Cross-Linked Starch Capsules Containing dsDNA Prepared in Inverse

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Biomacromolecules 2010, 11, 960–968

Cross-Linked Starch Capsules Containing dsDNA Prepared in Inverse Miniemulsion as “Nanoreactors” for Polymerase Chain Reaction Grit Baier,†,‡ Anna Musyanovych,*,†,‡ Martin Dass,†,‡ Sonja Theisinger,‡ and Katharina Landfester†,‡ Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and Institute of Organic Chemistry III - Macromolecular Chemistry and Organic Materials, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany Received December 11, 2009; Revised Manuscript Received February 12, 2010

Cross-linked potato starch nanocapsules with encapsulated dsDNA (with a defined number of base pairs, i.e., 286, 476, and 790 bp) were synthesized using the miniemulsion technique. The inverse (water-in-oil) miniemulsion system was applied to create stable aqueous nanodroplets of dissolved starch in cyclohexane as a continuous phase. The amphiphilic block copolymer poly[(ethylene-co-butylene)-b-(ethylene oxide)] was used as a surfactant to stabilize the droplets. After addition of the cross-linker, 2,4-toluene diisocyanate (TDI), the polyaddition reaction took place at the droplet’s interface, resulting in the formation of a polymeric cross-linked shell. The influence of starch, surfactant, and the amount of cross-linker on the average size, size distribution, and morphology of the capsules was studied by dynamic light scattering and electron microscopy. FTIR spectroscopy was used to identify the chemical composition of the capsule shell. The permeability of the shell was studied on the fluorescent dye (i.e., sulforhodamine 101) containing capsules using fluorescence spectroscopy. High thermal stability of the cross-linked capsules allows one to perform the polymerase chain reaction inside the core. The encapsulation of dsDNA and the efficiency of the PCR were confirmed by fluorescence spectroscopy after staining with the DNAselective dye (SYBRGreen).

Introduction The main advantage of nanocapsules for drug delivery is the efficient protection of therapeutic agents against degradation or oxidation. In addition, it is possible to achieve the nanocapsule durability and compatibility as well as a controlled release of the ingredients. More than 50 years ago, the preparation of soft gelatin capsules was reported by Weidenheimer and Callahan.1 Since then, the use of biocompatible or native polymers as shell materials is becoming more attractive. Starch in native as well as in modified forms has been employed for the encapsulation of fragrances,2,3 spices,4 drugs,5 and so on using the spray drying technology. Although this technology is well established, the number of suitable shell materials is limited and the resulting capsules generally are in the micrometer size range. Moreover, the process usually needs high temperatures of up to 200 °C for the evaporation of the solvents that might destroy the sensitive encapsulated materials. The use of the miniemulsion polymerization process offers the possibility to create stable capsules in a size range between 50 and 500 nm.6,7 The synthesis of nanocapsules can be accomplished by different methods using a variety of materials as “shells”.8 One possibility to synthesize polymeric nanocapsules based on the phase separation process. During the polymerization, the polymer and oil become insoluble and the phase separation takes place within the miniemulsion droplets.9,10 Under properly chosen reaction conditions (e.g., physicochemical properties of the monomers/encapsulated materials and their * Corresponding author. Tel.: +49(0)6131 379-248. Fax: +49(0)6131 379-100. E-mail: [email protected]. † Max Planck Institute for Polymer Research. ‡ University of Ulm.

corresponding ratios), a polymeric shell around a liquid core can be formed. Another possibility is the precipitation of the polymer onto stable nanodroplets in an inverse miniemulsion. In this way, poly(methyl methacrylate) nanocapsules with an antiseptic agent inside were produced.11 An efficient formation of nanocapsules could be achieved using different interfacial cross-linking or polymerization reactions. dsDNA molecules were successfully encapsulated into polybutylcyanoacrylate (PBCA) nanocapsules by anionic polymerization performed at the miniemulsion droplet’s interface.12 Scott et al.13 and Torini et al.14 described the synthesis of cross-linked PEG-1000 divinyl ether and polyurethane nanocapsules, respectively, using the direct oil-in-water miniemulsion process. When the polycondensation reaction was employed, cross-linked nanocapsules containing organic liquids were formed using isophorone diisocyanate and propanetriol.15 The formation of nanocapsules in the inverse water-in-oil miniemulsion system was previously studied in our group11,16–21 and by other authors.22,23 This approach was successfully applied for the encapsulation of silver salts with their subsequent reduction to silver particles within the capsules16 and for the encapsulation of hydrophilic contrast agents, that is, Magnevist or Gadovist for magnetic resonance imaging using polyurethane, polyurea, and cross-linked dextran shells.20 Furthermore, it was also shown that the nanocapsules can be functionalized and taken up by the cells efficiently. Neither the shell material nor the surfactant employed for the capsule synthesis (e.g., poly[(ethylene-co-butylene)-b-(ethylene oxide)], KLE) affected the cell vitality.24 The possibility of starch cross-linking at the interface of the miniemulsion droplets has recently been briefly described by our group.16 In the current paper, attention is focused on the formulation of capsules with controlled properties and their

10.1021/bm901414k  2010 American Chemical Society Published on Web 03/23/2010

Cross-Linked Starch Capsules as Nanoreactors

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Figure 1. Formulation process for the preparation of cross-linked starch capsules in an inverse miniemulsion.

utilization as “nanoreactors” for the amplification of dsDNA molecules by a polymerase chain reaction (PCR). The influence of different parameters such as the amount of starch, surfactant poly[(ethylene-co-butylene)-b-(ethylene oxide)] and cross-linker (2,4-toluene diisocyanate), reaction temperature on the capsule size and stability of the system was studied in detail. Kinetic investigations were carried out using infrared spectroscopy and the morphology of the synthesized nanocapsules was studied using TEM. Fluorescence spectroscopy was applied in order to determine the encapsulation efficiency of the dsDNA and the performance of the PCR inside the capsules. The fluorescence dye sulforhodamine 101 (SR101) was used to study the shell permeability of the nanocapsules.

Experimental Section Materials. All chemicals or materials were used without further purification. The hydrophilic water-soluble potato starch (Mw ) 15000 g · mol-1 (determined by GPC-MALLS) was purchased from Fluka. 2,4Toluene diisocyanate (TDI) and cyclohexane (>99.9%) were purchased from Sigma Aldrich. The osmotic reagent sodium chloride (>99%) salt was purchased from Fischer. The no-water-soluble block copolymer surfactant poly[(ethylene-co-butylene)-b-(ethylene oxide)], P(E/B-bEO), consisting of a poly(ethylene-co-butylene) block (Mw ) 3700 g · mol-1) and a poly(ethylene oxide) block (Mw ) 3600 g · mol-1) was synthesized starting from ω-hydroxypoly(ethylene-co-butylene), which was dissolved in toluene, by adding ethylene oxide under the typical conditions of anionic polymerization.25,26 P(E/B-b-EO) was vacuumdried prior to use. The anionic surfactant sodium dodecylsulfate (SDS) was purchased from Fluka. The dsDNA molecules with a defined number of base pairs (bp), that is, 276, 476, and 790 bp were obtained from genomic dsDNA through a polymerase chain reaction.27 In the dsDNA encapsulation experiments and PCR, the buffer consisted of 25 mM Tris · HCl (pH 8.4), 3.1 mM MgCl2, 62.5 mM KCl, 0.0125 mg · mL-1 gelatin, and water. The primers with the sequences forward (5′-CGC CAG CAA CAG CAG GT-3′), reverse-476 (5′-GCC TAG GGA TGA TCT TGC-′), and reverse-286 (5′-CCA CGC GAA TCA CTC TCA TCT-3′) were purchased from Thermo Electron (Ulm, Germany). The dNTP mix and Taq DNA polymerase were supplied from PecLab. The fluorescent dye SYBRGreen (M ) 509.27 g · mol-1), which is specific for dsDNA, was purchased from Molecular Probes. Sulforhodamine 101 (SR101) was purchased from BioChemica, Aldrich. Synthesis of Starch Nanocapsules. The starch capsules were prepared by a polyaddition reaction performed at the miniemulsion

droplet’s interface according to the previously published procedure.16 The formation steps are illustrated in Figure 1. Initially, different amounts of a soluble potato starch were mixed with 30 mg of sodium chloride and demineralized water (solution I). Then P(E/B-b-EO) at certain concentrations was dissolved in 7.5 g cyclohexane using an ultrasonication bath at 50 °C (solution II). Both solutions (I + II) were mixed together, stirred over 1 h at 25 °C, and then ultrasonicated for 180 s at 70% amplitude in a pulse regime (20 s sonication, 10 s pause) using a Branson Sonifier W-450-Digital and a 1/2′′ tip under ice cooling in order to prevent evaporation of the solvent. A clear solution (solution III) consisting of 5 g cyclohexane, 30 mg P(E/B-b-EO), and different amounts of TDI was prepared using an ultrasonication bath at 50 °C, as described before. This solution was added dropwise over 5 min to the earlier prepared mixture (I + II) maintaining the temperature at 60 °C. The mixture was stirred for 2 h at 60 °C (in some experiments at 40 or 25 °C, as stated in the text) and was kept over the night for further stirring at 25 °C. In general, the starch molecule consists of amylose and amylopectin polymers. The chemical structures of the cross-linker, 2,4-toluene diisocyanate, and starch molecule units are shown in Figure 2. The amounts of ingredients used for the capsule formation are summarized in Table 1. After the synthesis of the nanocapsules, they were transferred into the aqueous phase using the following procedure: 1 g (polymer solid content around 3%) of the capsule dispersion in cyclohexane was mixed with 5 g SDS aqueous solution (0.3 wt %) under mechanical stirring for 24 h at 25 °C. Then, the samples were redispersed for 5 min at 50 °C in a sonication bath (power 50%, 25 kHz). Encapsulation of DNA Molecules into the Cross-Linked Starch Capsules. For the encapsulation of dsDNA molecules with different numbers of base pairs (i.e., 286, 476, and 790 bp) the aqueous phase contained 0.1 g starch and 1300 µL of stock solution (buffer, 0.6 µL SYBRGreen (2.95 × 1016 molecules)) and 10 µL (65 ng · µL-1) dsDNA. The continuous phase consisted of 7.5 g cyclohexane and 100 mg of the surfactant P(E/B-b-EO). The cross-linker, 0.1 g TDI, was added together with 5 g cyclohexane and 30 mg P(E/B-b-EO). A sample without dsDNA was used as a control. The preparation and the ultrasonication procedure were the same as described above. PCR Amplification of a dsDNA Molecule Inside the Cross-Linked Starch Capsules. dsDNA molecules with 790 or 476 bp were encapsulated into the cross-linked starch capsules as described above. The aqueous phase consisted of 300 µL dsDNA (75 ng · µL-1), 12 µL dNTP mix (100 nmol · µL-1), 24 µL forward and reverse primers (each 100 pmol · µL-1), 12 µL Taq DNA polymerase (5 u · µL-1), and

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Figure 2. Chemical structures of the (a) cross-linker and (b) starch consisting of amylose and amylopectin. Table 1. Experimental Formulations for the Synthesis of Cross-Linked Starch Capsules sample

starch, g

water, g

P(E/B-b-EO), g

TDI, g

glucose unit/TDI, mol-ratioa

T, °C

GBSt1 GBSt4 GBSt13 GBSt11 GBSt12 GBSt14 GBSt5 GBSt6 GBSt7 GBSt9 GBSt10 GBStM-1 GBStM-2 GBStM-3

0.1

1.3

0.20

0.625:1

60

0.2

1.2

0.3

1.1

0.105 0.130 0.105 0.130 0.105 0.130

0.1

1.3

1.25:1 1.785:1 0.15 0.10 0.05 0.03 0.015 0.10

0.83:1 1.25:1 2.5:1 4.16:1 8.33:1 1.25:1

60 40 25

a The mol ratio was calculated for the TDI molecule and glucose unit, which contains on average 2.5 -OH groups (3 groups from amylose and 2 groups from brunched amylopectin). The hydrolysis of starch and the reaction of TDI with water as well as with the hydroxyl group from the surfactant were not considered for the calculations.

buffer up to a total volume of 1300 µL). The formation of nanocapsules was carried out at 25 °C for 24 h, and the obtained capsules were redispersed in 0.3 wt % SDS aqueous solution. Afterward, the capsule dispersion was divided between PCR tubes (80 µL each), and a PCR was carried out using a Thermal Cycler (Bio-Rad Thermocycler). The amplification program comprised of 35 or 10 cycles with the following steps: initial denaturation at 94 °C for 4 min; 35 or 10 cycles consisting of dsDNA denaturation at 94 °C for 30 s, primer annealing at 58 °C for 45 s, primer extension at 72 °C for 30 s, and final elongation at 72 °C for 2 min. A sample without dsDNA template was used as control. Characterization of Cross-Linked Starch Nanocapsules. The average size and the size distribution of the nanocapsules were analyzed by means of dynamic light scattering (DLS) at 25 °C using a Zeta Nanosizer (Malvern Instruments, U.K.) equipped with a detector at 173° to the incident beam. For the morphology observation, characterization using transmission electron microscopy (TEM) was carried out with Philips EM400 electron microscope operating at an acceleration voltage of 80 kV. Generally, the samples were prepared by diluting the capsule dispersion in cyclohexane or demineralized water (in the case of the redispersed samples) to about 0.01% solid content; then one droplet of the sample was placed on a 300 mesh carbon-coated copper grid and

left to dry overnight at room temperature. No additional contrasting was applied. For the cross-sectioned TEM, a chemical fixation method was used. The capsules were stained with ruthenium tetroxide, dehydrated in graded series of propanol, and embedded in EPON Ultrathin sections (80 nm) using a Leica microtome and imaged in a Zeiss EM 912 (Oberkochen, Germany) at an accelerating voltage of 120 kV. Scanning electron microscopy (SEM) images were recorded by using a field emission microscope (LEO (Zeiss) 1530 Gemini, Oberkochen, Germany) working at an accelerating voltage of 170 V. The samples were placed onto silica wafers and dried under ambient conditions. The solid content of the capsule dispersion was measured gravimetrically. The analysis of the polymer was performed by FTIR measurements. The sample powder was obtained by freeze-drying of the capsule dispersion for 48 h at -60 °C under reduced pressure. Then 3 mg of the dry sample was pressed with KBr to form a pellet, and a spectrum between 4000 and 400 cm-1 was recorded using the IFS 113v Bruker spectrometer. The thermal stability of the final capsules that were redispersed in the aqueous phase was studied in the temperature range from 25 to 95 °C. The average size of the nanocapsules was measured before and after 3 h of thermal treatment.



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Table 2. Characterization of the Polymer Capsules Obtained with Different Amounts of Starch and Surfactant (P(E/B-b-EO)) in Cyclohexanea DLS

TEM

sample

starch, g

total amount of P(E/B-b-EO), g

average diameter, nm

PDI


wall thickness, nm

GBSt1 GBSt13 GBSt12 GBSt4 GBSt11 GBSt14

0.1 0.2 0.3 0.1 0.2 0.3

0.105

920 775 485 405 400 320

0.34 0.36 0.32 0.23 0.26 0.26

362 381 366 286 378 325

23 24 26 20 20 21

a

0.130

The amount of the continuous phase (cyclohexane, 12.5 g) and cross-linker (TDI, 0.2 g) was identical in each sample.

The amount of encapsulated dsDNA was studied in the presence of the fluorescent dye SYBRGreen, which specifically intercalates between the DNA base pairs. This dye absorbs the blue light (λmax ) 488 nm) and emits the green light (λmax ) 522 nm). The measurements were performed using a FluoroMax-3 spectrofluorometer (HORIBA Jobin Yvon, Inc.) at 25 °C. The output signal of pure stock solution with a known amount of dsDNA was measured prior to encapsulation. After encapsulation, the capsules were redispersed in water and subjected to the fluorescence measurements. Then, the capsules were sedimented by centrifugation (20 min at 14000 rpm) and the fluorescence signal of the nonencapsulated SYBRGreen present in the supernatant was measured. The permeability of the capsule’s shell was studied on SR101containing capsules redispersed in water using a fluorescence spectrometer (NanoDrop ND-3300, PEQLab). After the encapsulation and redispersion process the polymeric nanocapsules were sedimented by centrifugation (20 min at 14000 rpm). The capsules prepared without fluorescent dye, but redispersed in an aqueous SDS solution containing SR101 (the amount is equal to SR101 amount taken in the encapsulation experiments) were used as a control sample. The total release of SR101 from the capsule was calculated as a difference between the fluorescent intensities of the supernatant obtained from the sample and the control sample. The fluorescence signal of the control sample was set as 100%. The polymeric nanocapsules were shaken gently for 40 days at 37 °C. After a certain period of time the amount of released SR101 was determined in the supernatant of the sedimented capsules and compared with the initial value. For each sample the encapsulation efficiency was calculated from six single measurements and the whole experiment was repeated three times.

Results and Discussion A series of cross-linked starch nanocapsules obtained by using different amounts of starch, surfactant, and cross-linker were synthesized by a polyaddition reaction performed in inverse miniemulsions at the water-in-oil droplet’s interface, created by the miniemulsion technique. The formulation process is shown in Figure 1. The aqueous phase consisting of dissolved starch and sodium salt was added to the nonaqueous phase which was composed of a water immiscible solvent (cyclohexane) and a nonionic surfactant. The sodium salt serves as a hydrophilic agent in order to suppress the Ostwald ripening.17 After the formation of miniemulsion droplets using ultrasonication, the cross-linker TDI dissolved in a mixture of cyclohexane and surfactant was added dropwise to the miniemulsion and the reaction took place. The interfacial cross-linking of OH groups from starch and surfactant molecules with NCO groups of TDI is responsible for the final capsule morphology. Employing various amounts of starch, surfactant, or TDI results in the formation of capsules with different properties (average size, size distribution, wall thickness, and stability). Influence of Starch and Surfactant Amounts on the Capsule Size. In the following set of experiments, the amount of starch and surfactant was varied in the range from 0.1 to

0.3 g and from 0.105 to 0.130 g, respectively, keeping the amount of the continuous phase (cyclohexane, 12.5 g) and crosslinker (TDI, 0.2 g) constant. The experimental solid content of the capsule dispersion mainly depends on the used amount of starch or surfactant and was within 3.0 and 3.5 wt % (theoretically calculated values are between 3.2 and 4.6 wt %). The obtained capsules were characterized in terms of their size, size distribution (polydispersity index, PDI) and the results are presented in Table 2. The average capsule size and the size distribution values in cyclohexane were determined by DLS. It can be seen that the capsule size measured using DLS is in the range of 485 to 920 nm, using 0.105 g surfactant, and between 320 and 405 nm in the case of capsules obtained with 0.130 g of surfactant. Generally, a higher amount of starch and surfactant results in smaller capsule sizes. It could also be seen that all samples have relatively high PDI values (>0.23), indicating a broad size distribution of the capsules. This could be due to the presence of the surfactant on the capsule surface. The mobile chains of the surfactant molecules are free to move, and therefore, they protrude differently far into the continuous phase, causing the diameter variability. Irrespective to the amount of starch used, the final capsules show good stability. No precipitation or coagulation was observed within 6 months of storage. TEM and SEM measurements were performed to study the capsule morphology and wall thickness. As an example, the images of cross-section TEM and SEM of cross-linked starch capsules obtained with 0.130 g surfactant and 0.1 g of starch are shown in Figure 3. From the TEM images, capsule sizes and the wall thicknesses obtained are quite similar in all samples. Due to the drying effects, the average size of the capsules is small as compared to DLS values and in some cases deformed capsules could be seen. Effect of the Cross-Linker (TDI) Amount on the Capsule Size. To achieve a better biocompatibility of the final capsules, it is essential to reduce the excess amount of TDI. Therefore, the smallest amount of cross-linker sufficient to produce stable capsules was determined in further experiments. The studied range of the TDI amount was between 0.015 and 0.2 g, which corresponds to a glucose unit (with two to three OH groups) to TDI (with two NCO groups) ratio of 8.33:1 to 0.625:1. This means that in the case of the 0.625:1 ratio a high number of starch OH groups could participate in the reaction with the NCO groups of the TDI. The amounts of TDI and the obtained capsule sizes are shown in Table 3. The amount of starch (0.1 g), water (1.3 g), P(E/B-b-EO) (0.13 g), and cyclohexane (12.5 g) were kept constant throughout the experiments. The DLS results show that with a decrease in the amount of TDI until 0.05 g stable capsules in the size range between 260 and 405 nm are obtained. With the TDI amount below 0.05 g, the DLS measurements were not reproducible. The TEM studies reveal the capsules formation with the concentrations

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Figure 3. Cross-section TEM (left) and SEM (right) images of cross-linked starch capsules in cyclohexane synthesized with 0.130 g surfactant and 0.1 g of starch. Table 3. Characterization of the Polymer Capsules Obtained with Different Amounts of Cross-Linker (TDI)a in SDS solution (after redispersion)c

in cyclohexane DLS sample

TDI, g

GBSt4 GBSt5 GBSt6 GBSt7 GBSt9 GBSt10

0.20 0.15 0.10 0.05 0.03 0.015

TEM

DLS

TEM


PDI


wall thickness, nm


PDI


wall thickness, nm

405 270 330 260

0.23 0.18 0.23 0.20

286 183 249 216

20 15 17 15

120 215 235 200

0.24 0.22 0.21 0.18

289 256 274 266

18 16 18 16

b

b

b

b

b

b

b

b

b

b

b

b

b

b

b

b

a The amount of starch (0.1 g) was identical in each sample. b The measurements were not possible due to the absence of the capsules. c Capsules were redispersed in aqueous SDS solution (0.3 wt %) at 1:5 ratio.

Figure 4. TEM images of cross-linked starch capsules in cyclohexane, obtained with different amounts of TDI: (a) GBSt5, (b) GBSt6, (c) GBSt7. Scale bar corresponds to 500 nm.

of TDI down to 0.05 g (Figure 4) and some aggregates instead of capsules were found after the reaction with the amounts of TDI below 0.05 g. Therefore, it can be concluded that the lowest amount of TDI required for the successful formation of the capsule (under given experimental conditions) is 0.05 g. The stability of the cross-linked starch nanocapsules (sample GBSt4) redispersed in an aqueous medium was studied over 12 months under ambient conditions. The size of the capsules and the polydispersity were measured before and after storage. It can be concluded that after storage the average diameter of the nanocapsules increased only by 10 nm in comparison to the one measured directly after redispersion (230 vs 220 nm). The polydispersity values in both cases were equal to 0.24. A clear capsule structure was also seen on the TEM images (not shown), indicating good stability of the cross-linked starch capsules in aqueous dispersions.

Characterization of the Shell Composition by FT-IR. FTIR spectroscopy was performed on the capsules to identify the chemical reaction between the cross-linker TDI and the OH groups of starch and surfactant or water inside the capsules. The studied range of the TDI amount was between 0.05 and 0.2 g (samples GBSt4 to GBSt7, see Table 3). The amount of starch and surfactant was kept constant at 0.1 and 0.13 g, respectively. The characteristic intensities of the isocyanate bands (2250 cm-1) are shown in Figure 5. Figure 5 shows FT-IR spectra of cross-linked starch nanocapsules obtained with different amounts of TDI. After removal of the continuous phase, the product was identified as polymer consisting of urethane and urea units.28,29 All samples have strong bands characteristic for an oxygen-bonded O-H stretching vibration at 3450 cm-1. The N-H valence vibration which


Figure 5. FT-IR spectra of cross-linked starch nanocapsules with different amounts of cross-linker (TDI): (a) 0.05 g (sample GBSt7), (b) 0.10 g (sample GBSt6), (c) 0.15 g (sample GBSt5), (d) 0.20 g (sample GBSt4).

is also expected in this area (3300 cm-1) is overlapped by the O-H vibration band. The C-H valence vibration of the aromatic system is known to show up in the same area (3000 cm-1). The characteristic NCO band is observed at about 2250 cm-1. The two bands at 1720-1700 cm-1 result from the CdO vibration. The band at 1720 cm-1 results from the free CdO and at 1700 cm-1 from the polyurethane.30 The polyurea peak is located next to these bands at about 1640 cm-1. Urethane carbonyl groups and urea can be divided into free carbonyl groups and hydrogen bonded ones. The peaks’ locations can vary depending on the type of monomer.31,32 In the area of 1600 cm-1 the CdC valence vibration of the aromatic system is usually visible. From all these characteristic peaks, it is evident that poly(urethane/urea) capsules have been successfully synthesized. The ratio between urethane and urea units is quite similar in all four samples independent of the TDI used in the synthesis, that is, GBSt4 0.54, GBSt5 0.54, GBSt6 0.52, and GBSt7 0.52. This result shows that the polymeric wall contains equal amounts of urea and urethane units. The presence of a small residual peak of the isocyanate group, which is visible after the reaction, can be attributed to the groups, which are incorporated into the capsule shell only by a one-side reaction with the starch molecule. After transferring the capsules into an aqueous phase, the isocyanate peak totally disappears, which shows that a hydrolysis reaction occurs between isocyanate groups and water to form amine groups via formation of an unstable carbamic acid intermediate. Synthesis of Cross-Linked Starch Nanocapsules at Lower Temperatures. Performing the synthesis of cross-linked starch nanocapsules at lower temperatures provides a good opportunity to encapsulate temperature sensitive molecules, for example, therapeutics, and so on. Additionally, low temperatures facilitate the capsule formation process which is an advantage for an upscaling production. Considering this, the polyaddition was carried out at 60, 40, and 25 °C, keeping the used ratio between the starch and TDI amount constant throughout the experiments. After the synthesis, the capsules were redispersed in 0.3 wt % aqueous SDS at the ratio 1:5. No significant effect of the reaction temperature on the capsule size, size distribution, and morphology was observed. The DLS values were in the range between 300 and 325 nm. The influence of temperature on the urethane to urea ratio of redispersed and freeze-dried capsule samples was recorded by FT-IR spectroscopy. The intensity of the isocyanate vibration band located at 2250 cm-1


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was used to follow the urethane/urea formation at different temperatures. The ratio between urethane and urea units was estimated from the characteristic bands that are assigned to the urethane (1724 and 1696 cm-1) and urea (1668 and 1646 cm-1) groups. The obtained results are shown in Figure 6 and it can be seen that the amount of residual NCO groups after the synthesis at 60 °C is close to that at 25 °C. This means that more NCO groups are consumed during the polyaddition reaction performed at 60 °C. Moreover, the formation of urethane units is favored at 25 °C, which is due to the difference in the reaction rates with the water. Permeability of the Capsule Shell. For the efficient delivery of the encapsulated material to the place of interest it is very important to avoid the leakage of the material through the capsule’s wall as much as possible. From our experiments (not included in the current paper) we know that poly(urethane/urea) capsules synthesized from diol and TDI (1:1.5 diol to TDI ratio) in the presence of block copolymer poly[(ethylene-co-butylene)b-(ethylene oxide)] (P(E/B-b-EO), show a leakage of more than 50 wt % related to the introduced amount. Keeping in mind that the starch molecule consists of amylose (three OH groups) and amylopectin (two to three OH groups), it can be assumed that the grade of the capsule’s shell cross-linking is higher as compared to the one obtained in the presence of diol (two OH groups). Due to the stability of the fluorescence intensity over the broad range of pH, the SR101 as a model substance was chosen to study the diffusion of the material through the capsule’s wall. Previously it was shown that this dye does not influence the reaction mechanism and the kinetic of the polyaddition reaction, as well as the final size and morphology of the capsules.24 Moreover, SR 101 dye does not interact with the cross-linker TDI,33 and therefore, it is a good candidate to be used as a model hydrophilic substance. Redispersed in the SDS aqueous solution, cross-linked starch capsules (produced at 25 °C from 0.1 g TDI, 0.1 g starch, and 0.130 g P(E/B-b-EO)) containing SR101 were left for 40 days at 37 °C. After certain periods of time, the capsules were precipitated by centrifugation and the amount of released SR101 was determined in the supernatant and compared with the initial value. The results of the fluorescence measurements are shown in Figure 7. After the synthesis and redispersion of the capsules, approximately 9% of the dye was found outside, which corresponds to an encapsulation efficiency of 91%. It can be noticed that until day eight the fluorescence signal increases by 5% points and stays on that level until day 32. Afterward (until day 40), another increase of 2% points can be observed. The obtained results indicate that the capsule’s polymeric shell obtained from the cross-linked starch and TDI is complete and possesses high compactness and resistance against leakage over the time. Encapsulation of dsDNA Molecules into the CrossLinked Starch Capsules. Previously, we have shown that dsDNA molecules could be efficiently encapsulated into the aqueous core of polybutylcyanoacrylate nanocapsules using the miniemulsion technique and interfacial anionic polymerization.12 In this experiment, the encapsulation of dsDNA into the crosslinked starch capsules obtained by the polyaddition reaction was studied. The amount of used TDI was 0.1 g and the aqueous phase of the miniemulsion contained 0.1 g starch and 1.3 g of a stock solution (buffer and 0.6 µL SYBRGreen (2.95 × 1016 molecules)). A total of 10 µL of dsDNA of different base pair amounts, that is, GBSt8-1 (476 bp, 65 ng · µL-1), GBSt8-2 (790

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Figure 6. FT-IR spectra analysis of cross-linked starch nanocapsules synthesized at different temperatures: (a) area of NCO-stretching; (b) polyurethane and polyurea shell composition ratio.

Figure 7. Fluorescence intensity of SR101 in the continuous phase after different storage times of the capsules at 37 °C.

bp, 65 ng · µL-1), and GBSt8-3 (286 bp, 65 ng · µL-1) were added to the 1.3 g of stock solution. Sample GBSt8-4 without any dsDNA served as a control sample. The obtained capsules were redispersed in a 0.3 wt % aqueous SDS solution. The ratio of the capsule dispersion in cyclohexane to the aqueous SDS solution was 1:5 (wt%/wt%). The characterization data of the obtained capsules after redispersion in an aqueous phase are presented in Table 4. All synthesized samples were colloidally stable during and after the polyaddition reaction. The produced capsules after redispersion in the aqueous phase were in the size range between 300 and 380 nm and the PDI values were between 0.25 and 0.42. Generally, the capsule diameter was bigger without the presence of dsDNA molecules. This fact can be due to the difference in the concentration of hydrophilic compounds. Without dsDNA molecules, the effect of Ostwald ripening is less suppressed, and therefore, the droplet collisions take place leading to larger droplets formation. To semiquantify the encapsulation of DNA the aqueous redispersed samples (GBSt8-1, GBSt8-2, and GBSt8-3) containing intercalated SYBRGreen were analyzed by fluorescence spectroscopy. Prior to encapsulation the output signal of a stock solution was measured and referred to as 0. Afterward, 1.3 g of this stock solution was mixed with 10 µL dsDNA (65 ng · µL-1) and used for the synthesis of the cross-linked starch capsules. The fluorescence intensity of the capsules dispersion was measured after encapsulation and redispersion in water. Additionally, the fluorescence signal of the supernatant was measured after centrifugation of the capsules. An encapsulation

of SYBRGreen without dsDNA (GBSt8-4) was performed and used as a control sample. The obtained results are summarized in Figure 8. The results in Figure 8 show that after the encapsulation process about 15% of dsDNA from the initial amount did not combine with SYBRGreen. This might be due to the destruction of DNA molecules during the ultrasonication step. The low fluorescence values of the supernatant after centrifugation (hatched columns) indicate that DNA and SYBRGreen have been encapsulated to the extent of 85%. However, it is not possible to state whether the DNA molecules are present in a “free form” inside the capsules cavity or embedded together with SYBRGreen into the capsule shell from the performed measurements. Here, polymerase chain reactions (PCR) have to be performed. Polymerase Chain Reaction (PCR) inside the CrossLinked Starch Capsules. In the following experiments, the amplification of dsDNA inside the cross-linked starch nanocapsules was performed using PCR. The PCR reagents were encapsulated into the capsules (0.1 g starch, 0.1 g TDI, 0.1 g surfactant) and redispersed in water as described above. Afterward, the capsules were subjected to the thermo cycles for PCR. The average capsule size was about 350 nm before and after PCR, confirming that no capsule destruction has taken place during the PCR process. The PCR efficiency was studied in the presence of the fluorescent dye SYBRGreen. Although the mechanism is not well understood,34 Zipper et al.35 showed the sequence specific bindings of SYBRGreen. This dye absorbs blue light (λmax ) 488 nm) and emits green light (λmax ) 522 nm). The fluorescence intensity of the stock solution with a known amount of dsDNA was measured prior to encapsulation. After the encapsulation, the capsules were redispersed in a water phase and subjected to the fluorescence measurements. The resulting intensities of the redispersed samples (before the PCR) were referred to as 0%. Afterward, one part of the capsules was precipitated by centrifugation and the fluorescence intensity of the supernatant was measured to determine the amount of nonencapsulated dsDNA/SYBRGreen (if any). The second part of the capsules was subjected to PCR using the program described in the experimental part. After PCR the fluorescence intensities of supernatant and redispersed capsules were measured as well. All experiments as well as the fluorescence measurements of each sample were performed three times and the averaged values are summarized in Table 5. The obtained results reveal that the amplification of dsDNA molecules took place within the capsule. The concentration of



967

Table 4. Characterization Data of Cross-Linked Starch Capsules with Entrapped dsDNA in Watera DLS

TEM

sample

dsDNA


PDI


wall thickness, nm

GBSt8-2 GBSt8-1 GBSt8-3 GBSt8-4

790 bp (7.6 × 1011 molecules per 10 µL) 476 bp (1.2 × 1012 molecules per 10 µL) 286 bp (2.1 × 1012 molecules per 10 µL) without dsDNA

300 330 345 380

0.34 0.25 0.38 0.42

285 324 334 382

14 15 15 18

a

The amount of DNA corresponds to 10 µL with a solid content of 65 ng · µL-1.

Figure 8. Fluorescence intensity of the capsule dispersion with encapsulated dsDNA and SYBRGreen (gray columns) and supernatant after sedimentation of the capsules (hatched columns). Table 5. Composition of the Samples and Fluorescence Intensities after PCRa fluorescence intensity sample

dsDNA, bp

GBPCR1 GBPCR2 GBPCR3 GBPCR4 GBPCR5

790 476 790 476

a

amt of PCR of the cycles after PCR, % supernatant, % 35 10 35

47.6 52.1 44.8 51.7 7.1

0.6 0.6 0.6 0.5 0.2

The signal of the redispersed samples before PCR was referred to

0%.

newly produced DNA molecules was slightly higher when short DNA templates (476 bp) were used. An increase in the fluorescence intensities to about 48 and 52% was observed for the samples with 790 and 476 bp, respectively. The amount of newly formed DNA molecules reaches the maximum value already after 10 cycles and afterward stays constant. The obtained results are in agreement with the theoretical calculations of the PCR ingredients distribution between the droplets. Each droplet contains approximately five primer molecules, therefore, the amplification should reach maximum already after three PCR cycles under ideal conditions. Performing PCR in nanocapsules reduces the reaction time, thereby allowing one to realize the advantage of the nanoreactor concept. The fluorescent signals measured in the supernatant could be referred to the trace DNA molecules released from the capsules, which were broken during the redispersion step.

Conclusion In the present work, the formulation process of stable crosslinked starch nanocapsules for the encapsulation of dsDNA using the miniemulsion technique is discussed. The nanocapsules were made of potato starch cross-linked with 2,4-toluene diisocyanate. The influence of different parameters such as the amounts of monomer, surfactant, and cross-linker on the capsule size and

colloidal stability of the system was studied. The obtained capsules were in a size range between 320 and 920 nm, depending on the ratio of reaction components. The higher amounts of starch and surfactant result in a smaller capsule size. Performing the encapsulation process at 25 °C did not affect the capsule size, and therefore it could be applied for the encapsulation of temperature sensitive molecules. The formation of the polyurethane/polyurea nanocapsules at 25, 40, and 60 °C was studied by FT-IR spectroscopy. The obtained data revealed that the shell of the capsules synthesized at 60 °C composed of almost equal amounts of urethane and urea units. Furthermore, it is shown that the smallest amount of TDI that is required to produce stable and homogeneous capsules can be as low as glucose unit to TDI ratio of 2.5 to 1. The permeability experiment performed with the capsules containing fluorescent dye revealed that in contrast to diols, the crosslinkage between the starch molecules and TDI results in a more compact polymeric shell. Less than 10% of the dye was released within 40 days of storage at 37 °C. The dsDNA molecules with different amounts of base pairs were encapsulated up to 85%. Furthermore, due to the high thermal stability of the capsules, amplification of dsDNA through PCR was successfully performed inside the capsules. These results showed the feasibility of cross-linked starch capsules to be used as “nanoreactors” for performing biomolecular reactions. With the proper stimulus for the capsule’s wall, controlled release of the “core” material can be realized for gene delivery applications. Acknowledgment. The authors thank Elvira Kaltenecker (University of Ulm) for FTIR measurements and Gunnar Glasser (Max Planck Institute for Polymer Research) for his help in TEM and SEM measurements. This study was supported by the German Research Foundation.

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BM901414K

Cross-Linked Starch Capsules Containing dsDNA Prepared in Inverse

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