Polymer particles that switch shape in response to a stimulus

Abstract

Particle engineering for biomedical applications has unfolded the roles of attributes such as size, surface chemistry, and shape for modulating particle interactions with cells. Recently, dynamic manipulation of such key properties has gained attention in view of the need to precisely control particle interaction with cells. With increasing recognition of the pivotal role of particle shape in determining their biomedical applications, we report on polymeric particles that are able to switch their shape in real time in a stimulus-responsive manner. The shape-switching behavior was driven by a subtle balance between polymer viscosity and interfacial tension. The balance between the two forces was modulated by application of an external stimulus chosen from temperature, pH, or chemical additives. The dynamics of shape switch was precisely controlled over minutes to days under physiological conditions. Shape-switching particles exhibited unique interactions with cells. Elliptical disk-shaped particles that are not phagocytosed by macrophages were made to internalize through shape switch, demonstrating the ability of shape-switchable particles in modulating interaction with cells.

Interactions of polymeric particles with various cells, including macrophages, in the form of endocytosis and phagocytosis determine the effectiveness of carriers used for drug delivery and medical imaging (1, 2). The outcome of these interactions relies on optimal selection of key particle properties including surface chemistry, size, and shape (3, 4). Accordingly, numerous studies have reported on methods to synthesize materials with precisely engineered functional attributes such as size, surface chemistry, mechanical properties, and shape to facilitate or mitigate interactions with various cell types (3–5).

For a given application, particle properties are optimized through extensive experimentation, and a set of fixed values are then chosen for further development. In reality, however, the optimal values of parameters may vary with time depending on the application. This variation has motivated the need to gain dynamic control over key particle properties so as to achieve an interactive interface between the particles and the complex biological milieu. For example, studies have reported on stimulus-responsive type control over the size of particles, and such particles have been used for the triggered release of encapsulated drugs (6–8). In another study, the surface chemistry of polymeric micelles has been controlled by using the environmental pH of solid tumors so as to enhance the cellular uptake and release of anticancer agents (9). Studies have also reported on achieving dynamic control of surface properties through the use of electric fields (10). Relatively little attention, however, has been devoted to switching particle’s shape.

Here we report poly(lactide-co-glycolide) (PLGA) particles whose shape can be switched in real time from an elliptical disk to a sphere in response to a stimulus selected from temperature, pH, or a chemical. The time scale of switching was controlled over a wide range, from minutes to days, by appropriate selection of polymer molecular weight, particle size, and the strength of the stimulus. We also demonstrate that these particles can modulate their interactions with macrophages through shape switch.

Results

PLGA was selected as a polymer because of its biocompatibility and proven record in polymer-based medical products (11, 12). Elliptical disk (ED) was chosen as a model particle shape, which was then switched to a sphere upon application of a stimulus including temperature, pH, and chemicals (Fig. 1A). Switching of particle shape was driven by a subtle balance between interfacial tension and polymer viscosity. Specifically, the interfacial tension (σ) between PLGA and the surrounding aqueous liquid drives the relaxation of a disk-shaped particle to an energetically favorable spherical shape (Fig. 1B and Movie S1); however, this relaxation is resisted by the high viscosity of PLGA (μ). The dynamics of particle relaxation can be described by solving the equation of motion assuming that the polymer can be modeled as a Newtonian fluid, a reasonable assumption at temperatures above polymer’s glass transition temperature T_g (13–15). PLGA indeed exhibits Newtonian behavior near T_g and at low shear rates (10^-4–10^-1 s^-1), which is the relevant range of shear rates for shape switching reported here (Fig. S1). A quasi-steady Stokes equation has been numerically solved for interfacial tension-driven relaxation of ellipsoidal droplets of Newtonian fluids, and a scaling parameter describing time constant of shape change, τ, was given by (16)

[1]

where L is the characteristic particle size and λ = μ/μ_L, where μ is PLGA viscosity and μ_L is the viscosity of the surrounding solution, typically an aqueous medium with viscosity comparable to water. Because PLGA is highly viscous compared to water, μ≫μ_L, thus resulting in τ ∼ Lμ/σ. By appropriately controlling the values of L, μ, and σ, we were able to control the dynamics of shape change over a few minutes (rapid change) to weeks (nearly static shapes) (Fig. 1C).

Fig. 1.

Design of stimulus-responsive shape-switchable PLGA particles. (A) Mechanism of shape switch. The balance between viscosity of polymer (μ) and interfacial tension (σ) between the particle and surrounding media determines the extent and dynamics of shape switch. When the interfacial tension overwhelms the polymer viscosity, an ED undergoes shape switch to a sphere in response to temperature (T), environmental pH, and chemicals (C). (B) Shape switching of PLGA particles (see also Movie S1). PLGA-ester EDs [molecular mass ∼5.2 kDa, T_g(mid) = 28 °C, AR = 5, switched in deionized water at 37 °C] (Left: 0 min; Center: 2 min; Right: 5 min). (Scale bar: 5 μm.) (C) For Newtonian fluids, shape switching can be described by the quasi-steady Stokes equation, and the scaling factor for shape switch (τ) is given by Eq. 1.

The first demonstration of shape switching was obtained via temperature-induced change of particle viscosity (μ) by increasing temperature above T_g, which in turn, was controlled through polymer molecular mass. We first measured the viscosity of PLGA (ester end, molecular mass 29.8 kDa, T_g(mid) = 40 °C) as a function of temperature. These measurements indicated that PLGA viscosity at T_g and at low shear rates is about 1.5 × 10⁶ Pa·s, which is generally consistent with the literature data for other polymers (14). The viscosity of PLGA decreased dramatically with temperature above T_g. The dependence of viscosity on temperature was consistent with the Vogel–Fulcher–Tammann (VFT) equation, which is routinely used to describe polymer viscosities near T_g (Fig. S1) (17). EDs with various aspect ratios (AR; the ratio of major axis to minor axis) were prepared from different types of PLGAs by using a previously reported method (Table S1) (18). For the temperature-induced shape switch, EDs (AR = 5.5) were fabricated by using PLGA-ester (PLGA with an ester [1-dodecanol] end group) of two molecular masses (29.8 kDa, Fig. 2A, top panels, and 52.7 kDa, Fig. 2A, bottom panels). At room temperature (∼25 °C) both particles maintained their ED shape for prolonged times (Fig. S2), indicating that the shape does not switch in a glassy state of an amorphous polymer. The temperature was then raised abruptly to various levels. Detectable shape switch for EDs of molecular mass 29.8 kDa PLGA occurred once the temperature reached 32 °C, which is the onset T_g as determined from differential scanning calorimetry (Fig. S2). At a temperature of 40 °C (mid-T_g), the shape was nearly completely switched in about 4 h (Fig. 2B, Closed Squares). For EDs of molecular mass 52.7 kDa, the particles gradually changed their shape to spheres over a period of 24 h upon increasing the temperature to their mid-T_g (43 °C, Fig. 2B, Open Squares). Although the shape change for both particles was studied at their respective mid-T_g, the switching time increased with increasing molecular mass, because of the higher viscosity of high molecular mass polymer. The characteristic switch time (T_1/2, defined as the time required for a 2-fold reduction of AR) decreased rapidly with an increase in the operating temperature, and for a 10 °C differential between the operating temperature and mid-T_g, the switch was completed in less than 10 min (Fig. 2C and Fig. S2). Particle size is another important parameter to determine the shape-switch time. A 2-fold increase or 10-fold decrease in the diameter of the initial sphere, compared to the baseline case of 1.6 μm, respectively, led to an around 3-fold increase and around 10-fold decrease in the switch time of EDs (Fig. 2A, bottom panels, Fig. 2D and Fig. S3). These results also demonstrated that the shape-switching phenomenon also occurs in nanosized particles.

Fig. 2.

Shape switching via temperature-induced change of polymer viscosity. (A) SEM images of shape-switching PLGA particles with different molecular masses. PLGA-ester EDs [Top, molecular mass 29.8 kDa, T_g(mid) = 40 °C, AR = 5.5 (scale bar: 5 μm)]; [Middle, molecular mass 52.7 kDa, T_g(mid) = 43 °C, AR = 5.5 (scale bar: 5 μm)]; and [Bottom, molecular mass 29.8 kDa, T_g(mid) = 40 °C, AR = 4 (scale bar: 500 nm)] were incubated at their mid-T_gs. (Scale bar: 5 μm.) (B) AR of the EDs decreased nearly exponentially to 1 (sphere) in a molecular-mass-dependent manner at their mid-T_g (closed squares: molecular mass 29.8 kDa; open squares: molecular mass 52.7 kDa). Higher molecular mass particles went through slower shape switch (T_1/2) because of their higher viscosity. (C) Temperature-responsive shape switching of PLGA-ester EDs (molecular mass 29.8 kDa). Whereas shape switch slowly occurred at onset T_g (32 °C), the T_1/2 remarkably decreased at a temperature 10 °C above mid-T_g. (D) Size-dependent shape switch of PLGA-ester EDs (molecular mass 29.8 kDa). The T_1/2 increased as the initial sphere’s particle size increased.

In order to validate the proposed mechanism for shape switching, the switching time for PLGA (ester end, molecular mass 29.8 kDa, T_g(mid) = 40 °C) was calculated by using a viscosity of 1.5 × 10⁶ Pa·s at T_g, an interfacial tension of 5.4 mN/m derived from contact angle measurements, and a particle diameter of 1.6 μm (Fig. S1). The estimated T_1/2 value under these conditions was in the range of 22–75 min, which is of the same order of magnitude as the experimentally observed value of 38 min (Fig. 2B, Closed Squares).

The dependence of T_1/2 on temperature and particle size is also consistent with the proposed mechanism of shape switch. For example, the combination of Eq. 1 and VFT theory suggests that the (Log [switching time]) should scale as (1/temperature). Indeed, the data in Fig. 2C are consistent with this trend. A plot of log (T_1/2) with 1/temperature yields a good linear fit with r² = 0.97. Similarly, Eq. 1 suggests that the switching time should scale linearly with the particle size. Indeed, the data in Fig 2D confirm this trend (r² = 0.96).

The results shown in Fig. 2 suggest that the dynamics of shape switching of PLGA EDs can be controlled over 1,000-fold by appropriate combination of molecular mass, temperature, and particle size. For biological applications, the shape change, however, must occur near physiological temperatures. For this purpose, we studied shape switch of PLGA-ester EDs at 37 °C with either blends of various molecular masses or various sizes and demonstrated that shape switch can be obtained at physiological temperatures (Fig. S4).

We next investigated whether the pH of the surrounding aqueous phase can be used as a stimulus to induce shape switch by affecting the interfacial tension of PLGA particles, σ (Eq. 1). Despite the general hydrophobic nature of PLGA, its surface properties can be tuned by choosing the appropriate end group, acid, or ester and by adjusting environmental pH (19–21). Specifically, the surface charge of PLGA-acid (PLGA with a carboxylic acid end group), and hence the interfacial tension of PLGA particles, can be controlled through ionization of its end carboxylic acid groups. At around physiological pH (∼7.4), the carboxyl groups of PLGA (pK_a = 3.85) are largely charged (-COO^-), leading to low hydrophobicity and thus low interfacial tension. Lowering the pH protonates the acid groups (-COOH), thus increasing the hydrophobicity and induces shape switch (Eq. 1). Measured contact angles of droplets with different pH on PLGA films demonstrated that the interfacial tension of PLGA-acid substantially decreases with increase in pH, whereas that of PLGA-ester was not significantly changed (Fig. S5 a and b). The zeta potential of PLGA-acid spheres [molecular mass 4.1 kDa, T_g(mid) = 27 °C], which was highly negative at pH 7, increased to almost zero as pH approached to 3.0, supporting the hypothesis that acidic pH protonates the acid end group of PLGA (Fig. S5c).

To assess the ability of pH to induce shape switch, EDs (AR = 4) were prepared by using PLGA-acid (molecular mass 4.1 kDa) and incubated in a buffer solution at pH 7.4 at 37 °C. A low molecular mass PLGA was chosen for these experiments so as to maintain a relatively low viscosity at the operating temperature of 37 °C. The ability of the particle to switch shape is then limited by the driving force, that is, interfacial tension. At pH 7.4, the EDs did not exhibit any significant shape switch. Upon lowering the pH, however, the shape changed to sphere in an exponential manner (Fig. 3A). Although the hydrophilic surface of PLGA-acid particles at pH 7.4 tends to uptake water because of reduced interfacial tension (22), no morphological deformation of particles such as swelling was found during pH-induced shape switch (Fig. S6), indicating that the shape switch was induced predominantly by the modulation of interfacial tension with pH change. Whereas interfacial tension may provide the primary driving force for shape switch in low molecular mass PLGA, high molecular mass PLGA may possess potential contributions from strain-induced chain orientation. The characteristic switch time (T_1/2) depended on the pH, with a T_1/2 value of 38 min at pH 5.0 (Fig. 3B). Change of ionic strength of buffer solutions did not alter the dynamics of shape switch (Fig. S7a). The pH-dependent shape switching is exhibited only by PLGA-acid particles and not PLGA-ester particles. Specifically, PLGA-acid particles exhibited over 10,000-fold faster switching at pH 4.0 compared to that at pH 7.4 (Fig. 3C). On the other hand, PLGA-ester particles exhibited a relatively rapid shape switch at both pH values because of their hydrophobic surface.

Fig. 3.

pH-induced shape switch of PLGA particles. (A) The shape-switching study was performed in a buffer solution at pH 7.4 at 37 °C by using PLGA-acid EDs [molecular mass 4.1 kDa, T_g(mid) = 27 °C, AR = 4]. There was no detectable shape switch at pH 7.4 because of low interfacial tension of hydrophilic surface originating from ionized acid end groups. Shape switch occurred upon pH reduction, which led to protonation of the acid end and exhibited a near-exponential decrease of AR. Data are shown as mean ± SEM (n > 30). (B) The T_1/2 of PLGA-acid EDs (molecular mass 4.1 kDa) depended strongly on pH. The T_1/2 gradually increased as pH increased from 3.0 to 4.5 and abruptly increased above pH 5.0. (C) Whereas PLGA-acid EDs showed over 10,000-fold increase of T_1/2 from pH 4 to 7.4, PLGA-ester ED [molecular mass 6.5 KDa, T_g(mid) = 28 °C, AR = 4] switched shape quickly (T_1/2 < 1 min) at both pHs, indicating that there was negligible change in the interfacial tension on PLGA-ester particles by pH.

On the basis of pH-sensitive shape switch, we hypothesized that the shape of PLGA particles can also be switched by using exogenous chemicals. Specifically, PLGA-acid particles possess a charged surface at around physiological pH (∼7.4). We hypothesized that immobilization of certain chemicals on PLGA particles can be used to enhance their surface hydrophobicity and induce shape switch through increased interfacial tension. Azure C was chosen as a model chemical because of its cationic charge as well as the presence of a hydrophobic group (Fig. 4A). PLGA-acid particles (molecular mass 4.1 kDa) were used in these experiments so as to maintain high ionization at pH 7.4 and low viscosity at 37 °C to facilitate switching. Incubation of these particles with Azure C (2 femtomoles per particle) led to shape switch of the EDs, which otherwise retain their shape (Fig. 4A and Fig. S7b). Azure C-induced shape switch was observed at very low Azure C amounts (< 1 femtomole per particle) and was concentration-dependent up to about 5 femtomoles per particle, after which the effect appears to have saturated (Fig. 4B). The effect of Azure C on shape switch was, to some extent, pH-dependent (Fig. 4C). Specifically, the degree of shape switch by Azure C was much lower at pH 5.5 compared to that at 7.4, which is consistent with the hypothesis that the particle surface is weakly ionized at lower pH, thereby limiting the binding of Azure C.

Fig. 4.

Chemical-induced shape switch. PLGA-acid EDs [molecular mass 4.1 kDa, T_g(mid) = 27 °C, AR = 4] were incubated at pH 7.4 and 37 °C. Cationic Azure C possessing a hydrophobic group was used as a trigger for shape switch. (A) PLGA-acid particles, whose shape remained the same, started switching shape upon exposure to Azure C (2 femtomoles per particle), indicating that Azure C binds to particles and reduces the interfacial tension. AR decreased nearly exponentially. Data are shown as mean ± SEM (n > 30). (b) The Azure C-induced shape switch was concentration-dependent up to 5 femtomole per particle, above which the effect seemed to have saturated. (C) Fold difference in the characteristic switch time between Azure C-treated and nontreated EDs. The degree of shape switch by Azure C was higher at high pH than at low pH because of greater availability of Azure C binding sites (i.e., ionized end carboxylic acids) on the particles at higher pH.

The ability of shape-switching particles to modulate their interactions with cells was next assessed. Macrophages were used as a model cell for these experiments. Macrophages, an essential component of the immune system, play a major role in governing the fate of polymeric particles administered into the body for drug delivery or diagnostic applications (23–25). It has been shown that particles with high AR, such as EDs, have the potential to mitigate phagocytosis (26). We hypothesized that the ability of particles to switch shapes may be used to achieve precise control over the extent and duration of mitigation of phagocytosis. PLGA-acid and -ester EDs (AR = 5, Table S1) were opsonized with mouse IgG and incubated with mouse peritoneal macrophages. PLGA-ester EDs switched their shape to spheres in due time, after which they were internalized (Fig. 5A, Movie S2, and Fig. S8a). As a control, PLGA-acid EDs, which are designed not to switch shape, were not phagocytosed by macrophages because of large AR (Fig. 5B, Movie S3, and Fig. S8b). The duration for which the particles resist phagocytosis depends directly on the dynamics of shape switch, which can be controlled over a wide range of times, from minutes to days, by choosing appropriate parameters.

Fig. 5.

Time-lapse video microscopy clips of shape-dependent phagocytosis by macrophage. (A) A shape-switching PLGA-ester ED (mixture of two PLGAs, AR = 5) was initially attached on a macrophage and not phagocytosed. The macrophage then quickly internalized the particle once shape switched to near-sphere shape. (B) Macrophage spread on a PLGA-acid ED [molecular mass 4.1 kDa, T_g(mid) = 27 °C, AR = 5], which do not switch shape at pH 7.4, but could not complete phagocytosis. All particles were opsonized with mouse IgG before the experiments. (Scale bar: 10 μm.)

Discussion

PLGA particles reported here exhibit stimulus-dependent shape switching. Yang et al. have previously reported shape switching of nanoparticles made from liquid crystalline polymers (27); however, those polymers are not physiologically compatible and the shape change occurred only under nonphysiological conditions (∼95 °C) with no temporal control. Studies have also reported on change in shape of hydrogel microparticles through their natural erosion behavior (28); however, synthesis of biocompatible polymeric particles with stimulus-controlled, physiologically compatible, switchable shape and their ability to modulate cellular interactions has not been previously demonstrated.

The results presented here demonstrate that shape of particles can be switched in real time in response to a wide range of stimuli, and the dynamics of shape switch can be controlled over a wide range of times by modulating physico-chemical properties of particles, such as molecular mass, size, and surface chemistry, as well as the strength of the external stimulus. The stimuli used here are also amenable to in vivo applications. For example, local elevation of temperature can be achieved by using ultrasound (29) or photothermal activation mediated by gold nanoshells (30), which can be incorporated in the particles. pH may also be used as a stimulus, especially for targeting tumors, which possess an acidic environment, or late endosomes and lysosomes, which also possess a distinctively acidic environment (31). Shape-switching particles can also be prepared at nanoscale. We prepared elongated particles from 160-nm PLGA nanoparticles and found that these particles switched their shape in 20 min upon temperature elevation from 25 to 40 °C (Fig. 2A, Bottom). Nanometer-sized particles changed shape in response to temperature in the same way as larger, micron-sized particles. Shape-switching nanoparticles could have potential applications for tumor targeting. Specifically, elongated (rods or elliptical disks) particles can be used to target tumors because of their prolonged circulation and better accumulation in tumors compared to their spherical counterparts (32–34). However, they exhibit slower internalization compared to spheres (34–37). Shape-switching nanoparticles can address this conflict by allowing the rods to accumulate in tumors and then shifting them to spheres to facilitate internalization. This shape shift can be induced by thermal or pH triggers as mentioned above.

The motivation to develop particles with switchable shapes comes from the fact that shape has recently emerged as a crucial design parameter of micro- and nanoparticles (3, 4, 33, 38, 39). Studies have demonstrated that departure from the conventional spherical shape of particles brings unique and improved functionalities to the particulate systems, resulting in improved biological responses. For example, macrophage-mediated phagocytosis is governed by the local geometry of the particles (40), and elongated particles exhibit reduced phagocytosis, longer circulation in the blood, and higher accumulation in target tissues (26, 32, 34, 41). The same shape, however, impedes particle internalization into target cells compared to spheres (34–36). Such conflicts in shape requirements can potentially be addressed through “switchable particles” whose shape can be switched in response to a stimulus.

Shape-switching particles follow the paradigm adopted by natural cells including erythrocytes and platelets as well as cancer cells, all of which undergo shape switch in response to a physical or chemical stimulus to adapt their properties and achieve advanced functions (42–44). Particles described here switch shape in one direction, and further studies are necessary to assess the feasibility of reversible shape switch. Upon future studies focused on cell-specific interactions and in vivo studies, shape-switching particles may open additional opportunities in biomedical applications.

Materials and Methods

Particle Preparation.

The characteristics of different types of PLGA used in this study are summarized in Table S1. Relatively uniform-sized PLGA spheres were synthesized by using a membrane emulsification technique (45). EDs were prepared by using previously published stretching method with some modifications (40). See SI Text for details of preparation of all particles.

Characterization of PLGAs.

Contact angle studies were performed by using a goniometer, and interfacial tension of PLGA in aqueous condition was calculated from the contact angles by using Young’s equation and equation of state approach (46). See SI Text for details. Viscosity of PLGA and zeta potential of PLGA particles was measured by using methods described in SI Text.

Shape-Switch Study.

The freshly prepared EDs were dispersed in deionized water, phosphate/citrate buffer, or Azure C solution prepared in phosphate/citrate buffer (10⁷ particles in 40 μL solution) in a polypropylene tube. The buffer solutions were normalized to 0.5 M of ionic strength by using NaCl. The particle dispersion was incubated in a temperature-controlled water bath (Isotemp Digital Model 210; Fisher Scientific). For temperature-induced switch, the temperature was adjusted to various levels. For pH-based switching, the pH of phosphate/citrate buffer was lowered to a specific pH by addition of a precalculated amount of 0.1 M citric acid. At a determined time point, the sample was immediately cooled down to 4 °C in the ice-cold water to stop further shape switch, and the AR of the particles was analyzed under the Axiovert 25 microscope at 100× or by SEM.

Scanning Electron Microscope (SEM).

SEM was used for confirmation of shape change progression of the particles at various time points. During the shape-change study, particle suspension was collected and dried at 4 °C to prevent further shape switch. To verify particle morphology, particles were coated with palladium (Hummer 6.2 Sputtering System; Anatech Ltd.) and imaged with a Sirion 400 scanning electron microscope (FEI Co.) at 3 eV.

Phagocytosis.

Mouse peritoneal macrophage cells (J774, ECACC) were used as model macrophages. The cells were cultured in DMEM (Gibco BRL) supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, and 10% fetal bovine serum under standard culture conditions (37 °C, 5% CO₂, humidified). Cells (2 × 10⁵ cells/mL) were allowed to attach in dishes lined with coverslip glass in the culture medium. The dishes were placed on the Axiovert 25 microscope at 100× with phase contrast filters and equipped with Bioptechs Delta T Controlled Culture Dish System® (Bioptechs Inc.) to keep the cells at 37 °C. For opsonization, EDs were incubated with 0.25 mg/mL mouse IgG (Sigma-Aldrich) for 60 min at 37 °C. The particles were washed twice with PBS. Opsonization was verified with fluorescently tagged IgG (Molecular Probes). EDs (one particle per cell) were added to the dishes and bright-field images were collected every 30 s for 20 min by a CoolSNAPHQ CCD camera (Roper Scientific) connected to the Metamorph® software. Observed cells were randomly chosen from the entire population, thus discounting potential bias because of heterogeneity in macrophage size (radius 7.5 ± 2.5 μm). Images were condensed into movies and analyzed for phagocytic events. Successful phagocytosis exhibited membrane ruffling at the site of attachment, blurring the crisp boundary of the membrane, and subsequent reforming of the membrane boundary after internalization.