Polymer modification can improve protein stability at high temperatures and pH extremes. Protein drugs are widely used in the field of medicine. However, changes in the environment during storage and transportation often lead to the reduction of their activity and poor efficacy. Human growth hormone (hGH) is a commonly used drug for the treatment of short stature and other diseases, which also has the above problems. In order to improve the stability of hGH, Grigoletto et al. Attached PEG chains to the N-terminal of hGH and Glutamine (Gln) at position 141 mediated by Glutamine amidase, and synthesized PEG-N-terminal-hGH and PEG-Gln₁₄₁-hGH^[49]. The thermal denaturation temperatures of hGH, PEG-Gln141-hGH and PEG-N-hGH were 82, 83.8 and 86 ℃, respectively, measured by circular dichroism spectroscopy, indicating that the polymer could improve the thermal stability of the enzyme. In addition, they found that the conjugate was able to restore its secondary structure after heat denaturation at high temperature, which was beneficial for the protein to recover its specific function after heat denaturation. Of course, in addition to high temperature, polymer modification can also improve stability to pH. Sharma et al. Covalently coupled α-chymotrypsin to a methyl methacrylate polymer via carbodiimide to improve the stability of the enzyme to pH^[50]. They placed the conjugate and the natural enzyme in phosphate buffer of pH = 5.7 ~ 8.0, and by measuring the residual activity of the enzyme, they found that the optimum pH range of the conjugate was wider than that of the natural enzyme, and the conjugate had better stability at pH = 6.5 and above. At pH = 5.6, the residual activity of the conjugate was about 80%, while that of the free enzyme was about 40%, indicating that the modification of the polymer could improve the stability of the enzyme to pH.

Polymer modification can simultaneously improve protein stability to temperature and pH. Papain is a highly active proteolytic enzyme, which is widely used in industrial and food processes. Verduzco et al. coupled poly (vinyl-2-pyrrolidone) (PVP) synthesized by Reversible addition-fragmentation chain transfer (RAFT) with papain using the grafting-to method^[51]. They studied the catalytic activity of the conjugate with the native enzyme at different pH values and temperatures. Under acidic conditions, with the increase of pH value, the activity of the conjugate was similar to that of the native enzyme. However, at pH = 9.0, the conjugate still retained about 70% of its activity, while the native enzyme had only about 28% of its residual activity. In addition, they found that the conjugate was more active than the native enzyme at different temperatures, and at 70 ℃, the conjugate still had about 60% activity, while the native enzyme had only about 29% activity. The results showed that polymer modification could improve the stability of papain to pH and high temperature.

Proteins are susceptible to enzymatic hydrolysis and loss of activity, while polymer modification can mask the relevant reaction sites of proteins or hinder the binding of enzymes to proteins so that proteins are protected from enzymatic hydrolysis. Catalase is a commonly used antioxidant in industry, which can remove toxic substances in bleaching wastewater to achieve the effect of water purification. However, due to the high sensitivity of catalase to protease, its application is greatly limited^[52]. Riccardi et al. Synthesized a catalase-PAA conjugate by linking the carboxyl group of Poly (acrylic acid) (PAA) with the amine group on catalase, thereby improving the stability and activity of the enzyme^[53]. They used trypsin and chymotrypsin to digest and hydrolyze the conjugate and the native enzyme, and found that the conjugate retained more than 80% of its activity under the action of both enzymes, while the activity of the native enzyme was greatly reduced. Therefore, the stability of catalase modified by PAA against protease hydrolysis was significantly improved. For this phenomenon, they suggest that PAA creates a protective cross-linked network and acts as a physical barrier around the protein, thereby preventing proteases or bacteria from approaching the enzyme embedded in it (Figure 3).

Polymer coupling can also increase protein stability to chemical denaturants. Cellulase can degrade the β-1,4-glycosidic bond in the main chain of cellulose, so that cellulose can be used as an energy source. However, cellulase is easy to be inactivated in practical application due to the addition of reagents such as dimethyl sulfoxide or dimethylformamide during the hydrolysis of cellulose^[54]. Wright et al. Used the RAFT method to graft a variety of polymers onto thermophilic cellulase to improve its stability to chemical denaturants^[55]. They tested the residual activity of the enzyme after incubating the various conjugates in a medium consisting of 76 vol% dimethylformamide and 24 vol% pH = 5 buffer for 2 H. The results showed that the polymer-modified enzyme had higher residual activity than the native enzyme. In addition to this, they found that the acrylamide- and N, N-dimethylacryloyl-modified enzyme showed a 50% increase in activity over the native enzyme. This shows that polymer modification can not only improve the stability of the enzyme in extreme environments, but also improve the activity of the enzyme.

Polymer modification in most cases leads to a decrease in enzyme activity. Therefore, researchers try to improve the stability and activity of polymer modification at the same time, so as to improve the performance of conjugates in all aspects. The work of Cui et al. Successfully achieved this goal^[56]. Cysteine (Cys) was introduced into PPase by site-directed mutagenesis. Then 2-methacrylamido glucopyranose (MAG) was synthesized by the substitution reaction of glucosamine and methacryloyl chloride, and PMAG with different molecular weight and ratio was synthesized by RAFT polymerization by controlling the ratio of chain initiator and MAG. Finally, PMAG reacts with pyridine dithioester and introduces a disulfide bond at the terminal position, which is exchanged with the Cys-introduced PPase by disulfide to give PMAG-PPase. The activity of PMAG-PPase with different molecular weights was increased by nearly 30% compared with PPase. In addition, the activity of PPase modified by 8000 Da PMAG was well retained under extreme pH conditions, high salt solution and trypsin. However, the synthesis steps of the polymer are complex, the cost is high, and it is difficult to be widely used. Therefore, in order to simplify this process, Kovaliov et al. Successfully synthesized N- (iso-butoxymethyl) acrylamide (NIBMA) and N- [3- (dimethylamino) propyl] acrylamide (DMAPA) modified Candida Antarctica lipase and Candida albicans lipase with different molecular weights by controlling the illumination time through the photoinduced electron transfer RAFT method proposed by Tucker et al^[39][57]. They found that the catalytic activity of the enzyme was significantly improved after the two polymers were coupled. This may be due to the increased solubility and affinity of the hydrophobic substrate in the polymer shell, which increases the effective concentration of the substrate. At the same time, the longer the chain length of DMAPA, the greater the activity of the conjugate, because the tertiary amine part of the polymer increases the enzyme activity by pulling water molecules away from the hydrophobic region of the protein. In addition, the conjugates also showed different degrees of resistance to high temperature.

Polymer-modified proteins can realize enzyme cascade reactions, thereby enhancing the activity of enzymes. When two or more enzymes are immobilized in adjacent spaces, they can catalyze multi-step reactions together, inducing two or more consecutive processes without separation of reaction intermediates, which is called enzyme cascade reaction^[58]. The enzyme cascade can reduce the separation and purification steps of the subsequent reaction, so that the upstream reaction product can be directly transferred to the nearby enzyme catalyzing the downstream reaction as a substrate without passing through the equilibrium, thereby increasing the reaction rate^[58,59]. Polymer modification is an important method to realize enzyme cascade process, and the formed cascade enzyme has the advantages of adjustable activity, good stability and high biocompatibility^[60⇓~62]. Glucose oxidase (GOX) and Horseradish peroxidase (HRP) are often used in combination to detect blood Glucose. Chiang et al. First attached initiators to GOX and HRP respectively, and then mixed the two enzymes attached to the initiators, and carried out Atom-transfer radical-polymerization (ATRP) reaction at the same time to obtain GOX/HRP-Poly (2-hydroxypropyl methacrylate) (PHPMA) conjugate self-assembled into micelle structure, as shown in Figure 4^[62]. They regulate the activity of the complex enzyme by regulating the ratio of GOX to HRP. Although both GOX-PHPMA and HRP-PHPMA decreased compared with the native enzyme after coupling with the polymer, the activity of GOX/HRP-PHPMA complex was significantly higher than that of the native enzyme at a specific ratio of GOX to HRP, and the glucose detection speed was faster than that of the commercial glucose analysis kit. Therefore, polymer modification to achieve enzyme cascade can increase the rate of enzymatic reaction, thereby enhancing the activity of the enzyme.

In the process of using enzymes, it is sometimes necessary to adjust their activity so that they can carry out the required catalytic reaction at the right place and time to avoid the occurrence of side reactions or slow down the "aging" process of enzymes. The polymer can regulate the activity of the enzyme in two ways: first, the polymer can be reversibly linked to the enzyme, and the structure of the enzyme is affected by the coupling and shedding of the polymer, thereby regulating the activity of the enzyme^[63]; Secondly, stimuli-responsive polymers can be used to regulate enzyme activity by responding to external environmental conditions^[64,65]. Wang et al. Introduced Cys to PPase by site-directed mutagenesis, and coupled the thiol group with poly (2-hydroxyethyl methacrylate) (PHEMA) functionalized with pyridine disulfide to form PPase-PHEMA^[63]. PPase activity was almost completely lost after PHEMA coupling, but was restored to a level comparable to that before coupling after reduction of the disulfide bond between PHEMA and PPase with 8 mmol/L dithiothreitol. Li et al. Used the photoelectron transfer RAFT method to couple the thermosensitive polymer poly (N-isopropylacrylamide) (PNIPAAM) to the Cys far away from the active center of PPase by site-directed mutagenesis to obtain PPase-PNIPAAM^[64]. The catalytic activity of the conjugate has a clear difference between the two sides of the ambient temperature at the phase transition temperature of PNIPAAM. It is speculated that the principle of thermal response of enzyme activity may be that PNIPAAM is in a stretched state below the phase transition temperature, shielding the active center of the enzyme, while the polymer chain is compressed and folded above the phase transition temperature, exposing the active center.

Polymer-coupled proteins can self-assemble into specific spatial structures to load drugs. Albumin is easy to accumulate in tumor tissue and be taken up by tumor cells, so it is often used as a targeting carrier of anti-tumor drugs^[106,107]. As shown in fig. 6, Liu et al. Linked hydrophobic poly (ε-caprolactone) (PCL) with hydrophilic BSA through maleimide-thiol reaction, and prepared degradable BSA-PCL vesicles through amphiphilic self-assembly of the material^[99]. The hydrophobic core of the vesicle can be loaded with the anti-tumor drug doxorubicin, and the surface can be conjugated with antibodies to further improve the targeting. In cell and animal experiments, the in vitro anti-tumor properties of the system are significantly enhanced relative to doxorubicin. In addition, the system has high selectivity and biodegradability, and reduces the in vivo toxicity of doxorubicin^[108]. On the basis of the above, many studies have further analyzed the performance of BSA-polymer conjugates^[107]. The coupling of the polymer can enhance the absorption of BSA by tumor cells. Piperazine derivatives are chemical permeation enhancers that alter the integrity of tight junctions between epithelial cells and increase permeation through the paracellular pathway. Cummings et al. Used ATRP method to grow phenylpiperazine acrylamide monomer on the surface of BSA, and prepared BSA-polymer conjugates containing phenylpiperazine^[109]. At non-cytotoxic doses, the conjugate increased the permeability of Caco-2 monolayer by about 30-fold compared with BSA, which mimics the absorption of small intestine, and has potential prospects for oral drug delivery.

Stimulus-responsive polymer-coupled protein-loaded drugs can control the time and site of drug release according to the change of environment, and enhance the selectivity of drugs^[110]. Vanparijs et al. Grown [ (2,2-dimethyl-1,3-dioxolane) methyl] acrylamide (DMDOMA) on lysine residues of BSA by RAFT method to form acid-soluble BSA-PD MDOMA conjugate^[111]. They encapsulated immunostimulatory molecule CL075 in the hydrophobic inner core of BSA-PDMDOMA, so that the nanoparticles were dissolved and activated in the acidic environment of the endosome after uptake by dendritic cells, and enhanced the immune response to tumors and intracellular pathogens. Inspired by the above work, Viana et al. Used ATRP to simultaneously grow temperature-sensitive polymers N-vinylcaprolactam and N, N-dimethylaminoethyl methacrylate on lysine residues of BSA to prepare BSA-block polymer conjugates^[106]. By controlling the ratio of the two thermosensitive polymers, they can adjust the phase transition temperature of the conjugate to 40 ℃, which has a potential targeting effect on tumor tissues above physiological temperature. However, they have no relevant animal experiments to prove that the material can achieve tumor tissue aggregation.

Some proteins themselves can be used as carriers, and after self-assembly, they form hollow spaces inside, which can load drugs, such as viral capsid, ferritin, chaperone and so on. This kind of protein is named protein cage, and some researchers have made a good summary of the related research^[112,113]. Due to the defects of the protein application process mentioned above, we can imagine that coupling polymers on the surface or inside of the protein cage can improve its corresponding properties, such as surface modification of PEG to reduce its immunogenicity. Many studies have modified protein cages from different perspectives to increase the possibility of their clinical application as drug carriers. Nussbaumer and his group coupled the dendrimer polyacrylamide to the pore size of the chaperone Thermosome (THS) through a bisarylamine linker, and used the charge effect to load the positively charged polymer with negatively charged siRNA^[114]. The vector system can protect the siRNA from being degraded and can play a better gene silencing effect on U87 tumor cells. However, the use of THS in drug delivery vehicles is relatively rare, and its safety and efficacy in clinical studies remain to be investigated. Viral capsid is a commonly used vector for gene therapy, in which AD is the focus of research. However, the application of AD vector in gene therapy is limited by some problems, such as easy uptake and inactivation by immune cells during intravenous injection, toxicity caused by interaction with platelets and erythrocytes, hepatotoxicity caused by accumulation in the liver, and low expression of AD receptor on the surface of tumor cells, which leads to low gene transduction efficiency^[115,116]. In order to solve these problems, Kim et al. First attached Arginine-grafted bioreducible polymer (ABP) to the surface of AD through charge interaction, which improved the transduction efficiency of AD, reduced the immunogenicity and erythrotoxicity. However, the particle size of the vector system was as high as 500 nm, and it was prone to aggregation, so it could not be administered by intravenous injection^[117]. They therefore further developed an AD-ABP system chemically coupled through a disulfide bond. The size of the conjugate is less than 150 nm, which is suitable for intravenous administration, and the surface is positively charged, which prevents the aggregation of the conjugate and solves the above problems^[115]. Thambi et al. And Sun et al. Also reviewed the corresponding studies on the coupling of AD with different types of polymers^[116][118]. Apoferritin is also a protein shell of natural origin, which dissociates 24 subunits at pH = 2 and restores the shell structure at neutral pH, and is a potential pH-responsive drug delivery carrier. Luo et al. Modified the polysaccharide, Hyaluronic acid (HA), on the surface of apoferritin by EDC-NHS method, and used the targeting effect of HA on the highly expressed CD₄₄ receptor on the surface of tumor cells to achieve selective drug delivery^[119]. They encapsulated daunomycin and negatively charged macromolecule polyaspartic acid in the protein cage, and the drug was stably encapsulated in the protein cage through the attraction between polyaspartic acid and positively charged daunomycin. The drug delivery system constructed by them has a fast release rate at low pH, a good response behavior to the acidic tumor microenvironment, and a dual-targeted killing effect on lung cancer cell models due to the binding of HA and CD₄₄ receptors.

With the development of biopharmaceuticals in recent years, especially the increasing proportion of protein drugs in the drug market share year by year, researchers hope to have an efficient protein labeling technology to better understand the dynamic distribution of protein drugs in the body. Among all kinds of labeling methods, fluorescent polymer-based labeling technology has attracted the attention of researchers in recent years because of its excellent performance. Its advantages include smaller fluorescent polymer chain size compared to inorganic or organic nanoparticles, brighter brightness compared to organic dyes, and significantly enhanced resistance to photobleaching^[120,121]. In addition, due to the biocompatibility, water solubility and the ability to modify multifunctional groups on the surface of polymers, researchers can use bioorthogonal reactions to achieve protein-specific fluorescent labeling on the surface of living cells, thus realizing applications in cell imaging and other fields^[122]. Duret et al. Synthesized a block copolymer of 4-acryloylmorpholine and N-acryloxysuccinimide (NAS) by RAFT method^[120]. They successfully labeled streptavidin by multi-site binding of an amino-modified fluorescent dye on the side chain of the N-hydroxysuccinimidyl activated ester of NAS in the polymer backbone, followed by covalent binding of the activated ester at the polymer terminus to primary amines on multiple lysine residues on the protein. Finally, the material was combined with biotin-labeled anti-CD₄₀ antibody by bioaffinity reaction. After co-incubation with dendritic cells, flow cytometry showed that dendritic cells had strong fluorescence brightness. Although the material has good performance, the synthesis steps of the material are complicated and the cost is high, which limits its popularization and application. Some researchers have also directly linked fluorescent polymers to antibody molecules to obtain antibody-polymer-dye conjugates with enhanced fluorescence signals^[123⇓~125]. Herceptin is an anti-tumor monoclonal antibody drug, which can treat breast cancer, ovarian cancer, gastric cancer and other diseases. Zhang et al. Combined ATRP initiator on Herceptin, and simultaneously grew POEGMA and rhodamine-modified POEGMA on this site to obtain site-specific antibody-polymer-dye conjugate^[125]. They obtained three types of Herceptin-POEGMA-rhodamine conjugates by adjusting the ratio of dye-modified monomer and antibody during ATRP. Compared with the rhodamine dye directly coupled with Herceptin disulfide bond, the signal intensity of antigen detection was significantly enhanced, and the specificity of Herceptin antibody binding to antigen was not changed. This study combines the targeting and therapeutic properties of antibodies and the imaging properties of fluorescent polymers, which helps to achieve the integration of diagnosis and treatment, and is one of the key development directions of modern medicine.

The specific spatial structure formed by the self-assembled protein-polymer can also load dye molecules, and the dye molecules can be released by using the structural change of the responsive polymer in a specific environment to realize the corresponding imaging process. poly (2- (diisopropylamino) ethyl methacrylate) (PDPA) is a pH-responsive polymer with an isoelectric point of about 6.5. When the pH is lower than the isoelectric point, the tertiary amino group of PDPA is protonated and hydrated to form a hydrophilic polymer. Li et al. Linked the maleimide-functionalized ATRP initiator to the thiol group on the Cys of Human serum albumin (HSA), and the DPA monomer grew on this site, and the conjugate self-assembled to form HSA-PDPA micelles of various shapes^[30]. They obtained HSA-PDPA spherical micelles by controlling the critical micelle concentration, and incorporated Indocyanine green (ICG) into the core of the micelles to form pH-responsive fluorescent nanoprobes. At blood pH, quenching of fluorescent molecules occurs due to aggregation within the micelle. At the pH of the tumor site, the nanoprobe dissociates into positively charged monomers, resulting in enhanced fluorescence and uptake of the monomers by tumor cells, as shown in fig. 7. They proved that the nanoprobe had excellent tumor fluorescence imaging performance after intravenous injection through corresponding mouse experiments, which provided the possibility of tumor-specific imaging.