Synthesis, Properties and Applications of Enzyme-Responsive Linear-Dendritic Block Copolymers

Lijun Bao; Junwu Wei; Yangyang Qian; Yujia Wang; Wenjie Song; Yunmei Bi

doi:10.7536/PC220414

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (8) : 1723-1733 DOI: 10.7536/PC220414

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Synthesis, Properties and Applications of Enzyme-Responsive Linear-Dendritic Block Copolymers

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Abstract

Linear-dendritic block copolymers (LDBCs) are composed of a linear polymer and dendrimers (dendrons). They not only combine the characteristics of the two types of polymers, but also have unique structures and properties. Furthermore, stimuli-responsive LDBCs can be generated through the selection and structural modification of their linear chains and dendrons. Stimuli-responsive LDBCs have attracted much attention in recent years because of combining the ability to respond to a specific stimulus and different polymeric architectures for a broad range of applications, including drug delivery, gene therapy and materials science. Among them, enzyme responsive LDBCs are able to specifically respond to endogenous enzymes. Compared with the polymers that are able to only respond to exogenous stimuli such as temperature, light, etc, enzyme responsive LDBCs have high selectivity and better biocompatibility. Therefore, they are more suitable for application in in vivo as biomedical materials. Amphiphilic enzyme responsive LDBCs are capable of self-assembling in aqueous solution into nano-aggregates and disassembling to release payloads upon enzymatic stimuli. Additionally, the self-assembly and enzyme responsive disassembly properties of amphiphilic enzyme responsive LDBCs can be adjusted by changing the enzyme-responsive groups, linear chains and dendrons of LDBCs. Due to their unique enzyme response properties and excellent biocompatibility, enzyme responsive LDBCs have broad application prospects in drug delivery, biomedical imaging, etc. Herein, this review summarizes the synthetic approaches of enzyme responsive LDBCs, the effect of the structures such as the length and structure of linear chain, and hydrophobicity of dendrons of the LDBCs on their self-assembly properties and enzyme-responsive properties. The applications of these copolymers are also introduced. Finally, the research prospect of enzyme responsive LDBCs are proposed.

Contents

1 Introduction

2 Synthetic approaches for enzyme responsive linear-dendritic block copolymers(LDBCs)

2.1 The divergent approach

2.2 The convergent approach

2.3 The coupling approach

3 Effect of LDBCs structures and enzyme concentration on the properties of enzyme responsive LDBCs

3.1 the effect of different enzyme responsive groups in The LDBCs

3.2 the effect of linear chain length of The LDBCs

3.3 the effect of linear chain structure and type of The LDBCs

3.4 the effect of hydrophobicity of dendrons in The LDBCs

3.5 The effect of enzyme concentration

4 Application of enzyme responsive LDBCs

4.1 As a drug carrier

4.2 Application in the field of fluorescent imaging probes

5 Conclusion and outlook

Graphical abstract

Keywords

enzyme responsiveness / linear-dendritic block copolymers (LDBCs) / synthetic approaches / structures and properties / drug carriers

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Lijun Bao, Junwu Wei, Yangyang Qian, Yujia Wang, Wenjie Song, Yunmei Bi. Synthesis, Properties and Applications of Enzyme-Responsive Linear-Dendritic Block Copolymers. Progress in Chemistry, 2022, 34(8): 1723-1733 DOI:10.7536/PC220414

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1 Introduction

Linear-dendritic block copolymers (LDBCs) are composed of a flexible Linear polymer and a semi-rigid spherical dendrimer (dendron) with a highly branched structure and a high density of surface functional groups.It not only combines the characteristics of the two kinds of polymers to make them have unique structure and performance, but also can produce LDBCs with environmental stimulus responsiveness by selecting and modifying the linear chain and dendronization motif^[1⇓⇓~4]. LDBCs with temperature, pH, light, enzyme and other stimuli have broad application prospects in the field of biomedicine, in which enzyme as a stimulus has more advantages than light, temperature and other stimuli^[5,6]^[7,8]^[9,10]^[11,12]^[13,14]. First, multiple enzymes exist in the human body, and stimulation does not need to be added externally. Moreover, enzymes are highly selective and efficient, and the expression disorder or activity imbalance of enzymes usually occurs at the lesion site, such as the overexpression of Cathepsin B, Matrix metalloproteinase (MMPs) and phosphatase in different types of malignant tumors has been confirmed^[15,16]^[17,18]^[19,20]. Overexpression of disease-related enzymes can be used to trigger the specific release of drugs at the lesion site and reduce adverse reactions to other tissues and organs. Enzyme-responsive amphiphilic LDBCs can form many enzyme-responsive self-assembled supramolecular structures by changing the structure and chain length of flexible linear polymers and the number of generations and surface functional groups of rigid dendrimers. The dendritic rigid structure enhances the stability of the self-assembly aggregate of LDBCs in aqueous solution, and the dendritic rigid structure constructs a highly ordered spatial structure in the center of the aggregate, which can not only load a large number of guest molecules in the internal gaps of the dendrite,But also can accommodate a large number of guest molecules in the gap between the single branches, thereby improving the encapsulation efficiency and the drug loading of the drug, and having potential application value in the fields of biomedicine, new material design and the like^[21]. In recent years, some enzyme-responsive LDBCs have been designed and synthesized, and gratifying progress has been made. In this paper, the research progress of enzyme-responsive LDBCs is introduced based on the work of our group. The synthesis methods, factors affecting the performance and applications are discussed, and the opportunities and challenges faced by enzyme-sensitive LDBCs are prospected.

2 Synthesis of enzyme-responsive LDBCs.

At present, the reported synthesis methods of enzyme-responsive LDBCs are still the conventional divergent method, convergent method and coupling method, but the enzyme-cleavable functional groups (such as peptide bond, phenylacetamide group, ester group, etc.) are introduced into the linear chain, dendronization motif or the periphery of dendronization motif of LDBCs (Figure 1).

2.1 Divergence method

The divergent method is also called Chain-first method, which first synthesizes linear polymers with active end groups, and then uses the active end groups to iteratively synthesize dendritic motifs to obtain LDBCs. This method is most suitable for the mass synthesis of LDBCs and the preparation of high-generation LDBCs, and is also the most commonly used method for the synthesis of enzyme-responsive LDBCs^[2]. For example, Harnoy et al. Used three kinds of polyethylene glycol (PEG) with different molecular weights and single terminal amino group as raw materials, coupled with 3,5-dipropargyl p-nitrophenyl benzoate to prepare PEG with two alkynyl groups at the end, and then carried out mercapto-alkyne click reaction with N-tert-butoxycarbonyl cysteamine.The LDBCs with four functional groups at the end of the dendron were obtained, and the four functional groups were coupled with 4-nitrophenyl phenylacetate after removing tert-butoxycarbonyl (Boc) to obtain the amphiphilic LDBCs with hydrophilic PEG as the linear chain and the second generation cysteamine with four lipophilic phenylacetamide terminal groups as the dendron^[22]. These LDBCs have four phenylacetamide terminal groups, which can be digested by penicillin G amidase (PGA) to show enzyme responsiveness. The synthesis of these enzyme-responsive LDBCs was made simple and efficient by using PEG as the hydrophilic linear chain and applying the thiol-alkyne click reaction.

By introducing living/controlled radical polymerization into the synthesis of linear chains of LDBCs, enzyme-responsive LDBCs with non-PEG linear chains can be obtained with more flexible design of linear chain length. For example, Wei et al. Synthesized poly (N-vinylpyrrolidone) (PNVP) with a single terminal amino group by combining xanthate-mediated reversible addition-fragmentation chain transfer (MADIX/RAFT) radical polymerization and mercapto-olefin Michael addition reaction.Three generations of enzyme-responsive LDBCs with PNVP as the hydrophilic linear chain and 9-fluorenylmethoxycarbonyl (Fmoc) -terminated phenylalanyl-lysine (Phe-Lys) dipeptide as the hydrophobic dendronization motif were synthesized by stepwise condensation-directed peptide synthesis (Fig. 2)^[11]. The Phe-Lys dipeptide can be digested by cathepsin B and trypsin^[24,25]. The synthesis of enzyme-responsive LDBCs by divergent methods is relatively time-consuming, requiring a large number of functional group transformations of dendronized motif end groups in the preparation of high-generation products, requiring the reaction to have a very high yield, otherwise it will cause structural defects, and it is not easy to purify and characterize^[2,4].

2.2 Convergence method

The convergence method is also called Dendron-first method, in which a dendrimer with an initiating center group is synthesized first and used as a macroinitiator to initiate the polymerization to obtain LDBCs. Compared with the divergent method, the convergent method is always limited to several active centers in each step of the reaction, which avoids the reaction of excessive reagents with terminal groups in the divergent method, and reduces the dendritic structural defects caused by incomplete reactions, so that the dendritic structure of LDBCs is more complete and orderly, and the purification and characterization are also easy. However, it also has some disadvantages, for example, with the increase of the generation of dendrimers, the steric hindrance gradually increases, and the activity of the functional group initiating polymerization decreases, resulting in a decrease in the conversion rate of polymer monomers^[26]. At present, there is only one report on the synthesis of enzyme-responsive LDBCs by convergent method. Qian et al. First synthesized cysteamine or 2,2-dimethylolpropionic acid (Bis-MPA) aliphatic polyester dendronized macroinitiator with primary amino group as the central group, and then used the terminal amino group to initiate the ring-opening polymerization of benzyl glutamate-N-carboxylic anhydride (NAC).Finally, the benzyl ester group on the linear chain of polybenzyl glutamate was aminolyzed with ethanolamine to obtain LDBCs with poly [N- (2-hydroxyethyl-L-glutamine)] (PHEG) as the hydrophilic linear chain and cysteamine or Bis-MPA aliphatic polyester as the hydrophobic dendronized motif (Fig. 3)^[23]. PHEG is capable of enzymatic cleavage by papain, pronase, and cathepsin B^[27].

2.3 Coupling method

The coupling method is to synthesize dendrimers and linear macromolecules with active groups at the end respectively, and then couple them to obtain LDBCs. The coupling method has many advantages. The pre-designed dendrimer and linear macromolecule are synthesized at the same time, and the coupling is carried out at last, which simplifies the synthesis steps and shortens the synthesis cycle.These pre-designed macromolecules can be strictly purified and characterized before coupling, which effectively solves the problems of difficult purification and characterization in the synthesis of high-generation LDBCs by divergent method^[2,4,28]. However, the synthesis of LDBCs by coupling method requires an effective coupling reaction. In recent years, the application of Click reaction, which has the characteristics of quantitative, rapid, efficient and high selectivity, mild reaction conditions, single product and easy separation, has greatly improved the effectiveness of coupling.These click reactions include alkynyl/azide 1,3-dipolar cycloaddition (CuAAC), thiol-ene/yne click, Diels-Alder cycloaddition, etc^[9,29,30]^[31,32]^[33,34]. For example, Slor et al. Attached lipophilic dendronized motifs with enzyme-responsive groups on the periphery to three different hydrophilic polymers PEG, poly (2-ethyl-2-oxazoline) (PEtOx), or polyacrylic acid (PAA) via CuAAC click reaction^[35]. Although the hydrophilic block composition and dendrimer structure are different, the enzyme-responsive LDBCs prepared by CuAAC click reaction have a clear structure, and all the polymers have good reaction and high conversion rate during synthesis. Segal et al. Used CuAAC click reaction to click one PEG (10 kDa) linear chain or two PEG (5 kDa) linear chains onto hydrophobic dendronized motifs with four esterase responses to obtain enzyme-responsive LDBCs with linear or V-shaped PEG linear chains^[36]. The molecular weights of the two copolymers are almost the same, indicating that the azide end group of the linear chain and the terminal alkyne group of the dendronized motif are effectively click-coupled, and the structure of the synthesized enzyme-responsive LDBCs is clear.

3 Factors affecting the performance of enzyme-responsive LDBCs

Firstly, the response of LDBCs to enzymes is specific because of the difference of enzyme response groups. Secondly, most of the reported enzyme-responsive LDBCs are amphiphilic and can self-assemble into supramolecular aggregates in selective solvents, which can be deassembled by enzymes. The structure of LDBCs (such as the length and structure of the linear chain and the hydrophobicity of the dendron) has an effect on its self-assembly performance and enzyme response performance. In addition, the concentration of enzyme also has an effect on the enzyme-responsive disassembly performance of amphiphilic LDBCs aggregates.

3.1 Effect of different enzyme-responsive groups

Enzyme-responsive polymers usually respond to enzyme stimulation through groups in the molecule that specifically respond to a certain enzyme. At present, enzyme-responsive phenylacetamido, Phe-Lys dipeptide, ester and other groups have been introduced into LDBCs (Table 1).These groups respond specifically to different enzymes, for example, LDBCs micelles with phenylacetamide terminal groups are only deassembled by PGA, but not by esterase^[22].

3.2 Effect of linear chain length

The length of the hydrophilic linear chain in the enzyme-responsive amphiphilic LDBCs has a great influence on the stability and enzyme-responsive performance of the self-assembled aggregates. Harnoy et al. Studied the effect of the length of PEG linear chains on the enzyme-responsive disassembly rate of amphiphilic LDBCs micelles, and unexpectedly found that the disassembly rate of LDBCs micelles with longer PEG chains was faster^[22]. To explain this result, they proposed a mechanism for the disassembly of LDBCs micelles with PEG as the linear chain and the second generation cysteamine with four phenylacetamide terminal groups as the dendronization motif under the action of PGA.That is, the enzyme cannot penetrate through the PEG shell of the micelle into the micelle core by osmosis to hydrolyze the phenylacetamide group at the end of the dendronized motif of LDBCs, and the enzymatic hydrolysis occurs on the unassembled monomolecule in the "monomolecule-aggregate" equilibrium of amphiphilic LDBCs (Fig. 5). The critical micelle concentration (CMC) of LDBCs with longer PEG chains is larger, more LDBCs exist in the form of single molecules, and the enzymatic hydrolysis reaction is more likely to occur. Song et al. Also observed in the enzyme-responsive LDBCs synthesized with poly (N-vinylcaprolactam) (PNVCL) as the linear chain and Phe-Lys dipeptide as the dendronization motif that the micelles formed by the self-assembly of LDBCs with longer PNVCL linear chains were more easily deassembled by cathepsin B^[40].

3.3 Effect of structure and type of linear chain

The hydrophilic linear chain structure in enzyme-responsive amphiphilic LDBCs molecules has a significant effect on the stability and enzyme-responsive performance of their self-assembled aggregates. Segal et al. Studied the effects of hydrophilic PEG blocks with similar molecular weight but different molecular structures (linear and V-shaped) on the self-assembly and disassembly behavior of enzyme-responsive LDBCs, and found that although the two enzyme-responsive LDBCs had very similar CMC values,However, the particle size of LDBC micelles with V-shaped PEG blocks is smaller than that of LDBC micelles with linear PEG blocks, and the change of hydrophilic PEG blocks from linear to V-shaped leads to a great decrease in the aggregation number of amphiphilic molecules^[36]. In the presence of pig liver esterase, the disassembly of V-shaped copolymer micelles is faster than that of linear copolymer micelles, but the enzymatic hydrolysis is slower. This is because the enzymatic hydrolysis occurs on the free single molecule in the "monomolecule-aggregate" equilibrium of amphiphilic LDBCs. After the ester group in the single molecule is hydrolyzed, the amphiphilicity of LDBCs is destroyed, resulting in partial decomposition and assembly of micelles. Compared with linear PEG, V-shaped PEG has greater steric hindrance, and the enzyme is not easy to approach the ester group on the single molecule of LDBCs, so the rate of complete enzymatic hydrolysis is slowed down (Fig. 6)^[22,41].

PEG is a nonionic water-soluble polymer with low toxicity to human body and no antigenicity and immunogenicity, which makes it the most commonly used linear chain to constitute enzyme-responsive LDBCs with drug carrier as the main application purpose.PEG-modified nanocarriers tend to be enriched in liver and spleen tissues due to the non-biodegradability of PEG, thermal instability, unpredictable changes in pharmacokinetic behavior, and hypersensitivity after intravenous and oral administration, and PEG antibodies are produced in vivo by multiple intravenous injections of PEG-modified nanocarriers^[42]^[43]; Moreover, PEG has only one modifiable reactive functional group at the end, which limits its application as a hydrophilic segment of polymer carrier for targeted drug delivery. Therefore, alternative polymers that can retain the advantages of PEG and overcome its disadvantages must be explored^[44,45]. PNVP has the same excellent water solubility and biocompatibility as PEG, and has been widely used in biology, medicine, food, materials and other fields. In the last decade, great progress has been made in the living/controlled radical polymerization of non-conjugated N-vinylpyrrolidone (NVP). Through the molecular design of chain transfer agents, it is possible not only to control the molecular weight of PNVP by reversible addition-fragmentation chain transfer (RAFT) polymerization of NVP, but also to introduce modifiable reactive groups at the chain end of PNVP^[46,47]. In addition, the polymeric micelles with PNVP shell do not have the aggregation problem during lyophilization. Therefore, as an amphiphilic block copolymer drug carrier constructed by hydrophilic segments, PNVP is one of the promising alternatives to PEGs^[48]. For example, the enzyme-responsive LDBCs designed and synthesized by Wei et al., with PNVP as the hydrophilic linear chain and Phe-Lys dipeptide as the hydrophobic dendronized motif, have good biocompatibility.The spherical micelles formed by self-assembly in aqueous solution can be deassembled to release the poorly soluble anticancer drug doxorubicin (DOX) under the action of trypsin, which is expected to become a targeted release carrier of poorly soluble drugs^[11].

Although PNVP and PEG have good biocompatibility, they are not biodegradable after all. To this end, Qian et al. Designed and synthesized enzyme-responsive LDBCs with biodegradable and hydrophilic poly [N- (2-hydroxyethyl-L-glutamine)] (PHEG) as the linear chain and cysteamine or bis-MPA aliphatic polyester as the hydrophobic dendronized motif^[23]. These block copolymers have good biocompatibility, can self-assemble into spherical nanomicelles or vesicles in aqueous solution, and can release the entrapped hydrophobic small molecules under the action of enzymes, which has potential application value in drug targeting release.

Multiple stimulus-responsive LDBCs can also be obtained by changing the hydrophilic linear chain. For example, Qian et al. Introduced temperature-responsive PNVCL into the linear chain of LDBCs, and designed and synthesized amphiphilic LDBCs with PNVCL as the hydrophilic linear chain and bis-MPA-derived aliphatic polyester as the hydrophobic dendronized motif. After the addition of papain, the permeability of these LDBCs in aqueous solution decreased, while the lower critical solution temperature (LCST) increased^[12]. Because papain hydrolyzes the amide bond and ester bond in LDBCs, the hydrophobic dendronized motif is separated from the hydrophilic linear chain PNVCL and precipitated, which reduces the transmittance of the solution^[49,50]. The hydrophobic arborization motif in LDBCs is partially or completely hydrolyzed, which increases the hydrophilicity of the molecule and increases the LCST. Therefore, LDBCs have both enzyme and temperature responsiveness, and the self-assembled aggregates of LDBCs can release the entrapped lipophilic small molecules after the addition of papain.

3.4 Effect of hydrophobicity of arborization motif

Small changes in the hydrophobicity of the peripheral groups of the dendronized motif also have a great impact on the self-assembly behavior and enzyme-responsive disassembly performance of amphiphilic LDBCs. For example, Segal et al. Found that the hydrophobicity of the peripheral group of the dendronized motif had no significant effect on the CMC of amphiphilic LDBCs with PEG as the linear chain and the second generation cysteamine as the dendronized motif, but had a greater impact on the size and aggregation number of self-assembled micelles, and LDBCs with smaller hydrophobic peripheral groups were hydrolyzed faster^[38]. They also found that the polydispersity of materials may be a key factor in controlling the drug release rate of drug delivery systems and the degradation rate of biodegradable materials. Harnoy et al. Studied the effect of the number of peripheral hydrophobic groups of dendronized motifs on the self-assembly behavior and enzyme-responsive disassembly performance of amphiphilic LDBCs, and the results showed that the more peripheral hydrophobic groups of dendronized motifs, the smaller the CMC of amphiphilic LDBCs, the more stable the micelles formed by self-assembly, and the slower the degradation rate by enzymes^[37].

The number of lipophilic dendronized motifs also has a great influence on the self-assembly behavior and enzyme-responsive disassembly performance of amphiphilic LDBCs. For example, Song et al studied the performance of amphiphilic LDBCs with PNVCL as the linear chain and Phe-Lys dipeptide as the dendronized motif.When the length of the linear chain of PNVCL was the same, the CMC and micelle size of LDBCs decreased with the increase of the number of dendron generations, and the drug release rate of LDBCs loaded micelles became slower with the increase of the number of dendron generations^[26].

Slor et al. compared the micelle size, critical micelle concentration, drug loading, stability and cell internalization of amphiphilic LDBCs based on peripheral dendrimers with 6 or 9 carbon atoms and different hydrophilic polymers PEG, poly (2-ethyl-2-oxazoline) (PEtOx) and polyacrylic acid (PAA).The LDBCs micelle diameter was found to be almost completely dependent on the hydrophobicity of the dendritic hydrophobic block, whereas the enzymatic degradation rate was strongly dependent on the composition of both blocks^[35].

3.5 Effect of enzyme concentration

The concentration of enzyme has an important effect on the disassembly of LDBCs self-assembly aggregates. The higher the concentration of enzyme, the faster the disassembly rate of LDBCs self-assembly aggregates and the more complete the enzymatic hydrolysis. For example, Harnoy et al. studied the effect of the concentration of PGA on the enzyme-responsive disassembly of LDBCs micelles with four phenylacetamide terminal groups, and the results showed that when the concentration of PGA was 0.14 μmol/L, the enzyme-responsive LDBCs micelles could not be completely hydrolyzed^[22]; However, when PGA was added at a high concentration (1. 4 μmol/L), LDBCs micelles could be completely disassembled in the same enzymolysis time. Qian et al. Also showed that with the increase of papain concentration, the disassembly rate of self-assembled aggregates (micelles or vesicles) of LDBCs with PHEG as hydrophilic linear chain and cysteamine or Bis-MPA aliphatic polyester as hydrophobic dendronized motif was accelerated^[23].

4 Application

4.1 As drug carrier

Cancer is one of the diseases that seriously endanger human health. Tumor cells often overexpress some biological enzymes based on the need of proliferation and diffusion. Therefore, enzyme-responsive LDBCs, as an anticancer drug carrier, have important application value in the targeted delivery system of anticancer drugs. Cai et al. Prepared a polymeric prodrug based on LDBCs loaded with doxorubicin (DOX)^[39]. The hydrophobic dye photosensitizer chlorin E6 (Ce6) was encapsulated in prodrug nanoparticles (NPs) to obtain a drug delivery system LD-DOX/Ce6 based on enzyme-responsive LDBCs, which is enzyme- and pH-responsive and can be used for combination therapy of tumors. Due to the existence of Gly-Phe-Leu-Gly tetrapeptide and hydrazone bond in the prodrug structure, the structure of the nanoparticle is disintegrated in the tumor microenvironment with high expression of cathepsin B and under weak acidic conditions, DOX and Ce6 are released at the same time, and Ce6 is further irradiated by laser to generate reactive oxygen species (ROS).So as to significantly improve the synergistic therapeutic effect, reduce systemic toxicity, improve the synergistic therapeutic effect by regulating apoptosis, DNA repair and cell cycle arrest, and inhibit tumor metastasis by inhibiting epithelial mesenchymal transition pathway, transforming growth factor-beta pathway, angiogenesis and hypoxia pathway. The results of this study indicate that enzyme-responsive LDBCs prodrugs can be used as a new class of multifunctional drug carriers, which has broad application prospects in the treatment of cancer, and enzyme-responsive LDBCs combined with a variety of therapeutic agents can be used in the multimodal treatment of cancer, providing a new idea for the treatment of cancer.

The loading of hydrophobic drugs by LDBCs can be roughly divided into two ways: drugs are embedded in the core of micelles formed by the self-assembly of amphiphilic LDBCs through non-covalent interactions such as electrostatic, hydrophobic and hydrogen bonds; Hydrophobic drugs are linked into LDBCs molecules by covalent bonds^[51]. Rosenbaum et al. Prepared two kinds of LDBCs micelles based on PEG hydrophilic linear chain and esterase-responsive lipophilic dendronization motif. In the first micelle, the hydrophobic coumarin-derived dye was non-covalently embedded in the hydrophobic core of LDBCs micelle^[52]; In the second micelle, the hydrophobic dye molecule is covalently linked to the end group of the hydrophobic tree via an enzyme-cleavable bond. The effects of two different drug loading modes on drug loading, stability and release rate were studied. The results showed that the loaded dye molecules were almost completely released from the two micelles under the action of pig liver esterase. The micelle loaded with drug in a non-covalent manner releases drug faster, while the micelle loaded with drug in a covalent manner is more stable and has a larger drug loading, but releases drug slower. The results of this study show that the payload capacity and release rate of micelles can be adjusted by adjusting the loading mode of hydrophobic drugs (Fig. 7).

4.2 Application in the field of fluorescence imaging probes

Fluorescence technology has been widely used in various fields such as optical materials, analytical chemistry, protein conformation and biological analysis, due to its advantages of high sensitivity, good selectivity, low cost and simple operation, as well as excellent spatial and temporal resolution^{[53⇓⇓~56]}. However, there are limited reports on the deassembly and fluorescence modification of synthetic amphiphilic block copolymer self-assembled micelles by enzymes^[41]. Buzhor et al. Designed and synthesized amphiphilic LDBCs with a single fluorescent dye at the junction of a hydrophilic PEG linear chain and a lipophilic dendronized motif with a benzoate terminal group, which can self-assemble into enzyme-responsive micelles with tunable fluorescence response^[57]. In micelles, the fluorescence of the dye is quenched or shifted due to intermolecular interactions. When pig liver esterase was added, the hydrophobic end group benzoate of amphiphilic LDBCs was hydrolyzed into hydrophilic molecules, the micelle was disassembled, the interaction between dye molecules disappeared, the fluorescence of the dye itself recovered, and the fluorescence intensity increased and blue-shifted. In this study, supramolecular assembly and disassembly are converted into spectral response, and the enzyme activation and spectral response capabilities of enzyme-responsive LDBCs can be adjusted by selecting enzyme substrates and fluorescent labeling dyes, which is expected to be used to manufacture advanced enzyme-activated fluorescent imaging probes.

5 Conclusion and outlook

LDBCs combine the advantages of linear polymers and dendrimers, and have unique structures and properties. Enzymes, as endogenous substances in organisms, have significant differences in distribution and expression between normal cells and diseased sites. Enzymes are highly specific, so highly specific substrates (or groups containing enzyme cleavage sites) can be selected and introduced into LDBCs to endow LDBCs with enzyme stimulation response performance. In addition, these copolymers generally have good biocompatibility and can be used in biomedical fields such as drug delivery, disease diagnosis and treatment, and tissue engineering. In recent years, great progress has been made in the synthesis, properties and applications of enzyme-responsive LDBCs, but compared with LDBCs that can respond to non-specific stimuli such as environmental temperature, pH, light and electric field,However, the study of LDBCs responsive to specific enzymes started relatively late, and the relevant literature reports are relatively limited, which also brings opportunities and challenges to the study of these biomolecular-responsive polymers.

In the synthesis of enzyme-responsive LDBCs, the divergent method is the most widely used at present. As mentioned above, the divergent method is cumbersome, and when preparing high-generation products, the yield is not high, and it is not easy to purify and characterize. However, there are only two reports on the synthesis of enzyme-responsive LDBCs by the more advanced coupling method, and both of them use the CuAAC click reaction as the coupling method. Due to the use of Cu (Ⅰ) as a catalyst in CuAAC reaction, the residual heavy metal copper ions in the product have high cytotoxicity, and it is difficult to remove them completely in the post-treatment. This has become a bottleneck in the application of enzyme-responsive LDBCs as drug carriers, so some new synthetic reactions and means, new product purification methods need to be applied to the synthesis of enzyme-responsive LDBCs.The biomolecular responsive polymer can give full play to the advantages of good biocompatibility and specific response, and can be practically applied in the field of biomedicine.

In terms of the molecular composition design of enzyme-responsive LDBCs, at present, the hydrophilic linear chain of enzyme-responsive amphiphilic LDBCs is mainly PEG, which has shown some defects in the application of biomedicine.Therefore, it is also an important topic in the research of enzyme-responsive LDBCs to select some materials with better biocompatibility and biodegradability, such as polypeptides, to replace the hydrophilic linear chain PEG in traditional enzyme-responsive LDBCs. At present, RAFT polymerization is mainly used in the synthesis of non-PEG linear chain enzyme-responsive LDBCs. If more living/controlled polymerization technologies can be applied to the synthesis of enzyme-responsive LDBCs linear chain, the structure of this kind of copolymer can be enriched, and it is expected to obtain biomolecule-responsive polymers with more excellent properties. In addition, because the types of enzyme-responsive groups that have been introduced into LDBCs are still relatively small, only a few enzyme-responsive LDBCs have been reported to respond specifically to cathepsin B, pig liver esterase and penicillin G amidase. If more enzyme-responsive groups can be introduced into LDBCs, it is expected to obtain LDBCs with specific response to more enzymes, especially those overexpressed in cancer cells, which can be used as targeted release carriers of anticancer drugs to improve the therapeutic effect of anticancer drugs and reduce their toxic and side effects.

In terms of the properties and applications of enzyme-responsive LDBCs, the current research mainly focuses on the effects of the structure of amphiphilic LDBCs (such as the length and properties of linear chains and the hydrophobicity of dendronized motifs) on their self-assembly properties and enzyme-responsive properties, and their applications as drug carriers, while there are few reports on the biocompatibility of such copolymers. In a word, there is still a huge space for the study of enzyme-responsive LDBCs. It is hoped that through reasonable molecular design and simple and effective synthesis methods, enzyme-responsive LDBCs with more excellent performance can be developed, and their application scope can be expanded, so that enzyme-sensitive LDBCs can play a unique role in more fields.

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