# Advances on the Use of Biodegradable Proteins/Peptides in Photothermal Theranostics

Authors: Sheng Wang; Jing Lin; Peng Huang
Journal: Journal of Nanomaterials
 (2016)

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## Abstract

Recently, photothermal therapy (PTT) which employs light-induced heating to destroy cancer tissues/cells has received tremendous attention due to its improved selectivity and minimal invasion to surrounding healthy tissues. A variety of photothermal conversion agents (PTCAs) with high near-infrared (NIR) light absorbance have been widely explored for NIR light-induced PTT. However, many of them cannot be used directlyin vivo owing to their nonbiodegradability, immunogenicity, poor pharmacokinetics, or potential long-term toxicity. Proteins and peptides with inherent biocompatibility and biodegradability have been used as delivery vehicles for PTCAs or used as biotemplates to direct the synthesis of PTCAs. In this review, we will summarize recent advances in the development of protein/peptide-based photothermal cancer theranostics. The perspectives and challenges of these nanoplatforms will also be discussed.


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## Body

## 1. Introduction

Photothermal therapy (PTT), which employs light-induced heating to destroy cancer tissues/cells, is a new promising strategy to treat cancer due to its improved selectivity and minimal invasion to surrounding healthy tissues [1]. In general, PTT involves noninvasive light and photothermal conversion agents (PTCAs) that show low toxicity in dark and therapeutic effect under light irradiation. So the PTT can achieve superimposed targeting effect: on the one hand, the photoabsorbing agent can be designed to nanosized and ligand-modified particle so as to target cancer via passive and active targeting; on the other hand, the light can be applied only in cancer site to reduce side effect. Particularly, near-infrared (NIR) light (650–950 nm) induced PTT holds much promise because of the relatively low absorption/scattering of skin, tissue, blood, and water in the NIR region [2]. In the NIR light-induced PTT, PTCAs are crucial elements that absorb NIR light energy and convert it into heat and thus raise the local temperature of tumor to kill them. In the past decades, a variety of inorganic/organic nanomaterials, such as noble metal nanomaterials (Au, Ag, and Pt) [3–7], carbon-based nanomaterials (graphene oxide and carbon nanotube) [8–10], semiconductor nanomaterials (WS2, CuS, and C
u
2
-
xSe) [11–16], and NIR dyes (indocyanine green, cypate, IR780, and IR825) [17–19], as well as semiconducting polymer nanomaterials (polyaniline and polypyrrole) [20–22], have been widely explored as PTCAs for PTT. These PTCAs exhibit high NIR absorbance; however, many of them cannot be used directlyin vivo owing to nonbiodegradability, immunogenicity, poor pharmacokinetics, or potential long-term toxicity. Therefore, development of novel photothermal nanosystems is highly desirable to accelerate the clinical translation of PTT.In the past few years, proteins and peptides have been developed as excellent delivery materials for many diagnostic/therapeutic agents due to their inherent biocompatibility and biodegradability [23–25] (Figure 1). On the one hand, proteins/peptides can encapsulate hydrophobic nanoparticles or molecules through hydrophobic interactions and thus improve the biocompatibility and water solubility of the nanoagents [26–28]. On the other hand, proteins/peptides can be used as biotemplates to synthesize nanoparticles directly [29–33]. Meanwhile, proteins/peptides contain a large number of functional groups (i.e., carboxylic and amino groups) that can easily conjugate functional substances, such as drugs, NIR dyes, photosensitizers, targeting ligands, and imaging agents, to achieve “All-in-One” multifunctional theranostics [34].Figure 1
A scheme showing applications of proteins.Considering the unique properties of proteins/peptides, recently, various protein-based photothermal nanoplatforms, which can combine the advantages and functionalities of both proteins/peptides and PTCAs, have been developed for PTT (Table1). Herein, we will give a brief review on recent advances in the development of protein-based photothermal cancer theranostics. The perspectives and challenges of these nanoplatforms will also be discussed.Table 1
A summary of the various kinds of protein-based photothermal theranostics.
Proteins
PTCAs
Laser irradiation
Model
Other functions
Ref.
Serum albumins
HSA
ICG
808 nm, 0.4 W/cm2, 10 min
4T1 tumor-bearing mice
FL imaging; chemotherapy
[38]
ICG
808 nm, 0.8 W/cm2, 5 min
4T1 tumor-bearing mice
FL and PA imaging; PDT
[37]
IR825
808 nm, 0.8 W/cm2, 5 min
4T1 tumor-bearing mice
FL and MR imaging
[39, 40]
IR780
808 nm, 1 W/cm2, 5 min
CT26 tumor-bearing mice
FL imaging; PDT
[41]
CySCOOH
808 nm, 1 W/cm2, 5 min
4T1 tumor-bearing mice
FL and PA imaging
[42]
Gold nanoshell, ICG
808 nm, 2 W/cm2, 15 min
HeLa and MDA-MB-231 cells
FL imaging; PDT; chemotherapy
[43]
Gold nanorod
808 nm, 2 W/cm2, 15 min
4T1 cells
Chemotherapy
[44]
BSA
Cypate
785 nm, 1.5 W/cm2, 3 min
4T1 tumor-bearing mice
FL, MR, and PA imaging
[46]
CuS
808 nm, 0.7 W/cm2, 15 min
HeLa tumor-bearing mice
N/A
[47]
rGO
808 nm, 0.6 W/cm2, 5 min
MCF-7 tumor-bearing mice
PA imaging
[48]
IR825
808 nm, 0.5 W/cm2, 5 min
4T1 tumor-bearing mice
FL and MR imaging; PDT
[28]
Squaraine
680 nm, 20 W/cm2, 30 min
KB tumor-bearing mice
FL imaging
[49]
Polypyrrole
808 nm, 0.5 W/cm2, 5 min
4T1 tumor-bearing mice
FL and MR imaging; PDT
[50]
Other proteins
Ferritin
IR-820
808 nm, 0.5 W/cm2, 10 min
4T1 tumor-bearing mice
FL and PA imaging
[51]
CuS
808 nm, 0.8 W/cm2, 5 min
U87MG tumor-bearing mice
PET and PA imaging
[52]
HBV core protein
Gold dot
655 nm, 24 W/cm2, 20 min
MDA-MB-468 tumor-bearing mice
FL imaging
[53]
Gelatin
CuS
808 nm, 6 W/cm2, 5 min
HeLa cells
PA imaging; chemotherapy
[54]
Polyaniline or Cu9S5
808 nm, 0.5 W/cm2
H22 tumor-bearing mice
PA imaging; chemotherapy
[55, 56]
Antibody
ICG
808 nm, 6 W/cm2, 200 s
1483 cells
Cell targeting
[57]
Gold nanorod
808 nm, 3 W/cm2, 8 min
Hep-2 tumor-bearing mice
Cell targeting
[58]
Phycocyanin
Carbon nanotube
808 nm, 0.5 W/cm2, 5 min
MCF-7 cells
PDT
[59]
Peptides
NapFFKYp
ICG
808 nm, 0.8 W/cm2, 5 min
HeLa tumor-bearing mice
FL and PA imaging
[60]
PAsp
ICG, cypate
785 nm, 1 W/cm2, 5 min
A549 tumor-bearing mice
FL imaging
[61]
PLL-PLLeu
ICG
808 nm, 1 W/cm2, 5 min
H460 cells
FL imaging
[62]


## 2. Serum Albumin-Based Photothermal Theranostics

Serum albumin, the major component of serum proteins, is an attractive biomacromolecule with the merits of biodegradability, nontoxicity, nonimmunogenicity, and easily biological production and purification [35]. Among the available serum albumin, human serum albumin (HSA) and bovine serum albumin (BSA) are two of the most used proteins.


### 2.1. HSA

HSA, which contains 585 amino acid residues with a relative molecular weight of 66.5 KDa, is the most abundant protein in human plasma (~42 g/L) and plays an important role in transporting fatty acids, amino acids, steroids, and metal ions [36]. It is an endogenous protein, which has been approved by the US Food and Drug Administration (FDA) for intravenous administration. Furthermore, some studies showed that HSA coated nanoparticles possess active tumor-targeting abilitiesvia gp60 and SPARC receptor-mediated transcytosis [36].Recently, numerous studies have reported HSA-based photothermal nanosystems for PTT. For example, Sheng et al. reported a kind of smart nanoparticle based on HSA and indocyanine green (ICG) for dual-modal imaging-guided synergistic phototherapy [37]. ICG is a FDA-approved multifunctional NIR dye which can be used not only for NIR fluorescence (FL) imaging and photoacoustic (PA) imaging but also for PTT and photodynamic therapy (PDT). In this work, the disulfide bonds of HSA were firstly cleaved by adding excessive reducing agent to obtain free sulfhydryl groups. Then the reduced HSA can reassemble into HSA nanoparticles through the formation of intermolecular disulfide bonds. During this reassembling process, the ICG molecules were loaded into HSA nanoparticles. Thus the final HSA-ICG nanoparticles are composed of HSA and ICG without any other toxic chemicals, which show excellent biosafety.In vivo dual-modal imaging could clearly differentiate tumor tissue with normal tissue and thus precisely guide PTT/PDT cancer treatment. In a more recent work, Chen and coworkers reported a nanodrug that obtainedvia self-assembly of HSA, ICG, and paclitaxel (PTX, an effective antitumor drug) at a predetermined molar ratio (Figure 2(a)) [38]. The structure of this nanodrug is similar to a FDA-approved formulation (trade name: Abraxane), which takes advantages of imaging-guided PTT and chemotherapy. Both HSA-ICG nanoparticle and HSA-ICG-PTX nanodrug are composed of FDA-approved components and demonstrated great antitumor performance which may be promising photothermal theranostics for clinical applications.Figure 2
Schematic illustrations to show the formation of HSA-ICG-PTX (a), HSA@CySCOOH (b), and Au NR-PTX-HSA (c).
(a)
(b)
(c)HSA has also been exploited as a nanocarrier for other NIR dyes, such as IR825 and IR780 [39–41]. These nanosystems also showed high antitumor effects in animal experiments. Rong et al. designed a NIR cyanine dye by introducing a rigid cyclohexenyl ring to the heptamethine chain to obtain a heptamethine dye CySCOOH with higher fluorescence quantum yield and greater stability than ICG [42]. By covalent conjugation of CySCOOH onto HSA (Figure 2(b)), the as-prepared HSA@CySCOOH nanoplatform is highly efficient for multimodality imaging-guided PTT. Compared with dye-encapsulated HSA-based photothermal theranostics, the obtained HSA@CySCOOH conjugates showed several advantages such as good stability without the risk of dye leakage in the blood circulation and high dye loading efficiency.HSA contains a variety of hydrophilic and hydrophobic amino acids and a large number of functional groups, so it also can be used to modify inorganic nanoparticles by hydrophobic interactions or cross-link. Topete et al. reported a multifunctional nanotheranostic platform, which is composed of PLGA/doxorubicin (DOX)-core Au-branched shell nanostructure (BGNSHs) and HSA/ICG/folic acid complex (HSA-ICG-FA) surface coating [43]. The nanotheranostic platform would achieve multifunctional abilities, such as PTT provided by the gold nanolayer, fluorescence imaging and PDT provided by the ICG, chemotherapy provided by the DOX, and active targeting provided by the FA. Peralta et al. employed a desolvation and cross-linking approach to develop gold nanorods (Au NRs)-PTX-loaded HSA nanoparticles [44]. As shown in Figure 2(c), the PTCAs (Au NRs) and antitumor drug (PTX) were encapsulated into the HSA nanoparticles simultaneously. The resulting Au NR-PTX-HSA nanoparticles demonstrated great antitumor effect of combination therapy.


### 2.2. BSA

BSA, which derived from cows, is also a widely used carrier for cancer theranostics due to its abundance and low cost. BSA can sequester inorganic ions through a simulative biomineralization process, which trigger the formation of nanoclusters in mild conditions [45]. In a recent work, Wang et al. developed smart cypate-grafted gadolinium oxide nanocrystals (Cy-GdNCs) [46]. BSA-based biomineralization approach was employed to synthesize GdNCs, followed by the covalent conjugation of Cy to BSA. This Cy-GdNC can achieve tumor-targeting trimodal FL/PA/magnetic resonance (MR) imaging and pH-responsive PTT, resulting in effective tumor ablation. BSA also can be used as the biotemplate to synthesize inorganic PTCAs. For example, Zhang et al. synthesized copper sulfide (CuS) nanoparticles using BSA as a template [47]. This method has several benefits such as high efficiency and mild synthesis condition.In vitro andin vivo results showed that the BSA-CuS nanoparticles had excellent biocompatibility and efficient PTT effect. In addition, BSA can be used as a reductant and stabilizer for the synthesis of PTCAs. Sheng et al. developed a facile fabrication method of nanoscale reduced graphene oxide (nano-rGO) [48]. BSA, as a reductant and coating material, can reduce nano-GO to nano-rGO with high NIR absorption, high stability, and low cytotoxicity. The BSA-based nano-rGO showed excellent performance forin vivo PA imaging and PTT.BSA is also a kind of commonly used vehicle for organic PTCAs (NIR dyes, polymeric nanoparticles, etc.) delivery. For example, the squaraine, IR825, or polypyrrole loaded BSA nanoplatforms were developed for imaging-guided therapy [28, 49, 50]. Thein vivo results evidenced the feasibility of using those nanoplatforms as photothermal theranostics for multimodality imaging and phototherapy.


## 2.1. HSA

HSA, which contains 585 amino acid residues with a relative molecular weight of 66.5 KDa, is the most abundant protein in human plasma (~42 g/L) and plays an important role in transporting fatty acids, amino acids, steroids, and metal ions [36]. It is an endogenous protein, which has been approved by the US Food and Drug Administration (FDA) for intravenous administration. Furthermore, some studies showed that HSA coated nanoparticles possess active tumor-targeting abilitiesvia gp60 and SPARC receptor-mediated transcytosis [36].Recently, numerous studies have reported HSA-based photothermal nanosystems for PTT. For example, Sheng et al. reported a kind of smart nanoparticle based on HSA and indocyanine green (ICG) for dual-modal imaging-guided synergistic phototherapy [37]. ICG is a FDA-approved multifunctional NIR dye which can be used not only for NIR fluorescence (FL) imaging and photoacoustic (PA) imaging but also for PTT and photodynamic therapy (PDT). In this work, the disulfide bonds of HSA were firstly cleaved by adding excessive reducing agent to obtain free sulfhydryl groups. Then the reduced HSA can reassemble into HSA nanoparticles through the formation of intermolecular disulfide bonds. During this reassembling process, the ICG molecules were loaded into HSA nanoparticles. Thus the final HSA-ICG nanoparticles are composed of HSA and ICG without any other toxic chemicals, which show excellent biosafety.In vivo dual-modal imaging could clearly differentiate tumor tissue with normal tissue and thus precisely guide PTT/PDT cancer treatment. In a more recent work, Chen and coworkers reported a nanodrug that obtainedvia self-assembly of HSA, ICG, and paclitaxel (PTX, an effective antitumor drug) at a predetermined molar ratio (Figure 2(a)) [38]. The structure of this nanodrug is similar to a FDA-approved formulation (trade name: Abraxane), which takes advantages of imaging-guided PTT and chemotherapy. Both HSA-ICG nanoparticle and HSA-ICG-PTX nanodrug are composed of FDA-approved components and demonstrated great antitumor performance which may be promising photothermal theranostics for clinical applications.Figure 2
Schematic illustrations to show the formation of HSA-ICG-PTX (a), HSA@CySCOOH (b), and Au NR-PTX-HSA (c).
(a)
(b)
(c)HSA has also been exploited as a nanocarrier for other NIR dyes, such as IR825 and IR780 [39–41]. These nanosystems also showed high antitumor effects in animal experiments. Rong et al. designed a NIR cyanine dye by introducing a rigid cyclohexenyl ring to the heptamethine chain to obtain a heptamethine dye CySCOOH with higher fluorescence quantum yield and greater stability than ICG [42]. By covalent conjugation of CySCOOH onto HSA (Figure 2(b)), the as-prepared HSA@CySCOOH nanoplatform is highly efficient for multimodality imaging-guided PTT. Compared with dye-encapsulated HSA-based photothermal theranostics, the obtained HSA@CySCOOH conjugates showed several advantages such as good stability without the risk of dye leakage in the blood circulation and high dye loading efficiency.HSA contains a variety of hydrophilic and hydrophobic amino acids and a large number of functional groups, so it also can be used to modify inorganic nanoparticles by hydrophobic interactions or cross-link. Topete et al. reported a multifunctional nanotheranostic platform, which is composed of PLGA/doxorubicin (DOX)-core Au-branched shell nanostructure (BGNSHs) and HSA/ICG/folic acid complex (HSA-ICG-FA) surface coating [43]. The nanotheranostic platform would achieve multifunctional abilities, such as PTT provided by the gold nanolayer, fluorescence imaging and PDT provided by the ICG, chemotherapy provided by the DOX, and active targeting provided by the FA. Peralta et al. employed a desolvation and cross-linking approach to develop gold nanorods (Au NRs)-PTX-loaded HSA nanoparticles [44]. As shown in Figure 2(c), the PTCAs (Au NRs) and antitumor drug (PTX) were encapsulated into the HSA nanoparticles simultaneously. The resulting Au NR-PTX-HSA nanoparticles demonstrated great antitumor effect of combination therapy.


## 2.2. BSA

BSA, which derived from cows, is also a widely used carrier for cancer theranostics due to its abundance and low cost. BSA can sequester inorganic ions through a simulative biomineralization process, which trigger the formation of nanoclusters in mild conditions [45]. In a recent work, Wang et al. developed smart cypate-grafted gadolinium oxide nanocrystals (Cy-GdNCs) [46]. BSA-based biomineralization approach was employed to synthesize GdNCs, followed by the covalent conjugation of Cy to BSA. This Cy-GdNC can achieve tumor-targeting trimodal FL/PA/magnetic resonance (MR) imaging and pH-responsive PTT, resulting in effective tumor ablation. BSA also can be used as the biotemplate to synthesize inorganic PTCAs. For example, Zhang et al. synthesized copper sulfide (CuS) nanoparticles using BSA as a template [47]. This method has several benefits such as high efficiency and mild synthesis condition.In vitro andin vivo results showed that the BSA-CuS nanoparticles had excellent biocompatibility and efficient PTT effect. In addition, BSA can be used as a reductant and stabilizer for the synthesis of PTCAs. Sheng et al. developed a facile fabrication method of nanoscale reduced graphene oxide (nano-rGO) [48]. BSA, as a reductant and coating material, can reduce nano-GO to nano-rGO with high NIR absorption, high stability, and low cytotoxicity. The BSA-based nano-rGO showed excellent performance forin vivo PA imaging and PTT.BSA is also a kind of commonly used vehicle for organic PTCAs (NIR dyes, polymeric nanoparticles, etc.) delivery. For example, the squaraine, IR825, or polypyrrole loaded BSA nanoplatforms were developed for imaging-guided therapy [28, 49, 50]. Thein vivo results evidenced the feasibility of using those nanoplatforms as photothermal theranostics for multimodality imaging and phototherapy.


## 3. Other Proteins-Based Photothermal Theranostics

Besides serum albumin, several other proteins with special molecular structures or functions, such as ferritin, hepatitis B virus (HBV) core protein, gelatin, antibodies, and phycocyanin (PC), have been used as building blocks for the construction of protein-based photothermal theranostics.


### 3.1. Ferritin

Ferritin, a protein of 450 kDa consisting of 24 subunits, has a nanocage structure with external and internal diameters of 12 and 8 nm, respectively. Ferritin serves to store iron in a nontoxic form, to deposit it in a safe form, and to transport it to areas where it is required. Because of the cage-like structure, ferritin has also been successfully used as nanocarrier of therapeutic agent or biotemplate for the fabrication of nanoparticles [23, 24]. The internal cavity allows high loading efficiency of therapeutic agents and the protein shells as a coating to prevent coagulation/aggregation between nanoparticles.Recently, Huang et al. reported a novel theranostic platform based on IR820 (NIR dye)-loaded ferritin nanocages with strong absorbance in the NIR region [51]. IR820 molecules were loaded into the internal cavity by “opening” and “closing” the ferritin nanocages. Under 550 nm light irradiation, the theranostic platform can achieve FL imaging with high fluorescence quantum yield, while under 808 nm NIR light irradiation, the theranostic platform can achieve PA imaging and PTT with high photothermal conversion efficiency.In vivo results showed that 100% tumor elimination was achieved by intravenous injection of IR820-loaded ferritin nanocages, under a low laser power density of 0.5 W/cm2. In another work, Wang et al. reported biomineralization-inspired synthesis and photothermal application of CuS/C
u
S
64-ferritin nanocages [52]. As shown in Figure 3(a), Cu and C
u
64 ions were firstly encapsulated into the ferritin cages. After the addition of S ions, CuS/C
u
S
64-Fn nanocages were obtained. Thein vitro (Figure 3(b)) andin vivo (Figures 3(c)–3(e)) results indicated that the CuS-Fn nanocages achieved superior tumor therapeutic efficiency without systemic toxicity.Figure 3
(a) Schematic of the synthesis process. (b) Fluorescence images of Calcein AM/PI costained U87MG cells after different treatments. Tumor growth (c), body weight changes (d), and survival rate (e) of U87MG tumor-bearing mice after different treatments. Copyright from American Chemical Society (ACS), 2016 [52].
(a)
(b)
(c)
(d)
(e)


### 3.2. HBV Core Protein

Protein-based particles that are self-assembled inside cells have been developed for a variety of bioassays and biomedical applications [63]. For example, when expressed inEscherichia coli, the HBV core protein forms a core shell capsid with a diameter of 36 nm. Based on the core shell capsid, Kwon et al. developed a protein/gold core/shell nanoparticle (PGCS-NP) that can be used as a PTCA for targeted cancer therapy withoutin vivo toxicity (Figure 4) [53]. Hexahistidine (H6), biotinylated peptide (BP), hexatyrosine (Y6), and affibody peptides for human epidermal growth factor receptor I (EGFR) were used to modify the HBV core protein. Then the modified HBV core proteins were self-assembled into the engineered particles, and gold ions are reduced to gold dots (1–3 nm) by the reduction potential of Y6 residues. The resulted nanoplatform has uniform particle size distribution, high colloidal stability, and excellent photothermal activity. Most importantly, as the nanoplatform was disassembled with time, the small gold dots were released and rapidly excreted from liver and kidney, indicating that the nanoplatform can be used as safe PTCAs without concern of long-term toxicity.Figure 4
Schematic illustration showing the biosynthesis of PGCS-NP. Copyright from Wiley, 2014 [53].


### 3.3. Gelatin

Gelatin, an irreversibly hydrolyzed form of collagen, is a mixture of proteins and peptides obtained from the skin, bones, and connective tissues of various animals. Gelatin has many excellent properties such as easy functionalization and good biocompatibility and can be effectively digested by gelatinase into nontoxic peptides. Zha et al. developed multifunctional CuS nanoparticles stabilized with DOX-conjugated gelatin (CuS@Gel-DOX) [54]. In this case, CuS nanoparticle was used as the PTCA and PA contrast agent; DOX was used as a model antitumor drug. Thus the as-prepared CuS@Gel-DOX nanosystem can achieve simultaneous PA imaging, PTT, and enzyme-responsive drug release. Lin et al. developed nanocomposites that incorporated Cu9S5@mSiO2 nanoparticles or polyaniline nanoparticles into poly(ε-caprolactone) and gelatin [55, 56]. The as-prepared nanocomposites were surgically implanted into tumors in mice and followed by efficient PTT treatment.


### 3.4. Antibody

Antibody, also known as immunoglobulin, is a kind of large, Y-shape protein produced by plasma cells that is used by the immune system to identify and neutralize pathogens. In recent years, some antibodies (anti-EGFR) were also used as tumor-targeting ligands, since the EGFR is overexpressed in many malignant cells. Yu et al. reported a kind of anti-EGFR-coated ICG-loaded polymer/salt nanocapsule forin vitro PTT [57]. The results indicated that the anti-EGFR-coated nanocapsules had significant thermal toxicity upon 808 nm NIR light irradiation. In another work, Zhang et al. developed the EGFR monoclonal antibody (mAb) modified AuNRs (EGFRmAb-AuNR) for targeted PTT on Hep-2 cells [58].


### 3.5. PC

PC, a pigment-protein complex from the light-harvesting phycobiliprotein family, is a water-soluble protein and is frequently used as a coloring agent in food and cosmetics. It also can be used as a photosensitizer for generating reactive oxygen species (ROS) under excitation of appropriate light [64]. Recently, Liao and Zhang developed a multiwalled carbon nanotube-chitosan-PC complex (MWNT-CS-PC) for PTT and PDT [59]. Both CS and PC improve the water solubility and stability of MWNTs. Additionally, PC is a photosensitizer that is able to generate cytotoxic ROS for PDT, and MWNT is a PTCA that is able to generate heat for PTT.


## 3.1. Ferritin

Ferritin, a protein of 450 kDa consisting of 24 subunits, has a nanocage structure with external and internal diameters of 12 and 8 nm, respectively. Ferritin serves to store iron in a nontoxic form, to deposit it in a safe form, and to transport it to areas where it is required. Because of the cage-like structure, ferritin has also been successfully used as nanocarrier of therapeutic agent or biotemplate for the fabrication of nanoparticles [23, 24]. The internal cavity allows high loading efficiency of therapeutic agents and the protein shells as a coating to prevent coagulation/aggregation between nanoparticles.Recently, Huang et al. reported a novel theranostic platform based on IR820 (NIR dye)-loaded ferritin nanocages with strong absorbance in the NIR region [51]. IR820 molecules were loaded into the internal cavity by “opening” and “closing” the ferritin nanocages. Under 550 nm light irradiation, the theranostic platform can achieve FL imaging with high fluorescence quantum yield, while under 808 nm NIR light irradiation, the theranostic platform can achieve PA imaging and PTT with high photothermal conversion efficiency.In vivo results showed that 100% tumor elimination was achieved by intravenous injection of IR820-loaded ferritin nanocages, under a low laser power density of 0.5 W/cm2. In another work, Wang et al. reported biomineralization-inspired synthesis and photothermal application of CuS/C
u
S
64-ferritin nanocages [52]. As shown in Figure 3(a), Cu and C
u
64 ions were firstly encapsulated into the ferritin cages. After the addition of S ions, CuS/C
u
S
64-Fn nanocages were obtained. Thein vitro (Figure 3(b)) andin vivo (Figures 3(c)–3(e)) results indicated that the CuS-Fn nanocages achieved superior tumor therapeutic efficiency without systemic toxicity.Figure 3
(a) Schematic of the synthesis process. (b) Fluorescence images of Calcein AM/PI costained U87MG cells after different treatments. Tumor growth (c), body weight changes (d), and survival rate (e) of U87MG tumor-bearing mice after different treatments. Copyright from American Chemical Society (ACS), 2016 [52].
(a)
(b)
(c)
(d)
(e)


## 3.2. HBV Core Protein

Protein-based particles that are self-assembled inside cells have been developed for a variety of bioassays and biomedical applications [63]. For example, when expressed inEscherichia coli, the HBV core protein forms a core shell capsid with a diameter of 36 nm. Based on the core shell capsid, Kwon et al. developed a protein/gold core/shell nanoparticle (PGCS-NP) that can be used as a PTCA for targeted cancer therapy withoutin vivo toxicity (Figure 4) [53]. Hexahistidine (H6), biotinylated peptide (BP), hexatyrosine (Y6), and affibody peptides for human epidermal growth factor receptor I (EGFR) were used to modify the HBV core protein. Then the modified HBV core proteins were self-assembled into the engineered particles, and gold ions are reduced to gold dots (1–3 nm) by the reduction potential of Y6 residues. The resulted nanoplatform has uniform particle size distribution, high colloidal stability, and excellent photothermal activity. Most importantly, as the nanoplatform was disassembled with time, the small gold dots were released and rapidly excreted from liver and kidney, indicating that the nanoplatform can be used as safe PTCAs without concern of long-term toxicity.Figure 4
Schematic illustration showing the biosynthesis of PGCS-NP. Copyright from Wiley, 2014 [53].


## 3.3. Gelatin

Gelatin, an irreversibly hydrolyzed form of collagen, is a mixture of proteins and peptides obtained from the skin, bones, and connective tissues of various animals. Gelatin has many excellent properties such as easy functionalization and good biocompatibility and can be effectively digested by gelatinase into nontoxic peptides. Zha et al. developed multifunctional CuS nanoparticles stabilized with DOX-conjugated gelatin (CuS@Gel-DOX) [54]. In this case, CuS nanoparticle was used as the PTCA and PA contrast agent; DOX was used as a model antitumor drug. Thus the as-prepared CuS@Gel-DOX nanosystem can achieve simultaneous PA imaging, PTT, and enzyme-responsive drug release. Lin et al. developed nanocomposites that incorporated Cu9S5@mSiO2 nanoparticles or polyaniline nanoparticles into poly(ε-caprolactone) and gelatin [55, 56]. The as-prepared nanocomposites were surgically implanted into tumors in mice and followed by efficient PTT treatment.


## 3.4. Antibody

Antibody, also known as immunoglobulin, is a kind of large, Y-shape protein produced by plasma cells that is used by the immune system to identify and neutralize pathogens. In recent years, some antibodies (anti-EGFR) were also used as tumor-targeting ligands, since the EGFR is overexpressed in many malignant cells. Yu et al. reported a kind of anti-EGFR-coated ICG-loaded polymer/salt nanocapsule forin vitro PTT [57]. The results indicated that the anti-EGFR-coated nanocapsules had significant thermal toxicity upon 808 nm NIR light irradiation. In another work, Zhang et al. developed the EGFR monoclonal antibody (mAb) modified AuNRs (EGFRmAb-AuNR) for targeted PTT on Hep-2 cells [58].


## 3.5. PC

PC, a pigment-protein complex from the light-harvesting phycobiliprotein family, is a water-soluble protein and is frequently used as a coloring agent in food and cosmetics. It also can be used as a photosensitizer for generating reactive oxygen species (ROS) under excitation of appropriate light [64]. Recently, Liao and Zhang developed a multiwalled carbon nanotube-chitosan-PC complex (MWNT-CS-PC) for PTT and PDT [59]. Both CS and PC improve the water solubility and stability of MWNTs. Additionally, PC is a photosensitizer that is able to generate cytotoxic ROS for PDT, and MWNT is a PTCA that is able to generate heat for PTT.


## 4. Peptides-Based Photothermal Theranostics

Peptides are short chains of amino acid monomers linked by peptide bonds. Proteins are composed of one or more peptides arranged in a biologically functional way. The boundaries that distinguish peptides from proteins are not absolute: long peptides such as amyloid beta have been referred to as proteins, and smaller proteins like insulin have been considered peptides. Like proteins, peptides were also employed as building blocks for the development of photothermal theranostics. Huang et al. developed an endogenous alkaline phosphatase- (ALP-) triggered coassembly strategy to form tumor-specific nanofibers, which based on ICG and NapFFKYp (an ALP-responsive peptide), for FL/PA dual-modality imaging-guided PTT (Figure5) [60]. The ICG-doped nanofiber showed significantly enhanced NIR absorbance and unique PA and PTT properties. Synthetic polypeptides are also promising nanocarriers of PTCAs because they own similar component and structure to natural proteins. For example, Yang et al. reported an ICG and cypate-loaded poly(L-aspartic acid)-based micelle system with multiple advantages for FL imaging and PTT [61]. These micelles exhibit high loading capacity, good stability, enhanced cellular uptake efficiency, and efficient photothermal efficacy. Wu et al. reported ICG-loaded polymeric micelles, which self-assembled from amphiphilic poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-leucine) (PEG-PLL-PLLeu), for tumor imaging and PTT [62]. Compared with free ICG, PEG-PLL-PLLeu-ICG micelles significantly improved the quantum yield and fluorescent stability of ICG.In vitro photothermal ablation studies also proved the feasibility of the PEG-PLL-PLLeu-ICG for tumor PTT.Figure 5
(a) Scheme ofin situ conversion of micelles to nanofiber after intravenous injection. TEM images of the negatively stained (b) micelles (scale bar: 1 μm) and (c) nanofiber (scale bar: 100 nm). (d) Calcein AM and PI costaining of HeLa cells incubated with nanofiber for 4 h with or without 808 nm laser irradiation (1 W/cm2, 5 min). (e) Photographs of HeLa-tumor-bearing mice at different days after PTT treatment. (f) Tumor growth curves of different groups of HeLa-tumor-bearing mice after PTT treatment. Copyright from American Chemical Society (ACS), 2015 [60] p
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## 5. Conclusion and Perspective

Natural proteins and synthetic peptides are playing important roles in cancer theranostics. A variety of protein-based theranostics have emerged in the past few years. The capabilities of proteins to load different PTCAs endow them with many advantages, such as improved water solubility and biocompatibility, prolonged blood circulation time, and multifunctional modification.However, the clinical translation of protein-based photothermal theranostics is still facing many challenges. Firstly, as natural polymers, proteins are heterogeneous mixtures and exhibit batch-to-batch variation, which may hinder the industrial application of protein-based nanoplatforms [35]. Secondly, although many photothermal nanoplatforms have demonstrated their high PTT efficacy in preclinical animal experiments, there are rarely FDA-approved proteins and PTCAs that are safe for use in humans. Thirdly, the poor photothermal stability of protein-organic agents and the potential long-term toxicity of protein-inorganic agents severely limit their future clinical applications. In addition, the PTCAs loading efficiency of most protein-based nanoplatforms are often too low, resulting in limited PTT efficacy.Studies will continue to further improve the clinical applicability of protein-based photothermal theranostics. For example, the recombinant protein technology is a promising strategy to overcome the batch-to-batch variation of protein-based platforms. More and more protein-based photothermal theranostics with high photothermal stability, great long-term biosafety, and high tumor accumulation have been developed for PTT. Although the development of PTT is still at an early stage, protein-based photothermal theranostics may have great potential for clinical applications to treat cancer patients in the near future.



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