Tunable stimuli-responsive self-assembly system that forms and stabilizes nanoparticles by simple mixing and heating/cooling of selected block copolymers

Abstract

We propose here a unique protocol to produce stimuli-responsive self-assemblies using two block copolymers, poly(N-isopropylacrylamide) (PNIPAAm)-b-P(NIPAAm-co-N-(hydroxymethyl)acrylamide (HMAAm)) and PNIPAAm-b-P(NIPAAm-co-sodium 2-acrylamido-2-methylpropane sulfonic acid (AMPS)). These block copolymers were synthesized by an atom transfer radical polymerization (ATRP) method. PNIPAAm was selected as the common block to trigger macromolecular assembly in an aqueous environment above the lower critical solution temperature (LCST). Stable core–shell assemblies were, therefore, produced only by mixing two block copolymers above the LCST of PNIPAAm (the first LCST) when the common blocks became hydrophobic. Upon heating above the LCST of P(NIPAAm-co-HMAAm) (the second LCST), the second block became dehydrated and the size growth of assemblies was observed. The P(NIPAAm-co-AMPS), however, still formed a hydrated shell that prevented further aggregation and precipitation due to electrostatic stabilization through the anionic groups of AMPS. In other words, nanoassemblies, assembled above the second LCST, could be stabilized at the desired size by P(NIPAAm-co-AMPS). The second LCST and the resulting nanoassemblies diameter were controlled by varying the HMAAm content. Nanoassemblies can also be reversibly disentangled at temperatures below the LCSTs, with recovery of soluble block copolymer chains. Thus, the proposed protocol enables the facile preparation of stimuli-responsive nanoassemblies and customization of their size by simple mixing and heating/cooling of the selected block copolymers. Using temperature as a single on–off parameter to induce self-assembly in water circumvents the need for using organic solvents. The system reported here may be potentially useful for a range of applications, including drug and gene delivery, biosensing, or separation of biological molecules.

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Introduction

There has been increased interest in amphiphilic block copolymers due to their potential to form core–shell type nanoassemblies in aqueous solutions.^1–8 Stimuli-responsive “smart” polymers enable nanoassembly applications in biomedicine, in particular drug delivery systems, because they can sense specific environmental changes in biological systems, and adjust in a predictable manner.^9–15 Recent progress in smart polymer synthesis has led to intriguing new amphiphilic polymers that respond to double or multiple stimuli. For example, various AB or ABC types of linear or branched stimuli-responsive copolymers have been synthesized by living radical polymerization (LRP).^16–18 These block copolymers can form core–shell micellar assemblies with the stimuli-responsive building block working as either the hydrophilic shell or the hydrophobic core i.e., if hydrophilic blocks (B and C block) are coupled to the hydrophobic block (a common A block), a mixing of AB and AC type block copolymers results in forming micelles with the common A block working as the hydrophobic core.^19–22 In the case of block copolymers of hydrophobic poly(styrene) (PSt) with a poly(N-isopropylacrylamide) (PNIPAAm) block and poly(ethylene oxide) (PEO) block, for example, stable core–shell nanoparticles were produced via hydrophobic interaction of the common PSt blocks in water.²³ The other interaction can also be used for producing core–shell assemblies. A mixing of poly((3-acrylamidopropyl)trimethylammonium chloride) (PAMPTMA)-b-PEO and poly(sodium 2-acrylamido-2-methylpropane sulfonic acid) (PAMPS)-b-PNIPAAm resulted in forming assembliesvia electrostatic interaction between PAMPTMA and PAMPS in the core.²⁴ Poly(D-lactide) (PDLA)-b-PEO and poly(L-lactide) (PLLA)-b-PNIPAAm also assembled into micelles via stereocomplexation between PDLA and PLLA.²⁵ Although the PNIPAAm shell permits the aqueous solubilization as well as temperature responsive destabilization of the micelle that triggers drug release, these methods rely on the use of organic solvents.^26–28 To avoid use of those violate conditions, PNIPAAm and hydrophilic polymers have been combined as a block copolymer in which PNIPAAm blocks are converted into a water-insoluble block by a thermal stimulus and thus the copolymer forms micelles in aqueous solutions above the LCST of PNIPAAm.^29–32 We have further developed a series of double thermo-responsive block copolymers with tunable LCSTs, which allows for the construction of reversible nanoassembly forming species.^33–35 The block copolymer can be transformed from a double-hydrophilic to an amphiphilic and finally to a double-hydrophobic block copolymer in bio-inert conditions. Therefore, nanoassembly formation and drug encapsulation occurred by simple mixing of polymer solutions with the drug below the LCSTs of both blocks, and upon heating above the second LCST, the nanoassembly aggregated and the drug was released from the micelles. This process was perfectly reversible.

Herein, we propose the facile preparation of a multi-functional assembly by simple mixing of plural functional block copolymers that are containing PNIPAAm block as the common block. In particular, we investigated the effect of addition of an electrolytic block copolymer to the double thermo-responsive system on their micelle formation at different temperatures by employing PNIPAAm in common (Fig. 1). A copolymer composed of NIPAAm and N-(hydroxymethyl)acrylamide (HMAAm) was successfully synthesized by one pot atom transfer radical polymerization (ATRP) via sequential NIPAAm and HMAAm addition to PNIPAAm solution, which has enabled to obtain double-responsive block copolymers as well as well-defined structure and narrow molecular weight distribution. The charged copolymer was also synthesized by the same method using NIPAAm and AMPS, which is known as a permanently charged anionic monomer.^36–39 A mixing of PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) solutions above the LCST of PNIPAAm (the first LCST) leads to the formation of micelles with the common PNIPAAm block forming the inner core. To tune the characteristics of the core–shell micelle, the solution temperature was raised above the LCST of P(NIPAAm-co-HMAAm) (the second LCST) to change the nature of the shell to hydrophobic. Because the P(NIPAAm-co-AMPS) block remained hydrated at the second LCST, the micelles remained stabilized because the P(NIPAAm-co-AMPS) block formed a shell that prevented further aggregation and precipitation due to the electrostatic stabilization through the anionic group of AMPS. The aggregation behavior dependence of the block copolymers on the polymer composition, concentration and temperature has been studied in detail by ¹H nuclear magnetic resonance (¹H NMR), transmittance measurement, and dynamic light scattering (DLS).

Fig. 1 Schematic representation of the tunable characteristics of nanoassemblies by mixing of the selected block copolymers and heating/cooling. PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) are abbreviated as NbNH and NbNA, respectively.

Experimental

Materials

NIPAAm was kindly provided by Kohjin (Tokyo, Japan) and recrystallized from a mixture of benzene and hexane. HMAAm was obtained from Wako Pure Chemical Industries (Osaka, Japan) and purified by recrystallization from ethanol. Tris(2-(dimethylamino)ethyl)amine (Me₆TREN) was synthesized and purified according to the protocol given in previous reports.^40,41 AMPS, ethyl 2-bromoisobutyrate (EBB) and copper(I) bromide (CuBr) were obtained from Sigma-Aldrich (Missouri, USA) and were used as received. Water used in this study was purified with a Millipore Milli-Q system. Other chemicals and solvents were used as received.

Synthesis of PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS)

PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) were synthesized by one pot ATRPvia sequential monomer addition as described previously (Scheme S1†).³⁵ Briefly, NIPAAm (1.1 g, 10 mmol) and EBB (2.4 mg, 0.013 mmol) were dissolved in a mixture of water/DMF (3 : 5 v/v%). The NIPAAm solutions were added to the water solutions of Me₆TREN (6.9 μl, 0.025 mmol) and CuBr (3.6 mg, 0.025 mmol) after three freeze–pump–thaw cycles. The mixture was allowed to polymerize at 20 °C until its conversion reached approximately 30% (precursor solution). For synthesis of the second P(NIPAAm-co-HMAAm) block (HMAAm content: 20 mol%), a mixture of water/DMF (1 : 1 v/v%) containing NIPAAm (71 mg, 0.62 mmol) and HMAAm (0.19 g, 1.9 mmol) was added to the precursor solution. The polymerization was started again and carried out at 20 °C for 24 h. The obtained block copolymers were purified by dialysis against ethanol for three days and pure water for four days at ambient temperature. PNIPAAm-b-P(NIPAAm-co-AMPS) was also synthesized by the same polymerization method described above. The PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) are abbreviated as NbNH and NbNA respectively. We also synthesized random copolymers of P(NIPAAm-co-AMPS)s with various AMPS contents by ATRP for the control study (Scheme S2†). P(NIPAAm-co-AMPS)s are abbreviated as NA_X, where X is the mole percent of AMPS in the feed.

¹H nuclear magnetic resonance (¹H NMR)

¹H NMR spectra of copolymers were taken with a JNM-GSX300 spectrometer operating at 300 MHz (JEOL, Tokyo, Japan) to confirm successful synthesis and determine the chemical composition of the synthesized copolymers. Monomer conversions were determined by comparing the integration of the ¹H NMR signals of the vinyl group of the residual monomers with the integration of the characteristic ¹H NMR signals of the copolymer.

Gel permeation chromatography (GPC)

The molecular weight and polydispersity of the synthesized copolymers were determined by gel permeation chromatography (GPC) at 40 °C with a TOSOH TSK-GEL α-2500 and α-4000 and (Tosoh, Tokyo, Japan) connected to a RI-2031 refractive index detector (JASCO International Co., Ltd., Tokyo, Japan).

Cloud point determination

The transmittance of the solution at 500 nm was continuously recorded at a heating rate of 1.0 °C min⁻¹ by a UV-Vis spectrometer V-550 (JASCO International Co., Ltd., Tokyo, Japan). Synthesized copolymers were dissolved in 100 mM NaCl_aq. at the given concentration. LCSTs of copolymers were determined from 50% transparency.

Dynamic light scattering (DLS)

DLS was performed with a FPAR-1000 spectrometer (Otsuka Electronics Co., Ltd., Osaka, Japan) using a light-scattering apparatus equipped with a 10 mW He–Ne laser and a temperature controller. Prior to measurement, the copolymer solutions were filtered using a 0.45 μm disposable filter (Millipore). The copolymer solutions were then mixed at room temperature and the mixed solution was submitted to a temperature jump at 40 °C. All samples were kept in given temperatures to reach the equilibrium prior to the measurements.

Results and discussion

Synthesis and characterization of PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS)

Because the controlled polymerization of acrylamide monomers, including NIPAAm, by ATRP has been well-studied previously,³³ PNIPAAm-b-P(NIPAAm-co-HMAAm) (NbNH) and PNIPAAm-b-P(NIPAAm-co-AMPS) (NbNA) were synthesized by one pot ATRPvia sequential monomer addition. During the preparation of the NbNH and NbNA, the NIPAAm was polymerized first, and the monomer conversion was monitored by ¹H NMR. The obtained PNIPAAm (the N-block) was then employed as a macroinitiator for the second block, leading to the preparation of NbNH and NbNA (Scheme S1†). The ¹H NMR spectra of NbNH and NbNA reveal the presence of characteristic signals of all blocks i.e., methylene group of HMAAm peak and methylene group of AMPS peak were assigned at 4.7 ppm and 3.3 ppm, respectively (Fig. S1†). The HMAAm and AMPS contents in the P(NIPAAm-co-HMAAm) (NH-block) and P(NIPAAm-co-AMPS) (NA-block) were determined to be 21 and 19 mol% by ¹H NMR, respectively (Table 1). Narrow and unimodal molecular weight distributions were observed by GPC with the good correlation between theoretical and experimental M_n values, demonstrating the controlled nature of the polymerization. The M_n of N-block, NbNH and NbNA were 21 700, 75 100, and 118 900, respectively. We also synthesized a series of P(NIPAAm-co-AMPS)s (NA_X) with various AMPS content by ATRP as the control (Scheme S2†). AMPS contents of NA_5, 10 and 20 were estimated to be 5, 11 and 19 by ¹H NMR, respectively, based on the integral ratio of resonances at 3.9 ppm and 0.5–2.3 ppm (Table S1†). GPC analysis revealed that the M_n of NA_5, 10 and 20 were 28 000, 34 600 and 43 300, respectively.

Table 1 Characteristic data of the PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) synthesized by one pot ATRP method

Code^a	HMAAm content of the NH-block in feed/mol%	HMAAm content in the NH-block^b/mol%	AMPS content of the NA-block in feed/mol%	AMPS content in the NA-block^b/mol%	M n,theo ^b	M n ^c	M w/Mn^c
a PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) were abbreviated as NbNH and NbNA respectively. b Estimated by ^1H NMR. c Determined by GPC using DMF with 10 mM LiBr.
N	0	0	0	0	28 300	21 700	1.14
NbNH	20	21	0	0	87 100	75 100	1.58
NbNA	0	0	20	19	105 100	118 900	1.54

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Temperature-dependent behavior in aqueous solution

In our previous study, random copolymers of NIPAAm and HMAAm exhibited LCST values between 33 °C and 74 °C when varying the content of HMAAm from 10 to 50 mol%.³³ For NbNH with HMAAm content of 19 mol%, therefore, the second LCST occurs at approximately 40 °C. Fig. 2 shows the temperature dependence of transmittance at 500 nm for NbNH and NbNA. The transmittances for both NbNH and NbNA began to decrease around 34 °C. This was due to the aggregation of collapsed PNIPAAm chains. For NbNH, the transmittance decreased from 100% at room temperature to ∼40% at 34 °C. It can be expected that NbNH formed assemblies consisting of a dehydrated N-block core surrounded by a soluble NH-block corona when the temperature was raised above the first LCST. Upon heating to over 40 °C, the transmittance decreased slightly and the aggregates eventually precipitated. This transition corresponds well to the LCST of the NH-block (i.e., the second LCST). Fig. 3(A) shows the size distribution histogram for NbNH at 40 °C and indicates assembly formation, at which an average size of 148 ± 36 nm was found. We had previously confirmed the detailed assembly formation mechanism for this double-responsive copolymer system by temperature-dependent ¹H NMR measurements and this initial assembly of the block copolymers was surely attributed to the dehydration of the N-block.³⁴

Fig. 2 Temperature-dependent behavior of 0.5 w/v% solutions of (○) NbNH and (●) NbNA (100 mM NaCl_aq.).

Fig. 3 Size distribution histograms for 0.1 w/v% of (A) NbNH and (B) NbNA solutions (100 mM NaCl_aq.) at 40 °C.

For NbNA, on the other hand, the transmittance decreased to ∼70% at 34 °C, but no further decrease in transmittance was observed. Because AMPS is a permanently charged anionic monomer, the dehydration and aggregation of the NA-block were prohibited by the repulsion force of AMPS. To elucidate the effects of AMPS, we prepared random copolymers of NIPAAm and AMPS, and measured their transmittance changes in aqueous media (Fig. S2†). The turbidity of an aqueous solution of P(NIPAAm-co-AMPS) containing 5 mol% of AMPS decreased only when heating beyond 50 °C. For a composition with greater than 10 mol% AMPS, the LCST disappears because AMPS conveys sufficient solubility to offset the aggregation of the NIPAAm components. Although the transitions tend to be sharp by addition of sodium chloride (NaCl),^42–44 the LCST also disappears when AMPS content is higher than 20 mol%. Thus, 20 mol% of charged AMPS ensures sufficient electrostatic repulsion for the intended micelle stabilization. Fig. 3(B) shows the size distribution histogram for NbNA at 40 °C and an average size of 240 ± 59 nm was found. No increase in light scattering was observed upon further heating.

Micelle formation by employing PNIPAAm as the common block

To investigate the effect of an electrolytic block copolymer on the double thermo-responsive micelle system, NbNH and NbNA were mixed at 40 °C where PNIPAAm dehydrates and can be employed as the common block in both copolymers. First, turbidimetry measurements were performed to assess the macroscopic phase separation. Fig. 4(A) shows the transmittance changes for a mixed solution of NbNH and NbNA (NbNH : NbNA = 90 : 10, 80 : 20 and 50 : 50 v/v%, respectively). The first decrease in transmittance was observed around 34 °C for all solutions, independent of NbNA content. The second decrease in transmittance, however, became much smaller when the amount of NbNA increased. This is because increasing NbNA concentration prevents further assembly aggregation due to the repulsion force of AMPS. Fig. 4(B) shows the phase transition behavior of NbNH in the presence of a random copolymer of P(NIPAAm-co-AMPS) which does not have the common N-block. Addition of P(NIPAAm-co-AMPS) did not affect the double thermo-responsive behavior of NbNH even when the content was 20 v/v%. Therefore, this result confirms that mixed NbNH and NbNA blocks form an assembly with the common N-block as a hydrophobic core.

Fig. 4 Temperature-dependent behavior of 0.5 w/v% solutions of (A) NbNH and NbNA mixtures (NbNH  NbNA = (○) 90   10 v/v%, (△) 80 : 20 v/v%, and (□) 50 : 50 v/v%) and (B) NbNH and NA_20 mixture (NbNH : NA_20 = 80 : 20 v/v%) (100 mM NaCl_aq.).

DLS was used to examine in detail the assembly structure and size distribution of the mixture of NbNH and NbNA. Samples were initially equilibrated at room temperature in order to assure complete dissolution. Thereafter, NbNH and NbNA solutions were mixed at various ratios and the mixed solution was heated to 40 °C. The size distribution of the obtained assembly (NbNH : NbNA = 50 : 50 v/v%) is shown in Fig. 5(A). The size distribution is relatively narrow with 178 ± 53 nm in an average diameter. Most importantly, the monodisperse distribution confirms that a mixing of NbNH and NbNA solutions above the LCST of N-block successfully leads to micelle formation with the collapsed common N-block forming the inner core. Fig. 5(B) shows the effect of NbNH and NbNA mixing ratio on the average diameter of the resulting assemblies. Although diameters increased slightly with increasing of NbNA content, the obtained sizes were found to be in the range 148–240 nm, which corresponds well to those of NbNH and NbNA micelles shown in Fig. 3. The obtained distributions were also monodisperse (data not shown). These results clearly supported the cartoon in Fig. 1 where mixed blocks form an assembly with the common PNIPAAm as a hydrophobic core. By using this system, we will also be able to prepare promising nanoassemblies having multiple functions.

Fig. 5 (A) Size distribution for the mixture solution of NbNH and NbNA (NbNH : NbNA = 50 : 50 v/v%) in 100 mM NaCl_aq. at 40 °C, (B) diameters for 0.1 w/v% of mixed block copolymers in 100 mM NaCl_aq. at 40 °C.

Fig. 6 shows the change in assembly size distribution of 0.1 w/v% solution of the mixture when heated to 45 °C from 40 °C. An increase in the assembly size from 156 ± 43 nm to 256 ± 38 nm was observed at 45 °C due to dehydration of NH-block. It is also noteworthy that the obtained size distribution was monodisperse and the transition between these two monodisperse assemblies was rapid and reversible. We also observed that the assembly size was constant when NbNA concentration was increased to more than 50 v/v% (data not shown). In other words, increasing NbNA concentration appears to prevent further assembly aggregation, indicating that a balance between repulsion force of permanently hydrophilic AMPS and hydrophobic interaction of dehydrated NH-block is important. The transition behavior corresponds well to the results from turbidimetry in Fig. 4(A).

Fig. 6 Size distribution histograms for 0.1 w/v% of mixed block copolymers (NbNH : NbNA = 95 : 5 v/v%) in 100 mM NaCl_aq. at (A) 40 °C and (B) 45 °C.

Conclusion

We propose herein an original method to produce double responsive and electrostatically stabilized assemblies by employing PNIPAAm as the common block. PNIPAAm-b-P(NIPAAm-co-HMAAm) and PNIPAAm-b-P(NIPAAm-co-AMPS) were successfully synthesized by a one pot ATRP method. PNIPAAm-b-P(NIPAAm-co-HMAAm) demonstrated a two step transmittance change, with one change beyond the LCST of each block, while PNIPAAm-b-P(NIPAAm-co-AMPS) formed a stable assembly above the LCST of the PNIPAAm block due to electrostatic repulsion of AMPS. A mixing of both block copolymers above the LCST of PNIPAAm successfully leads to the formation of micelles with the collapsed common PNIPAAm block forming the inner core. The size of stabilized nanoassemblies increased with increasing PNIPAAm-b-P(NIPAAm-co-AMPS) content. Complete disentanglement of the nanoassemblies was achieved by decreasing the temperature. The proposed system reported here may be potentially useful for a range of applications including drug delivery and biosensing because functional nanoassemblies can be formed in aqueous medium by simple mixing and heating without the use of organic solvent. Assemblies with a desired size can be obtained by fine tuning the balance between electrostatic repulsion and polymer hydrophobicity.

Acknowledgements

We are grateful to Dr John M. Hoffman (Biomaterials Center, National Institute for Materials Science) for continued discussion. This study was partially supported by the Japan Society for the Promotion of Science.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1py00004g

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