Cooling-Triggered Release from Mesoporous Poly(N-isopropylacrylamide) Microgels at Physiological Conditions.

Poly(N-isopropylacrylamide) (pNIPAM) hydrogels have broad potential applications as drug delivery vehicles because of their thermoresponsive behavior. pNIPAM loading/release performances are directly affected by the gel network structure. Therefore, there is a need with the approaches for accurate design of 3D pNIPAM assemblies with the structure ordered at the nanoscale. This study demonstrates size-selective spontaneous loading of macromolecules (dextrans 10-500 kDa) into pNIPAM microgels by microgel heating from 22 to 35 °C (microgels collapse and trap dextrans) followed by the dextran release upon further cooling down to 22 °C (microgels swell back) . This temperature-mediated behavior is fully reversible. The structure of pNIPAM microgels was tailored via hard templating and cross-linking of the hydrogel using sacrificial mesoporous cores of vaterite CaCO3 microcrystals. In addition, the fabrication of hollow thermoresponsive pNIPAM microshells has been demonstrated, utilizing vaterite microcrystals that had narrower pores. The proposed approach for heating-triggered encapsulation and cooling-triggered release into/from pNIPAM microgels may pave the ways for applications of pNIPAM hydrogels for skin and transdermal cooling-responsive drug delivery in the future.

Introduction

Temperature-sensitive polymers are widely recognized as one of the most actively explored stimuli-responsive materials for drug release. In the majority of reported systems, the drugs are released in response to the heating to approximately 40 °C.[1,2] Meanwhile, for some clinical applications (e.g., skin delivery) cooling-triggered release is often more favorable because the temperature can be safely decreased over a wider temperature range than that being raised by heating. Local overheating may result in the damage of healthy tissues. For instance, cooling of the human skin with gel packs or ice bags for less than half an hour leads to a drop of skin-surface temperature below 25 °C, wherein no occurrence of any undesirable side effects is reported.[3−5] This makes cooling as a temperature control over the release safer and superior over heating. However, until now there have been only a few reports of cooling-triggered release[6] that is mainly caused by the challenges in the design of such formulations.

Among the variety of thermosensitive polymers, poly(N-isopropylacrylamide) (pNIPAM) is considered as one of the most popular materials.[2,7] pNIPAM exhibits a reversible coil-to-globule phase transition at about 32 °C, which is highly attractive in a physiological context. There is no unique mechanism in which pNIPAM liberates its payload; the release is largely governed by the drug–pNIPAM interactions and depends on the nature of the drug, pH, ionic strength, etc.[8] Most commonly, heating of pNIPAM above its lower critical solution temperature (LCST) swiftly reduces the volume of the gel, which results in a fast ejection of preloaded drugs. This mechanism has been demonstrated for molecules of a different nature, for example, small hydrophobic drugs[9,10] and proteins.[11,12] At the same time, the most common mechanism of drug release from swollen pNIPAM gels at low temperatures is passive diffusion.[7,13] It has been demonstrated that some drugs may be liberated from a pNIPAM matrix upon cooling.[14−16] Cooling-mediated release of macromolecules from pNIPAM gels still remains a challenge; it has been achieved via 3D design of pNIPAM hydrogel, such as the fabrication of core–shell structures,[13] the use of interpenetrating polymer networks,[17] or the coating of metal–organic frameworks.[14]

Depending on the desired features, pNIPAM gels of various geometries can be fabricated, wherein spherical pNIPAM nano- and microgels are of special interest in controlled drug release.[18−23] Importantly, internal structure and mechanical and temperature-responsive properties of pNIPAM microgels can be tuned in a wide range.[24,25] For instance, large loading capacity and quick temperature response are pivotal characteristics for drug delivery applications. Both can be improved via the templating of porous pNIPAM gels on sacrificial matrixes that act as porogens.[26−30]

Recently, mesoporous crystals of vaterite CaCO3 were used as porogens to fabricate pNIPAM mesoporous microgels (hereinafter denoted as MM-gels).[30] Particles made of other biologically relevant functional molecules, for example, PEG[31] and proteins,[32,33] have also been assembled using this approach, giving an inverted replica of the mesoporous vaterite crystals. The vaterite CaCO3 crystals are beneficial, in contrast to many other decomposable matrixes because they can be eliminated at truly mild conditions (slightly acidic pH or the presence of chelating agents like EDTA or citric acid).[32] Besides this, the size, porosity, and shape of the crystals can be tuned.[34,35] These biocompatible crystals were successfully utilized for encapsulation of both small compounds[36,37] and biomacromolecules.[38−40]

At neutral pH, the MM-gels[30] shrunk under heating above the LCST by a factor of about 2, which corresponds to the shrinkage of conventional nonporous pNIPAM microgels. At acidic pH, the MM-gels have a reduced internal charge, allowing them to shrink to a much higher extent, that is, almost by an order of magnitude compared to their initial size before heating. This reversible temperature-mediated behavior demonstrates a high potential to utilize the MM-gels for drug delivery applications. In addition, the MM-gels represent an inverted CaCO3 replica with porosity governed by the size of CaCO3 nanocrystallines (10s of nm[34]) as building blocks from which the CaCO3 vaterite crystals are made. This mesoporous structure makes the MM-gels idoneous for impregnation with large therapeutic macromolecules like proteins and nucleic acids.

In the present study, we verified whether the MM-gels can serve as temperature-responsive material for entrapment and release of macromolecular compounds at physiologically relevant conditions via a cooling-triggered release. Dextran (DEX) molecules served as model uncharged macromolecules to avoid electrostatic interactions between the pNIPAM matrix and the payload and assess the entrapment driven only by the MM-gels phase-transition-induced shrinkage/swelling behavior.

Experimental Section

Materials

The materials used include calcium chloride dihydrate (CaCl2·2H2O; Sigma-Aldrich, Japan), sodium carbonate (Na2CO3, anhydrous; Sigma-Aldrich, Germany), phosphate buffered saline (PBS buffer, tablets, 0.01 M; Sigma, USA), fluorescein isothiocyanate-DEX [mol wt (MW): 10, 70, and 500 kDa; Sigma, USA], rhodamine 6G (Rho6G) chloride (Sigma, England), ethanol for spectroscopy (99.9%; Merck, Germany), Fluoresbrite calibration grade 6.0 μm microspheres (Polysciences Inc., UK), and hydrochloric acid fuming (37%, 0.05 M in all experiments; Merck, Germany). The water used in all experiments was prepared via a Millipore Milli-Q purification system and had a resistivity higher than 18.2 MΩ-cm.

Poly(N-isopropylacrylamide)-4-acryloylbenzophenone (pNIPAM-ABP) was kindly provided by Dr. Leonid Ionov, Germany. Briefly, pNIPAM-ABP was synthesized from 4-benzophenone acrylate (0.28 g, 1.12 mmol) and NIPAM (6 g, 51.57 mmol) dissolved in 30 mL of ethanol. The mixture was purged with nitrogen for 30 min. The polymerization was carried out at 70 °C under a nitrogen atmosphere with mechanical stirring overnight. The cooled mixture was poured into 750 mL of diethyl ether, and the precipitate was filtered and dried in vacuum at 40 °C.

Fabrication of CaCO3 Crystals

Three milliliters of Na2CO3 (1 M) was rapidly added to 12 mL of CaCl2 (0.25 M) solution at constant stirring at a moderate agitation speed; the time mixing was 30 s. The agitation temperature was kept at either 22 or 45 °C. In this process, calcium carbonate appeared as a white precipitate immediately after two salt solutions were mixed. The incubation was 15 and 30 min at 22 and 45 °C, respectively. CaCO3 crystals were rinsed thoroughly three times with water followed by the removal of the supernatant and drying at 90 °C overnight. The powdered calcium carbonate was stored at room temperature.

Synthesis of pNIPAM-ABP Microgels

The scheme of pNIPAM-ABP cross-linking is shown in Figure . pNIPAM-ABP was mixed with the suspension of CaCO3 crystals (2.2 mg) in 10 mL of ethanol under the polymer/CaCO3 weight ratio of 3:7 or 2:8 for the crystals prepared at 45 and 22 °C, respectively. The mixture was stirred for 10 min at 25 °C followed by a rotary evaporation of the ethanol (75 mbar, 90 min, 25 °C, Rotavapor R-200 Buchi). Polymerization of pNIPAM-ABP inside the CaCO3 templates was initiated by UV-light irradiation (254 nm, 120 min, 25 °C). Samples were dispersed in 1 mL of water and stained with 0.1 mg/mL Rho6G for 15 min, if required. CaCO3 cores were dissolved by the addition of hydrochloric acid (150 mM) directly before further experiments.

Figure 1

Radical cross-linking of pNIPAM-ABP under irradiation with UV light.

Mechanical Trapping of DEX

Forty microliters of the suspension of prepared pNIPAM/CaCO3 particles (3 mg/mL) was placed into a μ-Slide 2 well (ibidi, Germany) closed with a cap and supplied with a thermocouple element (Voltcraft ME-22T, Korea) and peristaltic pump P720 (Instech, USA) for removal of extra solution from the well (Figure S1, Supporting Information). At all experimental steps, exchange of the solutions was performed by pumping out solution into the well and adding a new one. After core removal with HCl, the solvent was exchanged to PBS (10 mM, pH 7.4), which was used as a solvent for all the next steps. pNIPAM microgels were incubated with the solution of DEX of different molecular weights (0.1 mg/mL) at 22 °C. Then the temperature was sharply increased until 33–37 °C inside the well by rapid addition of the double volume of PBS preheated to 70 °C (Thermo Bath Sonorex Super 10P, Bandelin, Germany). The temperature was kept constant by exchanging the solution in the well with hot PBS solution during the observation. Similarly, the fast addition of a 2-fold volume of the PBS solution with the temperature of 7.5 °C allowed us to regain the temperature of 22 °C.

Confocal Laser Scanning Microscopy (CLSM)

CLSM images were obtained on a Zeiss LSM 510 Meta (Zeiss, Germany), equipped with a 63× (N.A. 1.4) oil-immersion objective. Standard filter settings for excitation and emission of FITC or Rho6G were used for a laser source of 488 or 543 nm, respectively, and for transmission images at 633 nm. Images were acquired in series of 0.3–0.5 μm optical sections and figures were presented as xy images. Images were processed in a Zeiss LCM Image browser, ImagePRO, and ImageJ (Adobe Systems Inc.) to enhance the brightness and color and to take fluorescence profiles.

Scanning Electron Microscopy (SEM)

SEM images were recorded by means of a LEO 1550VP (Zeiss, Germany) electron microscope at an accelerating voltage of 3 kV. Samples were lyophilized; conductive 5 nm coating of Au/Pd was applied.

Brunauer–Emmett–Teller (BET) Analysis

N2 adsorption–desorption analysis of vaterite crystals was carried out using a QUADRASORB SI-MP (Quantachrome Instruments, USA) at 77.3 K. The samples were degassed at 150 °C for 20 h prior to the measurements. Porosity analysis was performed using the Barret–Joyner–Halenda model.

Results and Discussion

Fabrication and Internal Structure of MM-Gels

Figure demonstrates the main principle of MM-gels design. Vaterite crystals have pores (white areas, a) that were filled in with pNIPAM (blue areas, b). Cross-linking and elimination of the vaterite template (gray areas in a,b) leads to the formation of pure pNIPAM microparticles (c) responsive to heating and cooling (d).

Figure 2

Principal scheme of formulation of MM-gels and temperature-triggered loading/release of a payload (DEX). (a) Vaterite CaCO3 crystal. (a, b) Loading of pNIPAM-ABP into the crystals followed by UV-cross-linking and formation of pNIPAM/CaCO3 hybrids. (b, c) Dissolution of the CaCO3 crystal followed by a washing step with PBS. (c) pNIPAM MM-gel. (c, d) Loading of MM-gel with DEX by increasing the temperature above LCST followed by microgel shrinkage. (d, c) Release of DEX from the MM-gel by cooling below LCST due to the microgel swelling back.

For preparation of MM-gels in this study, spherical vaterite CaCO3 crystals with a diameter of 10 ± 2 μm were loaded with pNIPAM-ABP by the solvent evaporation technique followed by UV-light-initiated cross-linking of the polymer entrapped in the crystal pores as described elsewhere.[30] CaCO3 crystals were prepared at 45 °C[34] and had pores in the range of 5–35 nm, as was confirmed by BET analysis. Confocal laser scanning microscopy (CLSM) of pNIPAM/CaCO3 hybrids (step b, Figure b) stained with the fluorescent probe Rhodamine 6g (Rho6G) demonstrates a uniform distribution of pNIPAM inside the entire volume of the crystals (Figure a–c). The changes in the transparency of pNIPAM/CaCO3 hybrids occurring upon the addition of HCl are attributed to the elimination of CaCO3 matrix while the formation of spherical gel-like structures on hybrid crystals prove the formation of the MM-gels (Figure S2). Complete dissolution of the CaCO3 matrix using HCl resulted in the formation of one-component pNIPAM MM-gels (Figure d–f) with a diameter about 2 times smaller than that of the initial CaCO3 crystals. This may be attributed to a partial closure of the pores of the MM-gels after template removal. The fluorescence profile (Figure f) confirms that pNIPAM matrix remains uniformly distributed within the MM-gel after the reorganization induced by CaCO3 dissolution. It is also of note that without UV irradiation pNIPAM microparticles are not formed (data are not shown). In contrast to pNIPAM and pNIPAM/CaCO3 hybrids, bare CaCO3 crystals do not bind Rho6G because of electrostatic Rho6G-CaCO3 repulsion, which provides additional evidence of the successful formulation of MM-gels (Figure S3).

Figure 3

Fluorescence and transmittance of CLSM images and corresponding fluorescence profiles of pNIPAM/CaCO3 hybrids (a–c) and MM-gels (d–f) templated on CaCO3 crystals with pores in the range of 5–35 nm. pNIPAM is stained with Rho6G. Scale bars are 10 μm.

The internal structure of CaCO3 templates and formed MM-gels was analyzed by SEM. Typical morphology of the CaCO3 vaterite crystals is depicted in Figure a,b. The crystals were comprised from nanocrystallines and possess a highly developed mesoporous structure that is typical for the vaterite polymorph. Figure a also illustrates the presence of nonporous rhombohedral calcite crystals; however, their content was estimated as less than 5%.

Figure 4

SEM images of (a) pure CaCO3 crystals (overview) and (b) the crystal surface; (c, d) MM-gels after one heating/cooling cycle (22–35–22 °C), overview (c), and a single MM-gel particle (d).

Images of the dried MM-gels (after one heating/cooling cycle) shown in Figure c,d display the mesoporous nature of the MM-gels with clearly visible pores in the range 10–60 nm, which should be suitable for the loading of macromolecules. A smaller fraction of large pores in the 100–200 nm range was also identified.

Interestingly, the shape of the microgels slightly varied from the spherical one. It was estimated by laser scanning microscopy using a z-stack (Figure ). Since the pronounced difference in lateral (xy) and axial (z) resolution of CLSM leads to artificial extension of an image in the z-direction, the 3D shape of MM-gels was compared to the shape obtained for incompressible polystyrene particles 6 μm in diameter. Typical CLSM image in all xy-, xz-, and yz-planes with a schematic presentation of this image are shown in Figure a. As expected, spherical particles had an ellipsoidal shape in the z-plane because of the difference in diffraction-limited resolutions in xy- and z-planes. Particle sizes (α and β) were calculated as the full widths of fluorescence profiles in xy- and z-planes, respectively. The ratio between the z and xy particle size was calculated as k0 = β/α = 2.0 ± 0.1 (n = 3, ±s.d.). CLSM image of a representative MM-gel particle and its schematic explanation are presented in Figure b. In this case, the ratio between the z and xy particle size (k) was found to be 1.5 ± 0.2 (n = 3, ±s.d.). Calculating the ratio k/k0 allows one to estimate that MM-gels on a glass surface have an ellipsoidal shape flattened by a factor of 1.33. This is most probably driven by their interaction with the glass surface. Porous and, as a result, very soft MM-gels can flatten to increase contact with the surface driven by an entropy increase because of the release of bound water and counterions. An understanding of the initial shape of the MM-gels is important for their possible use in microgel-based stretchable reservoir devices responsive to mechanical forces.[41,42]

Figure 5

z-Stacking using confocal laser scanning microscopy. Left: incompressible spherical 6 μm polystyrene. Right: MM-gel stained with Rho6G. Corresponding xy and yz fluorescence profiles are given below. Scale bars for both confocal images are 10 μm.

Temperature-Mediated Loading/Release of DEXs-FITC

Furthermore, we investigated the temperature-mediated entrapment of DEXs-FITC by MM-gels. Sharp temperature changes from 22 to ca. 35 °C and back to 22 °C were achieved by the rapid exchange of the solvent. This procedure was designed to ensure the transition through the LCST of the MM-gels, which was reported in a previous study as 31–32 °C.[30] It is of note that the LCST of pure pNIPAM-ABP, which was found to be lower than that of pNIPAM-ABP MM-gels (ca. 29–30 °C in aqueous solution), was also crossed over (Figure S4). The heating of the swollen MM-gels above their LCST resulted in reversible reduction of the size by 50 ± 8%. Importantly, the presence of DEX-FITC of different molecular weights has no effect on the MM-gel shrinkage/swelling behavior (Figure ). Thus, the presence of DEXs-FITC does not hinder the shrinkage of the gels and macromolecules can potentially be trapped during the shrinkage.

Figure 6

Average diameters of CaCO3 pNIPAM/CaCO3 hybrids and corresponding MM-gels in the presence of DEX of different molecular weights at a temperature variation from 22 to 35 °C and back to 22 °C. Error bars are s.d. for n = 20–30.

In addition to the estimations made on the basis of SEM imaging (Figure ), the porosity of the swollen MM-gels was estimated on the basis of the diffusion of DEXs of different molecular weights into the gel matrix as examined by CLSM (Figures –9). The 10, 70, and 500 kDa DEXs-FITC having hydrodynamic radii of 2.3, 5.6, and 13.8 nm, respectively, were used as probes. The loaded amounts of DEXs-FITC were inversely proportional to their molecular weights, indicating diffusion imitations for larger probes. We assume that the MM-gels had pores 10–60 nm and the proportion of the narrow pores (not accessible for 500 kDa DEX-FITC) was rather high since the relative fluorescence of 500 kDa DEX-FITC accumulated inside the microgels was much less (rough estimation gives a factor of 3) compared to that for DEX with smaller molecular weights. The range of pores of 10s of nanometers corresponds well to the dimensions of nanocrystallites the crystals are made of, therefore proving that the MM-gels is likely an inverted replica of the crystals.

Figure 7

(a) CLSM (fluorescence/transmittance) images of pNIPAM MM-gels during incubation with 0.1 mg/mL DEX-FITC of molecular weight 10 kDa at 22 °C (upper images), exposed to 35 °C by preheated PBS solution (middle), followed by cooling back down to 2 °C (bottom). Scale bar is 10 μm for all images. Corresponding fluorescence radial profiles are shown in (b).

Figure 9

(a) CLSM (fluorescence/transmittance) images of pNIPAM MM-gels during incubation with 0.1 mg/mL DEX-FITC of molecular weight 500 kDa at 22 °C (upper images), exposed to 35 °C by preheated PBS solution (middle), followed by cooling back down to 22 °C (bottom). Scale bar is 10 μm for all images. Corresponding fluorescence radial profiles are shown in (b).

A swift increase of the temperature from 22 to ca. 35 °C by the addition of preheated PBS solution to MM-gels preincubated with DEXs-FITC resulted in a shrinkage of the gels and the entrapment of DEX in the MM-gels (Figures –9). This was accompanied by a partial release of the DEX-FITC molecules due to a dilution by the buffer. This release was more pronounced for 10 and 70 kDa DEX-FITC (Figures and 8, respectively) and was marginal for 500 kDa DEX-FITC (Figure ). Further cooling back down to 22 °C led to the swelling of the MM-gels, which triggered almost complete release of the entrapped DEX-FITC (Figure –9).

Figure 8

(a) CLSM (fluorescence/transmittance) images of pNIPAM MM-gels during incubation with 0.1 mg/mL DEX-FITC of molecular weight 70 kDa at 22 °C (upper images), exposed to 35 °C by preheated PBS solution (middle), followed by cooling back down to 22 °C (bottom). Scale bar is 10 μm for all images. Corresponding fluorescence radial profiles are shown in (b).

DEX loading upon the swift transition of the temperature from 22 to 35 °C and its release upon a reverse change occurred in the time scale of less than a minute, which testifies to the fast kinetics of DEX loading and release. It was observed for all tested DEX regardless of their molecular size.

It can be assumed that the swelling results in opening of the pores of the microgels, allowing free outward diffusion of the entrapped macromolecules. The degree of DEX derivatization with FITC labels ranged from 0.002 to 0.008 (according to the information provided by Sigma-Aldrich), introducing minimal changes to the DEX structure, which suggests that hydrophobic interactions between the FITC label and pNIPAM matrix unlikely influence the uptake and release of DEXs as model compounds. In other words, it can be postulated that the mechanism that underlies the uptake/release of the DEXs is defined by the passive diffusion of the macromolecules rather than by their interactions with the pNIPAM matrix. In this context, the partitioning coefficient between the microgels and surrounding medium should be the determinative for DEX loading and release.

Interestingly, while the entrapment of DEX-FITC is size-selective, the release of DEX-FITC seems independent of their molecular weights and nearly all DEX-FITC molecules are released in a burst manner after the cooling step (Figures –9), as can be concluded by a comparison of corresponding fluorescence profile. The results described above prove the concept of cooling-triggered release by temperature variation in the range 22–35 °C, acceptable for a number of biomedical applications. Finally, we verified whether the internal structure of the MM-gels could be tuned since this can provide future opportunities to control the entrapment and release of cargo.

Fabrication of MM-Shells

pNIPAM microgels with a shell-like structure (MM-shells) were synthesized using the same procedure as described above but utilizing the vaterite crystals that have smaller pores. These crystals were fabricated at 22 °C, as described elsewhere,[34] and had pores in the range of 3–15 nm (confirmed by BET). CLSM demonstrated that MM-shells have nonuniform shell-like distribution of pNIPAM that is caused by less uniform loading of pNIPAM into the crystals with smaller pores and its predominant localization on the surface of the crystals (Figure ). LCST of pNIPAM MM-shells (33–34 °C) was higher than that determined for MM-gels (31–32 °C[30]), Figure S5. Both MM-gels and MM-shells possessed a reversible thermal response at least in one heating/cooling cycle. Heating of the MM-shells above the LCST resulted in the reduction of their size by 70 ± 6%, which is more significant shrinkage compared to that of MM-gels (size reduction by 50 ± 8%). Temperature-dependent size change and other properties of the MM-shells and MM-gels may depend on the polymer/CaCO3 weight ratio used for their fabrication. Indeed, it was partially demonstrated for the MM-gels in a previous report where the optimal ratio was determined by a comparison of the size of the formed MM-gels as a function of the pNIPAM/CaCO3 mass ratio.[30] In this study polymer/vaterite ratios were slightly different for MM-shells and MM-gels (2:8 and 3:7, respectively). The ratio of 2:8 was chosen because its further increase did not influence the size of the MM-shells, indicating the achievement of saturation of the crystals with the polymer (Figure S6). The opportunity to modulate the geometry of the formed pNIPAM microgels itself is an important milestone in the development of temperature-sensitive drug delivery systems. In this work we forwent from further investigation of temperature-triggered loading and release from the MM-shells, keeping this question for further studies.

Figure 10

Fluorescence and transmittance CLSM images and corresponding fluorescence profiles of pNIPAM-CaCO3 hybrids (a–c) and MM-gels (d–f) templated on CaCO3 crystals with pores in the range 3–15 nm. pNIPAM is stained with Rho6G. Scale bars are 10 μm.

Future works will focus on the design of tailor-made porous pNIPAM micro- and nanogels to establish a tool for advanced cooling-triggered drug delivery. Below the mechanism of the temperature-triggered release from the MM-gels is discussed.

Conclusion

Cooling-mediated release from a pNIPAM microgels template on sacrificial vaterite cores can be attributed to the heterogeneous structure of the microgels tailored by two orthogonal parameters, that is, the templated structure due to the microgel synthesis and the external stimulus (temperature) applied to manipulate the phase transition of the gels. MM-gels have two populations of pores: (i) 1–10 nm associated with the pNIPAM network itself and (ii) a few 10s of nanometers that correspond to the size of CaCO3 nanocrystallines, which are solubilized during microgel synthesis and define the pore sizes. The temperature of reversible volume phase transition is preserved, as known for conventional microgels with only small pores of population (i). We believe that the heating of the MM-gels above the LCST is accompanied by the closure of both populations of the pores. If only pores of the population (i) would collapse, the shrinkage would not be as pronounced as that for nonporous microgels because of less material in the MM-gels compared to that for nonporous gels. At the same time, the collapse of the larger pores of the population (ii) is proven by the trapping of DEX molecules, for which the size significantly exceeds the dimensions of the smaller pores of population (i). After the heating above LSCT, the cooling down below the LCST leads to a release of the trapped DEX molecules because of the reversible opening of the pores of both populations because the size change of the MM-gels upon the temperature variation is completely reversible. We assume that the entrapment/release of DEX is based on its passive diffusion in/outward of the microgels and therefore DEX distribution between the microgels and the solution depends on the partitioning coefficient. When switching from model DEX molecules to therapeutic macromolecular drugs, one can likely expect more sophisticated mechanisms of drug uptake and release, which should be largely defined by interactions between the pNIPAM matrix and the macromolecules.

Many studies reported the possibility to produce nano-CaCO3. Among others, our recent results demonstrated the approach to fabricate nanocrystals that have a similar internal structure as large microsized CaCO3.[43] This opens avenues for the fabrication of mesoporous pNIPAM nanogels analogous to that of MM-gels. Potentially, such nanoformulations could be utilized for other administration routes such as intravenous injections.

In the future, the findings of this study could be broadened toward other external stimuli like pH, light, and alternating magnetic field, which allow the uptake and release of drugs on demand,[18] which will be the focus of our upcoming studies. Several populations of pore sizes in the microgels offer a unique option for the size-selective uptake of drugs and other bioactives of different sizes that can be released stepwise at physiologically relevant conditions to achieve various biological targets.