Synthesis of hybrid microgels by coupling of laser ablation and polymerization in aqueous medium

Zinc plays an important role in the field of wound healing, evidenced by involvement in a high number (ca. 300) of enzymatic reactions.¹ As a consequence of the antioxidant properties of zinc and metalloenzymes in which zinc acts as cofactor, the medicinal importance of zinc increases in the case of thermal injuries.^2–4 However, local wound treatment with zinc should be achieved without risking intoxication as consequence of high concentrations of zinc (ions). It was shown that when zinc oxide is used for topological wound care, the zinc ion concentration never reaches a toxic level because of the release kinetics. Meanwhile, the concentration is sufficient for wound healing effects.^5–8 

The increasing interest in aqueous colloidal nanogels or microgels⁹ in the past decades is due to many useful applications such as coatings, agriculture, medicine, etc., which microgels with their unique properties can enhance. Recently, numerous reports on the nanogel/microgel synthesis,¹⁰ characterization,^11–13 modification^14,15 and application in drug delivery,^16,17 design of biomaterials,¹⁸ development of sensors¹⁹ are emerging, thereby illustrating the potential of the crosslinked polymer colloids in a wide range of applications (and fundamental research).

Many studies show that properties such as chemical structure, size, and morphology of nano and microgels can be easily controlled by crosslinking processes such as physical crosslinking,^20–22 irradiation-induced crosslinking,^23–25 chemical crosslinking based on polyaddition reactions,^26–29 and polymerization methods (precipitation polymerization^30–34 or miniemulsion polymerization^35–39).

Based on the typical microgel properties, one can predict the following advantages when using them as microreactors or -carriers in comparison with other template systems: (1) easy preparation; (2) variable size and flexible functionalization by reactive groups; (3) highly porous structure with adjustable degree of crosslinking; (4) enhanced colloidal stability; and (5) stimuli-responsive change of the microgel dimension (T-, pH-sensitivity). With regard to those features, the preparation of different materials in form of nanoparticles (NPs) inside the microgels can offer: (1) controlled NP synthesis in microgels (localization of reactive sites and controlled growth, homogeneous distribution within the microgel); (2) adjustable NP properties (particle size and morphology control); (3) separation and stabilization (chemical grafting) in polymer network; (4) NP accessibility (high surface area, no diffusion limitation); (5) control of the distance between NPs by swelling or collapsing of the microgel as its responses to environmental conditions; and (6) enhanced colloidal stability provided by microgel particles. Different approaches to incorporate nanoparticles into aqueous nanogels and microgels were recently reviewed.^15,40 A large variety of nanoparticles have been incorporated into microgels, including NPs of conducting polymers,^41,42 noble metals,^43,44 metal oxides,⁴⁵ metal sulfides,^46,47 and biominerals.^48,49

The incorporation of different nanomaterials into microgel particles is a topic under intense study. There are three profoundly distinct approaches for incorporating NPs into microgels: (1) The first utilizes the microgel as a template for in-situ preparation of nanoscale materials such as inorganic NPs.¹⁴ (2) The second approach involves filling the microgels by diffusion of preformed nanoparticles into the microgel, accompanied by trapping due to the electrostatic interactions or hydrogen bonding with polymer chains.⁵⁰ (3) The third approach utilizes the incorporation of preformed nanoparticles with reactive surface in microgels directly during their synthesis. The advantage of this method is high loading efficiency of NPs and their fixation by covalent bonds in microgel network to prevent nanoparticle leakage.⁵¹ Therefore, this last approach is the most suitable for the design of composite colloids for medical applications.

Such nanoparticles should be available well dispersed in colloidal state, allowing sorption and binding to the microgels. Hence, wet chemical or physical methods could be the method fabricating the aqueous colloids. In contrary to chemical precipitation of sol-gel method, pulsed laser ablation in liquid has been demonstrated as a method that allows to fabricate nanoparticles in water and organic liquids free of impurities^52,53 even in gram scale,⁵⁴ in particular, if ultrashort laser pulses are used.^55,56

In this study, we aim to combine NP synthesis using laser ablation in liquids with using the synthesized colloidal NPs in the polymerization process to form hybrid microgels. Herein, we report the synthesis of microgels with encapsulated laser-generated ZnO NPs. The polymer chains confined to the network act both as capping agent and to prevent the particles from agglomeration. Additionally, we demonstrate that the polymerization can be initiated without the use of chemical initiators. An additional and important feature of the microgels is that they are intended to act as a container for the ZnO NPs, thereby allowing the controlled release of zinc ions. In our group, hybrid microgels modified with ZnO nanoparticles were successfully prepared previously via wet chemical route ZnO (Ref. 57). However, in this previous work, an organic solvent (ethanol) was used for the incorporation of ZnO NPs into microgels. In this study, we report a method for the preparation of ZnO NPs with high purity in an aqueous medium using laser ablation, followed by the in-situ incorporation of the freshly synthesized ZnO NPs in microgels.

All the analytical grade chemicals were purchased from Aldrich. Acetoacetoxyethyl methacrylate (AAEM) was passed through a column filled with Al₂O₃ to remove the inhibitor (AMPA). N-Vinylcaprolactam (VCL) was purified by vacuum distillation under high vacuum and in nitrogen atmosphere. Crosslinker 2,2-azobis-(2-methylpropyonamidine) dihydrochloride (AMPA) and N,N-methylen-bis-acrylamide (BIS) were used as received. Millipore water was employed as the medium.

Pulsed laser ablation was performed ablating zinc in a solution of the respective organic microgel precursors in water (Milli Q 18.2 MΩ cm). The laser parameters (Ekspla laser, Atlantic series) employed are pulse length 10 ps, wavelength 1064 nm, pulse energy 80 μJ, repetition rate 300 kHz, and irradiation time 30 min.

Human dermis samples were obtained from patients undergoing elective plastic surgery. The isolation and use was approved by the local ethics committee. Primary cell culture of human dermal fibroblasts (hd-FBs) is obtained through enzymatic digestion of fresh dermis with collagenase (Biochrom AG, Berlin, Germany; collagenase type CLS). Cells were cultured in media (PAA, DMEM/F-12 (1×) liquid 500 ml) supplemented with mammalian blood-derived nutrition (Biochrom AG, Berlin, Germany; 10% fetal bovine serum superior 500 ml) and 1% antibiotics (Biochrom AG, Berlin, Germany; Penicillin/Streptomycin 100 ml) at 37 °C with 5% CO₂ atmosphere.

Hd-FBs were maintained in culture till third passage and then transferred into microchambers (Ibidi, Matrinsried, Germany; μ-chamber 12 well). Zinc chloride (Sigma-Aldrich, ZnCl₂) was mixed to the culture medium at concentrations from 100 to 500 μM. A standard control without ZnCl₂ was prepared in each slide to assure standardization. After 18 h incubation, an optical microscopy control was made under an Olympus CKX41 light microscope, followed by a cell viability assay (Invitrogen; LIVE/DEAD viability/cytotoxicity kit for mammalian cells) performed as promoted by Invitrogen. In principle, Calcein bis-acetoxymethylester (AM) diffused into living cells is cleaved by endogenous esterases resulting in a bright fluorescence (494/517 nm) while membrane-impermeable ethidum homodimer-1 fluoresces at 528/617 nm (red) when bound to deoxyribonucleic acid (DNA) in cells weakened membranes indicating cell death. Fluorescence microscopy was obtained under an ZEISS Axiovert 200 M equipped with the appropriate barrier filters. Digital photographs were taken with Axiovision 6.0 software and analyzed manually due to the ratio between ethidium homodimer-1 positive nuclei on total Calcein AM marked cells. Experiments were performed with three different patients’ cells, each in triplicates. Statistic significance was proofed with Bonferroni test in statistical package for the social sciences.

The polymerization of microgels was performed during the laser ablation process, using the ablation chamber as reaction vessel (Fig. 1). Two approaches were employed; the reaction conditions are listed in Table I.

In the first approach (1, Table I), 548 mg of the monomer (VCL) and 16 mg of the crosslinker (BIS) were dissolved in 40 ml water. The solution was placed into the ablation chamber and stirred with a mechanical stirrer. The ablation process was initiated by switching on the laser and left for 30 min. After the reaction, the solution turned slightly turbid. In order to enhance the polymerization process, the sample was subsequently annealed at 70 °C for 2–3 h. The obtained colorless solid was characterized after centrifugation.

In a second approach (2, Table I), 548 mg of the monomer (VCL), 44 mg of an additional comonomer (AAEM), and 16 mg of the crosslinker (BIS) were dissolved in 35 ml water. The solution was placed into the same ablation chamber as before and heated up to 70 °C, while stirring with a mechanical stirrer. Immediately after ablation process had started, 5 ml of an aqueous solution of the radical initiator (AMPA) (5 g/l) was added through a canule which was left between chamber-walls and -cover plate. After addition, the canule was removed and the cover was completely closed. The process was stopped after 30 min. The freshly formed colorless microgel dispersion was collected and characterized without subsequent annealing or further purification.

Fourier transform infrared (FTIR) measurements were recorded with a Thermo Nicolet Nexus 470 instrument. Low amounts of the dried samples were mixed with KBr to form a pellet which acts as specimen.

The size of the hybrid microgels was measured with ALV/LSE-5004 Light Scattering Multiple Tau Digital Correlator at the scattering angle set at 90°. The samples were measured at 20 °C, and the temperature fluctuations were below 0.1 °C. Prior to the measurement, microgel samples were diluted with doubly distilled water and filtered through 2.5 μm filter.

Transmission electron microscopy (TEM) measurements were performed with a, Zeiss LIBRA 120 (80 kV, >10⁻⁷ bar). One drop of the dispersion or suspension was added on a Formvar/carbon or silica grid (which was used for annealing) placed on filter paper. The zinc yield was determined by weighing the metal foil before and after ablation. It was presumed that the loss of weight directly correlates to the amount of zinc deposited. The product yield was weighed after centrifugation of the dispersion at 10 000 rpm for 1 h and drying at 40 °C over-night. The polymer yield could be obtained via subtracting the zinc mass from the total product mass.

The polymerization of VCL in an aqueous medium during laser ablation without addition of an initiator was confirmed by FTIR spectroscopy (Fig. 2). The spectrum shows two distinctive peaks, one appearing at 460 cm⁻¹ which corresponds to ZnO,^58,59 and the second one appearing at 1640 cm⁻¹ which is characteristic for poly(N-Vinylcaprolactam) (PVCL). The TEM micrograph [Fig. 3(a)] reveals the formation of a hybrid material composed of ZnO NPs (dark small crystals) and polymer (large grey areas). It also shows that the hydrogel is able to cap the ZnO NPs. As shown in Fig. 3(a), the ZnO NPs are mostly situated at the surface of the polymer beads and we could successfully show that it is possible to initiate the radical polymerization of VCL by use of a laser and subsequent annealing at 70 °C for 2–3 h. Nevertheless, further investigations in case of the influence of laser irradiation and the reaction conditions on the polymerization, especially with respect to the formation of colloidal systems, will have to be performed and are already in progress.

Creating colloidal dispersions of microgels loaded with ZnO NPs was accomplished using laser ablation under standard polymerization conditions.⁶⁰ The solution containing VCL, AAEM, and BIS was preheated up to 70 °C. Then the initiator (AMPA) was added, and the reaction left for 30 min. Even without flushing the solution with nitrogen, it was possible to prepare microgel dispersion with a yield of 68% related to the initial amount of monomer mass within 30 min. It is evident that during this process the in-situ formation of initiator (ZnO NPs), as observed in the case where no chemical initiator (AMPA) was added [Figs. 2(a) and 3(a)], succeeds and aids the polymerization together with the added chemical initiator (AMPA). This would explain why the reaction could be successfully accomplished within such a short reaction time in presence of inhibiting oxygen. The dispersion was characterized by FTIR spectroscopy and shows a typical spectrum with the characteristic shoulder at 1750 cm⁻¹ assigned to the carbonyl group of AAEM [Fig. 2(b)].

The TEM micrograph [Fig. 3(b)] confirms the formation of typical PVCL–AAEM microgel colloids. In order to confirm the capping of ZnO NPs which were formed by laser ablation, TEM specimen were heated up to >300 °C leading to a degradation and collapse of the colloids. TEM images [Fig. 3(c)] of the specimen after annealing shows "flat" microgel spheres including inorganic NPs. Additionally, the TEM investigation clearly shows that nanoparticles are not completely capped by the microgel colloids and microgels free of inorganic material are gained after reaction [Fig. 3(d)]. At this point, further research is required in order to clarify the optimal capping parameters and to succeed to a complete capping of all nanoparticles.

In order to validate the existence of zinc within the colloidal sphere of the microgels, energy dispersive X-ray spectroscopy measurements were performed. Unfortunately, detection of zinc was impossible due to the fact that the limit of determination lies at 1 mass-% minimum. The amount of zinc which was reached in this experiment lies beneath this limit (0.9 mass-%, see Table I). Nevertheless, the TEM images showing particles within (and nearby) the spheres demonstrate that nanoparticles can be capped by microgels during their formation using laser ablation and by in-situ polymerization of colloidal crosslinked polymers.

Extensive purification normally required to ensure the formation of a hybrid material which is completely free of toxic species is not required, since the employed method does not include the use of organic solvents, precursors, etc. Unquestionably, this is a requirement for any method aiming at the synthesis of materials in the field of medical technology.

In order to determine a therapeutical window for future zinc ion release tests of the here prepared durg-release systems, cytotoxicity and vitality tests were perfromed with zinc chloride solutions at different concentrations. Optical microscopy (Fig. 4) shows under 100 μM ZnCl₂ typical FB elongated shapes similar to the control culture. We observe for zinc concentration above 200 μM atrophic cytoplasm, inducing a reduced cell covered area compared to lower zinc concentrations.

Cells also present weaker adherence in presence of the higher molarity of zinc with concentrations above 300 μM. Breakdown of cell membrane integrity is a typical symptom of cytotoxicity visualized by ethidium bromide staining which is not diffusible into healthy cells. Fluorescence microscopy (Fig. 5) shows significant increases of ethidium bromide positive nuclei from less than 1% at 0 and 100 μM zinc concentrations, 32% at 200 μM, and 81% at 300 μM zinc to 100% ethidium bromide positive cells at 400 and 500 μM zinc. Our study indicates that zinc ion concentrations between 200 and 500 μM have a cytotoxic effect on primary fibroblasts. Remarkably, concentrations of 100 μM were tolerated by the cells indicating a sufficient resistance of primary human fibroblasts against zinc ion exposition. Zinc ions exert a number of effects on cell cultures including antioxidation³ when administered in dosages comparable to the study presented here, and protects against DNA damage in concentration of 22.6 μM.⁶¹ Higher concentrations have been shown to be cytotoxic for a number of cells including neuronal cells and a murine fibroblast cell line Balb/c 3T3 (Ref. 62) confirming our results. Thus, these first tests with hd-FB show that cell integrity reduces over 200 μM zinc. The cell damage increases with the concentration of zinc demonstrating a dose-dependent effect. Therefore, later tests should focus on below 200 μM concentration of zinc. In summary, an aqueous synthesis route of hybrid microgels loaded with inorganic nanoparticles has been demonstrated based on pulsed laser ablation of zinc in microgel monomer solution. Microgels were partially loaded with ZnO and spectroscopic measurements verified the formation of polymer colloids, indicating application potential of laser ablation in microgel precursor solution for fabrication of colloidal microgel matrices loaded with inorganic nanoparticles.

This work was performed in the framework of the Priority Program 1327, “Optisch erzeugte sub-100 nm Strukturen für biomedizinische und technische Applikationen.” Authors acknowledge DFG for financial support.