Microgels for Cell Delivery in Tissue Engineering and Regenerative Medicine

Microgels prepared from natural or synthetic hydrogel materials have aroused extensive attention as multifunctional cells or drug carriers,that are promising for tissue engineering and regenerative medicine.Microgels can also be aggregated into microporous scaffolds,promoting cell infiltration and proliferation for tissue repair.This review gives an overview of recent developments in the fabrication techniques and applications of microgels.A series of conventional and novel strategies including emulsification,microfluidic,lithography,electrospray,centrifugation,gas-shearing,three-dimensional bioprinting,etc.are discussed in depth.The characteristics and applications of microgels and microgel-based scaffolds for cell culture and delivery are elaborated with an emphasis on the advantages of these carriers in cell therapy.Additionally,we expound on the ongoing and foreseeable applications and current limitations of microgels and their aggregate in the field of biomedical engineering.Through stimulating innovative ideas,the present review paves new avenues for expanding the application of microgels in cell delivery techniques.

Application of Monodisperse Thermo-Responsive Composite Microgels with Core-Shell Structure Based on Au@Ag Bimetallic Nanorod as Core in Surface Enhanced Raman Spectroscopy Substrate

The monodisperse Au@Ag bimetallic nanorod is encapsulated by crosslinked poly( N-isopropylacrylamide)( PNIPAM) to produce thermo-responsive composite microgel with well-defined core-shell structure( Au@ Ag NR@ PNIPAM microgel)by seed-precipitation polymerization method using butenoic acid modified Au @ Ag NRs as seeds. When the temperature of the aqueous medium increases from 20℃ to 50℃,the localized surface plasmon resonance( LSPR) band of the entrapped Au @ Ag NR is pronouncedly red-shifted because of the decreased spatial distances between them as a result of shrinkage of the microgels,leading to their plasmonic coupling. The temperature tunable plasmonic coupling is demonstrated by temperature dependence of the surface enhanced Raman spectroscopy( SERS) signal of 1-naphthol in aqueous solution. Different from static plasmonic coupling modes from nanostructured assembly or array system of noble metals,the proposed plasmonic coupling can be dynamically controlled by environmental temperature. Therefore, the thermo responsive hybrid microgels have potential applications in mobile LSPR or SERS microsensors for living tissues or cells.

Structured Microgels through Microfluidic Assembly and Their Biomedical Applications

Droplet based microfluidic is an effective, versatile and scalable approach which can be used to produce structured microgels with desirable features. The high degree of control endowed with microfluidics enables the formation of various functional microgels such as multicompartment encapsulations, Janus shaped particles and non spherical microgels. These microgels have aroused great interest in biological engineering aspect, since they outperform their counterparts produced from other techniques and have been applied in drug delivery, 3 Dimensional cell culture, micro tissues, single cell assay, tissue engineering and bioimaging. In this review, we will summarize the fabrication processes, technology comparisons and the usages in biomedical applications.

Preparation of Manganese Oxide Hollow Spheres Using pH-responsive Microgels as Templates

Manganese oxide hollow spheres were prepared by a novel and facile approach using pH- responsive microgels as templates. The final products were thoroughly characterized with X-ray powder diffraction, thermogravimetric analysis, scanning electron microscopy, Fourier transform infrared, and transmission electron microscopy. The results reveal that the shell thickness of manganese oxide hollow spheres increased with the dosage of KMnO4, which implies that a controllable and feasible strategy for manganese oxide hollow spheres prepa- ration has been established. Further studies on the microgels template showed some of them had an irreversible swelling/deswelling transition due to the uneven cross-link extent. Based on the results, a probable formation mechanism for the hollow spheres was proposed.

Application of reactive acrylate microgels in water-base coatings

Reactive acrylate microgels with different reactive groups such as carboxyl, hydroxide groups had excellent properties such as quick-dry, low viscosity, high adhesion and hardness, which made them extensively used in preparing paints or in coating-modification. Reactive acrylate microgels were prepared by emulsion co-polymerization with zwitterions surfactant, anionic surfactant and nonionic surfactant as co-emulsifier. The water-base baking paints made from reactive acrylate microgels and melamine-formaldehyde resin had excellent combination properties. The aluminium powder can be well-dispersed in the paints. The influences of monomer components on the properties of the water-base baking paints were discussed in this paper. And the baking paints were also compared with the marketing solvent acrylate baking paints. It was found that the water-base acrylate amino baking paints had better combination properties than the organic solvent acrylate baking paints, which means that the water-base baking paints had a bright marketing future.

Preparation and Properties of Hydroxyethyl Methacrylate Chemically Modified Temperature-Sensitive Microgels

A novel series of poly(N-isopropylacrylamide-co-hydroxyethyl methacrylate) (p(NIPAM-co-HEMA)) microgels were prepared through precipitation polymerization. Nuclear magnetic resonance (NMR), transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV) and dynamic light scattering (DLS) were employed to characterize the microgels. The experimental results indicate that the prepared microgels with narrow distribution remain good temperature sensitivity after incorporation of functional-OH groups. In marked contrast to the general rule, incorporation of hydrophilic HEMA makes the volume-phase-transition temperature shift to the lower temperature due to the strong intermolecular H-bonding between amide and -OH groups, -OH and -OH groups.

Surface Properties of the Microgels Based on N-isopropylacrylamide and Tert-butyl Acrylate by Using N,N′-methylene-bis(acrylamide) and Clay as Cross-linker

Adopting N, N＇-methylene-bis （aculamide） （MBA） and inorganic clay （hectorite） as chemical and physical crosslinking agent, respectively, a series temperature sensitive microgels, based on N-isopropylacrylamide （NIPAM） as a main monomer and tert-butyl acrylate （tBA） as a comonomer were synthesized by surfactant-free emulsion polymerization （SFEP）. The microgel particle size and morphology was investigated by means of Atomic Force Microscope （AFM）. The surface tension of latex particles was measured by OCA 40 Micro Video based contact angle measuring device. The results showed that the particle size of the microgels with clay as cross-linker was smaller than that using MBA as chemical cross-linker, but exterior morphology of physical microgels is not as clean and neat as chemical microgels. In general, surface tension decreases with increasing hydrophobic tBA content. These smart microgels varied with tetnperature have the potential applications in the field of drug delivery and intelligent gel fiber.

Phototactic Poly(N-isopropyl acrylamide)Microgels with Photoresponsive Property

Smart functional microgels hold great potential in a variety of applications,especially in drug transportation.However,current drug carriers based on physiological internal stimuli cannot efficiently orientate to designated locations.Therefore,it is necessary to introduce the self-propelled particles to the drug release of the microgels.In order to study self-propulsion of microgels induced by light,it is also a challenge to prepare micronsized microgels so that they can be observed directly under optical microscopes.In this work,phototactic microgels with photoresponsive properties are prepared.The microgel particles can be observed by confocal laser scanning microscopy.The photoresponsive properties of microgels are fully investigated by various instruments.Light can also regulate the state of the microgel solution,making it switch between turbidity and clarity.The phototaxis of particles irradiated by UV light was studied,which may be used for microgels enrichment and drug transportation and release.

STABILITY OF POLY(N,N'-METHYLENEBISACRYLAMIDE-co-4-VINYLPYRIDINE)MICROGEL DISPERSION AND ADJUSTMENT OF THE HYDROPHOBICITY IN THE MICROGELS

In the UV-Vis spectra of pure light-scattering systems, there is an exponential relationship between absorbance and wavelength （A = Kλ^-n）. Here, the exponent n is named as flocculation-coagulation parameter. In the present paper, the effects of different additives on the stability of poly（N,N＇-methylenebisacrylamide-co-4-vinylpyridine） （poly（Bis-co-4-VP）） microgel dispersion were studied in detail via this parameter. The results showed that the stability of the dispersion mainly comes from the ionization of pyridine groups, making the microgel positively charged on its surface. This was confirmed by the measurement of Zeta potential and the result of conductometric titration. The result of fluorescence analysis indicated that the hydrophobicity in the microgels is enhanced with the increase in total 4-VP unit content.

PARAMETERS AFFECTING PARTICLE SIZE OF POLYBUTYL-ACRYLATE MICROGELS

The factors affecting particle size of reactive microgels formed during the self-emulsifying copolymerization of unsaturated polyester (UP)with butyl acrylate (BA)have been studied. The parameters discussed are: the proportion of the UP in the monomer mixture, the molecular weight and the carboxyl value of the UP, the phase ratio, the electrolyte concentration and the polar solvent additive. The seeding emulsion polymerization is discussed as well.It turned out that the particle size of the reactive microgels can be controlled in a definite range by changing the experimental conditions. However the particle size distribution becomes broader as the average diameter increases. It is suggested that the agglomeration of primary particles plays an important role during the growth of microgel particle.

Interferometers Utilizing Reversible X-ray-induced Chemical Changes in Poly(N-isopropylacrylamide)Microgels

Photonic materials,which react to light,have garnered interest due to their capability to exhibit adjustable structural colors.Typically,light targets the UV,visible,or near-IR spectrums.In this study,microgel-based photonic materials that are capable of reversibly responding to Xrays have been engineered.To accomplish this,azobenzene(Azo)-containing poly(N-isopropylacrylamide)(pNiPAm)-based microgels are synthesized.Subsequently,ZnS scintillator and Cr/Au are applied on each side of the poly(methyl methacrylate(PMMA)substrate.Subsequently,the Azo MG monolayer is deposited onto the Au surface,followed by the deposition of an additional layer of Cr/Au.This process generates ZnS/PMMA/Cr/Au/Azo MG/Cr/Au or ZnS/Au-Azo MG-Au structure.Functioning as a typical interferometer,ZnS/Au-Azo MG-Au demonstrates tunable colors based on the separation distance between the two Au layers.The ZnS scintillator can absorb and convert X-rays into Uv light,initiating the transition of the Azo groups from a trans to a cis state.Consequently,this transition causes the Azo MG to swell.As Azo MG swells,the distance between the two Au layers increases,resulting in a red-shift of approximately 350 nm in the optical signal of the ZnS/Au-Azo MG-Au interferometer.Remarkably,this X-ray responsivity of the interferometer is reversible,as it returns to its initial state after being stored in the dark for 24 h.To demonstrate its capabilities,the ZnS/Au-Azo MG-Au interferometer successfully releases a drug when triggered by X-ray stimulation,thus validating its potential.The microgel-based interferometers hold significant promise for applications in chemoradiotherapy,radiobiology,and actuators in space.

Alveolar macrophage phagocytosis-evading inhaled microgels incorporating nintedanib-PLGA nanoparticles and pirfenidone-liposomes for improved treatment of pulmonary fibrosis

Idiopathic pulmonary fibrosis(IPF)is a chronic inflammatory and fibrotic response-driven lung disease that is difficult to cure because it manifests excessive profibrotic cytokines(e.g.,TGF-β),activated myofibroblasts,and accumulated extracellular matrix(ECM).In an attempt to develop an inhalation formulation with enhanced antifibrotic efficacy,we sought to fabricate unique aerosolizable inhaled microgels(μGel)that contain nintedanib-poly(lactic-co-glycolic acid)(PLGA)nanoparticles(NPs;n-PN)and pirfenidone-liposomes(p-LP).The aero-μGel was~12μm,resisted phagocytosis by alveolar macrophages in vitro and in vivo,and protected inner-entrapped n-PN and p-LP.The n-PN/p-LP@aero-μGel caused enhanced/extended antifibrotic efficacy in a bleomycin-induced pulmonary fibrosis mouse presumably due to prolonged lung residence.Consequently,the results obtained by intratracheal aerosol insufflation of our n-PN/p-LP@aero-μGel twice a week were much better than those by as many as seven doses of single or mixed applications of n-PN or p-LP.The antifibrotic/pharmacokinetic results for the n-PN/p-LP@aero-μGel included reduced fibrosis progression,restored lung physiological functions,deactivated myofibroblasts,inhibited TGF-βprogression,and suppressed ECM component production(collagen I andα-SMA)along with prolonged lung retention time.We believe that our n-PN/p-LP@aero-μGel increased the local availability of both nintedanib and pirfenidone due to evasion of alveolar macrophage phagocytosis and prolonged lung retention with reduced systemic distribution.Through this approach,our inhalation formulation subsequently attenuated fibrosis progression and improved lung function.Importantly,these results hold profound implications in the therapeutic potential of our n-PN/p-LP@aero-μGel to serve as a clinically promising platform,providing significant advancements for improved treatment of many respiratory diseases including IFP.

Fluorescent Nile blue-functionalized poly(N-isopropylacrylamide)microgels responsive to temperature and polyamines

Fluorescent poly(N-isopropylacrylamide-co-Nile blue)(pNIPAm-co-NB)microgels were synthesized that exhibited fluorescence intensity changes in a water temperature-dependent fashion.NB is well known to exhibit fluorescence intensity that depends on the hydrophobicity of the environment,while pNIPAm-based microgels are well known to transition from swollen(hydrophilic)to collapsed(relatively hydrophobic)at temperatures greater than 32℃;hence,we attribute the above behavior to the hydrophobicity changes of the microgels with increasing temperature.This phenomenon is ultimately due to NB dimers(relatively quenched fluorescence)being broken in the hydrophobic environment of the microgels leading to relatively enhanced fluorescence.We went on to show that the introduction of cucurbit[7]uril(CB[7])into the pNIPAm-co-NB microgels enhanced their fluorescence allowing them to be used for polyamine(e.g.,spermine[SPM])detection.Specifically,CB[7]forms a host–guest interaction with NB in the microgels,which prevents NB dimerization and enhances their fluorescence.When SPM is present,it forms a host–guest complex that is favored over the CB[7]-NB host–guest interaction,which frees the NB for dimerization and leads to fluorescence quenching.As a result,we could generate an SPM sensor capable of SPM detection down to~0.5µmol/L in complicated matrixes such as serum and urine.

Large-sized bone defect repair by combining a decalcified bone matrix framework and bone regeneration units based on photo-crosslinkable osteogenic microgels

Physiological repair of large-sized bone defects is great challenging in clinic due to a lack of ideal grafts suitable for bone regeneration.Decalcified bone matrix(DBM)is considered as an ideal bone regeneration scaffold,but low cell seeding efficiency and a poor osteoinductive microenvironment greatly restrict its application in large-sized bone regeneration.To address these problems,we proposed a novel strategy of bone regeneration units(BRUs)based on microgels produced by photo-crosslinkable and microfluidic techniques,containing both the osteogenic ingredient DBM and vascular endothelial growth factor(VEGF)for accurate biomimic of an osteoinductive microenvironment.The physicochemical properties of microgels could be precisely controlled and the microgels effectively promoted adhesion,proliferation,and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro.BRUs were successfully constructed by seeding BMSCs onto microgels,which achieved reliable bone regeneration in vivo.Finally,by integrating the advantages of BRUs in bone regeneration and the advantages of DBM scaffolds in 3D morphology and mechanical strength,a BRU-loaded DBM framework successfully regenerated bone tissue with the desired 3D morphology and effectively repaired a large-sized bone defect of rabbit tibia.The current study developed an ideal bone biomimetic microcarrier and provided a novel strategy for bone regeneration and large-sized bone defect repair.

Bidirectionally aligned MXene hybrid aerogels assembled with MXene nanosheets and microgels for supercapacitors

MXene nanomaterials are one of the most promising electrode material candidates for supercapacitors owing to their high conductivity,abundant surface functional groups and large surface area.However,electrodes based on MXene may result in low ion-accessible surface area and blocked ion transport pathways because of the self-restacking of MXene nanosheets.It is essential to suppress the self-res tacking of nanosheets and increase the electrochemical active sites in order to optimize the electrode.In this work,bidirectionally aligned MXene hybrid aerogel(A-MHA)assembled with MXene nanosheets and microgels is prepared using a facile bidirectional freeze casting and freeze-drying method.The bidirectionally aligned structure together with the three-dimensional structured microgels in the A-MHAs,can improve the ionaccessible surface area and provide more barrier-free channels by exposing more active sites and ensuring electrolyte transport freely.The A-MHA with MXene microgels content of 40 wt%exhibits a high specific capacitance of 760 F·g^(-1)at 1 A·g^(-1)and a remarkable cyclic performance of 97%after 10,000 cycles at100 mV·s^(-1)in 1 mol·L^(-1)H_(2)SO_(4)electrolyte.A-MHAs show remarkable electrochemical properties and are of potential application in energy storage.

Synovial fluid-inspired biomimetic lubricating microspheres:Zwitterionic polyelectrolyte brushes-grafted microgels

Synovial fluid is made up of various biomacromolecules,including hyaluronic acid,aggrecans,lubricins,and phosphatidylcholine lipid,which are assembled onto the surface of articular cartilage in a gel state.Among them,brush-like biomacromolecules or assemblies have a vital effect on human joint lubrication.Inspired by this,the combination of brush-like molecular structures and gel-like assembly may be an efficient approach for the synthesis of biomimetic lubricating matters.Learning from the lubrication system of human joints,poly(2-methacryloyloxyethyl phosphorylcholine)(PMPC)brushes grafted poly(N-isopropylacrylamide-co-acrylic acid)(poly(NIPAAm-co-AA))microgels,abbreviated as MBs-g-MGs,were synthesized as one kind of biomimetic lubricating additives.It is worth noting that this bionic strategy considered both molecular structure and assembled form,which enabled this hairy microgel to achieve low friction in aqueous medium.Meanwhile,the effective lubrication was still achieved when using MBs-g-MGs at high temperature,indicating that this microgel maintains a good lubricating effect over a wide range of temperature.In addition,this kind of microgel possessed good biocompatibility,which laid the foundation for potential biomedical applications.Looking beyond,these biomimetic microgels may provide an effective lubricating effect for water-based sliding interfaces,especially in biomedical systems.

Assembly of Preformed Gold Nanoparticles onto Thermore- sponsive Poly（N-isopropylacrylamide）-Based Microgels on the Basis of Au-thiol Chemistry

The assembly of preformed gold nanoparticles （AuNPs） onto the thermoresponsive poly（N-isopropylacrylamide） （PNIPAM）-based microgels was achieved on the basis of the driving force of Au-thiol chemistry. The loading amount of AuNPs can be controlled by varying the ratio of AuNPs relative to PNIPAM-based microgels. The as-prepared PNIPAM/Au hybrid microgels showed well-defined reversible swelling/deswelling transition in re- sponse to temperature, which can be employed to tune the plasmonic property of hybrid microgels. As the tempera- ture was increased, the position of localized surface plasmon resonance （LSPR） band red-shifted to some extent mainly due to the increase in the local refractive index around AuNPs.

Temperature-sensitive molecularly imprinted microgels with esterase activity

Temperature-sensitive molecularly imprinted microgels(MIGs)exhibiting esterase activity were prepared by a reverse emulsion method using dialdehyde dextran-histidine conjugate(PAD-His)as the functional macromonomer and p-nitrophenyl phosphate(NPP)as the stable transition state analogue(TSA)as well as Co2+as the coordination center.The catalytic activity of MIGs was greatly influenced by the amount of the template,and could be modulated by temperature.The hydrolysis kinetics of p-nitrophenyl acetate(NPA)in the presence of MIGs could be described by the Michaelis-Menten equation.The MichaelisMenten constant and maximum velocity were found to be 2.2×105mol/L and 2.04×10 -8mol/h,respectively.In addition,the MIGs were found to have a high catalytic selectivity to NPA.

Poly(N-isopropylmethacrylamide-co-4-acrylamidobenzo-18-crown-6)microgels with expanded networks for excellent adsorption of lead(Ⅱ)ions

Lead(Ⅱ)(Pb2+)ions are toxic heavy metal ions that can accumulate in the human body through water and cause severe health problems,including neurotoxicity,nephrotoxicity,hematological toxicity and even genotoxicity effects.To remove Pb2+selectively and effectively from aqueous solutions,we develop a novel type of Pb2+-recognizable microgels with excellent adsorption capacity to Pb2+,which are fabri-cated from 4-acrylamidobenzo-18-crown-6(B18C6Am)and N-isopropylmethacrylamide(NIPMAM)monomers by precipitation copolymerization method.The prepared poly(N-isopropylmethacrylamide-co-4-acrylamidobenzo-18-crown-6)(PNMB)microgels exhibit expanded structures,because the electron-donating methyl groups atα-carbon could descend the polarity of C=O in the NIPMAM monomers and thus weaken the polymer segment…segment interactions.The expanded structures of PNMB microgels are beneficial for adsorption of Pb2+due to the low steric hindrance in the polymeric networks.The Pb2+adsorption isotherms of PNMB microgels are consistent with the Langmuir model for monolayer adsorption.The results indicate that the prepared Pb2+-recognizable PNMB microgels are highly promising for the selective removal of lead(Ⅱ)ions from aqueous solutions.

Responsive Hybrid Poly(vinyl alcohol)Hydrogel Membranes with Embedded Microgels as Valves

The research and application of responsive materials have long been hampered by their complicated designs and tedious construction processes.Besides,many current responsive materials show retard or weak responsiveness.In this study,responsive hybrid poly(vinyl alcohol)hydrogel membranes with embedded poly(N-isopropylacrylamide-acrylic acid)microgels as valves were constructed by simple mixing and subsequent freezing-thawing process.In the structure of the membranes,the matrix poly(vinyl alcohol)chains thread through and entangle with the microgels,and the microgels are firmly constrained within the hybrid hydrogel network.The fast and sharp temperature responsiveness of the embedded microgels was largely retained and endowed the hydrogel membrane with excellent temperature and pH responsiveness.Moreover,the hydrogel membrane showed excellent fatigue resistance in both temperature and pH-responsive flux examination.This study presented the great potential of these hybrid hydrogel membranes in biomedical applications and provided a new strategy for the future design and construction of responsive biomaterials.