Doxorubicin‐Loaded Nanogel Assemblies with pH/Thermo‐triggered Payload Release for Intracellular Drug Delivery

For improving intracellular doxorubicin (DOX) delivery, DOX‐encapsulated nanogel assemblies with pH/thermo‐responsive drug release are developed. DOX and a graft copolymer comprising acrylic acid (AAc) and 2‐methacryloyloxyethyl acrylate (MEA) units as the backbone and with poly(N‐isopropylacrylamide) (PNIPAAm) and monomethoxy poly(ethylene glycol) as the grafts at pH 7.4 and 4 °C undergo electrostatically induced co‐association into copolymer/DOX nanocomplexes. After being crosslinked by polymerization of the MEA moieties, the complex nanoconstructs exhibit a unique nanogel‐like architecture. Taking advantage of the extensive electrostatic attraction of the DOX molecules with ionized AAc residues and π–π stacking among copolymer‐bound DOX molecules, the DOX‐loaded nanogels show a relatively high payload content. With the milieu pH being reduced from 7.4 to 4.7, the drug release is appreciably promoted due to the massive disruption of ionic AAc/DOX pairings. The thermo‐evolved phase transition of the PNIPAAm grafts further accelerates drug elution, particularly at pH 4.7. In vitro characterization indicates that the DOX‐embedded nanogels endocytosed by HeLa cells can progressively release DOX within acidic organelles. As a result, the viability of cancer cells treated with DOX‐loaded nanoparticles can be further reduced by prolonging incubation time. This work demonstrates the great potential of the DOX‐loaded nanogel assemblies for effective intracellular drug delivery.

     

In vivo lifetime and anti-cancer efficacy of doxorubicin-loaded nanogels composed of cinnamoyl poly (β-cyclodextrin) and cinnamoyl Pluronic F127

Nanogel composed of rhodamine B isothiocyanate-labeled cinnamoyl polymeric β cyclodextrin and cinnamoyl Pluronic F127 was prepared by taking advantage of their self-assembling property in aqueous phase. The in vivo lifetime of the nanogel in the blood circulation system of hairless mouse was determined by fluorescence imaging method using rhodamine B isothiocyanate as a fluorescence probe, and it was more than 192 h. The in vivo clearance of doxorubicin loaded in the nanogel was observed by the same fluorescence imaging method. Doxorubicin loaded in the nanogel was cleared from the blood circulation system more slowly than free drug, indicating that the in vivo lifetime of the anti-cancer drug was prolonged by the nanogel. The fluorescence of doxorubicin was detected from the mouse even 200 h after the doxorubicin-loaded nanogel was intravenously injected into a mouse. According to the histological study of tumor tissue, the in vivo tumor growth-inhibiting efficacy of doxorubicin loaded in the nanogel was higher than that of free drug, possibly because of its longer in vivo lifetime.     

Multistructured Nanogel‐Based Networks Formed from Interfacial Redox Polymerizations for Modulating Small Molecule Release

Uniform, 3D nanogel‐based coatings are generated on polymeric substrates using an interfacial redox polymerization. Diffusion of a reducing agent from a core material into a solution of acrylate‐functionalized nanogels and an oxidizer generates free radicals that initiate nanogel crosslinking selectively at the gel surface. Coating thickness is controlled between 20 and 150 µm by varying reaction time and initiator concentration. Controlling the structure of the final nanogel‐based coating is demonstrated to predictably change the diffusion coefficient of a small molecule drug loaded into the core material. Multidrug release from the coating and the core is demonstrated with model dyes. This study showcases the ability to design multifunctional networks for controlled release using a simple, mild coating procedure.     

Tetraphenylsilane‐Cored Star‐Shaped Amphiphilic Block Copolymers for pH‐Responsive Anticancer Drug Delivery

Four‐arm star‐shaped poly[2‐(diethylamino)ethyl methacrylate]‐b‐poly[2‐hydroxyethyl methacrylate]s block copolymers using tetraphenylsilane (TPS) as a core with adjustable arm lengths are synthesized through two‐step atom transfer radical polymerizations. For comparison, a linear block copolymer with similar molecular weight is also prepared. The assembled star‐shaped copolymer micelles exhibit sizes of 102–139 nm and critical micelle concentrations of 1.49–3.93 mg L^?1. Moreover, the bulky and rigid TPS core is advantageous for propping up the four star‐shaped arms to generate large intersegmental space. As a result, the drug‐loading capacity in the micelles is up to 33.97 wt%, much surpassing the linear counterpart (8.92 wt%) and the previously reported star‐shaped copolymers prepared using pentaerythritol as the core. Furthermore, the micelles show sensitive pH‐responsive drug release when the pH changes from 7.4 to 5.0. The in vitro cytotoxicity to Hela cells indicates that the doxorubicin (DOX)‐loaded micelles have similar anticancer activity to the pristine DOX. The combination of excellent micelle stability, high drug‐loading, sensitive pH response, and good anticancer activity endows the copolymers with promising application in drug control delivery for anticancer therapy.     

Micelles with a Loose Core Self‐Assembled from Coil‐g‐Rod Graft Copolymers Displaying High Drug Loading Capacity

High drug loading capacity is one of the critical demands of micellar drug‐delivery vehicles; however, it is a challenging work. Herein, it is demonstrated that micelles self‐assembled from poly(ethylene glycol)‐graft‐poly(γ‐benzyl‐l ‐glutamate) (PEG‐g‐PBLG) coil‐g‐rod graft copolymers display high drug‐loading capacity for doxorubicin (DOX) model drugs. As revealed by a combination study of experiments and dissipative particle dynamics simulations, the high drug‐loading capacity of the micelles is related to the loose core structure of the micelles. In these micelles, the hydrophobic PBLG grafts randomly disperse in the micelle core due to their rigid nature and the coil‐g‐rod topology of the graft copolymers, which results in a loose core of the micelles. The structure of the graft copolymer, including the length of rod grafts, the length of coil backbone, and the grafting ratio of the rod grafts affecting the arrangement of the rod grafts in the micelle core has influence on the drug‐loading capacity of the micelles. Besides, the strong π–π stacking interaction between graft copolymers and DOX also plays an important part in achieving high drug‐loading capacity. In vitro studies reveal that these drug‐loaded micelles show good biocompatibility, and the DOX can be gradually released from the micelles.     

Acid‐Labile Copolymer Micelles Cross‐Linked by a Twin Ortho Ester Cross‐Linking Agent: Synthesis,Characterization, and Evaluation

In this work, a series of poly(ethylene glycol)‐block‐poly(N‐succinimidyl methacrylate) (PEG‐b‐PNSM) micelles with different length of PNSM are prepared by self‐assembly in phosphate buffer. In situ cross‐linked micelles (CLM) with high stability at neutral pH are obtained by adding a pH‐sensitive cross‐linker bearing two five‐membered ortho ester rings, 2‐{4‐[2‐(2‐amino‐ethoxy)‐[1,3]dioxolan‐4‐ylmethoxymethyl]‐[1,3]dioxolan‐2‐yloxy}‐thylamine. Nile Red as a model drug is easily loaded into CLM, and the release rate accelerate at acidic environments (pH 5). Cytotoxicity of PEG‐b‐PNSM and CLM against NIH3T3 cells is determined and favorable biocompatibility is possessed. Confocal laser scanning microscopy study shows that Nile Red‐loaded CLM has excellent cellular uptake by MCF‐7 cells. Paclitaxel (PTX) is successfully encapsulated into the cross‐linked micelles. In vitro cytotoxicity of free PTX, and PTX‐loaded CLM causes almost the same potency in killing MCF‐7 cells. These results suggest that cross‐linked micelles can be a potential vehicle for delivering hydrophobic anticancer drugs to tumors.

     

Photocrosslinkable Poly(ε‐caprolactone)‐b‐Hyperbranched Polyglycerol (PCL‐b‐hbPG) with Improved Biocompatibility and Stability for Drug Delivery

The major limitations of nanocarriers for drug delivery are the poor biocompatibility and low stability that may induce the unintended burst release of loaded drugs during blood circulation. To overcome these limitations, a photocrosslinkable amphiphilic block copolymer consisting of hydrophobic poly(ε‐caprolactone) (PCL) block and a hydrophilic hyperbranched polyglycerol (hbPG) block with improved biocompatibility and stability for drug delivery is developed. They are readily prepared via UV‐triggered chemical crosslinking with 4‐hydroxycinnamic acid (CA) modification in the hbPG block. The photocrosslinked hbPG‐b‐PCL‐CA nanoparticles are spherical and the size of nanoparticles is increased to 60 ± 30 nm. Photocrosslinked hbPG‐b‐PCL‐CA nanoparticles exhibit significantly high stability in a physiological buffer and the loaded drug in sustained manner. In addition, photocrosslinked hbPG‐b‐PCL‐CA nanoparticles show good biocompatibility in vitro and in vivo. These data imply the promising potential of photocrosslinked hbPG‐b‐PCL‐CA nanoparticles as nanocarriers for drug delivery.

     

In Situ Encapsulation of Nile Red or Doxorubicin during RAFT‐Mediated Emulsion Polymerization via Polymerization‐Induced Self‐Assembly for Biomedical Applications

Hydrophobic agents, a fluorescent dye (Nile red, NR) or an anticancer drug (doxorubicin, DOX), are encapsulated into poly((N‐[3‐(dimethylamino) propyl] methacrylamide)‐b‐poly (methyl methacrylate) (PDMAPMA‐b‐PMMA) nanoparticles (NPs) via one‐pot reversible addition‐fragmentation chain‐transfer (RAFT)‐mediated emulsion polymerization in water. The macroRAFT, PDMAPMA, is chain‐extended with the methyl methacrylate (MMA), with the hydrophobic agents soluble in MMA, resulting in loaded NPs, with either NR or DOX via polymerization‐induced self‐assembly (PISA). The NR‐loaded NPs are visualized by structured illumination microscopy (SIM), thus indicating the successful loading of the fluorescent dye into the PMMA core. The DOX‐loaded NPs exhibit a sustained release profile over 5 d, showing a small burst effect during the first 2 h, as compared with the free DOX. The DOX‐loaded NPs show higher cell toxicity than the free DOX in RAW 264.7 cell line. The results demonstrate the potential of using emulsion polymerization for synthesis of tailored and reproducible NPs encapsulating hydrophobic agents.     

Facile one-pot synthesis of glucose-sensitive nanogel via thiol-ene click chemistry for self-regulated drug delivery

A novel glucose-sensitive nanogel was conveniently prepared through one-pot thiol-ene copolymerization of pentaerythritol tetra(3-mercaptopropionate), poly(ethylene glycol) diacrylate, methoxyl poly(ethylene glycol) acrylate and N-acryloyl-3-aminophenylboronic acid. The formation of core–shell nanogel was verified by proton nuclear magnetic resonance, dynamic laser scattering (DLS) and transmission electron microscopy. The successful incorporation of phenylboronic acid (PBA) in the nanogel was confirmed through Fourier transform infrared spectroscopy, inductively coupled plasma mass spectrometry and fluorescence technology. Owing to the presence of PBA, the nanogel exhibited high glucose sensitivity in phosphate-buffered saline determined by DLS and fluorescence technology. The increased amount of glucose causes an increase in the hydrodrodynamic radius and a decrease in the fluorescence intensity of PBA–alizarin red S (ARS) complex in the nanogel at pH 7.4 because of the competitive substitution of ARS to form the hydrophilic PBA–glucose complex. ARS and insulin were loaded into this glucose-sensitive nanogel. In vitro release profiles revealed that the drug release from the nanogel could be triggered by the presence of glucose. The more glucose in the release medium, the more drug was released and the faster the release rate. Furthermore, in vitro methyl thiazolyl tetrazolium assay, lactate dehydrogenase assay and hemolysis test suggested that the nanogel was biocompatible. Therefore, the PBA-incorporated nanogel with high glucose-sensitivity and good biocompatibility may have great potential for self-regulated drug release.     

One‐Step Preparation of Reduction‐Responsive Biodegradable Polymers as Efficient Intracellular Drug Delivery Platforms

Reduction‐responsive biodegradable polymeric micelles based on functional 2‐methylene‐1,3‐dioxepane (MDO) copolymers are developed and investigated for triggered doxorubicin (DOX) release. The MDO‐based copolymers P(MDO‐co‐PEGMA‐co‐PDSMA) are synthesized via the simple one‐step radical ring‐opening copolymerization of MDO, poly(ethylene glycol) methyl ether methacrylate (PEGMA), and pyridyldisulfide ethylmethacrylate (PDSMA). The copolymers can self‐assemble to form micelles in aqueous solution. DOX, a model anticancer drug, is loaded into the micelles with the drug loading content (DLC) of 11.3%. The micelles can be disassembled under a reductive environment (10 × 10^?3m glutathione), which results in a triggered drug release behavior. The glutathione‐mediated intracellular drug release of DOX‐loaded micelles is investigated against A549 cells. Confocal laser scanning microscopy (CLSM) results demonstrated that DOX‐loaded micelles exhibits faster drug release in glutathione monoester (GSH‐OEt)‐pretreated A549 cells, compared with untreated and buthionine sulfoximine (BSO)‐pretreated A549 cells. Based on the facile synthetic strategy, the reduction‐sensitive biodegradable micelles with triggered intracellular drug release are promising for anticancer drug delivery.

     

Biocompatible Poly(D,L‐lactide)‐block‐Poly(2‐methacryloyloxyethylphosphorylcholine) Micelles for Drug Delivery

Well‐defined amphiphilic PLA‐b‐PMPC diblock copolymers were synthesized. Bimimetic micelles were prepared and applied for release of anti‐cancer drugs (DOX). TEM and DLS analysis revealed a regular spherical shape with small diameter (less than 50 nm) of the micelle. The biocompatibility of PLA‐b‐PMPC micelles was studied, and it was found that the micelles possessed excellent cytocompatibility due to the zwitterionic phosphorylcholine group. DOX could be efficiently loaded into the micelles with a loading efficiency of 44–67%. The DOX‐loaded micelles showed lower cytotoxicity than free drugs and efficiently delivered and released the drug into cancer cells. With these properties, the PLA‐b‐PMPC polymer micelles are attractive as drug carriers for pharmaceutical application.

     

Folate‐Conjugated Poly(N‐(2‐hydroxypropyl) methacrylamide)‐block‐Poly(benzyl methacrylate): Synthesis,Self‐Assembly,and Drug Release

Well‐defined amphiphilic diblock copolymers of poly(N‐(2‐hydroxypropyl)methacrylamide)‐block‐poly(benzyl methacrylate) (PHPMA‐b‐PBnMA) are synthesized using reversible addition–fragmentation chain transfer polymerization. The terminal dithiobenzoate groups are converted into carboxylic acids. The copolymers self‐assemble into micelles with a PBnMA core and PHPMA shell. Their mean size is <30 nm, and can be regulated by the length of the hydrophilic chain. The compatibility between the hydrophobic segment and the drug doxorubicin (DOX) affords more interaction of the cores with DOX. Fluorescence spectra are used to determine the critical micelle concentration of the folate‐conjugated amphiphilic block copolymer. Dynamic light scattering measurements reveal the stability of the micelles with or without DOX. Drug release experiments show that the DOX‐loaded micelles are stable under simulated circulation conditions and the DOX can be quickly released under acidic endosome pH.     

Synthesis and Properties of UCST‐Type Thermo‐ and Light‐Responsive Homopolypeptides with Azobenzene Spacers and Imidazolium Pendants

Homopolypeptide bearing azobenzene and triethylene glycol spacers, and 1‐butylimidazolium pendants, namely P(Azo‐OEG₃‐ImX) (X = Cl, I, BF₄) are prepared by 1,3‐dipolar cycloaddition and subsequent ion‐exchange reaction. Both ¹H NMR and FTIR confirm the polymer structures and reveal a high grafting efficiency (97%). P(Azo‐OEG₃‐ImI) shows reversible upper critical solution temperature (UCST)‐type thermoresponsive property in ethanol while P(Azo‐OEG₃‐ImI/BF₄) shows a UCST in ethanol/water solvent mixtures. P(Azo‐OEG₃‐ImI) shows UCST‐type phase transitions in ethanol or ethanol/water solvent mixtures. After UV irradiation, the UCST‐type cloud point temperature (T_cp) remains constant in ethanol. However, it decreases after UV irradiation and increases after visible light irradiation in ethanol/water solvent mixtures due to the trans–cis isomerization of azobenzene moieties. P(Azo‐OEG₃‐ImCl/I) shows NaI‐induced UCST in water. The T_cp of the P(Azo‐OEG₃‐ImCl/I) aqueous solution is highly related to the polymer/salt concentration and nature of counteranions. P(Azo‐OEG₃‐ImCl) also shows reversible light‐responsive properties in NaI aqueous solutions. Higher NaI concentration is needed to induce a UCST for P(Azo‐OEG₃‐ImCl) after UV irradiation. Moreover, ciprofloxacin‐loaded hydrogel containing P(Azo‐OEG₃‐ImCl) shows more efficient drug release while the UV irradiation leads to slightly low drug release.     

Poly(N‐isopropylacrylamide‐co‐N‐isopropylmethacrylamide) Thermo‐Responsive Microgels as Self‐Regulated Drug Delivery System

Poly(N‐isopropylacrylamide‐co‐N‐isopropylmethacrylamide) (poly(NIPAAm‐co‐NIPMAAm)) is synthesized as an attractive thermo‐responsive copolymer by an original procedure. Due to the similar structure of the two co‐monomers, the poly(NIPAAm‐co‐NIPMAAm) copolymer displays a very sharp phase transition, under physiological conditions (phosphate buffer solution at pH = 7.4). The copolymer, showing the 51/49 co‐monomer NIPAAm/NIPMAAm molar ratio, displays a lower critical solution temperature (LCST) close to that of the human body temperature (36.8 °C). The poly(NIPAAm‐co‐NIPMAAm) microgels obtained at the 51:49 co‐monomer ratio displays a volume phase transition temperature (VPTT) slightly smaller than LCST. The deswelling rate of the microgels is very high (k = 0.019 s^?1), the shrinkage occurring almost instantaneously, whereas the swelling rate is slightly lower (k = 0.0077 s^?1). The microgels are loaded with the model drug dexamethasone and the drug release is investigated at different temperatures, below and above the VPTT. Under thermal cycling operation between 32 and 38 °C, the pulsatile release of dexamethasone is observed.

     

Self‐assembled UCST‐Type Micelles as Potential Drug Carriers for Cancer Therapeutics

A methoxy‐poly(ethylene glycol)‐block‐poly(acrylamide‐co‐acrylonitrile) (mPEG‐b‐P(AAm‐co‐AN)) amphiphilic copolymer exhibiting upper critical solution temperature (UCST) behavior is synthesized, and micelles from this copolymer are fabricated. It is found that the thermal responses of these micelles are tunable through balancing the hydrophobic/hydrophilic blocks in the copolymer. The size of the doxorubicin (DOX)‐loaded micelles is dependent on the hydrophobic interaction as well as hydrogen bonding between polymer and drug molecules. As a proof of concept, the drug release behavior is studied in vitro, and the cumulative release of DOX increases at temperature above the UCST of blank micelles. 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assays indicate that these polymers are non‐toxic towards human hepatic carcinoma cells (Bel 7402 cells) as well as human embryonic hepatocytes (L02 cells). DOX‐loaded micelles could effectively enter Bel 7402 cells in 2 h, and display much lower half inhibitory concentration compared with free DOX. These micelles may be exploited as a promising drug carrier for cancer therapeutics.

     

Light‐Switchable Supramolecular Self‐Assembly of Soft Colloids

Self‐assembly is an efficient strategy of constructing microgel‐based intelligent materials. However, it remains a challenge to realize the reversible self‐assembly of microgels. Herein, a method to guide the self‐assembly of soft colloids with light‐stimuli is proposed, utilizing the light‐responsive host–guest interaction between an azobenzene functionalized nanogel (the guest colloid) and an α‐cyclodextrin functionalized microgel (the host colloid). The two colloids can form a stable colloid cluster when the surface of the host colloid is fully packed with the guest colloids. The colloid cluster can disassemble when irradiated with UV light and reassemble when irradiated with visible light. The reversible colloidal self‐assembly can be controlled by the interplay between the supramolecular and covalent crosslinking, and can also be adjusted by the addition of competitive host molecules. Besides the light‐sensitivity, the colloid cluster inherits the deformability and temperature‐sensitivity from its parent colloids. These features are different from the supramolecular self‐assembly of hard colloids or macroscopic gels, and manifest the as‐prepared colloid cluster potential building blocks of light‐responsive materials.     

SET‐LRP Synthesis of Well‐Defined Light‐Responsible Block Copolymer Micelles

Herein, the synthesis of well‐defined light‐sensitive amphiphilic diblock copolymers consisting of UV‐responsive poly(2‐nitrobenzyl acrylate) (PNBA) and hydrophilic poly(ethylene oxide) (PEO) blocks is reported. This is achieved by a single electron transfer living radical polymerization (SET‐LRP) of 2‐nitrobenzyl acrylate monomer initiated by PEO‐containing macroinitiator. Despite several reports on PEO‐b‐PNBA copolymers, this is the first time the PNBA block is synthesized by a controlled radical polymerization leading to the copolymers with low dispersity (Ð = 1.10). In water, the copolymers self‐assemble into well‐defined micelles with a hydrodynamic diameter of 25 nm. Upon irradiation with UV‐light, the PNBA units degrade to hydrophilic poly(acrylate) resulting in disassembly of the micelles. Considering the robustness of the reported synthetic protocol, the prepared polymers represent an interesting platform for the construction of new stimuli‐responsive drug delivery systems.     

Temperature‐Triggered Nanosphere Formation Through Self‐Assembly of Amphiphilic Polyphosphazene

Summary: An amphiphilic graft polyphosphazene with a molar ratio of poly(N‐isopropylacrylamide) (PNIPAm) to ethyl glycinate (GlyEt) of 0.54:1 was synthesized. This copolymer in aqueous solution exhibited two temperature induced phase transitions at 17.2 and 33.7 °C, which correspond to the transformation of primary aggregate morphology (at T_ph1) and the collapse of PNIPAm chains (at T_ph2) respectively. Network micelles were assembled in water at lower temperature (far below T_ph1), and then narrowly dispersed nanoparticles were formed above T_ph1, while inter‐nanoparticle aggregation occurred due to the collapse of PNIPAm chains surrounding the GlyEt core when the temperature was above T_ph2. Through solubilization of the hydrophobic drug ibuprofen into polymeric aggregates at lower temperature, drug loaded nanospheres were prepared successfully. In vitro release revealed that sustained drug release was achieved with this novel delivery system. These results suggest that this novel copolymer could be used as a potential drug carrier, especially for the delivery of hydrophobic biocompounds through parenteral administration.

Schematic illustration of the temperature‐triggered self‐assembly process of PNIPAm/GlyEt‐PPP in aqueous solution.     

A nanogel of on-site tunable pH-response for efficient anticancer drug delivery

A smart, soft and small nanoparticulate drug carrier that can efficiently transport therapeutics into tumor cells to control the intracellular drug concentration will enable major advancements in cancer therapy. To facilitate a remote modulation of the intracellular pH-regulated drug release, we have designed a new class of pH-responsive chitosan-based nanogels (<200 nm) by the physical interpenetration of chitosan chains into a nonlinear poly(ethylene glycol) (nonlinear PEG) chain network. The resultant PEG-chitosan nanogels not only respond to the changes in environmental pH over the physiologically important range of 5.0–7.4, but – more importantly – also enable us to remotely modulate the pH response by external cooling/heating. The nanogel, as well as the nanogel loaded with a model anticancer drug 5-fluorouracil (5-FU), is capable of varying its surface charge from nearly neutral to positive around tumor extracellular pH (～6.0–6.2) to facilitate cell internalization. Subsequently, the significantly increased acidity in subcellular compartments (～5.0) can trigger 5-FU release from the endocytosed drug carriers. While this nanogel serving as a drug carrier exhibits a reduced toxicity in combined chemo-thermo treatments, it has shown significantly enhanced therapeutic efficacy in combined chemo-cryo treatments of the model B16F10 melanoma cells, indicating its great potential for cancer therapy.     

Thermo‐Induced Double Phase Transition Behavior of Physically Cross‐Linked Hydrogels Based on Oligo(ethylene glycol) methacrylates

Double thermosensitive clay/P(MEO₂MA‐co‐POEGMA) nanocomposite (NC) hydrogels are prepared by in situ free radical polymerization of 2‐(2‐methoxyethoxy) ethyl methacrylate (MEO₂MA) and oligo(ethylene glycol) methacrylate (OEGMA) in the presence of physical cross‐linker clay. The temperature‐induced phase transition behavior of NC hydrogels is investigated by turbidity, temperature dependent nuclear magnetic resonance, and variable temperature Fourier transform infrared spectroscopy. 2D infrared analysis is employed to study the sequential order of changes of all groups in NC hydrogels during the heating and cooling process. The obtained novel clay/P(MEO₂MA‐co‐POEGMA) NC hydrogels exhibit an unusual thermally induced multistep aggregation process and successively undergo three consecutive microstructural changes: “loose clay/polymer microaggregation ? homogeneous network structure ? dense clay/polymer macroaggregation.” Dynamic light scattering and transmission electron microscopy measurements show similar results of the NC nanogel at the same temperature regions, confirming the three consecutive microstructural changes. This new generation of thermosensitive hydrogel offers promising potential for applications as smart devices, biomedical materials, and drug carriers.