Micro ﬂ uidic fabrication of composite hydrogel microparticles in the size range of blood cells †

The fabrication of alginate hydrogel microparticles with embedded liposomes and magnetic nanoparticles for radiofrequency controlled release of encapsulated chemical cargo was considered. An extractive gelation process was implemented in a micro ﬂ uidic device, which enabled the production of uniform composite microparticles of dimensions comparable to those of blood cells (between 5 and 10 m m). The critical parameters that control the extractive gelation process were systematically explored and feasible values that provide microgel particles of a de ﬁ ned size and morphology were identi ﬁ ed. First, the initial water-in-oil droplet is formed in a ﬂ ow-focusing junction whose size is controlled by the ﬂ ow-rate of the oil phase. Then, the train of droplets is sandwiched between two streams of oil containing calcium ions. In that way, a ﬂ ux of water molecules from the droplets towards the continuous phase as well as a transport of calcium ions towards the disperse phase are initiated. The ﬁ nal microparticle properties were thus found to be sensitive to three elementary sub-processes: (i) the initial droplet size; (ii) the extraction of water into the oil phase, which was controlled by the volume of the oil phase and its initial moisture content; and (iii) the kinetics of ionic cross-linking of the alginate matrix, which was controlled by the varying calcium concentration. The size and morphology of the ﬁ nal composite microgels were fully characterized.


Introduction
Recent research in the eld of drug delivery systems has resulted in the rst prototypes of chemical robotshydrogel composite microparticles, which are able to store, release and chemically process active components encapsulated in the particles as a result of external radiofrequency signals. 1 The functional components (liposomes, magnetic nanoparticles, enzymes) are encapsulated in the alginate gel (Fig. 1A).Alginate is a biopolymer soluble in water, which gels in the presence of multivalent ions like calcium. 2 The purpose of the alginate matrix is to hold all the functional components together in a single composite particle, and at the same time allow rapid diffusion of low molecular weight payloads out of the particle once they are released from the liposomes.In addition, alginate is biodegradable and can be relatively easily functionalized e.g. by the attachment of targeting antibodies.The functional components encapsulated into the alginate matrix were iron oxide nanoparticles (Fig. 1B) for radiofrequency heating 3 and MRI visualization, 4 liposomes (Fig. 1C) for storage and release of active molecules or their precursors, 5 and optionally also immobilized enzymes that facilitate the conversion of pro-drugs to active substances. 1 Liposomes are spherical structures formed by a phospholipid bilayer, which are able to release the active matter in response to a temperature stimulus (Fig. 1C). 6he permeability of liposomes can be signicantly increased once they are heated above their phase transition temperature.In the case of chemical robots, the needed temperature change is achieved by iron oxide nanoparticles upon exposure to external radio-frequency magnetic eld. 3arly prototypes of chemical robots were fabricated by the drop-on-demand inkjet printing method, 3,7 and have characteristic dimensions around 50 mm.However, for in vivo applications in drug delivery, it is necessary to further reduce their size to that of the blood cells 8 or lower, i.e. approximately by a factor of 10 in diameter (a factor of 1000 in volume), which is a challenging task.For instance, human erythrocytes (red blood cells) have a characteristic diameter of 6-8 mm and various types of leukocytes (white blood cells) can have characteristic diameters ranging from 10-12 mm for neutrophils up to 12-15 mm for large lymphocytes. 9][15][16][17][18][19][20][21][22] Overall, synthesis of the alginate microparticles on a custom design microuidic chip appears like the most feasible technique to synthesize monodisperse alginate microparticles.By appropriate design of the microuidic chip, it is possible to prepare a wide range of different alginate microparticles, although it turns out that the size of the microparticles is a limiting factor if blood cell-sized particles are desired.For example, Liu et al. produced monodisperse alginate microparticles with diverse shapes (such as plugs, disks, microspheres, rods or threads) in a microuidic chip with two individual ow-focusing channels and a synthesizing channel where the gelation of the microparticles was obtained.The smallest particles had a diameter of 30 mm. 15 Yeh et al. used a T-junction channel to produce a w/o emulsion, which was gelled in the bulk by CaCl 2 .They encapsulated nanoparticles into alginate microparticles with diameters ranging from 70 to 220 mm. 20Pectin-alginate Janus hydrogel microparticles with average diameter of 90 mm were synthesized by Marquis et al. 16 by a ow focusing channel and gelled by internal gelation. 22Finally, Zhang et al. prepared 40 mm alginate microparticles in a microuidic chip, where on-chip gelation was achieved by providing sufficient residence time in the microchannel. 21t has been shown that a further reduction of the particle size on the microuidic chip is possible by using an organic solvent with a non-negligible water solubility as the continuous phase.In that way, it enables partial extraction of water from the droplets, consequently reducing their size.Rondeau and Cooper-White 18 have demonstrated the principle of extractive gelation using dimethyl carbonate (DMS) as a continuous phase.By extractive gelation, Seki's group 19,23 has produced collagen and alginate microparticles in the size range lower than 10 mm.By varying the initial alginate concentration and using methyl acetate as the continuous phase they achieved pure alginate microparticles with a diameter of 6 mm.In addition to pure alginate microparticles, it was also shown that DNA molecules can be immobilized in the gel; however, the particle size with immobilized DNA increased up to 20 mm. 19The principle of extraction coupled with both internal and external gelation was also applied to pectin microparticles and the possibility to control not only size but also morphology was demonstrated. 17he objective of the present work was to achieve a reduction in particle size by the principle of extractive gelation, while exploring the possibility to incorporate functional components (magnetic nanoparticles and liposomes) into the hydrogel matrix.Previously, such functional components have never been incorporated into hydrogel microparticles produced by microuidics.We are introducing a method to produce monodisperse composite alginate microparticles with a sub 10 mm characteristic size using a custom-designed microuidic chip that implements extractive gelation (Fig. 2).First, droplets of aqueous solution of alginate in the continuous oil phase are formed in a ow-focusing channel.Second, gelation of the droplets is established in a long wavy channel by diffusion of calcium ions from the continuous phase.At the same time, the volume of the droplets is reduced by partial extraction of water into the continuous phase.Depending on the volumetric owrates of the aqueous and the oil phase, different conditions of the production were mapped and the effect of extraction on the achievable size of the microparticles was evaluated.The inuence of the calcium concentration on the buckling of the alginate microparticles and their nal morphology was also part of our investigation.The ideal conditions to produce monodisperse microparticles and to incorporate functional components (iron oxide nanoparticles, liposomes) were found.

Fabrication of microuidic chip
The chip was made by a standard so lithography technique.The master was fabricated from a photoresist on a silicon wafer according to a previously published protocol. 24The PDMSbased microuidic device was fabricated by pouring PDMS prepolymer in a 1 : 5 cross-linking ratio onto the master.Aer curing at 70 C for 22 minutes the polymer was lied off with the pattern on its surface.Separately, a cover glass was coated with PDMS in a 1 : 14 cross-linking ratio and cured at 70 C for 26 minutes. 25The thin layer of PDMS on the cover glass was achieved by spin-coating (Spin 150, 2000 rpm for 40 s).The nal microuidic chip was assembled by fusing the PDMS piece together with the PDMS-coated glass and le in an oven at 70 C overnight.The bonding was achieved due to diffusion of excess cross-linker from the PDMS piece to the thin layer of PDMS on the glass.The bonding achieved in this way was sufficient for the pressures used in subsequent uidic experiments.Being made from PDMS, the channel walls were hydrophobic.The width of the microchannel at the ow focusing orice was 50 mm, whereas the width of the rest microchannels was 85 mm.The height of the microchannels was 22 mm.A schematic of the microuidic chip layout and a capture of microparticle production can be found in ESI-1.†

Principle of alginate microparticle formation
Droplets of alginate aqueous solution were formed in the ow focusing section of the microuidic device, as shown in ESI-1.† The disperse phase was aqueous alginate solution (1% w/w) with a viscosity of 32 mPa s and the continuous phase was 1undecanol with 5% w/w of surfactant Abil Em 90.The chosen surfactant concentration was the result of a separate optimization study, detailed in ESI-2.† The ow focusing geometry offers a convenient method to produce monodisperse droplets (typical coefficient of variation 1.3%).Once droplets were formed, a second stream of immiscible liquid was introduced (1-undecanol with 5% w/w Abil Em 90 and 2% CaI 2 ) in the next section (Fig. 2) to initiate the gelation of the alginate droplets due to diffusion of calcium ions.The gelation then continued in the long wavy channel (130 mm) in the third section of the micro-uidic device (Fig. 2).Calcium ions have to be introduced aer the formation of the droplets to avoid pre-mature gelation of alginate in the ow-focusing orice, which would cause instabilities during the droplet formation.Following the introduction of the continuous phase, the alginate droplets decrease their volume not only due to cross-linking by Ca 2+ , but primarily due to the extraction of water from the disperse to the continuous phase (the solubility of water in 1-undecanol without surfactant at room temperature was found to be 2.7% v/v by FTIR spectroscopic measurements, which is consistent with values reported in the literature 26 ).At the same time, the solubility of 1-undecanol in water is negligible, therefore no mass transfer of the oil phase into the droplets takes place.

Parametric investigation of extractive gelation
The extraction of water from the droplets is affected by the volume ratio between the continuous and the disperse phase; this dependency was systematically investigated to control the size of the produced microparticles.The ow rate ratio, and thus the volume ratio, of the oil to the aqueous phase was investigated in a range lying from 10 to 100.The lower limit is given by the need to separate individual water droplets in the ow-focusing section, and the upper limit is given by the pressure conditions in the microuidic chip (to avoid back ow).However, not only the ratio but also the absolute values of the ow-rates of all phases inuence the morphology of the alginate microparticles.Different ow rates of the disperse phase (Q w ¼ 2-12 ml h À1 ) and the continuous phase (Q oil1 ¼ 50-190 ml h À1 and Q oil2 ¼ 10-150 ml h À1 ) were studied, where Q w denotes the ow rate of the alginate solution, Q oil1 refers to the oil ow rate into the ow focusing section, Q oil2 refers to the oil ow rate into the gelation section, and Q oil ¼ Q oil1 + Q oil2 will be used to denote the overall ow rate of the oil phase.For all the studied combinations of ow rates, the stable dripping regime was present but vastly different nal particle morphology was observed, as will be discussed in Section 3.2.The aqueous solution of alginate and both oils were supplied to the microchannels by means of digitally controlled syringe pumps (neMESYS, low pressure module, Cetoni) attached to PTFE poly(tetrauoroethylen) tubes (Adtech).The process of microparticles formation was observed and recorded by a high-speed camera (Redlake Motion Pro) attached to an inverted optical microscope (Olympus CKX 41).Video images taken during the microparticle formation process were analyzed by image analysis soware ImageJ.

Preparation of composite alginate-magnetic-liposome microparticles
To prepare alginate microparticles that also contain functional components (magnetic nanoparticles and liposomes), the disperse phase was prepared as follows: 1 ml of 2% w/w aqueous solution of alginate was ltered to remove any impurities (0.2 mm pore lter, Sartorius Biotech) and mixed with 500 ml of magnetic nanoparticle suspension in deionized water (10 g l À1 ) and 500 ml suspension of liposomes puried by gel chromatography and stirred for 10 minutes.The details of the preparation of the magnetic nanoparticles and the liposomes are provided in ESI-3, ESI-4 and 5. † Aer mixing of the alginate solution with the magnetic nanoparticle and the liposome suspensions, the nal alginate concentration was 1% w/w, i.e. the same as in the pure alginate experiments described above.The continuous phase was also identical as in the case of pure alginate experiments, i.e. 1-undecanol with 5% w/w Abil Em 90 for the ow-focusing section and 1-undecanol with 5% w/w Abil Em 90 and 2% CaI 2 in the gelation section.Both phases were introduced into the microuidic chip by PTFE tubes and the system was controlled as described above (Section 2.4).The microparticles were collected outside of the chip in a beaker connected with the outlet of the chip via PTFE tubes (0.33 mm ID Â 0.75 mm OD), separated from the oil phase by magnetic decantation and nally washed by deionized water three times.The internal structure of the microparticles was visualized by laser scanning confocal microscopy (Olympus Fluoview FV-1000).

Release kinetics measurement
In order to conrm the ability of the composite alginatemagnetic-liposome microparticles to store and release encapsulated chemical payload, the liposomes were loaded by a uorescent dye carboxyuorescein (CF) as described in ESI-5, † which has excitation/emission wavelength at 490/516 nm.Two types of release measurements were of interest: spontaneous leakage of liposomes during storage (which is undesirable), and controlled release of CF from liposomes by external stimuli (which is desirable).In both cases, the uorescence of the bulk solution containing the alginate-magnetic-liposome microparticles was measured by a uorescence spectrometer Cary Eclipse (Agilent Technologies).Typically, 5 ml of concentrated microparticle suspension was dispersed in 500 ml of isotonic 10 mM Tris-HCl buffer.The uorescence was always normalized to that corresponding to complete release of all encapsulated CF. 1 To determine the amount of encapsulated CF the liposomes were destroyed by surfactant Triton X-100 (50 ml of 1% solution).To conrm the destruction of all liposomes aer adding the Triton, the sample was heated to 65 C for one hour, no increase of uorescence was then observed, therefore the destruction was sufficient.The uorescence intensity was measured at 25 C for all samples.

Radiofrequency heating set-up
The radiofrequency heating of the composite microparticles was characterized by monitoring the temperature rise of a microparticle suspension exposed to alternating magnetic eld.10 ml of closely packed microparticles in an insulated plastic vial were placed into a double-turn (20 mm ID), watercooled copper coil induction head connected to 900 kHz RF generator (PowerCube 64/900, CEIA, Italy).The sample temperature was measured by a ber-optic temperature sensor (Neoptix).

Effect of ow rates on droplet shrinking
Droplets of water in oil (w/o) were produced in the ow focusing device under the stable dripping regime (Fig. 3A) corresponding to the shear induced droplet formation.In the shear driven droplet break-up the diameter of the produced droplets depends only on the capillary number: Ca ¼ Q oil m oil g , where Q oil is the ow rate of the continuous phase, m oil is the dynamic viscosity of the continuous phase and g is the interfacial tension between the two phases. 27The droplet diameter decreases continuously with increasing capillary number, therefore with increasing Q oil (Table 1).The ow rate of the disperse phase has a negligible effect on the droplet size, however it has an effect on the frequency of the break-up.As described above, particle size reduction was achieved by partially extracting water from the droplets formed in the ow focusing section of the chip into the surrounding oil phase.For extraction experiments described in this section, none of the oil phase contained calcium ions, so the size decrease of the microparticles was purely due to extraction (no gelation took place).We evaluated the volume change of the droplets by image analysis.Images of the droplets were taken in two locations (130 mm distant from each other), i.e. just behind the ow focusing section of the chip and just before the outlet from the chip (Fig. 3B).The droplet volume was calculated by considering the disk shape of the droplet as follows: , where the D is measured diameter of the droplet and h is the height of the channel, for D > h.For cases when D < h the droplet volume was evaluated simply as the volume of a sphere.The extent of shrinkage will be described by a ratio R V ¼ V nal /V orig , were V nal and V orig denote the nal and the original droplet volume, respectively.The volume change of the droplet by extraction depends on two factors: the ratio R Q between the continuous and the disperse phase, i.e.R Q ¼ Q oil /Q w and the contact time between the two phases.The ratio R Q determines the extraction capacity of the oil phase in the limiting case of sufficiently long residence time in the microuidic chip for extraction equilibrium to be established.Indeed, considering the solubility K of water in oil (on a volume basis) and the planar geometry of the system where capillary pressure is negligible, the theoretical volume ratio R eq V at equilibrium is R eq V ¼ 1 À KR Q .The evolution of the droplet shrinking ratio R V as a function of the ratio R Q is reported in Fig. 4. It can be seen that the droplets are systematically decreasing their volume with increasing amount of the continuous phase, with a possibility to decrease the volume of the droplets by as much as 60%.However, the observed volume reduction is also limited by kinetics (i.e., the residence time in the chip might not be sufficiently long for extraction equilibrium to be established).The trend of volume change was found to be almost identical regardless of whether the disperse phase was water or 1% w/w aqueous solution of alginate (Fig. 4).As a reference, experiments where the continuous phase was 1undecanol previously saturated by water were also performed.As expected, no extraction and therefore no shrinking was observed.

Effect of extractive gelation on microparticle size and morphology
The above section dealt with a non-gelling case, i.e. extractiononly without alginate cross-linking by Ca 2+ .In the present section, the second oil stream (Q oil2 ) already contained dissolved calcium ions and therefore the extraction and gelation Fig. 5 Different morphologies of alginate microparticles; the scale bar is 20 mm, the conditions (A-F) correspond to parameter values given in Table 1.
Fig. 4 Droplet shrinking ratio R V as a function of dilution R Q in dry 1undecanol (water ( ) and 1% alginate solution ( ) as the disperse phase), and in 1-undecanol pre-saturated by water ( ).The continuous line corresponds to the theoretical limit of equilibrium shrinkage ( ).
processes took place simultaneously.In that case, the morphology and monodispersity of the nal alginate microparticles are inuenced by several variables, i.e. ow rates of both continuous phases (Q oil1 and Q oil2 ), ow rate of the disperse phase (Q w ), ratio between the phases, concentration of calcium ions, concentration of surfactant, and concentration of alginate.To simplify the system, the concentration of surfactant (5% w/w) and aqueous alginate solution (1% w/w) were always kept the same, as well as the ratio R Q .The different concentration of calcium ions in the system can be established by changing the ow rates of the oil without (Q oil1 ) and with (Q oil2 ) calcium ions and by the initial concentration of CaI 2 , while maintaining the overall oil ow rate (Q oil ¼ Q oil1 + Q oil2 ) constant, as shown in Table 1.
Particles were produced under the stable dripping regime corresponding to the shear induced droplet formation, hence the initial droplet size is exclusively function of Q oil1 as discussed above.At the outlet from the chip all microparticles had a round shape, yet the nal microparticles collected in the reservoir had different morphologies.The morphology change is happening in the tube connecting the outlet of the chip with the reservoir.Three main morphologies were observed: round shaped microparticles, slightly deformed microparticles and microparticles with a collapsed structure.As the microparticles (emulsion) are pushed through the tube, they are exposed to shear stress.Compressed emulsion has an elastic behavior therefore it deforms its shape under the shear stress. 28he morphological changes of the microparticles in the tube are inuenced by the character of the partially gelled microparticles that are leaving the chip (diameter, shell thickness).Depending on the initial drop size (D in ) and calcium content (C Ca 2+ ) the morphology of the microparticles varies from the spherical to the folded object with a variable degree of deformation (Table 1 and Fig. 5).The morphology is quantied by the circularity dened as: Circ ¼ 4p area perimeter , with a value of 1 indicating a perfect circle.The inuence of calcium ions concentration and the initial droplet size on the nal morphology of the alginate microparticles was evaluated in order to explain the microparticle deformation.The rates of gelation and water extraction jointly determine the buckling (collapse of a spherical shell under external pressure) of the microparticles.The critical stress applied to a hollow sphere that leads to buckling is proportional to the ratio between the thickness of the shell and the diameter of the core (s c $ t D , where s c is critical external stress for buckling, t is thickness of the shell and D is diameter). 29In this case the shell is the cross-linked alginate gel and the core is the still un-crosslinked aqueous phase.If rapid surface gelation takes place before water extraction, such as in the case of high Ca 2+ concentration and/or large initial droplet size, the particle will buckle upon the nal extraction of water from the core (particles in Fig. 5A and D, mechanism in Fig. 6B and C).On the other hand, the combination of low initial droplet size and/or low calcium ion concentration (therefore low rate of crosslinking) leads to particles that have a sufficiently thick shell relative to the diameter of the core (or in the limit, are crosslinked throughout their volume), so that they can withstand the external pressure during water extraction and not buckle, retaining their spherical shape (particles in Fig. 5B, E and F, mechanism in Fig. 6A and D).
Even if the microparticles do not buckle under the external pressure in the tube they can still slightly deform their shape under the ow.When the concentration of calcium ions is very low, the formed shell is weak, therefore the particles behave more like a viscous liquid and they gell in this drop-shape deformation (Fig. 5E and F).The ideal conditions to obtain monodisperse microparticles with a high circularity were: ow rate of the disperse phase Q w ¼ 5 ml h À1 , ow rates of the continuous phases Q oil1 ¼ 150 ml h À1 and Q oil2 ¼ 50 ml h À1 , and concentration of calcium ions in the oil 0.5% w/w (Fig. 5B).Based on a sample of 500 particles, the mean diameter d ¼ 10.5 AE 0.5 mm and circularity Circ ¼ 0.93 AE 0.04 were evaluated, and  the coefficient of variation in diameter (dened as the standard deviation divided by the mean diameter) was as low as 4.7%.
For the particles with the highest circularity (Fig. 5B, Q oil1 ¼ 150 ml h À1 , Q oil2 ¼ 50 ml h À1 ), their mean diameter was evaluated for changing R Q ratio, which was achieved by changing the ow of the disperse phase.The mean particle diameter as function of the R Q ratio is plotted in Fig. 7.The observed asymptotic behavior of the size reduction is due to a rise of osmotic pressure induced by increased alginate concentration.For instance, for microparticle with a nal diameter 10 mm and initial diameter 33 mm the alginate concentration increased from 1% to roughly 36% aer extraction.Therefore, it is possible to achieve further reduction of the particle size by decreasing the initial alginate concentration below 1%.The nal microparticle diameter signicantly decreased with a lower initial alginate concentration as can be seen in Table 2.

Microstructure of alginate-magnetic-liposome microparticles
Once the parameters necessary for the formation of spherical and monodisperse alginate microgels in the desired size range have been found, the next step was the encapsulation of both functional components, i.e. magnetic nanoparticles and liposomes.Since the particles are prepared by extractive gelation, not just alginate but also liposomes and magnetic nanoparticles increase their concentration in the particle.This increase of concentration was found to lead to premature leakage of CF from the liposomes, caused by steric interactions inside of the microparticle (the liposomes are "squeezed" during the extraction phase).To minimaze the steric interaction, we decreased the concentration of the magnetite nanoparticle solution until no leakage of CF from the liposomes was observed and at the same time the composite microparticles still retained their magneto-responsive properties (i.e. they align and migrate when exposed to an external magnetic eld Fig. 8B) the nal composition of the microparticles was the following: 2.5% v/v of magnetite nanoparticle solution (10 g l À1 ), 25% v/v of liposome solution and 72.5% v/v of aqueous solution of alginate (0.025% w/w).The presence of intact uorescein-loaded liposomes in the composite alginate microparticles was visualized by confocal microscopy (Fig. 8C and D).The concentration of the encapsulated magnetite nanoparticles also has an effect on the extractive gelation; with a lower concentration of magnetite nanoparticles it is possible to further decrease the nal microparticle diameter to d ¼ 5.1 AE 0.3 mm.

Stimuli-responsive release from alginate-magneticliposome microparticles
To conrm the ability of the composite particles to store and release a chemical payload, the release kinetics of an encapsulated uorescence dye from the composite microparticles was evaluated.The phase transition of the phospholipid bilayer in the liposomes (made from DPPC and cholesterol in a molar ratio DPPC : cholesterol ¼ 2 : 1) is above 45 C. Therefore, the composite microparticles were exposed repeatedly to a temperature pulse of 45 C for 10 minutes and then cooled down and maintained at room temperature (25 C) for 15 min, while measuring the uorescence of the surrounding solution.As  shown in Fig. 9, a step increase of the released uorescein concentration was observed aer each temperature pulse in the sequence, while no uorescein was released during the periods at 25 C.This proves that by controlling temperature of the composite microparticles, a repeated on-demand release can be achieved and the diffusion process can be stopped and restarted several times.
To evaluate the ability of composite alginate microparticles with encapsulated iron oxide to heat up in the radiofrequency eld and to reach temperatures necessary for the phase transition of the liposomes, composite alginate microparticles were prepared from sodium alginate (0.025% w/w) dissolved in a concentrated (10 g l À1 ) solution of iron oxide nanoparticles.The temperature rise of the resulting microparticles was measured as described in Section 2.7 and the results are summarized in Fig. 10, which shows that the microparticles can indeed reach the desired temperature 45 C in less than 20 s at 100% power of RF generator.In addition, by modulating the power of the RF generator, the time at which the phase transition temperature is reached can be controlled.

Conclusion
The present work demonstrates the feasibility of microuidic fabrication of composite alginate microparticles with incorporated functional components (magnetic nanoparticles and liposomes that contain a chemical payload) in the size range comparable to that of blood cells.The extractive gelation process was used for down-scaling the droplet volume produced in a ow-focusing junction, and on-chip gelation with subsequent magnetic separation of the produced gel microparticles was employed as a separation and purication method.The ability to separate and purify the produced microparticles from the oil/surfactant mixture is oen neglected in the microuidic literature, but this is a crucial step for any further application of the microparticles (for example in biological applications).In the present work, the parametric sensitivity of the extractive gelation process was systematically explored and a feasible parameter range that ensured both sufficient quality of the particles (spherical, monodisperse) and acceptable production rate, was found.It was shown that by optimizing the initial concentration of all components (alginate, liposomes, magnetic nanoparticles), the nal composite microparticles can be both stable (i.e.no undesired leakage from the liposomes), magnetoresponsive, and provide on-demand release functionality when exposed to temperature cycles.

Fig. 1 (
Fig. 1 (A) Composite alginate microparticle; (B) iron oxide nanoparticles that locally dissipate energy in the radio-frequency alternating magnetic field; (C) liposomes that locally release encapsulated chemical payload in response to a temperature change.

Fig. 2
Fig. 2 Workflow principle of the production of composite alginate microparticles in the 5-10 micrometers size range.

Fig. 3 (
Fig. 3 (A) Droplet formation in the flow focusing device.(B) An example of droplet shrinkage; the scale bar is 50 mm.

C 4 Fig. 7 Fig. 6
Fig.7The mean particle diameter of the final alginate microparticles as a function of the ratio R Q ; for all the cases D in ¼ 33 mm.

Fig. 9
Fig. 9 Diffusion of CF from composite microparticles during the temperature burst release experiments.

Fig. 8
Fig. 8 Composite microparticles (A) and their orientation in the magnetic field (B); (C) an overlay of optical and confocal fluorescent image of microparticles; (D) confocal image of microparticle.

Fig. 10
Fig. 10 Temperature rise of microparticles in the alternating magnetic field for different powers (100%, 75% and 50%) of the RF generator.

Table 2
Final microparticles diameter for different initial concentration of alginate and constant R Q ¼ 50 and D in ¼ 33 mm.