Colorimetric Determination of Polyamidoamine

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Accuracy and precision of the colorimetric method were assessed by statistical analysis. Acetylated ... Analytical Letters, 41: 444–455, 2008. Copyright © Taylor ...
Analytical Letters, 41: 444–455, 2008 Copyright # Taylor & Francis Group, LLC ISSN 0003-2719 print/1532-236X online DOI: 10.1080/00032710701484350

ASSAYS

Colorimetric Determination of Polyamidoamine Dendrimers and their Derivates using a Simple and Rapid Ninhydrin Assay Zhenhua Xu,1 Tongwen Xu,2 Yiyun Cheng,1,2 Minglu Ma,1 Peng Xu,1 Haiou Qu,1 and Longping Wen1 1

Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China 2 Laboratory of Functional Membranes, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, China

Abstract: A simple, accurate and rapid colorimetric method using ninhydrin reagent was developed for the determination of polyamidoamine (PAMAM) dendrimers (G4, G5, and G6) and their derivates in aqueous medium. This method was based on the interaction of the primary amino group of PAMAM dendrimers with ninhydrin reagent to form a blue-colored product with lmax at 570 nm. Beer’s law was obeyed in the concentration range of 25 –200 mg/ml of all the three investigated PAMAM dendrimers. The effects of experimental parameters such as reagent concentration and reaction time were studied to optimize the colorimetric method. Accuracy and precision of the colorimetric method were assessed by statistical analysis. Acetylated G5 PAMAM dendrimers with various acetylated rates were simultaneously measured by the described ninhydrin assay and NMR studies and the data obtained by the two methods approximately accorded with each other. Results showed that Received 9 May 2007; accepted 28 May 2007 Financial supports from the One Hundred Talent Project and Nanomedicine Research Project (kjcx2-sw-h12-01) of the Chinese Academy of Sciences, Anhui Talent Fund (2004Z023), the National Natural Science Foundation of China (30470871), and the Innovation Foundation of Graduate Student in University of Science and Technology of China (KD2004035) were highly appreciated. Address correspondence to Longping Wen, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China. E-mail: [email protected] 444

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the suggested procedures were suitable for the determination of PAMAM dendrimers and their derivates in aqueous solutions with satisfactory accuracy and precision. Keywords: Spectrophotometry, PAMAM, dendrimer, ninhydrin assay

1 INTRODUCTION Dendrimers are new-artificial macromolecules topologically based on the structure of a tree. They are hyperbranched, monodisperse, three-dimensional molecules, having defined molecular weight and host-guest entrapment properties. Polyamidoamine (PAMAM) dendrimer with an ellipsoidal or spheroidal shape is one of the most-studied starburst macromolecules (Tomalia et al. 1985). Due to special synthesis in a step-wise manner from branched monomer units, PAMAM dendrimers have some interesting properties, which distinguish them from classical linear polymers. They allow the precise control of size, shape, dimensions, density, polarity, flexibility, solubility, and placement of functional groups by choosing of these building units and functional group chemistry. As a result, they combine typical characteristics of small organic molecules and polymers that result in special physical and chemical properties (Tomalia et al. 1985; Tomalia et al. 1986; Tomalia and Dewald 1986; Tomalia et al. 1990). Up to now, PAMAM dendrimers have already attracted increasing attention for their applications in many fields including model chemistry or combination chemistry, electrochemistry and photochemistry, nanoparticle synthesis template, water purification, dye decolorization, monomolecular membranes, curing agents in epoxy resin systems, catalyzer in extensive areas, drug delivery systems and gene transfection in biomedical fields. Currently, different analytical methods including high-performance liquid chromatography (HPLC), mass spectrometry (MS) (Mohammad et al.; Thorsten et al. 2005) have been described for the determination of PAMAM dendrimer in aqueous solutions. These methods have enough sensitivity to determine lower concentration of dendrimer. However, it is always required to develop analytical methods using low cost techniques. UV-Vis spectrophotometry is still considered a convenient and economical technique for routine analysis (Raham and Kashif 2003). This paper suggests simple and sensitive colorimetric procedure for the determination of dendrimer in aqueous solutions. The method we described here is based on the reaction of primary amino group of dendrimer with ninhydrin. The aim of the present work was (a) to develop a colorimetric method for the determination of PAMAM dendrimers and their derivates in aqueous medium; (b) to optimize the procedure by varying the experimental parameters; (c) to validate the accuracy and precision of the procedure by statistical analysis.

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2 EXPERIMENTS 2.1

Materials and Reagents

All reagents used in the present study were of analytical reagent grade. G4, G5, and G6 PAMAM dendrimers stored in methanol (G4, 10 wt.%; G5 and G6, 5 wt.%) were purchased from Sigma-Aldrich; 0.2 mg/ml PAMAM dendrimer stock solutions of each generation was prepared in doubledistilled water (DDW) and further diluted according to the need with DDW. Ninhydrin was obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Ethylene glycol monomethyl ether solution was purchased from Shanghai Lingfeng Chemical Reagent Co. (Shanghai, China). Acetic anhydride was obtained from Guangdong Xilong Chemical Co., Ltd (Guangdong, China) and triethylamine was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). 2.2

Preparation of Hydrindantin

500 mg ninhydrin was dissolved in 12.5 ml distilled water at 1008C to obtain a yellow solution. Then, 500 mg ascorbic acid solution (dissolved in 25 ml distilled water) was added dropwise during stirring. The reaction mixture was kept on stirring for 15 min at room temperature and then cooled in a fridge. The obtained white precipitation (hydrindantin) was filtrated, washed three times with cool water, and dried before ninhydrin assay. 2.3

Ninhydrin Assay (Leane 2004)

Sodium acetate buffer (2M, 100 ml) was prepared by dissolving 14.11 g sodium acetate in 86 ml distilled water. Then 14 ml glacial acetic acid (2M) was added. The pH of the resulting solution was adjusted to 5.4. The ninhydrin reagent was freshly prepared on the day of the assay by dissolving 85 mg ninhydrin and 15 mg hydrindantin in 10 ml ethylene glycol-monomethyl ether solvent. For the assay, 100 mL of the sample and 100 mL of the prepared sodium acetate buffer (pH 5.4) were mixed together in a 1.5 ml Eppendorf tubes, then 100 mL of the ninhydrin reagent was added. The tubes were heated in boiling water for 10 min. After cooling, 300 mL of 60:40 ethanol/water mixtures was added to each tube. The absorbance of each solution was finally measured at 570 nm against a reagent blank by a UV-Vis spectrophotometer. 2.4

Optimizing the Procedure

To optimize the ninhydrin assay procedure, the effects of experimental parameters such as ninhydrin reagent concentration and reaction time were

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studied. The optimum procedures were obtained by varying one of the experimental parameters and observing its effect on the absorbance of the final bluecolored product. Take G6 PAMAM dendrimer for an example, different volumes (20 –100 mL) of ninhydrin reagents were added to 100 mL G6 PAMAM dendrimer (0.15 mg/ml), other experimental parameters being constant. In another separate experiment, the heating time was ranging from 0 to 20 min, other experimental parameters being constant. After the reactions completed, the absorbance of each solution was measured by a UV-Vis spectrophotometer.

2.5 Preparation of Calibration Curve Different volumes (0, 12.5, 25, 37.5, 50, 62.5, 75, 87.5, and 100 mL) of the 0.2 mg/ml dendrimer stock solutions were transferred into a series of 1.5 ml Eppendorf tubes. To each tube 100 mL sodium acetate buffer and 100 mL ninhydrin reagent were added. The volume of the solutions in each tube was made up to 300 mL with DDW and the tubes were then heated in boiling water for 10 min. The later procedures were done as described in section 2.3. The calibration curves were prepared by plotting absorbance of the final products against concentration of G4, G5, and G6 PAMAM dendrimers, respectively.

2.6 Validation of Accuracy and Precision In order to determine the accuracy and precision of the described procedures, 100 mL solutions containing three different concentrations (0.03, 0.06, and 0.09 mg/ml) of G4, G5, and G6 PAMAM dendrimers were prepared and conducted in three replicates. The predicted results were obtained according to the calibration curves as described in section 2.5 and statistically analyzed to determine the accuracy and precision of the method.

2.7 Partial Acetylation of G5 PAMAM Dendrimer To determine whether the described ninhydrin assay is suitable for PAMAM dendrimer derivates, we synthesized partial acetylated G5 PAMAM dendrimer according to a previous publication (Choi et al. 2005). Briefly, 90, 101, and 145 equivalents of acetic anhydride (70.3, 78.9, and 113.3% ratio of primary amine of dendrimer) were slowly added to the G5 PAMAM dendrimer (100 mg, 3.47 mmol) in methanol (10 ml) in the presence of triethylamine (equivalents of acetic anhydride), respectively. The mixtures were then stirred under N2 atmosphere for 18 h at room temperature. After that, excess solvent and reagents were removed by rotary evaporation and extensive

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dialysis (MWCO ¼ 3500 Da) in PBS buffer for three days. The samples were lyophilized and stored in a dry place before NMR studies.

2.8

NMR Studies

H NMR spectra of the synthesized partial acetylated PAMAM dendrimers were obtained on a 500 MHz NMR spectrometer (Bruker, German) with samples issolved in acetone-D6 at an approximate concentration of 3 mg/ml. Acetylated rate of these PAMAM dendrimers can be calculated from the spectra of these samples. The results were further compared with the data obtained from the ninhydrin assay.

3 RESULTS AND DISCUSSION 3.1

Mechanism of Described Procedures

Ninhydrin assay is usually used for the determination of primary aliphatic amines or amino acid groups (Moore and Stein 1948; Hisham and Khalil 2003). These compounds with amino groups could react with ninhydrin reagent in aqueous medium via oxidative-reductive reactions to form the colored reaction product of Ruhemenn purple. The reaction product is measured between 550 and 580 nm depending on the reaction condition (Gorog 1995). In this present study, the PAMAM dendrimers have a much higher amino group density comparing with conventional macromolecules (Fig. 1 and Table 1), a third generation PAMAM prepared from ammonia core has 1.24  1024 amine moieties per unit volume (cubic Angstrom units) in contrast to the 1.58  1026 amine moieties per unit volume of a conventional

Figure 1.

Schematically molecular structure of the G2 PAMAM dendrimer.

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Table 1. The characteristic data of G4, G5, and G6 PAMAM dendrimers

Generation G4 G5 G6

Molecular formula

Molecular weight

Number of terminal amino/ester groups

Number of total amino groups

Radius ˚) (A

C622H1248O250N124 C1262H2528O506N252 C2542H5088O1018N508

14,215 28,826 58,048

64 128 256

124 252 508

22.5 27 33.5

star polymer (Tomalia et al. 1985; Tomalia et al. 1986). The high density of primary amino groups of PAMAM dendrimers could react with hydrindantin (2-hydroxyindan-1,3-dione) in aqueous medium to form an amino compound which further reacted with ninhydrin to produce diketohydrindylidene-diketohydrindamine (Fig. 2). This product could finally interact with primary amino groups of PAMAM dendrimers followed by the production of a blue-colored complex with the maximum absorbance at 570 nm (Raham and Kashif 2003). Also, the generation of PAMAM dendrimers did not change lmax of the blue-colored products (Fig. 3).

3.2 Optimization of the Procedures To optimize the described procedure in section 2.3, different experimental parameters, which influence the absorbance of the blue-colored product, such as heating time and ninhydrin reagent concentration were investigated.

Figure 2. The proposed interaction mechanism between PAMAM dendrimers and ninhydrin reagent in aqueous solutions.

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Figure 3. Visible absorption spectrum of the blue-colored product between G4, G5, and G6 PAMAM dendrimers and ninhydrin reagent (G4, G5, and G6 concentration: 2.8 mM).

The optimum procedures were obtained by varying one of the experimental parameters and observing its effect on the absorbance of the final bluecolored product (Hisham and Khalil 2003). The optimum heating time was determined by heating the reaction system in boiling water. It is observed in Fig. 4 that maximum color intensity was obtained at 8 min of heating. So 10 min was selected as the optimum heating time during preparation of calibration and validation of accuracy and precision in the experiment. The effect of ninhydrin concentration on the color intensity was also investigated. As shown in Fig. 5, 80 mL of ninhydrin reagent was found optimum to

Figure 4. Effect of heating time on the absorbance of the blue-colored product (G6 PAMAM dendrimer concentration: 0.15 mg/ml).

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Figure 5. Effect of ninhydrin concentration on the absorbance of the blue-colored product (G6 PAMAM dendrimer concentration: 0.15 mg/ml).

maximize the color intensity, beyond which the intensity became constant. Therefore, 100 mL of the ninhydrin reagent was selected as an optimum experimental parameter for the described procedure (Raham and Kashif 2003).

3.3 Preparation of Calibration Curve By using the described colorimetric procedure with established optimum experimental parameters, the calibration curves shown in Figs. 6 –8 were prepared by plotting absorbance of the final products at 570 nm against concentration of G4, G5, and G6 PAMAM dendrimers, respectively. Optical characteristics for the

Figure 6. The calibration curve of G4 PAMAM dendrimer in aqueous medium obtained by ninhydrin assay.

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Figure 7. The calibration curve of G5 PAMAM dendrimer in aqueous medium by ninhydrin assay.

regression equations such as the molar absorptivity, slope, intercept, and correlation coefficient were given in Table 2. Correlation coefficients of the regression equations prove excellent linearity of the calibration curves.

3.4

Accuracy and Precision

In order to determine the accuracy and precision of the methods, solutions containing three different concentrations of G4, G5, and G6 dendrimers

Figure 8. The calibration curve of G6 PAMAM dendrimer in aqueous medium by ninhydrin assay.

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Table 2. Optical characteristics of the regression equations of G4, G5, and G6 PAMAM dendrimers Geeration

G4

Beer’s law range Molar absorptivity (1 mol21 cm – 1) Regression equationa Slope (b) Intercept (a) Correlation coefficient

G5

G6

0.181.41  1025 1.16  105

0.876.94  1026 1.95  105

0.433.45  1026 3.75  105

A ¼ 8.15 c 20.03

A ¼ 6.76 c 20.02

A ¼ 6.46 c 20.04

8.15 20.03 0.99961

6.76 20.02 0.99705

6.46 20.04 0.99892

a

A ¼ bc þ a, where c is the concentration in mg/ml.

were prepared and analyzed in three replicates. The analytical results obtained from this investigation are summarized in Table 3. The mean relative standard deviation (RSD) and the mean standard error (SE) can be considered to be very satisfactory.

3.5 Comparison of the Ninhydrin Assay with NMR Studies The ninhydrin assay was conducted to determine the acetylated rate of G5 PAMAM dendrimers as described above. While the NMR characterization of acetylated G5 PAMAM dendrimers was used to calculate the acetylated rate as described in the reference (Choi et al. 2005). The data calculated by NMR studies and ninhydrin assay were shown in Fig. 9. The results obtained by the two methods approximatively accorded with each other. It Table 3. Validation of accuracy and precision of the described procedures in dendrimer determination Generation G4

G5

G6

Added (mg)

Calculated (mg)

RSD (%)

SAE

0.003 0.006 0.009 0.003 0.006 0.009 0.003 0.006 0.009

0.00295 0.00621 0.00918 0.00313 0.00617 0.00901 0.00320 0.00631 0.00928

4.75 0.653 3.12 4.84 2.63 4.52 2.90 1.03 0.899

0.00008 0.00002 0.00017 0.00009 0.00009 0.00024 0.00005 0.00004 0.00005

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Figure 9. Comparison of the acetylated rate of G5 PAMAM dendrimers measured by NMR studies and ninhydrin assay.

can be concluded that the ninhydrin assay is sensitive enough not only to PAMAM dendrimers but also their derivates such as acetylated PAMAM dendrimers.

4 CONCLUSION The ninhydrin assay described in this present study has proved to be a simple, accurate, and rapid method with satisfactory precision and accuracy for determination of PAMAM dendrimers and their derivates in aqueous solution. Beer’s law limit of the described procedure is quite reasonable for the assay of PAMAM dendrimers in aqueous medium. Future studies will focus on using the ninhydrin assay to determinate the modified PAMAM dendrimers such as folic acid, biotin, sugar, and other molecule conjugated PAMAM dendrimers.

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