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Abbreviation (ISO4): Prog Chem      Editor in chief: Jincai ZHAO

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Progress in Chemistry >

2025 , Vol. 37 >Issue 2: 211 - 225

DOI: https://doi.org/10.7536/PC240405

Review

Research Progress of Pseudo-Proteins as Drug Carriers

  • Xuan Zhang ,
  • Min Sun ,
  • Yunjiao Xue ,
  • Yuhuan Chen ,
  • Jing Fang ,
  • Fang Yang , *
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  • Applied Chemistry, School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China
* e-mail:

Received date: 2024-04-07

  Revised date: 2024-07-23

  Online published: 2025-02-07

Supported by

Introduced Overseas Students Funding Program of Hebei Province in 2022(C20220502)

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Abstract

Pseudo-protein materials have the advantages of high biocompatibility, biodegradability, and high tunability, and have attracted wide attention in the biomedical field as a drug carrier in recent years. Pseudo-protein molecules contain amide bonds, ester bonds and other active groups, compared with protein, not only retain the advantages of high tissue compatibility, and ester bonds and other active groups overcome the disadvantages of single protein structure, single function, make it have better mechanical properties and functionality, according to the actual demand for diversified morphology design and surface modification. The pseudo-protein drug carriers constructed by various methods such as self-assembly not only enhance the bioavailability of the drug in vivo, but also make the pseudo-protein drug carriers show ideal targeted controlled release performance with the help of specific signals at the focus. This paper focuses on the pseudo-protein drug delivery materials, introduces the construction and loading mode of pseudo-protein drug carriers, and summarizes the targeted release strategy of pseudo-protein drug carrier, and finally makes the prospect of pseudo-protein in the direction of controlled drug release, so as to provide reference for the subsequent research of pseudo-protein drug carriers.

Contents

1 Introduction

2 Construction of pseudo-protein drug carriers

2.1 The pseudo-protein itself constructs the drug carrier

2.2 Pseudo-protein with other substances to construct the drug carriers

3 Drug loading mode of pseudo-protein drug carrier

3.1 Physical coating

3.2 Preparation of sudden-release microcapsules

3.3 Chemical bonding

4 Targeted release of pseudo-protein drug carriers

4.1 Passive targeting

4.2 Active targeting

4.3 Stimulus-responsive targeting

Key words： pseudo-proteins; polymer drug carrier; drug loading; targeted release; stimulus response

Cite this article

Xuan Zhang , Min Sun , Yunjiao Xue , Yuhuan Chen , Jing Fang , Fang Yang . Research Progress of Pseudo-Proteins as Drug Carriers[J]. Progress in Chemistry, 2025 , 37(2) : 211 -225 . DOI: 10.7536/PC240405

1 Introduction

The rapid development of medicinal chemistry and molecular biology has promoted the production of a large number of highly effective drugs (such as small molecule drugs, proteins, nucleic acids). However, studies [1-2] have shown that under physiological conditions, many drugs are insoluble, aggregable, degradable, and impermeable to vascular endothelial cell layers. These properties result in a short half-life in the body's circulation and low bioavailability, seriously affecting their efficacy. In addition, environmental changes (such as pressure, temperature, humidity, and pH) can also affect drug efficacy. To improve the bioavailability of drugs, drug carriers based on biodegradable polymers have been extensively studied [3]. After encapsulation by carriers, drugs can effectively improve their solubility, degradability, and permeability, thereby significantly enhancing drug efficacy.
Amino acids are natural human metabolites, and proteins composed of amino acids as the basic units can be degraded by the human body, and the presence of amide bonds gives them excellent tissue affinity, thus they are widely used in biomedical applications[4-5]. However, the amino acids in protein molecules are linked by α-amino (head) and α-carboxyl (tail), as shown in Figure 1A, studies[6-7] have found that this "head-to-tail" orientation has enhanced immunogenicity, which may trigger an immune response in the human body. In addition, the protein molecules only contain amide bonds, and the single molecular structure leads to poor mechanical properties, which severely limits their application. Considering the limitations of the aforementioned natural proteins, Arabuli et al.[8-9] used α-amino acids as the basic units and synthesized a novel biodegradable polymer, amino acid-based polyester amide (AA-PEA), via solution polycondensation with aliphatic diols and dicarboxylic acids (Figure 1B), by introducing ester bonds into the protein molecules to break the original orientation of the protein, not only avoiding immune rejection with the human body but also making the mechanical properties and tunability of the polymer much higher than traditional proteins due to the addition of multiple components, which can be hydrolyzed and enzymatically degraded at physiological pH[10], serving as a potential material for drug delivery systems. Subsequent researchers synthesized a series of amino acid-based polymers such as amino acid-based polyester urea (AA-PEU) and amino acid-based polyester polyurethane (AA-PEUU) by condensation polymerization, interfacial polymerization, etc., with other components like diisocyanates and glycerol, due to their amino acid-based yet distinct characteristics from proteins, they are referred to as "pseudo-proteins"[11-12].
图1 蛋白质及伪蛋白的分子结构对比；（A）蛋白质分子结构；（B）伪蛋白分子结构

Fig. 1 Structural ison of proteins and pseudo-proteins; (A) general formula of amino acid structure; (B) molecular structure of protein

Pseudo-protein molecular structures are abundant and exhibit excellent biocompatibility. By encapsulating drugs through physicochemical interactions, the chemical stability of the drugs can be significantly improved. Additionally, its good mechanical properties allow it to be designed and processed into different structural drug carriers as needed. Moreover, the functionality of pseudo-proteins is considerably different from that of traditional proteins. The functionality of proteins is highly correlated with their three-dimensional spatial structure, and any subtle changes in the spatial structure may lead to the loss of their function, which also creates difficulties for protein design and application[13]. Pseudo-proteins are polymer chains formed by the polymerization of substances such as amino acids. Although they do not possess a three-dimensional spatial structure, their molecular composition is very flexible. By selecting different amino acids and linkers or employing methods such as surface modification, various functionalities (such as hydrophilicity, charge, and stimuli-responsiveness) can be introduced into the polymer chain. Their applications in drug delivery are more flexible and stable than proteins, making them one of the most promising drug delivery materials[14-15]. This article will summarize the progress of pseudo-protein materials in drug delivery from three aspects: the construction of pseudo-protein drug carriers (including preparation methods, morphology, surface modification methods, and modifiers), drug loading, and drug release, thereby providing references for the rational design and application of pseudo-protein drug carriers.

2 Construction of Pseudo-Protein Drug Carriers

Pseudo-proteins themselves possess good biocompatibility and degradability. When used as drug carriers, their diffusion and distribution in the body can be improved by adjusting physical properties such as size, shape, and surface charge. Moreover, the surface of pseudo-protein molecules contains many functional groups that are easy to modify, and their functionality can be enriched through surface modification. Therefore, the construction of pseudo-protein carriers can be roughly divided into two types: one is the drug carrier constructed by pseudo-proteins themselves; the other is the drug carrier co-constructed with other substances (including polymers and macromolecular materials) through surface modification.

2.1 Pseudo-Protein Self-Assembled Drug Carriers

The diversity of amino acids endows pseudoproteins with various properties, such as hydrophilicity, hydrophobicity, and charge. When pseudoproteins themselves serve as drug carriers, these characteristics can facilitate the formation of macromolecules and particles. Moreover, pseudoprotein molecules contain a large number of amide bonds, and their excellent mechanical properties allow them to be processed into drug carriers of different morphologies through diversified structural design, including microspheres[16⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓-27], nanospheres[28⇓⇓⇓⇓-33], micelles[34⇓⇓-37], capsules[38], and fibers[39⇓⇓⇓⇓⇓-45]. The preparation methods, principles, and characteristics of pseudoprotein drug carriers with different morphologies are summarized in Table 1.
表1 不同形貌的伪蛋白载体制备原理及特点

Table 1 Preparation principle and characteristics of pseudo-protein carriers with different morphologies

Morphology Preparation method Principle Peculiarity Ref.
Microsphere Emulsion process
Emulsification method is based on polymer and drug selection of incompatible two phase (continuous phase and discontinuous phase), usually water and oil, to make an emulsion system of single emulsion (W/O, O/O) or compound emulsion (W/O/W, O/W/O), and make the emulsion phase curing to obtain nanoparticles[46⇓-48]. Advantages: low preparation process requirements, good economic benefits, release with long-term effect, high efficiency and slow release;
Disadvantages: Its slow release rate is easily affected by environmental factors, and the micron size is large, so the targeting still needs to be further improved.		 16-27
Oil encapsulation emulsification method Different from the traditional emulsification method, the incompatible two phases selected by oil-coated solid emulsification are oil phases, and surfactant, block copolymer or solid particles are used to improve the emulsion stability[49]. Compared with conventional aqueous emulsion, oil-coated solid emulsion has a higher yield, and the hydrophobic oil phase hinders the distribution of water-soluble drugs in the outer phase, resulting in a high encapsulation efficiency[50].
Nanosphere Nano-precipitation method First, the hydrophobic polymer material and drugs are dissolved in volatile organic solvents, and surfactant or stabilizer is added to them to improve the stability of the emulsion. Then, the organic phase is dropped into the aqueous organic phase under continuous high speed stirring, and the organic solvent gradually diffuses into the aqueous phase. In this process, the self-assembly of the polymer forms the drug-carrying particles due to its hydrophobicity. Finally, the organic solvent was removed by centrifugation or dialysis to obtain the drug-loaded nanosphere[51-52]. Advantages: simple preparation process, mild conditions, release with high permeability long retention effect (EPR).
Disadvantages: Nanospheres do not improve drug water solubility, and the traditional spherical shape may weaken EPR effect[53], and also detrimental to endothelial cell adhesion[54].		 28-33
Micelle Self-assembly Amhiphilic polymers contain hydrophilic segments and hydrophobic segments, which are first dispersed or adsorbed on the surface in aqueous solution. When the concentration increases to the critical micelle concentration (CMC), in order to reduce the surface tension, the hydrophobic segments associate into the core by hydrophobic forces and van der Waals force, and the hydrophobic drug is wrapped in it, and the hydrophilic segments forms the shell and separates into separate spherical micelles under the action of electrostatic repulsion. Advantages: small particle size, enhanced EPR effect[55], and hydrophilic shell can significantly increase drug solubility.
Disadvantages: It is not easy to replicate the same quality and specifications of pseudo-protein micelles in mass production, and the self-assembled micelles are affected by the interaction between polymer and blood components, which may have a risk of drug leakage[56-57].		 34-37
Capsule Interfacial polymerization Interfacial polymerization refers to two monomers with high reaction performance dissolved in two incompatible solvents, and irreversible condensation reaction at the interface of the two liquid phases (or on the side of the organic phase). The resulting polymer is insoluble in the solvent and then precipitated in the interface. Advantages: The dense external film can effectively wrap the drug and reduce the initial burst release of the carrier in the body. The pseudo-protein has good biocompatibility and degradability, which can avoid the safety problems of traditional material capsules on the human body and the environment.
Disadvantages: between the preparation and characterization techniques, the research of capsule is more in the micron level, nano scale capsule is less research.		 38
Fiber Electrostatic spinning The polymer and drug are first dissolved in the organic solvent and added to the syringe, then the surface of the polymer and the drug solution at the end of the capillary is charged using a high pressure field and induced by liquid injection into the collector. By controlling the process conditions, changing the internal structure of the nozzle and using different receiving devices, different structures and orientations can be prepared. Advantages: high drug loading efficiency, large contact surface with tissue, its large aperture can enhance the diffusion of drug to surrounding tissues, has great advantages in the skin route of drug administration.
Disadvantages: its synthesis is easy to be affected by equipment conditions and has high requirements for production equipment.		 39-45
Drug carriers constructed by pseudo-proteins themselves have stable morphological structures and sizes, with relatively mature preparation technology and simple operation. However, such drug carriers are inevitably limited by the pseudo-protein materials themselves, including problems such as relatively single functionality, poor flexibility, and not comprehensive enough clinical applications.

2.2 Pseudo-Protein and Other Substances Construct Drug Carriers

Since the structure of pseudo-protein molecules is highly flexible, containing various functional groups (such as amino, carboxyl, and unsaturated C=C groups), other polymers can be introduced through surface modification (physical or chemical modification) to jointly construct better drug carriers. Surface modification methods are widely used in designing and constructing pseudo-protein drug carriers. Pseudo-protein molecules modified on the surface can significantly improve the biocompatibility of drug carriers, and specific functional modifications can promote endosomal escape, achieving effective intracellular drug release, thereby greatly enhancing therapeutic effects.

2.2.1 Physical Modification

The physical modification of pseudo-protein molecules mainly includes two types: electrostatic interaction and host-guest interaction. Amino acids such as lysine and arginine exhibit cationic characteristics due to the presence of amino or guanidino groups in their side chains, which makes the surface of the synthesized pseudo-protein molecules positively charged. When mixed with negatively charged polymers in solution, they interact to balance the charges and can form nanoparticles under appropriate conditions[58⇓⇓-61]. Wan et al.[60] synthesized phenylalanine-arginine-based polyester amides (Phe-Arg-PEA), where the guanidino groups on the arginine side chain provide positive charges. When this long chain is mixed with anionic hyaluronic acid in solution, charge balance promotes their interaction and forms nanomicelles under appropriate conditions. It has been demonstrated that these micelles have a bilayer or multilayer structure, with the outer layer composed of negatively charged HA and the inner layer made up of positively charged Phe-Arg-PEA. Ji et al.[61] synthesized ovalbumin-loaded polyester amide nanoparticles (Arg-Phe-PEA). The presence of arginine in the polyester amide component imparts a cationic charge to the particle surface, providing the ability to form electrostatic complexes with other anionic molecules. As shown in Figure 2, the two adjacent sulfonic acid groups on the photosensitizer AlPcS2a make it anionic in aqueous solution. By mixing the cationic OVA-loaded nanoparticles with AlPcS2a, an electrostatic complex can be formed, thereby achieving photochemical internalization and significantly enhancing drug delivery efficiency. Additionally, studies[12] have shown that since the outer layer of endosomal membranes consists of negatively charged phospholipids, the interaction between the captured cationic pseudo-proteins and the endosomal membrane may cause non-lamellar phase transitions and membrane destabilization, promoting endosomal escape and effective intracellular release.
图2 Arg-Phe-PEA聚合物的化学结构及静电配合物的形成[61]

Fig. 2 Chemical structure of the Arg-Phe-PEA polymer and the formation of the electrostatic complexes[61]

Host-guest complexes are formed by two or more molecules or ions combined through non-covalent interactions with a specific structural relationship[62]. In host-guest chemistry, the host is the larger of the two in size, and the guest is the smaller one; the host must be able to recognize those guests that match in arrangement and spatially[63]. As shown in Figure 3, cyclodextrin (CD) has a cavity that can accommodate molecules or ions and can co-build drug carriers with pseudo-protein molecules through host-guest interactions[64]. For example, Ji et al.[65-66] synthesized phenylalanine-based polyester amide (Phe-PEA) and cyclodextrin-hyaluronic acid grafted polymer (HA-CD). With the cavity of cyclodextrin, host-guest interactions can bind it with the benzene groups of CD and Phe-PEA. The hydrophilic hyaluronic acid (HA) serves as the outer layer of the carrier, while the Phe part provides hydrophobicity as the inner layer of the carrier, inducing the polymer to self-assemble into micelle structures in water through hydrophilic and hydrophobic forces.
图3 环糊精的结构[64]

Fig. 3 The structure of cyclodextrin[64]

2.2.2 Chemical Modification

In addition to the aforementioned physical modifications such as electrostatic adsorption and host-guest interactions, the ease of modifying pseudo-protein molecules allows functional groups to be attached at different positions on the main chain and side chains. These can be chemically modified to connect with other polymers for the co-construction of drug carriers. Based on the connection methods between them, chemical modifications can be roughly divided into three types: block modification, graft modification, and cross-linking modification.
(1) Block modification
Block modification refers to the process of connecting two polymers through chemical bonds by functionalizing the end groups of the polymer to form a linear polymer. Both ends of the pseudo-protein backbone are amino and carboxyl groups which allow covalent linkage with other substances to jointly form block copolymers[67⇓⇓-70]. For instance, Xu et al.[63] synthesized methionine-based polyester amide (Met-PEA), then linked carboxyl-functionalized polyethylene glycol to the terminal amino group of Met-PEA. The resulting block copolymer exhibits amphiphilicity and can self-assemble into stable spherical micelles in water. The modified drug carrier has a hydrophilic PEG shell, which can reduce the absorption of protein on the surface of nanoparticles in the blood, thereby prolonging the circulation time.
(2) Graft modification
When amino acids with reactive side groups (such as aspartic acid, lysine, arginine, and serine, etc.) are used as one of the components of pseudo-proteins, they can also provide reactive sites for pseudo-protein molecules[71⇓⇓⇓⇓⇓-77]. The addition of such side chains increases the complexity of pseudo-protein molecules, which may lead to changes in the solubility, charge, biocompatibility, and biodegradability of the polymers. For instance, Zilinskas et al[71] synthesized a polyesteramide backbone with side amine functional groups (Phe-Arg-PEA) (Figure 4), where arginine provides the side amine groups; then, 4-nitrophenyl carbonate-activated polyethylene oxide reacted with the amine to synthesize amphiphilic graft copolymers. Due to hydrophilic-hydrophobic interactions, this polymer can self-assemble in aqueous solutions, resulting in micelles with a diameter of approximately 60 nm. The incorporation of polyethylene oxide enhances the hydrophilicity of the drug carrier and prevents uptake by the reticuloendothelial system through shielding antigens and functional epitopes. Guo et al[77] selected fumaryl chloride/unsaturated diol to synthesize unsaturated polyesteramides (AA-UPEA) and polyetheresteramides (AA-UPEEA) with built-in reactive C=C bonds. In the presence of the radical initiator 2,20-azobisisobutyronitrile, through thiol-ene reactions, reactive groups (ACOOH or ANH2) can be linked to the C=C on the main chain, thus obtaining new PEA or PEEA with functional side chains. Due to the free volume expansion of the side chains, these new functional AA-PEA and AA-PEEA derivatives have lower glass transition temperatures than the original unsaturated AA-PEA and AA-PEEA polymers, and their solubility in certain organic solvents has been improved. Additionally, the incorporation of functional side chains increases the feasibility of covalently binding with bioactive substances, offering broader application prospects in the biomedical field.
图4 两亲性PEA接枝共聚物的制备及其胶束组装[72]

Fig. 4 Preparation of grafted copolymers from amphiphilic PEA and their micellar assembly[72]

(3) Crosslinking modification
Crosslinking modification refers to the process of adding a free radical initiator to the mixed gel precursor solution of pseudo-protein and other polymers, and using specific means (such as light irradiation, etc.) to generate free radicals in molecules, leading to polymerization and formation of crosslinked structures[78]. Free radical-induced crosslinking can form robust carbon-carbon bonds between pseudo-proteins and other substances instead of just forming hydrolysable amide, ester, or ether bonds, making the product more stable[79⇓⇓⇓⇓⇓-85]. As shown in Figure 5, Alapure et al.[85] dissolved unsaturated arginine-based polyester amides (UArg-PEA) and GMA-chitosan precursors in deionized water and performed photopolymerization in the presence of the photoinitiator Irgacure 2959. Both UArg-PEA and GMA-chitosan precursors have unsaturated double bonds that can form hydrogels through intermolecular crosslinking; the double bonds in UArg-PEA are located on the main chain of the polymer, while those in GMA-chitosan are located in the side groups. Under UV irradiation, the two polymer components can form stable crosslinked structures. The resulting hydrogel has excellent mechanical properties, accelerates wound healing, and does not produce cytotoxicity. By surface functionalization of the corresponding substances, they can bind to active sites on the surface of pseudo-protein molecules. Compared with physical modifications, this method allows a wider variety of substances to participate in constructing drug carriers together. In addition, by changing the composition of the pseudo-protein molecules, reactive sites can be introduced into the molecular backbone[86⇓⇓⇓⇓-91]. The resulting copolymer structures are diverse and complex, allowing for the construction of corresponding drug carriers according to clinical needs.
图5 光交联水凝胶的形成及透皮给药过程[85]

Fig. 5 Formation of a photocrosslinked hydrogel and the transdermal administration process[85]

3 Drug Loading Methods of Pseudo-Protein Drug Carriers

Loading drugs into drug carriers is one of the key steps in developing pseudo-protein drug delivery systems. When loading drugs onto pseudo-proteins, the main factors to consider are: how to achieve better encapsulation efficiency and how to maintain the activity of the drug. The drug-loading methods of pseudo-protein drug carriers mainly include physical coating and chemical bonding.

3.1 Physical Coating

Physical coating refers to the method of loading drugs by the combination of polymers and drugs through intermolecular forces, mainly including three types: hydrophobic interaction, electrostatic adsorption, and π-π stacking interaction.

3.1.1 Hydrophobic Effect

As shown in Figure 6, hydrophobic interaction drug loading utilizes the hydrophobic interaction and hydrogen bonding forces between the hydrophobic core of micelles and poorly soluble drugs to solubilize drugs within polymeric micelles, suitable for most hydrophobic drugs. The preparation process of drug carriers loaded by hydrophobic interactions is simple and low-cost, which can significantly improve the water solubility of drugs and is widely used in the controlled release of many hydrophobic drugs. For example, He et al[88] synthesized an amphiphilic arginine-leucine-based poly(ester urea-urethane) (Arg-Leu-PEUU), which can self-assemble into micellar form in water, where the leucine segment serves as the hydrophobic core that can encapsulate doxorubicin (DOX) inside and keep it stable through hydrophobic interactions.
图6 两亲性胶束负载疏水药物的过程

Fig. 6 The process of amphiphilic micelles loading the hydrophobic drugs

3.1.2 Electrostatic Adsorption Effect

Drug loading through electrostatic adsorption refers to the interaction where polymers and drugs with opposite surface charges attract and bind to each other under the effect of electrostatic forces. As shown in Figure 7, pseudo-proteins based on cationic amino acids often load negatively charged genetic drugs or other bioactive substances via electrostatic adsorption. Electrostatic adsorption can significantly increase drug loading capacity, and the drug release is facilitated by the pH differences within the intracellular environment. Moreover, the preparation conditions for drug loading using electrostatic adsorption are mild, simple, and feasible without the need for organic solvents, avoiding the residue of organic solvents and the damage caused by ultrasound. Wu et al.[92-93] synthesized a series of water-soluble, positively charged L-arginine-based polyesteramides (Arg-PEAs) through solution polycondensation. The results showed that by altering the number of methylene groups in the diol or dicarboxylic acid segments, the hydrophobicity and cationic properties of Arg-PEAs could be finely tuned, thereby influencing gene delivery efficiency.
图7 静电吸附作用负载药物

Fig. 7 Electrostatic adsorption of the loaded drugs

3.1.3 π-π Stacking Interactions

π-π stacking is a unique spatial arrangement of aromatic compounds and a weak interaction occurring between aromatic rings. When both polymers and drugs contain aryl groups simultaneously, the interaction between aromatic rings can lead to a spatial arrangement of π-π stacking between the two, thereby increasing drug loading and preventing premature drug release[94]. Chandra et al.[91] designed and synthesized a phenol and catechol functionalized polyester-polyurethane (PEUR) based on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA) (Figure 8), which self-assembles into nanoparticles of (100±10) nm in water and encapsulates polyaromatic anticancer drugs such as doxorubicin and topotecan using the electron-rich aromatic π core of L-DOPA. The quenching of characteristic emission peaks of the drug and L-DOPA in drug-loaded nanoparticles confirms the presence of strong π-π stacking between the drug and the electron-rich L-DOPA segments within the micellar nanoassemblies.
图8 基于L-氨基酸的芳香族π-π堆叠相互作用驱动的给药系统[93]

Fig. 8 Aromatic π-stack interaction-driven drug delivery system based on L-amino acids[93]

3.2 Chemical Bonding

In addition to the aforementioned methods, when there are functional groups available for covalent conjugation between the carrier and the drug, pseudo-protein drug carriers can also load drugs through chemical bonds, forming polymer-drug conjugates (PDC)[95]. For example, pseudoproteins synthesized based on amino acids with reactive side chains, such as L-serine, L-tyrosine, L-aspartic acid, L-lysine, and L-arginine, can be covalently conjugated with drugs. As shown in Figure 9, Soleimani et al.[75] synthesized an L-aspartic acid-based polyester amide random copolymer (Phe-Asp-PEA) and obtained Phe-Asp-PEA-COOH by removing the carboxylic acid protecting groups from the copolymer. Subsequently, under the presence of N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), each pendant carboxylic acid segment of Phe-Asp-PEA-COOH reacted with 0.5 equivalents of paclitaxel (PTX). Then, it was reacted with 0.5 equivalents (per side-chain carboxylic acid moiety) of amine-terminated PEO under the same conditions, resulting in hydrolysable ester linkages formed between the polymer's pendant carboxylic acid moieties and PTX’s 2-hydroxy group, providing the final amphiphilic PTX conjugate (PEA-PTX). When the micelles are disrupted, the exposure of the ester bonds to water accelerates drug release.
图9 PEA-PTX共轭物的形成以及PTX的结构[75]

Fig. 9 Formation of the PEA-PTX conjugate as well as the structure of the PTX[75]

Whether it is hydrophobic interaction, electrostatic interaction, or π-π stacking interaction, the drug is loaded by intermolecular forces. This purely physical coating method does not require the participation of chemical reagents, is simple to operate, and is suitable for conventional pseudo-protein drug carriers. However, the intermolecular forces are relatively weak, and the stability of drug delivery systems using physical adsorption is poor. Moreover, the drug release mechanism of carriers prepared by physical adsorption is diffusion, which results in a faster drug release rate and is significantly affected by the carrier properties. On the other hand, PDCs linked by chemical bonds are relatively stable, and a high drug loading can significantly enhance therapeutic effects, thereby increasing the efficacy of drugs reaching tumors. Nevertheless, one obvious drawback of PDCs is that not all drugs have chemical functional groups available for covalent conjugation, and the synthesis cost is relatively high.

4 Targeted Release of Pseudo-Protein Drug Carriers

For conventional drugs, only a small portion can actually reach the pathological site after entering the body, which is the root cause that limits drug efficacy and leads to drug toxicity and side effects. Therefore, enabling drugs to be precisely targeted and released at specific sites like missiles is one of the important goals in the development of drug carriers. Pseudo-protein drug carriers have excellent functional tunability and can be endowed with targeting properties through surface modification, thereby responding to specific signals at the target site (Fig. 10). According to the different targeting mechanisms, they can be divided into three major categories: passive targeting, active targeting, and stimuli-responsive targeting.
图10 伪蛋白药物递送系统的靶向策略（用BioRender.com创建）

Fig. 10 Targeted strategy for the pseudo-protein drug delivery system (Created with BioRender.com)

4.1 Passive Targeting

Passive targeting delivery refers to the process by which injected drug particles reach diseased tissues through the leaky vasculature system of tumors, infections, or inflammations, also known as the enhanced permeability and retention (EPR) effect[96]. Studies have shown that due to the abundance of new blood vessels in solid tumor tissues, the gaps in the normal microvascular walls are relatively wide, the structural integrity is poor, and the lymphatic drainage is missing. These differences lead to spherical particles with sizes between 50-200 nm being the easiest to achieve long circulation and can avoid being cleared by the kidneys[97-98]. For example, Chen et al[28] synthesized polyester amide (Phe-PEA) based on L-phenylalanine, and assembled it with docetaxel (Dtxl) into nanoparticles with a diameter of about 100 nm. In vitro release and cytotoxicity experiments showed that, compared with free Dtxl, the loaded Dtxl significantly reduced normal cell toxicity, demonstrating that nanoparticles could accumulate more in tumor tissues. However, despite this, the specificity of drug carriers achieving targeted release solely relying on the EPR effect is not high, and the high interstitial pressure of tumor neovascularization will also inhibit drug aggregation in tumors. Additionally, some studies have shown that the EPR effect varies with the physicochemical properties of nanocarriers, and nanoparticles with poor diffusion ability may find it difficult to penetrate into the tumor via the EPR effect[99-100].

4.2 Active Targeting

Active targeting means that the drug carrier is coupled with specific ligand or antibody components to bind to specific receptors on the cell surface, thereby achieving accumulation in the targeted tissue. The unlimited proliferation of tumor cells is closely related to the overexpression of some genes, cytokines, and proteins, which provides a basis for designing targeted drugs containing smart recognition sites. The current research on drug carriers prepared by pseudo-proteins is mainly focused on ligand-receptor interaction-mediated targeting, such as folic acid ligands, hyaluronic acid ligands, and other specific ligands.

4.2.1 Folic Acid Ligand

The folate receptor (FR) is a cell surface glycosylphosphatidylinositol-anchored glycopeptide characterized by binding to folic acid (FA) and transporting folic acid through a non-classical endocytic mechanism. It is known that many malignant tumors overexpress FR-α[90,101], including ovarian, endometrial and cervical adenocarcinomas, testicular choriocarcinoma, ependymal brain tumors, pleural mesothelioma, etc., thus allowing targeted delivery to tumor cells overexpressing the receptor by conjugating folic acid to the surface of drug carriers[102]. He et al.[88] synthesized folate-conjugated linear and branched arginine-based poly(ester-urea-polyurethane) (FA-Arg-PEUU) as a carrier for gambogic acid (GA). Compared with Arg-PEUU NP, GA delivered by the FA-Arg-PEUU-NP carrier exhibited higher cytotoxicity and induced a higher percentage of cancer cell apoptosis.

4.2.2 Hyaluronic Acid Ligand

Hyaluronic acid (HA) is a naturally biodegradable polysaccharide composed of D-glucuronic acid and N-acetylglucosamine forming glucosamine repeating units. Studies have shown that HA is the ligand molecule for the lymphocyte homing (CD44) receptor. Due to the overexpression of CD44 receptors on many types of tumor cells, HA-based drug carriers exhibit enhanced binding and endocytosis effects and have been explored to improve the efficacy of various therapeutic agents[103⇓-105]. As shown in Figure 11, Ji et al.[66] synthesized phenylalanine-based polyester amides grafted with CD and HA (HA-CD-4Phe4) as a carrier for gambogic acid. Multidrug-resistant cells overexpressing CD44 receptors and T3 fibroblasts were used as models to test the endocytosis and subcellular distribution of the carrier. Experiments demonstrated that HA-CD-4Phe4 was encapsulated within lysosomes to decompose the nano-carrier and accelerate release; the nano-complex significantly enhanced the endocytosis of multidrug-resistant cells overexpressing CD44, thereby achieving a targeting effect.
图11 HA-CD-4Phe4包合物的形成[66]

Fig. 11 Formation of the HA-CD-4Phe4 inclusion complex[66]

4.2.3 Other Specific Ligands

β-d-Galactose (Gal) is a ligand that specifically targets the insulin glycoprotein receptor (ASGP-R) on mammalian hepatocytes, and studies[106-107] have found that ASGP-R is overexpressed on the surface of hepatocellular carcinoma cells. Lv et al.[89] conjugated phenylalanine-cysteine-based polyester amide with thiol-functionalized galactose (Gal-SH) via Michael addition reaction to prepare the SSPEA-Gal copolymer (Figure 12). The results showed that DOX-loaded SSPEA-Gal42 exhibited significantly higher toxicity to human hepatocellular carcinoma cells (overexpressing ASGP-R) compared to human breast cancer cells (low expression of ASGP-R), supporting the specific recognition and efficient cellular uptake of SSPEA-Gal by ASGP receptor-overexpressing HepG2 cells, with better anti-tumor effects.
图12 基于SS-PEA-Gal共聚物的肝癌靶向还原性可降解纳米颗粒示意图[89]

Fig. 12 Illustration of hepatoma-targeting reductively degradable nanoparticles based on SSPEA-Gal copolymers[89]

Compared with passively targeted pseudo-protein drug carriers, ligand-modified pseudo-protein drug carriers can specifically bind to target cells, allowing drugs to accumulate in tumors, thereby enhancing therapeutic efficacy while reducing the total drug dosage and toxic side effects on normal tissues. However, during the modification of pseudo-proteins, many targeting ligands are prone to denaturation and inactivation, which may compromise the stability of their targeting function. Moreover, after pseudo-protein modification, changes in properties such as the size and surface charge of the drug carrier might make it more susceptible to capture by the reticuloendothelial system, potentially affecting the targeting efficiency.

4.3 Stimulus-Responsive Targeting

In addition to the two common targeting strategies mentioned above, stimulus-responsive targeting strategies that release drug carriers at specific sites through specific means have also been extensively studied in recent years. Relevant studies[108-109] have shown that solid tumors are characterized by high cell density, abnormal vasculature, hypoxia, acidosis, poor lymphatic drainage, abnormal expression of extracellular matrix-related receptors and enzymes, and elevated interstitial fluid pressure. Among these characteristics, hypoxia, acidosis, abnormal temperature, and overexpression of specific enzymes can be utilized to design stimulus-responsive targeting systems to enhance the effectiveness of anti-tumor drug delivery. In response to these signals, pseudo-proteins have also been constructed into many stimulus-responsive targeting systems, including pH-stimulus responsive, reactive oxygen species (ROS)-stimulus responsive, redox (GSH)-stimulus responsive, light-stimulus responsive, and temperature-stimulus responsive systems.Table 2 summarizes the stimulus signals and responsive components of different stimulus-responsive drug carriers.
表2 不同刺激响应性药物载体的刺激信号和响应组分

Table 2 Stimulation signals and response components of different stimulus-responsive drug carriers

Stimulus response type Spike Response components
pH stimulation response The tumor is rapidly affected by growth, and the large accumulation of lactic acid results in the microacid environment (pH is about 6.2-7.2), while the normal tissue cell environment pH is close to 7.3. When the polymer contains acid-sensitive bonds/groups, the tumor will cause bond breakage or protonation due to the acidic environment.
ROS stimulation response Due to increased tumor cell metabolism, mitochondrial dysfunction, produces higher levels of ROS. And ROS has a strong oxidation capacity, which can denature the easily oxidized substances.
REDOX stimulation response In order to prevent the induced damage of high concentration of reactive oxygen species, the cells produce a large number of reducing molecules such as glutathione (GSH) to produce a large redox potential difference between them and normal cells. When the polymer contains chemical bonds susceptible to breakage by GSH, high concentrations of GSH can accelerate drug release.
Light stimulation response External light, such as UV light irradiation. Because light is an additional signal in vitro, it has temporal and spatial control flexibility to modify the light-sensitive unit through appropriate illumination.
Temperature stimulation response The polymer containing a low critical solution temperature (LCST) portion is structurally stable below that temperature, and the hydrophilic-hydrophobic transition of the partial chain segment above that temperature leads to its phase separation for the release of the drug.

4.3.1 pH-Responsive Stimulation

pH-stimuli responsiveness refers to the cleavage or protonation of polymer drug carriers containing acid-sensitive chemical bonds (such as imine bonds, acetal bonds, hydrazone bonds, and borate ester bonds, etc.) in an acidic environment[110], which causes physical dissociation or internal structural changes of polymeric nanoparticles, triggering drug release. At the tumor organ level, the rapid growth of tumors and abnormal new blood vessels lead to insufficient oxygen supply around the tumor, producing the "Warburg" effect[111]. This effect forces tumors to undergo extensive glycolysis to provide the energy needed for their growth, thus generating a large amount of lactic acid, which keeps the tumor in an acidic microenvironment (pH 6.2~7.2) for a long time. This characteristic can distinguish the tumor environment from normal tissue environments, providing insights into pH-responsive drug carriers[112-113]. The pH-stimuli targeting mechanism of pseudo-protein drug carriers mainly involves the protonation of groups leading to decreased particle stability, thereby facilitating drug release. As shown in Figure 13, Yuan et al.[87] prepared degradable branched polyester amides (BPEA) using inositol and amino acids as raw materials, encapsulating paclitaxel within the formed nanoparticles (BPEA@PTX NP). The guanidino group undergoes protonation under acidic conditions, increasing the hydrophilicity of BPEA molecules, thereby promoting PTX release. Experimental results show that BPEA@PTX NP is stable in phosphate-buffered saline (PBS) at pH 7.4; however, when the pH is 5.0 or 6.5, approximately 80% of PTX is rapidly released into the medium within the first 10 hours, demonstrating its responsiveness to pH stimuli.
图13 BPEA@PTX NPs的构建和体内作用示意图[87]

Fig. 13 Schematic representation of the construction and in vivo action of BPEA@PTX NPs[87]

4.3.2 Reactive Oxygen Species Stimulus Response

Reactive oxygen species (ROS) are by-products of normal metabolism during oxygen formation, but malignant tumor cells produce high levels of ROS due to increased basal metabolic activity, mitochondrial dysfunction, uncontrolled growth factors, or oncogene stimulation[114-115]. Due to the high redox potential gradient between the extracellular and intracellular environments as well as between tumor tissues and normal tissues, particles containing sulfhydryl, selenyl, or boronate groups that are susceptible to ROS cleavage can rapidly and effectively release drugs in tumor cells[116]. Xu et al.[67] prepared an L-methionine-based ROS-responsive polyester amide nanoparticle carrier for loading gambogic acid. L-methionine (Met) is an important antioxidant in the body and can be oxidized by ROS into methionine sulfoxide. Experimental results showed that drug release from the nanoparticles was faster under high concentrations of H2O2, a phenomenon attributed to the oxidation of Met, where hydrophobic sulfides transform into hydrophilic sulfoxide groups, enhancing the polymer's hydrophilicity and accelerating micelle disintegration.

4.3.3 Redox Stimulus Response

Cells tend to maintain redox homeostasis, therefore they also highly express reduced glutathione (GSH), and the GSH concentration inside and outside of tumor cells may differ by thousands of times compared to normal cells. Since GSH can cleave chemical bonds such as disulfide bonds, diselenide bonds, and succinimide-thioether bonds, polymers containing such chemical bonds can exhibit responsive properties at the site of tumor cells. As shown in Figure 14, Sun et al[33] designed and synthesized an L-phenylalanine-based polyester amide (SS-PEA) containing disulfide bonds. Although the disulfide bonds are stable during circulation and in the extracellular environment, due to the presence of high levels of GSH, the disulfide bonds will rapidly break in the cytoplasm, accelerating drug release. In vitro drug release studies have shown that in a reductive environment containing 10 mM dithiothreitol (DTT), SS-PEAs underwent extensive degradation, indicating that SS-PEA has reduction responsiveness.
图14 还原性可降解SS-PEA聚合物的构成及药物传递过程[33]

Fig. 14 Composition of reducing-degradable SS-PEA polymer and the drug delivery process[33]

4.3.4 Light Stimulation Response

Among various stimuli-responsive polymers, photoresponsive polymers have received particular attention in recent years[117-118], because light stimuli can be localized and controlled in both time and space. Photoresponsive chemical moieties (such as coumarin, 2-nitrobenzyl, spiropyran, azide, 6-diazo, pyrene, cinnamyl, and N-alkyldimethylmaleimide) can be positioned as side chains or main chains. Under light irradiation, these photosensitive groups undergo reversible structural changes, such as photoisomerization[119] or cleavage reactions[120], thereby causing changes in the structure and properties of the polymer. Soleimani et al[75] designed and synthesized a photoresponsive amphiphilic micelle for loading the anticancer drug docetaxel. The o-nitrophenyl group was selected as the photodegradable unit and connected to each unit of the phenylalanine-based polyesteramide backbone to achieve complete degradation of the polymer upon light exposure. As shown in Figure 15, the results indicate that before light irradiation, the micelle diameters were relatively uniform ((60±11) nm), while after 20 minutes of UV irradiation, the micelle structure disappeared and only some loose aggregates were observed. This might be due to partial cleavage of the photostable o-nitrobenzyl ester, leading to a certain degree of micelle breakdown, increased contact between ester bonds and water, and accelerated hydrolysis.
图15 紫外光照射导致胶束解体的过程[75]

Fig. 15 The process by which UV light irradiation leads to micelle disassembly[75]

4.3.5 Temperature Stimulus Response

Temperature stimulus response refers to the presence of a certain proportion of hydrophilic and hydrophobic groups in the polymer structure, where the interaction between the polymer segments and the aqueous solution changes with temperature variations at a critical dissolution temperature, thus affecting the hydrophilicity and hydrophobicity levels of the polymer and the hydrogen bonding between the polymer segments, which in turn causes changes in the polymer structure. Thermoresponsive polymer materials have the ability to undergo phase separation at higher temperatures, providing additional advantages for targeted drug release[121]. Aluri et al[74] prepared high molecular weight amphiphilic polyester polyurethane (Lys-PEUR) and loaded DOX and CPT into the polymer micelles. As shown in Figure 16, in an aqueous medium, the PEG chains will be highly hydrophilic and protrude outward while the main chain will fold away from water, thus self-assembling into nanoparticles with dimensions <200 nm. The hydrophilic PEG chains have a lower critical solution temperature (LCST), above which they tend to undergo a thermoresponsive phase transition, transforming into a hydrophobic state. At normal body temperature (37 ℃), the polymer scaffold is stable; at cancer tissue temperature (approximately 42 ℃), 90% of the CPT was released within 12 hours. This phenomenon indicates that tyrosine-based polymers possess unique thermoresponsive phase separation capabilities and can deliver drugs through phase separation.
图16 两亲性聚酯聚氨酯的研制及其热刺激响应性能示意图（a）聚合物的合成路线；（b）聚合物在热刺激下靶向释药的过程[74]

Fig. 16 Schematic diagram of the development of amphiphilic polyester polyurethane and its thermal stimulation response performance (a) the synthetic route of the polymer; (b) the targeted drug release process of the polymer under thermal stimulation[74]

The nanoscale pseudo-protein drug delivery system has an enhanced EPR effect, which helps the drug escape endothelial cell capture. By specifically modifying the surface of pseudo-protein molecules, it can respond to special signals on the surface of pathological cells, thereby achieving targeted release. The targeted release of the pseudo-protein drug delivery system significantly increases the directionality and retention of the drug towards pathological tissues, thereby reducing the toxicity of the drug to normal cells, avoiding unnecessary off-target toxicity, and achieving maximum therapeutic effect through effective drug delivery and release.

5 Summary and Outlook

Pseudoproteins based on natural amino acids possess good biocompatibility, degradability, and tunability, showing great potential in drug delivery and controlled release. The ester bonds on their main chain can be enzymatically degraded, and the degradation products are non-toxic, while the amide bonds provide strong intermolecular forces, giving the polymer good mechanical properties. Moreover, functional pseudoproteins synthesized based on amino acids with reactive side chains have high tunability and can be linked with other substances to endow them with richer properties. This article provides a detailed introduction to the synthesis and drug loading of pseudoprotein drug carriers, as well as the targeted release mechanism of the pseudoprotein drug delivery system, and summarizes these aspects. Although pseudoproteins are currently widely researched and developed for drug delivery, there are still issues such as low drug-loading capacity, non-sensitive targeting sites, relatively cumbersome and time-consuming synthesis steps, and high costs. To address these problems, the following methods can be adopted for improvement: adjusting the composition design of pseudoproteins to synthesize multifunctional pseudoproteins and enhancing drug-loading capacity through surface modification; designing multi-stimuli-responsive drug-loading systems, or combining stimuli-responsive elements with ligands to enhance targeting.

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