig. 3a). However,the solubility values largely decrease in comparison with those under the basic condition. For example,about 60 mg/mL folic acid can dissolve at pH 11 in the presence of 10 mmol/L G0 dendrimer,while at pH 5 only ~2 mg/mL folic acid was determined in 10 mmol/L G0 solution. Exhibiting pKa value of 4.65 and 6.75, the two carboxylic acid groups of folic acid are practically not deprotonated at pH 5,whereas the primary amines in dendrimer are almost fully protonated because of the high pKa (9-10). The much lower solubility of folic acid under the acidic condition indicates that the interaction between these two species is remarkably weaker than that between ionized carboxylic groups and the primary amines at pH 11. This further suggests the significant impact of ionization of folic acid over its aqueous solubility.

Fig. 3

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Fig. 3.Solubility of folic acid at pH 5 in the presence of (a) varied concentration of G0/G1/G2 PAMAM dendrimers,(b) G0/G1/G2 PAMAM dendrimers with the same concentration (32 mmol/L) of primary amine groups,and (c) ethylenediamine/G2 PAMAM dendrimer with the same concentration (20 mmol/L or 80 mmol/L) of primary amine groups.

Aiming to discovery the dendritic effect of dendrimer on the dissolution of folic acid at pH 5,we subsequently compared the enhancing ability of G0/G1/G2 dendrimer at the same concentration of terminal primary amines. The solubility of folic acid in the presence of 8 mmol/L G0,4 mmol/L G1 and 2 mmol/L G2 are shown in Fig. 3b. In contrast to the results obtained at pH 11,the solubility of folic acid at pH 5 increases with generation even at identical amine concentration,giving ~2 mg/mL,~6 mg/mL and ~11 mg/mL for G0,G1 and G2 PAMAM dendrimer,respectively. This suggests that the interaction offered by the surface primary amines does not play the dominant role in the dissolution of folic acid. Accordingly,this process may be attributed to the interaction inside the dendrimer molecules,i.e.,the interaction with tertiary amine groups or/and hydrophobic encapsulation. Considering the concentration of tertiary amine (16 mmol/L,24 mmol/L,and 28 mmol/L for G0,G1,and G2 respectively),we then deduce that the hydrophobic encapsulation of dendrimer molecules accounts for their solubility enhancement of folic acid. A higher generation of dendrimer provides more compact internal cavities that may be more capable to encapsulate the folic acid molecule and help its solubilization process. For the G0 dendrimer,however,the structure is more open and cannot provide a significant encapsulation effect,showing a considerably lower enhancing capability. These results demonstrate the dendritic effect induced by the hyperbranched structure of the dendrimer molecules.

In order to verify the importance of the dendritic effect on the solubility of folic acid,ethylenediamine was further used as the control for comparison. As shown in Fig. 3c,ethylenediamine displays a much lower enhancer effect than G2PAMAM dendrimer, providing only 1/30 of dissolved folic acid at the amine concentration of 80 mmol/L,which is totally different from the case at pH 11. In addition,the solubility remains virtually unchanged when the concentration of ethylenediamine increases from 10 mmol/L to 40 mmol/L,suggesting the inactive effect of ethylenediamine on the dissolution of folic acid. On the contrary, the substance solubility markedly increases at higher concentration of G2 dendrimer,which further confirms the dendritic effect of PAMAM dendrimer caused by the internal encapsulation of folic acid under the acidic condition. 4. Conclusion

Low generational PAMAM dendrimers have the potential to increase significantly the aqueous solubility of poorly soluble drugs,such as folic acid. The improvement of drug solubility in PAMAM dendrimer solutions depends on the generation of the dendrimer,the concentration of dendrimer,and the pH of the medium. The solubility of folic acid increases with the concentration and the generation of the dendrimers,both at pH 5 and pH 11, however,the solubilization mechanism is different. At pH 11,the dendrimers greatly improved the dissolution of folic acid through the electrostatic interaction between the terminal primary amines in dendrimer molecules and the deprotonated carboxylic groups in folic acid,while no dendritic effect was observed. On the other hand,the solubility of folic acid with dendrimer enhancer at pH 5 is much lower than that at pH 11,owing to the non-ionization of folic acid. Interestingly,however,the positive dendritic effect could be observed which is attributed to the hydrophobic encapsulation related to the dendritic architectures. Further investigation,such as the dissolution assay of folic acid with higher generational PAMAM dendrimers and the in-depth examination on the interaction mechanism,is currently underway in our lab.

The work was supported by Wuhan Institute of Technology, Wuhan Chenguang Project (No. 200950431195) and the National Natural Science Foundation of China (No. 51003081).

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.02.004.