Protein Drug Carrier Research Articles

Develop targeted protein drug carriers through a high-throughput screening platform and rational design.

Protein-based drugs offer advantages such as high specificity, low toxicity, and minimal side effects compared to small molecule drugs. However, delivery of proteins to target tissues or cells remains challenging due to the instability, diverse structures, charges, and molecular weights of proteins. Polymers have emerged as a leading choice for designing effective protein delivery systems, but identifying a suitable polymer for a given protein is complicated by the complexity of both proteins and polymers. To address this challenge, we developed a fluorescence-based high-throughput screening platform called ProMatch to efficiently collect data on protein-polymer interactions, followed by in vivo and in vitro experiments with rational design. Using this approach to streamline polymer selection for targeted protein delivery, we identified candidate polymers from commercially available options and developed a polyhexamethylene biguanide (PHMB)-based system for delivering proteins to white adipose tissue as a treatment for obesity. We also developed a branched polyethylenimine (bPEI)-based system for neuron-specific protein delivery to stimulate optic nerve regeneration. Our high-throughput screening methodology expedites identification of promising polymer candidates for tissue-specific protein delivery systems, thereby providing a platform to develop innovative protein-based therapeutics. This article is protected by copyright. All rights reserved.

Anti-lung cancer targets of ellagic acid and biological interaction with a blood carrier protein

Ellagic acid (C14H6O8, 4,4′,5,5′6,6′-hexahydroxydiphenic acid 2,6,2′6′-dilactone) as a metabolite polyphenol compound has shown potential anticancer activities. However, its potential interaction affinity with plasma carrier proteins and antimetastatic activities are still unknown. Therefore, in this study, the anticancer and antimetastatic activities of ellagic acid in H358 metastatic lung cancer cells were assessed by MTT, qRT-PCR and western blotting. Then, the interaction of ellagic acid with human transferrin (HTf) as a drug carrier protein was measured. The results showed that ellagic acid-triggered cytotoxicity in H358 metastatic cells with an IC50 concentration of 7.18 µM. was mediated by overexpression of Bax/Bcl-2 ratio and caspase-3 mRNA as well as caspase-3 activity. Additionally, after being incubated with ellagic acid, the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), a hallmark of angiogenesis, and the metastasis markers matrix metalloproteinase-2 (MMP-2) and MMP-9 were noticeably reduced. In protein–ligand interaction section, we found that one molecule of ellagic acid potentially binds to one molecule of HTf mediated by the involvement of hydrogen bonds and van der Waals forces, supported by molecular docking simulations. In conclusion, our novel data suggested that ellagic acid can show promising antimetastatic activity against H358 lung cancer cells as well as ideal binding affinity to a drug carrier protein model.

The application of supramolecular chemistry in biomedicine

Nowadays, with the development of the supramolecular chemistry theory, more and more products based on Supramolecular chemistry are applied in people's life. Due to the good hydrophobic/hydrophilic structure, biocompatibility, spontaneous self-assembly and other characteristics of supermolecules, these applications are particularly prominent in the field of biomedicine. In this paper, the research status, development trend and potential problems of supermolecular materials in the field of biomedicine will be reviewed from the aspects of peptide/polyamino acid self-assembled nanomaterials for cancer diagnosis and treatment and soybean protein drug carriers. The early diagnosis and treatment of tumors and the delivery of drugs have always been challenges in the medical field. However, with the dynamic, reversible and controllable characteristics of supermolecules, as well as their excellent biocompatibility, sequence controllability and high biological activity, people can develop imaging systems and drug carriers based on supermolecule theory to improve the diagnostic ability of certain diseases and the biological accessibility of certain drugs.

Efficient Cytotoxicity of Recombinant Azurin in Escherichia coli Nissle 1917-Derived Minicells against Colon Cancer Cells.

Compared to chemical drugs, therapeutic proteins exhibit higher specificity and activity and are generally well-tolerated by the human body. However, the limitations, such as poor stability both in vivo and in vitro as well as difficulties in penetrating cell membranes, hinder their widespread application. To overcome the challenges, a highly efficient protocol was developed and implemented for the recombinant expression of the therapeutic protein azurin and secretion into minicells derived from probiotic Escherichia coli Nissle 1917. The novel coupled production with a delivery system of therapeutic proteins based on minicells was obtained through purification to enhance protein activity, circulation characteristics, and targeting specificity. This protein drug carrier integrates the production of carrier materials and the loading of expression proteins. The drug carrier also protects the encapsulated polypeptide drugs from enzymatic or gastric acid degradation until they are released. Escherichia coli Nissle 1917-derived minicells have natural targeting to colon cancer cells, low toxicity, and can accumulate for a long time after penetrating tumor tissue. This self-produced protein drug delivery system simplified the process of protein preparation, and its inhibitory effect on different types of colon cancer cells was verified by CCK-8 cytotoxicity assay, cancer cell invasion, and migration assay. This work provided a simple method to prepare minicell drug delivery systems for protein drug production and a novel approach for the transport of recombinant protein drugs.

Insights into soy Protein-Based drug delivery vehicles via molecular dynamic simulation

Protein-based drug carriers have emerged as promising candidates for efficient drug delivery among various colloidal carrier systems due to their low cytotoxicity, abundance, renewability, diverse functional groups and interactions, and high drug loading capacity. In this study, the molecular dynamics (MD) simulations are conducted to investigate the surface-governed mechanisms of soy protein as a drug delivery vehicle using allyl isothiocyanate (AITC) as the model drug. The interaction strengths between protein and AITCs are characterized, and the loading capacities of the protein molecules are calculated and compared with experimental results. Our findings reveal that both nonpolar and polar functional groups on the protein have the ability to adsorb AITCs, but the polar residues primarily facilitate stable attachment of the drug molecules through the electrostatic (dipole–dipole) interactions. Furthermore, tunable loading capability of soy protein drug carrier is observed by modifying its surface through two types of denaturation treatments: heat denaturation and breaking of disulfide bonds. Both denaturation approaches are found to increase the exposure of polar active sites, thereby enhancing the loading efficiency of the protein carriers. These findings contribute to the advancement and application of biodegradable protein-based drug carriers.

Common Self-Assembly Mechanisms and Their Applications in the Medical Field

Due to the different synthesis methods, organic materials made using self-assembly technology have many unique chemical properties compared to other organic materials. These specific chemical properties make these materials important in many fields, especially in the field of drug carrier design and protein structure simulation for pharmaceutical applications. Recently, more self-assembly techniques are being applied in completely new ways at different levels of discipline structure in order to produce results in the medical field. This paper will take the self-assembly method of non-covalent interaction as the starting point, introduce a variety of common mainstream self-assembly technologies from traditional hydrophilic hydrophobic self-assembly, hydrogen bond self-assembly to emerging charge transfer self-assembly, electrostatic self-assembly, and their application in medical treatment from drug transport to pathogenic mechanism simulation, discuss and consider the relationship between existing technological achievements and technical mechanisms themselves, and look forward to the future development and use of this technology and its subordinate categories.

Combination of Tissue Microarray Profiling and Multiplexed IHC Approaches to Investigate Transport Mechanism of Nucleoside Analog Drug Resistance.

Nucleoside analogs (NAs) are an established class of anticancer agents being used clinically for the treatment of diverse cancers, either as monotherapy or in combination with other established anticancer or pharmacological agents. To date, nearly a dozen anticancer NAs are approved by the FDA, and several novel NAs are being tested in preclinical and clinical trials for future applications. However, improper delivery of NAs into tumor cells because of alterations in expression of one or more drug carrier proteins (e.g., solute carrier (SLC) transporters) within tumor cells or cells surrounding the tumor microenvironment stands as one of the primary reasons for therapeutic drug resistance. The combination of tissue microarray (TMA) and multiplexed immunohistochemistry (IHC) is an advanced, high-throughput approach over conventional IHC that enables researchers to effectively investigate alterations to numerous such chemosensitivity determinants simultaneously in hundreds of tumor tissues derived from patients. In this chapter, taking an example of a TMA from pancreatic cancer patients treated with gemcitabine (a NA chemotherapeutic agent), we describe the step-by-step procedure of performing multiplexed IHC, imaging of TMA slides, and quantification of expression of some relevant markers in these tissue sections as optimized in our laboratory and discuss considerations while designing and carrying out this experiment.

Inhalable dry powder mRNA vaccines based on extracellular vesicles

Respiratory diseases are a global burden, with millions of deaths attributed to pulmonary illnesses and dysfunctions. Therapeutics have been developed, but they present major limitations regarding pulmonary bioavailability and product stability. To circumvent such limitations, we developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) as mRNA and protein drug carriers. Compared with standard synthetic nanoparticle liposomes (Lipos), Lung-Exos exhibited superior distribution to the bronchioles and parenchyma and are deliverable to the lungs of rodents and nonhuman primates (NHPs) by dry powder inhalation. In a vaccine application, severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein encoding mRNA-loaded Lung-Exos (S-Exos) elicited greater immunoglobulin G (IgG) and secretory IgA (SIgA) responses than its loaded liposome (S-Lipo) counterpart. Importantly, S-Exos remained functional at room-temperature storage for one month. Our results suggest that extracellular vesicles can serve as an inhaled mRNA drug-delivery system that is superior to synthetic liposomes.

Inhalable exosomes outperform liposomes as mRNA and protein drug carriers to the lung

Respiratory diseases are among the leading causes of morbidity and mortality worldwide, coupled with the ongoing coronavirus disease 2019 (COVID-19) pandemic. mRNA lipid nanoparticle (LNP) vaccines have been developed, but their intramuscular delivery limits pulmonary bioavailability. Inhalation of nanoparticle therapeutics offers localized drug delivery that minimizes off targeted adverse effects and has greater patient compliance. However, LNP platforms require extensive reformulation for inhaled delivery. Lung-derived extracellular vesicles (Lung-Exo) offer a biological nanoparticle alternative that is naturally optimized for mRNA translation and delivery to pulmonary cells. We compared the biodistribution of Lung-Exo against commercially standard biological extracellular vesicles (HEK-Exo) and LNPs (Lipo), where Lung-Exo exhibited superior mRNA and protein cargo distribution to and retention in the bronchioles and parenchyma following nebulization administration. This suggests that inhaled Lung-Exo can deliver mRNA and protein drugs with enhanced pulmonary bioavailability and therapeutic efficacy.

A Platelet-Mimicking Single-Atom Nanozyme for Mitochondrial Damage-Mediated Mild-Temperature Photothermal Therapy.

Single-atom nanozyme (SAzyme) systems have shown great potential in tumor therapy. A multifunctional SAzyme not only possesses high catalytic activity but also can be used as photothermal agents in photothermal therapy (PTT). Furthermore, it is also imperative to overcome tumor thermal resistance in SAzyme-based PTT so that PTT under a mild temperature is achievable. Herein, a novel platelet membrane (PM)-coated mesoporous Fe single-atom nanozyme (Fe-SAzyme) was formulated to solve these issues. The PM-coated mesoporous Fe-SAzyme (PMS) showed a satisfactory NIR-II photothermal performance, high peroxidase (POD) activity, and good tumor-targeting ability. In addition, PMS may be used as a carrier for protein drugs owing to its inner mesoporous structure. In vitro experiments showed that PMS could inhibit the expression of heat shock protein (HSP) by damaging the mitochondria, thereby finally improving the effect of mild-temperature PTT. Moreover, in vivo results showed that PMS could efficiently accumulate in tumor sites and suppress tumor growth with minimal toxicity in major organs. To the best of our knowledge, this study is the first report of a biomimetic mesoporous Fe-SAzyme used to achieve mitochondrial damage-mediated mild-temperature PTT. The study provides new promising ideas for designing other SAzyme systems for cancer treatment.

Correlation of the CTD structural domain of hepatitis B virus core protein with the encapsulation effect of indocyanine green

Hepatitis B virus core protein (HBc) has become a hot spot in drug carrier protein research due to its natural particle self-assembly ability and ease of modification. The truncation of the C-terminal polyarginine domain (CTD, aa 151-183) of HBc does not affect the self-assembly of the particles. However, it does affect the internal and external charges of the particles, which may subsequently affect drug encapsulation. Thus, the truncated C-terminal polyarginine domain (CTD) of HBc and the inserted RGD peptide were selected to construct and express three HBc variants (RH) encapsulated with ICG (RH/ICG) with different C-terminal lengths to compare the stability and drug activity of their nanoformulations. RH160/ICG was found to have a great advantages in encapsulation efficiency and biological imaging. Compared with other HBc variants, RH160/ICG significantly improved encapsulation efficiency, up to 32.77%±1.23%. Cytotoxicity and hemolysis assays further demonstrated the good biocompatibility of RH160/ICG. Cell uptake and in vivo imaging experiments in mice showed that RH160/ICG could efficiently deliver ICG in tumor cells and tumor sites with good imaging effect. This research provides a new direction for further expanding the diagnosis and treatment application of ICG and development of HBc-based nanoparticle drug carrier platform.

Coiled coil exposure and histidine tags drive function of an intracellular protein drug carrier

In recent years, protein engineering efforts have yielded a diverse set of binding proteins that hold promise for various therapeutic applications. Despite this, their inability to reach intracellular targets limits their applications to cell surface or soluble targets. To address this challenge, we previously reported a protein carrier that binds antibodies and delivers them to therapeutic targets inside cancer cells. This carrier, known as the Hex carrier, is comprised of a self-assembling coiled coil hexamer at the core, with each alpha helix fused to a linker, an antibody binding domain, and a six Histidine-tag (His-tag). In this work, we designed different versions of the carrier to determine the role of each building block in cytosolic protein delivery. We found that increasing exposure of the Hex coiled coil on the carriers, through molecular design or removing antibodies, increased internalization, pointing to a role of the coiled coil in promoting endocytosis. We observed a clear increase in endosomal disruption events when His-tags were present on the carrier relative to when they were removed, due to an endosomal buffering effect. Finally, we found that the antibody binding domains of the Hex carrier could be replaced with monomeric ultra-stable GFP for intracellular delivery and endosomal escape. Our results demonstrate that the Hex coiled coil, in conjunction with His-tags, could be a generalizable vehicle for delivering small and large proteins to intracellular targets. This work also highlights new biological applications for oligomeric coiled coils and shows the direct and quantifiable impact of histidine residues on endosomal disruption. These findings could inform the design of future drug delivery vehicles in applications beyond intracellular protein delivery.

Binding of fluorescent dansyl amino acids in albumin: When access to the protein cavity is more important than the strength of binding

Human serum albumin (HSA) is the primary drug carrier protein in the blood plasma. To perform this task, it has two cavities (sites I and II) where the pharmaceutical drugs can be bound. Dansyl amino acids are fluorescent markers widely used to identify the binding sites of new drugs. Here, we found intriguing differences between site I and site II for dansyl amino acids. These findings might open new perspectives for the studies of the interaction of drugs with albumin. In a comprehensive experimental and theoretical approach, six dansyl amino acids that are supposed to be specifically bound either to site I or site II were characterized. The interactions were evaluated by fluorescence quantum yield, fluorescence lifetime, anisotropy, association constant, induced circular dichroism, and isothermal titration calorimetry. All these techniques showed that dansyl amino acids of site II are significantly better ligands compared to the ones of site I. Oppositely, the binding free energies obtained by the molecular dynamics-based approaches suggested that dansyl amino acids bind stronger to site I. These results, contradictory at first glance, were clarified by calculating the Potential of Mean Force corresponding to ligand unbinding. These data suggested that the ligands could easier access site II than site I, explaining the experimental data. In conclusion, the access of a potential ligand of albumin to its binding site might be crucial for their interactions and in the design of new practical applicability as a drug-albumin.

Facile preparation of multi-stimuli-responsive degradable hydrogels for protein loading and release

Functional materials that can recognize the tumor microenvironment, characterized by acidic or reducing conditions, are needed for the designing of drug delivery carriers for cancer treatment. Hydrogels are potential protein drug carriers because they contain a large amount of water and stimuli-responsive functions can easily be introduced in them. However, it is difficult to introduce multi-stimuli-responsive functions and degradability at the same time. Here, we synthesized thermo- and pH-responsive hydrogels via a coupling reaction between poly(ethylene glycol) diglycidyl ether (PEGDE) and cystamine (CA). The prepared hydrogels showed lower critical solution temperature-type thermoresponsive behavior and pH-responsive swelling changes due to the protonation of secondary and/or tertiary amino groups arising from the crosslinking agent CA. Under reducing conditions, the hydrogels were degraded via the thiol exchange reaction in the presence of dithiothreitol or glutathione. The loading and release properties of FITC-labeled model proteins from the hydrogels were investigated. The loaded amount of the protein increased with decreasing molecular weight or hydrodynamic radius, which is based on the size of the network structure of the hydrogels. Notably, loaded proteins in the hydrogels were released only under reducing conditions, which mimic the tumor microenvironment. Thus, the prepared multi-responsive degradable hydrogels are expected to be used as functional drug delivery carriers for cancer treatment.

Preparation and characterization of pH-responsive microgel using arabinoxylan from wheat bran for BSA delivery

Wheat bran arabinoxylan (AX) discard from wheat production was utilized to form pH-responsive microgels. AX was modified by carboxymethylation, and the carboxymethylated arabinoxylans (CMAX) were characterized by FT-IR, NMR, gel permeation chromatography (GPC), and rheological analysis. The CMAX microgel was cross-linked by Fe3+ using an inverse emulsification polymerization. The morphology, particle size, pH sensitivity, and mechanism of cross-linking between COO− and Fe3+ of the CMAX microgel was investigated. The CMAX microgel was used to be an oral protein drug carrier. The CMAX microgel particles exhibited a stable spherical structure. FT-IR spectral analysis of the CMAX microgel indicated that the microgel was crosslinked by bridging Fe3+ and COO− with unidentate binding. The CMAX microgel exhibited good pH sensitivity and high stability in acid condition. Additionally, BSA was used as the embedding protein, and the controlled release effect of CMAX microgel was explored in gastrointestinal tract simulation.

The Hantzsch Reaction in Polymer Chemistry: From Synthetic Methods to Applications.

The Hantzcsh reaction is a robust four-component reaction for the efficient generation of 1,4-dihydropyridine (1,4-DHP) derivatives. Recently, this reaction has been introduced into polymer chemistry in order to develop polymers having 1,4-DHP structures in the main and/or side chains. The 1,4-DHP groups confer new properties/functions to the polymers. This mini-review summarizes the recent studies on the development of new functional polymers by using the Hantzsch reaction. Several synthetic approaches, including polycondensation, post-polymerization modification (PPM), monomer to polymer strategy, and one-pot strategy are introduced; different applications (protein conjugation, formaldehyde detection, drug carrier, and anti-bacterial adhesion) of the resulting polymers are emphasized. Meanwhile, the future development of the Hantzsch reaction in exploring new functional polymers is also discussed.

Evaluation of chitosan-ferulic acid microcapsules for sustained drug delivery: Synthesis, characterizations, and release kinetics in vitro

Herein, bovine serum albumin (BSA) as model protein drug was loaded on chitosan-ferulic acid (CS-FA) conjugants as protein drug carrier through the spray drying technique. The successful graft copolymerization of chitosan with ferulic acid was confirmed with FT-IR and XRD analyses. Additionally, the results of UV–Vis confirmed that the bovine serum albumin loading in the chitosan-ferulic acid-bovine serum albumin microcapsule was about 15.93%, respectively. The thermal properties of the conjugates were evaluated by means of thermogravimetric analysis and differential scanning calorimetry. The results revealed that the chitosan-ferulic acid conjugates exhibited low crystallinity but high thermal stability compared with that of chitosan. The pharmacokinetic study demonstrated that the chitosan-ferulic acid conjugates could provide a significantly higher swelling ratio and superior in vitro sustained release property. Furthermore, the results of drug release mechanism exhibited the corrode model was more accurately described the drug release behaviour involving a combination of drug diffusion and polymer relaxation. All these properties made chitosan-ferulic acid conjugates have potential to be developed as an oral carrier in functional foods and drug delivery systems.

Biscoumarin-pyrimidine conjugates as potent anticancer agents and binding mechanism of hit candidate with human serum albumin.

In our continuing efforts to develop therapeutically active coumarin-based compounds, a series of new C4-C4' biscoumarin-pyrimidine conjugates (1a-l) was synthesized via SN 2 reaction of substituted 4-bromomethyl coumarin with thymine. All compounds were characterized using spectroscopic techniques, that is, attenuated total reflection infrared (ATR-IR), CHN elemental analysis, and 1 H and 13 C NMR (nuclear magnetic resonance). In addition, the structure of compound 1d (1,3-bis[(7-chloro-2-oxo-2H-chromen-4-yl)methyl]-5-methylpyrimidine-2,4(1H,3H)-dione) was established through X-ray crystallography. Compounds 1a-l were screened for in vitro anticancer activity against C6 rat glioma cells. Among the screened compounds, 1,3-bis[(6-chloro-2-oxo-2H-chromen-4-yl)methyl]-5-methylpyrimidine-2,4(1H,3H)-dione (1c) was identified as the best antiproliferative candidate, exhibiting an IC50 value of 4.85 μM. All the compounds (1a-l) were found to be nontoxic toward healthy human embryonic kidney cells (HEK293), indicating their selective nature. In addition, the most active compound (1c) displayed strong binding interactions with the drug carrier protein, human serum albumin, and exhibited good solution stability at biological pH conditions. Fluorescence, UV-visible spectrophotometry and molecular modeling methodologies were employed for studying the interaction mechanism of compound 1c with protein.

Site-Specific Bioconjugation Approaches for Enhanced Delivery of Protein Therapeutics and Protein Drug Carriers

Proteins have the capacity to treat a multitude of diseases both as therapeutics and as drug carriers due to their complex functional properties, specificity toward binding partners, biocompatibility, and programmability. Despite this, native proteins often require assistance to target diseased tissue due to poor pharmacokinetic properties and membrane impermeability. Functionalizing therapeutic proteins and drug carriers through direct conjugation of delivery moieties can enhance delivery capabilities. Traditionally, this has been accomplished through bioconjugation methods that have little control over the location or orientation of the modification, leading to highly heterogeneous products with varying activity. A multitude of promising site-specific protein conjugation methods have been developed to allow more tailorable display of delivery moieties and thereby enhance protein activity, circulation properties, and targeting specificity. Here, we focus on three particularly promising site-specific bioconjugation techniques for protein delivery: unnatural amino acid incorporation, Sortase-mediated ligation, and SpyCatcher/SpyTag chemistry. In this review, we highlight the promise of site-specific bioconjugation for targeted drug delivery by summarizing impactful examples in literature, considering important design principles when constructing bioconjugates, and discussing our perspectives on future directions.

Application of poly(2-methyl-2-oxazoline) in protein separation by capillary electrophoresis

Capillary electrophoresis (CE), a commonly used liquid-phase separation technology, has many advantages such as high analysis speed, high separation efficiency, and low sample consumption. Hence, CE has gained popularity in food analysis, medical clinical diagnosis, environmental monitoring, and biological sample separation, especially in the field of protein separation and analysis. However, the fused silica capillaries that are commonly used in CE easily adsorb proteins, resulting in unstable electroosmotic flow and poor reproducibility of the separation results. In addition, due to the short optical path of the typical ultraviolet detectors employed in commercial CE, the detection sensitivity often does not meet the requirements for the direct analysis of low-abundance proteins. Therefore, developing a coating that can prevent protein adsorption and improve detection sensitivity is one of the important challenges in CE separation and analysis of proteins. Poly(2-methyl-2-oxazoline), a peptide-like hydrophilic polymer, not only has hydrophilicity, protein-repellent ability, and biocompatibility similar to the gold standard of the anti-protein adsorption polymer (polyethylene glycol), but also shows better stability than polyethylene glycol due to its peptide-like structure. Therefore, it has been increasingly used in biomass transfer, drug carrier, and impedance protein adsorption in recent years. This article aims to review the recent applications of poly(2-methyl-2-oxazoline) in CE from two standpoints. First, poly(2-methyl-2-oxazoline) was grafted onto the capillary inner wall using polydopamine as an anchor. The resulting coated capillary successfully separated a mixture of proteins (such as lysozyme, cytochrome C, ribonuclease A, and α -pancreas chymosinogen A), in addition to preventing the non-specific adsorption of other proteins during the quantitative analysis of melamine and lactoferrin in milk powder. Thus, the detection efficiency of melamine and lactoferrin in milk powder was improved. Second, poly(2-methyl-2-oxazoline) was used to produce a binary mixed brush coating with a stimulus-responsive polymer (such as polyacrylic acid). The capillary coated with the mixed brushes could adsorb high amounts of the target protein (such as bovine serum albumin and lysozyme) under certain pH and ionic strength conditions, and most of the adsorbed proteins could be desorbed by changing the pH and ionic strength. During the release, poly(2-methyl-2-oxazoline) present on the coating would prevent the adsorption of proteins. Under the dual effects of electroosmotic flow and electrophoresis, the released protein could migrate rapidly, and the instantaneous concentration of the protein reaching the detector could be greatly increased. Therefore, the target proteins could be on-line concentrated and the detection signals could be amplified, resulting in improved detection sensitivity for the protein. Future development trends in the function of poly(2-methyl-2-oxazoline) for the separation of proteins by CE are also discussed.