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.

Highly Sensitive Poly-N-isopropylacrylamide Microgel-based Electrochemical Biosensor for the Detection of SARS-COV-2 Spike Protein

Objective Late 2019 witnessed the outbreak and widespread transmission of coronavirus disease 2019(COVID-19),a new,highly contagious disease caused by novel severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Consequently,considerable attention has been paid to the development of new diagnostic tools for the early detection of SARS-CoV-2.Methods In this study,a new poly-N-isopropylacrylamide microgel-based electrochemical sensor was explored to detect the SARS-CoV-2 spike protein(S protein)in human saliva.The microgel was composed of a copolymer of N-isopropylacrylamide and acrylic acid,and gold nanoparticles were encapsulated within the microgel through facile and economical fabrication.The electrochemical performance of the sensor was evaluated through differential pulse voltammetry.Results Under optimal experimental conditions,the linear range of the sensor was 10-13-10-9 mg/m L,whereas the detection limit was 9.55 fg/mL.Furthermore,the S protein was instilled in artificial saliva as the infected human saliva model,and the sensing platform showed satisfactory detection capability.Conclusion The sensing platform exhibited excellent specificity and sensitivity in detecting spike protein,indicating its potential application for the time-saving and inexpensive detection of SARS-CoV-2.

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.

Sacrificial microgel‑laden bioink‑enabled 3D bioprinting of mesoscale pore networks

Three-dimensional(3D)bioprinting is a powerful approach that enables the fabrication of 3D tissue constructs that retain complex biological functions.However,the dense hydrogel networks that form after the gelation of bioinks often restrict the migration and proliferation of encapsulated cells.Herein,a sacrificial microgel-laden bioink strategy was designed for directly bioprinting constructs with mesoscale pore networks(MPNs)for enhancing nutrient delivery and cell growth.The sacrificial microgel-laden bioink,which contains cell/gelatin methacryloyl(GelMA)mixture and gelled gelatin microgel,is first thermo-crosslinked to fabricate temporary predesigned cell-laden constructs by extrusion bioprinting onto a cold platform.Then,the construct is permanently stabilized through photo-crosslinking of GelMA.The MPNs inside the printed constructs are formed after subsequent dissolution of the gelatin microgel.These MPNs allowed for effective oxygen/nutrient diffusion,facilitating the generation of bioactive tissues.Specifically,osteoblast and human umbilical vein endothelial cells encapsulated in the bioprinted large-scale constructs(≥1 cm)with MPNs showed enhanced bioactivity during culture.The 3D bioprinting strategy based on the sacrificial microgel-laden bioink provided a facile method to facilitate formation of complex tissue constructs with MPNs and set a foundation for future optimization of MPN-based tissue constructs with applications in diverse areas of tissue engineering.

Conformance control by a microgel in a multi-layered heterogeneous reservoir during CO_(2) enhanced oil recovery process

Injecting CO_(2)into the underground for oil displacement and shortage is an important technique for carbon capture,utilization and storage(CCUS).One of the main problems during the CO_(2)injection is the channeling plugging.Finding an effective method for the gas channeling plugging is a critical issue in the CO_(2)EOR process.In this work,an acid-resistance microgel named dispersed particle gel(DPG)was characterized and its stability was tested in the CO_(2)environment.The microgel size selection strategies for the homogeneous and heterogeneous reservoirs were respectively investigated using the single core flooding and three parallel core flooding experiments.Moreover,the comparison of microgel alternate CO_(2)(MAC)injection and water alternate CO_(2)(WAC)injection in the dual core flooding experiments were presented for the investigation of the role of microgel on the conformance control in CO_(2)flooding process.The results have shown that the microgel featured with ANH and CAN groups can keep its morphology after aging 7 days in the CO_(2)environment.Where,the small microgel with unobstructed migration and large microgel with good plugging efficiency for the high permeability zone were respectively featured with the higher recovery factor in homogeneous and heterogeneous conditions,which indicate they are preferred used for the oil displacement and conformance control.Compared to WAC injection,MAC injection had a higher incremental recovery factor of 12.4%.It suggests the acid-resistance microgel would be a good candidate for the conformance control during CO_(2)flooding process.

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.

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.

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.

Non-isothermal crystallization kinetics of reactive microgel/nylon 6 blends

The non-isothermal crystallization kinetics of reactive microgel/nylon 6 blends was investigated by differential scanning calorimetry(DSC). The Mo equation was employed to analyze the non-isothermal crystallization data. The crystallization activation energies were also evaluated by the Kissinger method. The results show that the crystallization onset temperature(T onset) and crystallization peak temperature(T p) decrease with the increase of the content of reactive microgel, while ΔT(T onset–T p), the crystallization half-time(t1/2) and the crystallization enthalpy(ΔH c) increase. The required cooling rates of blends are higher than that of neat nylon6 in order to achieve the same relative crystallinity in a unit of time. The crystallization activation energies of the reactive microgel/nylon 6 blends are greater than those of the neat nylon 6. When the content of reactive microgel is 30%, the relative crystallinity(X t) reaches the maximum.

CURE OF A MICROGEL-EPOXY RESIN TWO-PHASE POLYMER WITH ETHYLENE DIAMINE

The curing of a microgel-epoxy resin two phase polymer prepared by in situ copolymerization of unsaturated polyester with acrylic monomer was studied. The unsaturated unit reacted with N—H during the cure of the resin with ethylene diamine. The Michael type reaction was ten times more rapid than the addition of N—H to epoxide.This was accounted for the lower apparent activation energy of the curing of the two phase resin.

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.

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 OF MICROGEL-EPOXY RESIN TWO-PHASE POLYMERS BY IN SITU POLYMERIZATION~*

Microgel-epoxy resin two-phase polymers were prepared by in situ copolymerization of ethylenic monomers with unsaturated polyesters. The choice of monomers and the effect of monomer concentration on microgel particle size were discussed. Agglomeration of particles played a significant role in the early stage of polymerization. The microgel dispersion in epoxy resin was stable after the finish of polymerization. Upon curing the particles remained well dispersed.

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.

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.

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.

POLY(N-ISOPROPYL ACRYLAMIDE) MICROGEL DOPED WITH LUMINESCENT PBS QUANTUM DOTS

Thiol-stabilized PbS quantum dots （QDs） with dimensions 3-5 nm capped with a mixture of 1-thioglycerol/dithioglycerol （TGL/DTG） were coUoidally prepared at room temperature. Room temperature photoluminescence quantum efficiency of freshly prepared PbS QDs （7%-11%） remained higher than 5% upon aging for three weeks when the nanocrystals （NCs） were stored in an ice-bath in the dark, and higher than 5%for at least five weeks when extra DTG ligands were introduced into the nanocrystal solution followed by stirring every two weeks. Poly（N-isopropyl acrylamide） （PNIPAM） microgels were produced via precipitation polymerization with dimensions of ca. 230 nm and polydispersity of 3-5%. Incorporation of PbS QDs into PNIPAM microgels indicated that PbS can be incorporated into the interior of microgel particles and not at the microgel interface. The combination of reasonable room temperature quantum efficiency and strong, efficient luminescence covering the 1.3-1.55 μm telecommunication window makes these nanoparticles promising materials in optical devices and telecommunications.

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.

Microgel-based carriers enhance skeletal stem cell reprogramming towards immunomodulatory phenotype in osteoarthritic therapy

Skeletal stem cells(SSC)have gained attentions as candidates for the treatment of osteoarthritis due to their osteochondrogenic capacity.However,the immunomodulatory properties of SSC,especially under delivery operations,have been largely ignored.In the study,we found that Pdpn+and Grem1+SSC subpopulations owned immunoregulatory potential,and the single-cell RNA sequencing(scRNA-seq)data suggested that the mechanical activation of microgel carriers on SSC induced the generation of Pdpn+Grem1+Ptgs2+SSC subpopulation,which was potent at suppressing macrophage inflammation.The microgel carriers promoted the YAP nuclear translocation,and the activated YAP protein was necessary for the increased expression of Ptgs2 and PGE2 in microgels-delivered SSC,which further suppressed the expression of TNF-ɑ,IL-1βand promoted the expression of IL-10 in macrophages.SSC delivered with microgels yielded better preventive effects on articular lesions and macrophage activation in osteoarthritic rats than SSC without microgels.Chemically blocking the YAP and Ptgs2 in microgels-delivered SSC partially abolished the enhanced protection on articular tissues and suppression on osteoarthritic macrophages.Moreover,microgel carriers significantly prolonged SSC retention time in vivo without increasing SSC implanting into osteoarthritic joints.Together,our study demonstrated that microgel carriers enhanced SSC reprogramming towards immunomodulatory phenotype to regulate macrophage phenotype transformation for effectively osteoarthritic therapy by promoting YAP protein translocation into nucleus.The study not only complement and perfect the immunological mechanisms of SSC-based therapy at the single-cell level,but also provide new insight for microgel carriers in stem cell-based therapy.