Surfactant-free aqueous preparation from a star polymer of size-controlled nanoparticles with encapsulated functional molecules

Abstract

A simple method based on the vortex mixing of precursor chemicals in surfactant-free aqueous conditions has been developed for preparing nanoparticles that encapsulate functional molecules within a four-armed star polymer. Through investigation into the effects of altering such variables as the vortex intensity, chemical composition, temperature etc., it was found that the size of these nanoparticles could be controlled within a range of 20 to 200 nm. Furthermore, a variety of functional molecules could be encapsulated without changing the size of the nanoparticles, suggesting that this method has the potential to be used for a wide range of applications, such as medicine, industrial production and cosmetics.

---

Introduction

Functional molecule-containing nanoparticles are a promising tool for drug delivery systems (DDSs) and regenerative medicine, and could broaden the potential applications of nanoparticles.^1–6 However, as the functionality of any nanoparticle is greatly dependent on its size and the molecule it encapsulates, controlling these factors is vital to its practical application.^7–14

Although many different methods have been developed for the preparation of nanoparticles containing functional molecules, there are still difficulties in preparing nanoparticles with not only a controlled size, but also a capability to encapsulate a variety of molecules (including proteins). This stems from the fact that common preparation methods^15–20 such as free radical polymerization (emulsion polymerization), living polymerization, reversible addition–fragmentation chain transfer polymerization (RAFT), atom transfer radical polymerization (ATRP) and microwave polymerization utilize a surfactant and organic solvent that can denature proteins. Thus, there is a great need to develop a fast and simple procedure for the preparation of functional molecule-containing nanoparticles with a controlled size.

The authors have developed a method of preparing nanoparticles from a four-armed polyethylene glycol (PEG) star polymer, which can encapsulate a range of functional molecules such as proteins and siRNA by wrapping them in the mesh structure formed during polymerization.^5,6,21–23 This polymerization under surfactant-free physiological conditions (room temperature with no organic modifier) is ideal for the preparation of nanoparticles containing fragile biomolecules. In this study, we developed a preparation procedure that can control the type and quantity of the molecule encapsulated within size-controlled nanoparticles through a greater understanding of the polymerization reaction of the star polymer used.

Experimental

Chemicals

Pentaerythritol tetra(aminopropyl) polyoxyethylene (SUNBRIGHT PTE-050PA; Mn, 5328 g mol⁻¹) was purchased from NOF Corporation (Tokyo, Japan); N,N,N′,N′-tetramethylethylenediamine (TEMED), dichloromethane, acryloyl chloride, triethylamine, ammonium persulfate (APS), tris(hydroxymethyl) aminomethane (Tris), hydrochloric acid, methanol, diethyl ether, acetic acid, trypsin, phosphoric acid and boric acid were purchased from Wako Pure Chemical Industries (Osaka, Japan); and fluorescein (FLU) sodium was purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were dissolved water that was purified with a Milli-Q apparatus (Millipore, Bedford, MA, USA).

Preparation of nanoparticles with encapsulated functional molecules

The preparation schemes for the 4-armed PEG (PEG-Ac) and PEG-Ac with a photocleavable group (PEG-Photo-Ac) have been described in previous papers by the authors.²⁴ For this, a solution containing 37.5 μL of 100 mg mL⁻¹ PEG-Ac (or PEG-Photo-Ac), 30 μL of encapsulated molecule (FLU or trypsin), 52.5 μL of water, 15 μL of 20 mg mL⁻¹ APS, and 15 μL of 0.1 M TEMED in 1 M Tris/HCl buffer was added into a tube and stirred for 20 min using a vortex mixing device (VORTEX-GENIE 2, Scientific Industries, Inc., Bohemia, NY, USA). After mixing, the dispersed solution of nanoparticles was left for 30 min, after which 550 μL of water was added. The as-prepared nanoparticle solution was then sealed in a Spectra/Por dialysis bag (MW cutoff: 10 000; Spectrum Laboratories, Inc., Compton, CA, USA) and incubated in purified water for 1 day. The incubation solution was changed three times during dialysis.

Fluorescence analysis of the FLU-encapsulated nanoparticles

The measurement was performed the similar procedure that was described in our previous paper.²⁴ After purification of the nanoparticle by dialysis, the dispersed solutions (150 μL) of the nanoparticles that prepared from the different concentration of the FLU solution were dispensed to a well of the 96-well micro assay plates (BD, Franklin Lakes, NJ, USA). The fluorescence (excitation 494 nm, emission 521 nm) of the dispersed solution was measured by a multiplate reader (SH-9000, Corona Electric Co., Ibaraki, Japan).

Dynamic light scattering (DLS) analysis

Dynamic light scattering measurement was performed using a similar procedure to that described in a previous paper by the authors.²⁵ A Nanotrac Wave dynamic light scattering (DLS) instrument (Microtrac BEL Corp., Osaka, Japan) was used to measure the diameters of the nanoparticles, with all measurements being carried out room temperature using a 780 nm laser beam. At least three replicates were performed for each sample using 20 μL of sample solution each time. From this, size distribution graphs representing the dependence of the relative intensity of scattered light on the hydrodynamic diameter of the nanoparticles were obtained.

UV stimulation for photocleavage

An Aicure UJ20 (Panasonic, Osaka, Japan) UV curing apparatus was used as a light source for UV irradiation. The wavelength and time of irradiation were 365 nm and 1 min, respectively.

Measurement of trypsin activity using fluorescence

Trypsin reactions were performed in a 96-well micro assay plate, into which a dispersed solution of trypsin-containing nanoparticles (10 μL) was poured. The fluorescence signal of each well was measured before and after UV irradiation (E_x 485 nm, E_m 535 nm) using a multiplate reader (SH-9000, Corona Electric Co., Ibaraki, Japan).

Results and discussion

Functional molecule-encapsulated nanoparticles have been previously prepared by vortex mixing suitable precursor chemicals for 20 min, and then adding water after leaving the reaction tube undisturbed for 30 min (Fig. 1a–c).^5,6 However, the role played by each of these steps is not well understood. What is known is that although the viscosity of the initial solution becomes high when left without vortex mixing, there is no evidence of nanoparticles being formed when analysed using dynamic light scattering (DLS). Nevertheless, when this same solution was vortex mixed at different strengths for 20 min, the formation of nanoparticles was confirmed by DLS after 30 min (Fig. 1d). The vortex strength was controlled by a screw on the front of the VORTEX-GENIE 2 apparatus used, with 1 representing the lowest strength and 10 the maximum. Two different sizes of nanoparticles (40 and 120 nm) were produced when the strength of the vortex was weak (2), with the size distribution narrowing to a single size of 100 or 60 nm when the vortex strength was increased to 5 or 10, respectively. This indicates that a strong vortex is needed for nanoparticles to form, especially if a small particle size is required.

Fig. 1 a) Preparation of the nanoparticles, (b) TEM image of the nanoparticles, (c) error signal image of the nanoparticles measured by AFM and (d) DLS analyses of nanoparticles prepared using different vortex conditions.

The effect of changing the encapsulated molecule was also examined using three different concentrations (0.01, 0.05, and 0.1 mg mL⁻¹) of FLU, which was compared against using trypsin or nothing at all as the encapsulated molecule. As shown in Fig. 2a, the size distribution of the nanoparticles was essentially the same in all cases, with an average particle size of about 38 nm. In other words, the nature of the encapsulated molecule has no bearing on the size of the nanoparticles, which means that the effect of different molecules on the properties of the nanoparticle can be easily ascertained without worrying about size effects.

Fig. 2 DLS analyses of nanoparticles prepared using different (a) encapsulated molecules and concentrations, and (b) pH solutions.

To examine the effect of the pH, nanoparticles were produced under five different pH conditions (3–11) using a Britton–Robinson buffer. However, as shown in Fig. 2b, this had no discernible effect on the particle size distribution. This is a significant finding, as it means that the solution pH can be tailored to optimize the solubility and charge of the functional molecule without the fear of adversely affecting the size of the nanoparticles.

Encapsulation of the functional molecule within the nanoparticles was confirmed by using different concentrations of FLU, which was readily quantified within the nanoparticles by fluorescence analysis due to the PEG polymer of the nanoparticle exhibiting no fluorescence. Fig. 3 shows the relationship between the FLU concentration of the prepared solution and the fluorescence intensity of the dispersed solution, in which there is clearly a correlation between the two. This means that the quantity of functional molecule encapsulated can be easily controlled by simply changing its concentration in the initial solution.

Fig. 3 Fluorescence analysis of FLU-encapsulated nanoparticles prepared from different concentrations of FLU solution.

The polymer and APS concentration, as well as the ionic strength of the preparation solution, were examined to better understand the formation mechanism of the nanoparticles and how it could be used for size control. Three different concentrations (20, 25, and 30 mg mL⁻¹) were used for this, and in Fig. 4a, we see that a polymer concentration of 20 mg mL⁻¹ produced a particle size of 50 nm. This increased to 60 and 100 nm with polymer concentrations of 25 and 30 mg mL⁻¹, respectively, meaning that the polymer concentration had a large influence on the nanoparticle's size. In the case of APS concentration (Fig. 4b), the formation of nanoparticles was not observed by DLS analysis at low APS concentrations (10 mg mL⁻¹), but the particle size increased when the APS concentration was raised from 15 to 30 mg mL⁻¹, indicating that although APS is required for nanoparticle formation, a large quantity results in large nanoparticles. This differs from the results seen with nanoparticles prepared using radical polymerization, where the size becomes smaller with increasing APS concentration.^17,19 This opposite tendency is thought to derive from the large molecular size of the PEG star monomer, or possibly the four reaction groups within each monomer. With the ionic strength (Fig. 4c), values of 0.01, 0.1, and 1 M produced nanoparticle sizes of 130, 100 and 50 nm, respectively. Thus, the effect of ionic strength on size clearly becomes much more pronounced as the ionic strength is increased.

Fig. 4 DLS analyses of nanoparticles prepared using different (a) concentrations of the star polymer, (b) concentrations of APS, (c) ionic strengths, (d) holding temperatures, and (e) holding times.

Varying the temperature at which the mixture was left at prior to adding water (Fig. 4d) revealed that holding it at less than 10 °C for 15 min resulted in there being no evidence of nanoparticle formation by DLS analysis; however, when held at ∼20 or 30 °C, 50 nm diameter nanoparticles were confirmed. When the temperature was further increased to 40 and 50 °C the particle size distribution became much wider and the average size increased. This means that room temperature actually represents ideal conditions for achieving a homogeneous size distribution of nanoparticles. The effect of the time spent at room temperature was also examined, and as shown in Fig. 4e, this is clearly an important part of the process given that no nanoparticles are present immediately following vortex. Increasing the holding to 20, 30 and 60 min produced nanoparticles that were 30, 50, and 80 nm in size, respectively, clearly indicating that this represents the period of nanoparticle growth. In other words, increasing the time or temperature here increases the amount of polymerization that occurs. This process, however, is halted once water is added due to the concentration of unreacted star polymer becoming dilute to continue the reaction.

The inverse correlation between particle size and vortex intensity (Fig. 1d) can be explained by the fact that the quantity of APS consumed through reaction with oxygen in the reaction tube is increased with increasing vortex intensity, thereby reducing the effective quantity of APS that can be used for polymerization of the star polymer. This reduced quantity of APS results in the small nanoparticles seen in Fig. 4b. To confirm this hypothesis, nanoparticles were prepared under conditions in which all of the oxygen in the reaction tube was first removed by sonication and substituted by N₂, resulting in an almost consistent nanoparticle size (ESI Fig. 1†). By varying the preparation conditions, it proved possible to produce nanoparticles with a size of anywhere between 20 and 200 nm (Fig. 4f). Finally, the stabilities of the encapsulated molecule and the nanoparticles themselves were examined, and the effect of the formation process on the encapsulated molecule was evaluated using protein (trypsin)-containing photodegradable nanoparticles prepared from a star PEG monomer with a photocleavable nitrobenzyl group. The activity of the trypsin released upon light irradiation was correlated with the amount of trypsin used for the nanoparticle preparation (ESI Fig. 2†). As no change was observed by DLS when the dispersed nanoparticle solution was stored at 4 °C for 2 weeks (ESI Fig. 3†), it can be concluded that any degradation of the encapsulated molecule during nanoparticle formation or of the nanoparticle during storage are negligible.

Conclusions

This study has shown that nanoparticles with various encapsulated functional molecules can be easily prepared by vortex mixing suitable chemicals and allowing them to react at room temperature. Using this method, the type and concentration of the encapsulated molecule has no effect on the size distribution of the nanoparticles, but the size can be mainly controlled by varying the vortex intensity, the polymer and APS concentrations, and the duration of the post-mixing reaction. By allowing precise control over the quantity and type of encapsulated, as well as the nanoparticle size, this method has great potential for use in developing new materials for a wide range of applications.

Acknowledgements

This work was supported by grants (Kakenhi) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and JSPS Core-to-Core Program, A. Advanced Research Networks.

Notes and references

1. M. E. Davis, Z. Chen and D. M. Shin, Nat. Rev. Drug Discovery, 2008, 7, 771.
2. M. C. Hersam, Nat. Nanotechnol., 2008, 3, 387.
3. J. Ge, E. Neofytou, T. J. Cahill, R. E. Beygui and R. N. Zare, ACS Nano, 2012, 6, 227.
4. M. Murakami, H. Cabral, Y. Matsumoto, S. Wu, M. R. Kano, T. Yamori, N. Nishiyama and K. Kataoka, Sci. Transl. Med., 2011, 3, 64ra2.
5. S. Murayama, B. Su, K. Okabe, A. Kishimura, K. Osada, M. Miura, T. Funatsu, K. Kataoka and M. Kato, Chem. Commun., 2012, 48, 8380.
6. S. Murayama, T. Nishiyama, K. Takagi, F. Ishizuka, T. Santa and M. Kato, Chem. Commun., 2012, 48, 11461.
7. A. C. Malcolm, J. M. Parnis and A. J. Vreugdenhil, J. Non-Cryst. Solids, 2011, 357, 1203.
8. T. Yonezawa, K. Imamura and N. Kimizuka, Langmuir, 2001, 17, 4701.
9. D. Kim, J. Choi, J.-Y. Kim, Y.-K. Han and D. Sohn, Macromolecules, 2002, 35, 5314.
10. P. Sahu and B. L. V. Prasad, Nanoscale, 2013, 5, 1768.
11. M. R. Reithofer, A. Lakshmanan, A. T. K. Ping, J. M. Chin and C. A. E. Hauser, Biomaterials, 2014, 35, 7535.
12. K. Niesz, M. Grass and G. A. Somorjai, Nano Lett., 2005, 5, 2238.
13. H. Cabral, M. Murakami, H. Hojo, Y. Terada, M. R. Kano, U. Chung, N. Nishiyama and K. Kataoka, Proc. Natl. Acad. Sci. U. S. A., 2001, 110(3), 11397.
14. Y. Anraku, A. Kishimura, A. Kobayashi, M. Oba and K. Kataoka, Chem. Commun., 2011, 47, 6054.
15. G. Zhang, A. Niu, S. Peng, M. Jiang, Y. Tu, M. Li and C. Wu, Acc. Chem. Res., 2001, 34, 249.
16. G. Liua and Z. An, Polym. Chem., 2014, 5, 1559.
17. N. Sanson and J. Rieger, Polym. Chem., 2010, 1, 965.
18. Z. An, W. Tang, C. J. Hawker and G. D. Stucky, J. Am. Chem. Soc., 2006, 128, 15054.
19. T. Sato, N. Sato, M. Seno and T. Hirano, J. Polym. Sci., Part A: Polym. Chem., 2004, 42, 1609.
20. K. Min, H. Gao, J. Ae Yoon, W. Wu, T. Kowalewski and K. Matyjaszewski, Macromolecules, 2009, 42, 1597.
21. S. Murayama, P. Kos, K. Miyata, K. Kataoka, E. Wagner and M. Kato, Macromol. Biosci., 2014, 14, 626.
22. S. Murayama, J. Jo, Y. Shibata, K. Liang, T. Santa, T. Saga, I. Aoki and M. Kato, J. Mater. Chem. B, 2013, 1, 4932.
23. K. Takagi, S. Murayama, T. Sakai, M. Asai, T. Santa and M. Kato, Soft Matter, 2014, 10, 3553.
24. S. Murayama and M. Kato, Anal. Chem., 2010, 82, 2186.
25. M. Kato, M. Sasaki, Y. Ueyama, A. Koga, A. Sano, T. Higashi and T. Santa, J. Sep. Sci., 2015, 38, 468.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra12205h

---