Cancer is the second leading cause of death worldwide. Current drug-loaded nanocarriers used for cancer treatment can only mitigate the adverse effects, but they cannot improve the therapeutic efficacy of anticancer drugs. The main reason for this low therapeutic efficacy is that it is difficult to transfer the anticancer drugs into the target tumor cells as free molecules ^[1][30]. In 2017, Shen et al. analyzed the typical cancer-drug-delivery process of intravenously administered drug-loaded nanocarriers, and they concluded that the delivery involves five steps: blood circulation, accumulation at the tumor site, tumor internal penetration, cellular internalization, and intracellular drug release; this was named the CAPIR cascade^[2][31]. They believe that a high efficiency at every step is crucial to ensure a high overall therapeutic efficiency.

To achieve a high efficiency in the corresponding steps, a nanocarrier must have three specific capabilities: good drug-carrying characteristics, suitable surface properties, and sufficient diffusivity. For drug carrying, it should first hold the drug tightly before entering cancer cells, but it must release the drug quickly once inside the cells. For the nanocarriers’ surface, it should also be stealthy for a long time during blood circulation, to give time for tumor accumulation; on the other hand, it must be able to interact with tumor cells for efficient cellular uptake after reaching the tumor cells. For diffusivity, the nanocarrier must have sufficient mobility to penetrate deep into the tumor so that it can reach tumor cells remote from the blood vessels ^[1][30]. To meet these requirements, nanocarriers should exhibit different or even opposite properties at different CAPIR steps. However, they can be easily grouped into three transitions of the nanoproperties: stability, surface, and size, also known as the 3S transitions ^[2][31].

Polypeptides can be conveniently endowed with stimuli responsiveness by introducing natural amino acid residues with innate stimuli-responsive characteristics, or by introducing responsive moieties to their side chains using simple conjugating methods, which makes them suitable carriers of anticancer drugs to fulfill the 3S transitions [2]. In ^[3]the following, we will review the polypeptide carriers for anticancer drug delivery which fulfill the 3S transitions.

The stability transition means that nanocarriers must remain stable enough during blood circulation, but that they should release the drug quickly after entering the tumor cells. To achieve the transition from stable to unstable, many stimuli-responsive polypeptide-based nanocarriers have been prepared over the past 10 years. There are two main strategies to construct stimuli-responsive polypeptide-based nanocarriers. The first is to introduce blood-stable but intracellular-labile bonds between polypeptide–drug conjugates, including acid-labile bonds (such as hydrazine ^[4][5][32,33] or benzoic imine ^[6][34]) responding to lysosomal acidity, and glutathione (GSH)-sensitive disulfide bonds responding to elevated GSH levels in tumor cells ^[7][8][9][35,36,37]. Li et al. synthesized a tetra-doxorubicin-tailed polyethylene glycol via benzoic-imine bond linkage, which can self-assemble into a pH-sensitive prodrug micelle. The micelle can quickly release doxorubicin in tumor sites to exert anticancer activity according to the experiments^[6][34]. Li et al. conjugated disulfide-containing camptothecin to poly(L-glutamic acid)-graft-methoxy poly(ethylene glycol) to create an amphiphilic biodegradable prodrug, which could self-assemble into micellar nanoparticles and encapsulate doxorubicin. The drug-release experiment proved that camptothecin could be released quickly in a GSH water solution at a cytosolic concentration (10 mM), which is a promising GSH-triggered drug-release system ^[8][36].

The other strategy is to prepare polypeptide drug carriers that can disintegrate under some special stimulation conditions ^[3][10][2,38], including a low pH value ^[11][12][13][39,40,41], low oxygen concentration ^[14][15][42,43], high GSH concentration ^[16][44] or a high concentration of reactive oxygen species (ROS) ^[17][18][45,46]. Zhong et al. synthesized a pH-responsive block polymer consisting of PEG and poly(asparagyl diisopropylethylenediamine-co-phenylalanine), which can self-assemble into nanovesicles to encapsulate hydrophilic tirapazamine and the hydrophobic photosensitizer dihydrogen porphin ^[19][47]. These nanovesicles would disassemble when incubated in a solution of 10 mM phosphate-buffered saline (PBS) at pH 5.0 for 24 h, due to the pH sensitivity of the diisopropylethylenediamine segment, which can enhance the release of the encapsulated active ingredient. Hoang et al. synthesized a ROS-responsive poly(ethylene glycol)-poly(methionine) and prepared micelles via self-assembly with a hydrophobic pro-oxidant drug, piperlongumine ^[17][45]. The increased ROS content in cancer cells triggered a hydrophobic to hydrophilic transition of the polypeptide, which led to the disassembly of the micelles, and consequently, to efficient drug release, increasing the anticancer efficiency.

The surface transition means that the carriers should remain stealthy during blood circulation, but after they reach the tumor tissue, they should be sticky toward tumor cells for efficient internalization. To achieve a stealthy-to-sticky transition, the surface properties of polypeptide carriers could undergo several changes such as PEGylation/dePEGylation, and changes in the surface charge. Both alternatives are intensively studied in recent years.

Poly(ethylene glycol) (PEG) has been demonstrated to give nanocarriers a stealth property. Nanocarriers with a PEG shell will show long blood circulation times, which is essential for the nanocarriers’ passive tumor targeting/accumulation, through their enhanced permeability and retention (EPR) effects ^[20][48]. However, this stealth layer also slows the nanocarriers’ cellular uptake, which limits their therapeutic effect. Therefore, many polypeptide carriers with detachable PEG shells have been designed in recent years ^[21][22][49,50]. Jiang et al. prepared two kinds of cisplatin-loaded poly(glutamate-lysine) complex nanoformulations with detachable PEG grafted onto lysine segments through two different bridged chemical bonds, which are responsive to specific tumor tissue microenvironments, including low pH and matrix metalloproteinases-2/9 ^[21][49]. The nanoformulations with PEG showed a prolonged circulation time in the blood and increased drug accumulation in the tumor tissue compared to the nanoformulations without the PEG segment. After arriving at the tumor tissues, the nanoformulations also showed enhanced cell uptake and cytotoxicity, due to the cleavage of the bridged chemical bond between PEG and polylysine. Wu et al. fabricated a multivalent amphiphilic peptide dendrimer to form nanoparticles with two siRNAs and linked the aldehyde-alkylated aptamer–PEG to the amino group of the nanoparticles through an acid-sensitive Schiff base, to construct tumor-activatable nanoparticles ^[22][50]. After arriving at the tumor tissues, the PEG layer of the nanoparticles was removed due to the low pH values, which facilitated siRNA penetration into the tumor tissues.

Studies have shown that nanocarriers that are slightly negatively charged or uncharged can remain stable in the circulatory system, while positively charged nanoparticles are more likely to promote cellular internalization and transcytosis for deep tumor penetration ^[23][51]. Therefore, charge-reversal nanocarriers based on polypeptides have been intensively studied over the years ^{[24][25][26][27][28][29][30][31]}[52,53,54,55,56,57,58,59]. Li et al. fabricated a surface charge-reversible nonviral gene vector with PEG, polypeptide, and PEI ^[32][60]. Owing to the protonation of PEI, the vector was negatively charged during circulation, but positively charged once it entered the tumor tissue, which facilitates both long circulations in the bloodstream as well as easy cell uptake. Qu et al. synthesized pH-sensitive polypeptide hybrid terpolymers, poly (lysine-co-N,N-bis(acryloyl) cystamine-co-dimethylmaleic anhydride), which can self-assemble into spherical nano-micelles with a negative surface charge under normal physiological conditions ^[33][61]. After arriving at the slightly acidic tumor tissues, the surface charge of the micelles switched from negative to positive, due to the protonation of lysine residues to enhance cellular uptake. Later, Qu et al. also prepared dual pH and redox-responsive cross-linked polypeptides based on poly(L-lysine-co-N,N-bis(acryloyl)cystamine-co-γ-glutamic acid), which can self-assemble into nanoparticles with negatively charged surfaces under physiological conditions ^[34][62]. The surface charge of the nanoparticles can switch to positive in a slightly acidic tumor extracellular environment because of the protonation of lysine residues, which increases the cellular uptake efficacy.

Size transition requires a carrier size of around 100 nm during blood circulation, but this size should be reduced to approximately 30 nm when arriving at the tumor tissue. Studies have shown that nanoparticles with a size smaller than 30 nm or larger than 200 nm are easily cleared, while nanoparticles around 100 nm are most likely to achieve long circulation times, which are crucial for the passive targeting of EPR effects in tumor tissues. However, this size is too big for the nanocarriers to diffuse into the tumor, and only nanoparticles with a size that is smaller than 30 nm exhibit a high penetration ability ^[35][63]. Therefore, polypeptide carriers that can realize a size transition have been intensively studied over the years ^{[36][37][38][39][40][41]}[64,65,66,67,68,69]. Cun et al. prepared a size-switchable nanoplatform by conjugating small dendrigraft polylysine (DGL) to poly(ethylene glycol)-poly(caprolactone) micelles via a matrix metalloproteinase 2-sensitive peptide ^[38][66]. The nanoplatform had an initial size of 100 nm and a nearly neutral charge, which was suitable for a long circulation time. After arriving at the tumor tissues, small DGL/DOX nanoparticles, which were approximately 30 nm in size, were rapidly released from the nanoplatform due to the cleavage of enzyme-sensitive peptides, thus enhancing the penetration of tumor cells.

For the anti-tumor active agents that could have direct anti-tumor effects on tumor tissues, the steps of cell internalization and release are not required, which significantly simplifies the design idea for the polypeptide carrier. For photothermal therapy, the delivery of the photosensitizer, which can generate heat for the thermal ablation of cancer cells upon NIR laser irradiation, into tumor tissues, is sufficient for treatment, and thus only the circulation and accumulation steps are needed ^[2][44][45][31,73,74]. Therefore, many stable polypeptide carriers for photosensitizer delivery have been extensively studied in recent years ^[46][47][75,76]. Huang et al. fabricated photothermal therapeutic nanocarriers by conjugating indocyanine green dye with self-assembled polypeptides via chemical bonding. The drug-loaded nanocarrier showed a diameter of around 50 nm, and had an improved degree of tumor accumulation and photothermal effect compared to the free dye ^[46][75].

Photodynamic therapy (PDT) is a similar treatment in which photosensitizers are used to generate reactive oxygen species to induce oxidation stress to kill cancer cells, but therefore the photosensitizers still need to be delivered to the cancer cells. However, the therapeutic effect of PDT is frequently limited due to the hypoxic nature that is characteristic of many solid tumors ^[48][77]. Thus, numerous efforts have been made to increase the oxygen content in tumors to enhance PDT efficacy ^[49][72]. Feng et al. is now focusing on the synthesis of perfluorodecalin-filled oxygen carriers, using polypeptides as encapsulating shell materials, which allow for fast gas exchange ^[50][78]. The capsule is expected to be used to improve the hypoxic condition of tumor tissue to enhance photodynamic therapy in the future.