Research progress of natural silk fibroin and the application for drug delivery in chemotherapies

Silk fibroin has been widely used in biological fields due to its biocompatibility, mechanical properties, biodegradability, and safety. Recently, silk fibroin as a drug carrier was developed rapidly and achieved remarkable progress in cancer treatment. The silk fibroin-based delivery system could effectively kill tumor cells without significant side effects and drug resistance. However, few studies have been reported on silk fibroin delivery systems for antitumor therapy. The advancement of silk fibroin-based drug delivery systems research and its applications in cancer therapy are highlighted in this study. The properties, applications, private opinions, and future prospects of silk fibroin carriers are discussed to understand better the development of anti-cancer drug delivery systems, which may also contribute to advancing silk fibroin innovation.

Cytotoxic drugs are well-known anti-tumor drugs, such as cytarabine, gemcitabine, docetaxel, and paclitaxel. It has a very strong killing effect on tumor cells but will also kill normal cells in the human body (Al Alawi et al., 2020). This is especially obvious for the increase of toxic and side effects and the decline of quality of life in the later stage of patients. Therefore, reducing their inhibition and killing effects on normal cells and improving their targeting and therapeutic effects on tumor cells are the focus of current research. In recent years, research on drug preparation oriented to precise drug delivery is expected to solve the problem of cytotoxic drugs. It is of great value to develop new pharmaceutical strategies for tumor treatment, and the establishment and application of new drug delivery systems are exceptionally prominent for improving cytotoxic drug problems (Mazzaferro et al., 2013). For cytotoxic drugs, the new delivery systems can maintain or even increase the therapeutic efficacy and reduce toxic and side effects. Moreover, the targeted drug delivery system can accurately target tumor cells and improve bioavailability and selectivity, becoming one of the possible directions for cytotoxic drugs to be used for tumor treatment (Hitchcock et al., 2020).
Synthetic and natural polymers are primarily used as a carrier for drug delivery, including polylactic acid (PLA), lactic acid glycolic acid copolymer (PLGA), silk fibroin, chitosan, and corn protein (Estey et al., 2006;Pascoli et al., 2018). In general, natural polymer materials have good biocompatibility and biodegradability and are feasible to be a carrier for constructing nano-drug delivery systems (Mathur and Gupta, 2010;Mohammed et al., 2017). Silk fibroin, a medical material approved by the US Food and Drug Administration (FDA), has been widely used in the postoperative suture, tissue regeneration, and drug delivery system (Yucel et al., 2014). On the basis of the advantages of excellent mechanical properties and good biocompatibility and biodegradability, drug loaded nanoparticles can be prepared by using silk fibroin to acquire various biological functions (Qi et al., 2017). However, there are no

Types
Active pharmaceutic ingredient Trademark Types of tumor First approval anti-tumor nanomedicine with silk fibroin as drug carrier on the market yet. Currently, only albumin and silk fibroin are close in characteristics and categories among the anti-tumor nanomedicine approved for marketing, providing some reference and basis for the clinical transformation of silk fibroin in the future nanomedicine. Some of approved anti-tumor nanomedicines are shown in Table 1.
Herein the structure, properties, applications and preparation of silk fibroin nanoparticles were introduced to get better understanding of silk fibroin and its application in anti-tumor drug delivery system. The research progress and achievements of silk fibroin as a carrier material of drug delivery system in tumor drug therapy in the past decade were also summarized and discussed. Finally, we further analyzed the current problems and future application prospects of silk fibroin and put forward some personal ideas.
2 Major characteristics of silk fibroin

The chemical structure
Silk fibroin is the main component of silk, accounting for about 75% of the total weight of silk. Silk fibroin consists of two leading chains, a heavy (H-) chain (390 kDa) and a light (L-) chain (26 kDa), which are linked by disulfide bonds to form the FIGURE 1 Schematic diagram of the silk structure. (A) heavy chain (i.e., N-terminus, β-sheets, Amorphous and C-terminus). and light chain which linked via disulphide bonds. (B) silkworm thread, fibril overall structure and silk fibroin polypeptide chains. Notes: Sun, W., Gregory, D.A., Tomeh, M.A., Zhao, X., 2021. Silk fibroin as a functional biomaterial for tissue engineering. International Journal of Molecular Sciences 22, 1499. Reprinted with permission from references. Copyright 2021, MDPI.
HL complex ( Figure 1A) (Holland et al., 2019;Sun et al., 2021). P25 (25 kDa) is a glycoprotein, including ASN linked oligosaccharide chain, which is hydrophobically linked to H-L complex (Tanaka et al., 1999). Three structural protein subsets (heavy chain, light chain, and glycoprotein P25) comprise silk fibroin with a molecular ratio of 6:6:1 (Koh et al., 2015). Among them, the H-chain of silk fibroin has amphiphilic properties, including hydrophobic and hydrophilic blocks (Gholipourmalekabadi et al., 2020). The hydrophilic region is short and non-repetitive, but the hydrophobic region has repetitive sequences. It can be folded into a β-sheet to generate a crystal structure, so silk fibroin can be considered a hydrophobic glycoprotein and is insoluble in water (Bini et al., 2004).
Silk fibroin has two main crystal structures, including silk I and silk II ( Figure 1B). Silk I is a metastable crystal structure containing bound water molecules and is intermediate between α-helix and β-fold. Silk I is hydrophilic and thermodynamically unstable. Silk II is the most stable state and is an antiparallel β-folded lamellar structure due to strong hydrogen bonds between adjacent peptide blocks, resulting in increased mechanical properties, including stiffness and tensile strength (Sun et al., 2021). On the contrary, silk II is hydrophobic and thermodynamically stable. Besides, the water solubility of silk fibroin was significantly related to the crystalline state and environmental pH value. Because silk I is more soluble than silk II, and the methods of transforming insoluble silk II into silk I can increase the water solubility of silk fibroin (Pham and Tiyaboonchai, 2020). Similarly, silk I can also be converted into silk II by changing solution conditions and ambient temperatures, such as temperature, pH, methanol, and potassium chloride, which can induce this conversion (Rusa et al., 2005;Chen et al., 2007;Huot et al., 2015;Ming et al., 2015;Gregory et al., 2016). Due to the role of hydrogen bonds, it is relatively challenging to convert silk II to silk I, and it can only be converted after LiBr, strong acid or urea is used to break the hydrogen bonds (Chen et al., 2007;Liu et al., 2011;Zheng et al., 2016). The theoretical isoelectric point pI of silk fibroin is 4.53. When the environmental pH is near the isoelectric point, the water solubility of silk fibroin will decrease (Lammel et al., 2010). Based on silk fibroin's water solubility, organic solvents can be reduced during the preparation, thus reducing the impact on the encapsulated drugs. Nowadays, this technique is widely used for biomaterial engineering applications, including the silk fibroin nanoparticles based on transformation from silk I with a random coil and α-helix structure to the highly ordered βsheet structure of silk II (Ma et al., 2020). Moreover, the regenerated silk fibroin solution has a small amount of unstable silk III in the air/water interface, which is a triple helical crystal structure (Valluzzi et al., 1999;Pham et al., 2019).

Biocompatibility and safety
As a kind of natural fiber, the medical use of silk as a wound suture can be traced back hundreds of years. Nowadays, biocompatible and safety are the main concern for silk fibroin used as a drug delivery carrier. Degummed silk and regenerated silk fibroin are considered biological materials suitable for animal cell culture, which has been confirmed for a long time (Minoura et al., 1995;Lazaris et al., 2002). Wang et al. used a vacuum ultraviolet-ozone activation method to wrap silk fibroin on the surface of MgZnCa alloy as a protective barrier and then implanted it in cells and rabbits to evaluate the corrosion resistance and biocompatibility of MgZnCa alloy (Wang et al., 2019a). The tests showed that this silk fibroin-coated MgZnCa had improved biocompatibility with bone marrow mesenchymal stem cells. According to another study about the stents made of silk implanted in mice, almost no inflammation and immune response were found after 1 year of feeding (Wang et al., 2008). Silk fibroin is a natural high-purity protein secreted and synthesized by endothelial cells on the inner wall of silkworm silk gland. The body can absorb its final degradation product, and its molecular weight can also be adjusted by modifying the composition of silk fibroin to meet the requirements of different biological environments (Guo et al., 2021). Therefore, it has good biocompatibility.
Some researchers have discussed the safety and effectiveness of microparticle scaffold prepared from silk fibroin for bone void filling (Deshpande et al., 2022). In vitro and vivo studies showed that this scaffold was biocompatible through cytotoxicity, immunogenicity, genotoxicity, systemic toxicity, and implantation. At the same time, compared with commercial calcium-based bone gap fillers, microparticle scaffold prepared from silk fibroin is non-toxic for bone gap filling and contributes to the good healing of fracture defects. In addition, the effectiveness and safety of engineered silk fibroin and collagen tissue have been evaluated in patients with bone defects. The results showed that it is safe and effective and suggested that a larger sample of randomized controlled trials can be carried out in the next stage (Sharafat-Vaziri et al., 2020). The latest study reported that inhalable ciprofloxacin hydrochloride microparticles (CMs) based on silk fibroin and mannitol . A series of in vitro and in vivo performance evaluation tests demonstrated that CMs did not affect lung function nor induce the secretion of inflammatory cytokines in the lungs. CMs, thus would be a promising pulmonary drug delivery system for the treatment of non-cystic fibrosis bronchiectasis. All these findings revealed that silk fibroin is a kind of natural polymer material that has a prominent advantage in the application of drug carriers due to its excellent biocompatibility and biosafety.

Mechanical properties and biodegradability
Four secondary structures of silk fibroin exist, including βsheet, random coil, α-helix, and β-turn (Choi et al., 2018). Among them, the β-sheet structure in the crystalline region of silk fibroin is the main factor that plays a vital role in the excellent flexibility and tensile resistance of silk, and the toughness is higher than other synthetic fibers, such as carbon fiber, collagen (Gosline et al., 1999;Cheng et al., 2014;Choi et al., 2018;Blamires et al., 2020). Although natural silk fibroin has good mechanical properties, the biological materials made of silk fibroin, such as scaffolds, are made by regenerating silk fibroin solution. However, these scaffolds are highly fragile because the regenerated silk fibroin lacks appropriate secondary and hierarchical structures (Altman et al., 2002). In order to deal with the problem of poor mechanical properties of regenerated silk fibroin, some technology methods are used to manipulate the regeneration process to improve its mechanical capabilities, such as porogen and 3D bioprinting technology (Nazarov et al., 2004;Floren et al., 2016).
When exogenous substances enter the body, they generally cause immunogenic reactions. However, after degumming, silk fibroin does not contain sericin, so it will not produce immunogenicity (Florczak et al., 2021). Some studies have found that pure sericin can be used as a material with good biocompatibility, so the coexistence of silk fibroin and sericin may lead to an immune response (Chirila et al., 2013). On the contrary, silk fibroin is a polymer that enzymes can degrade, and its degradation products also do not have immunogenicity (Cao and Wang, 2009). Although these views are not consistent, they allow people to achieve different purposes of use by regulating the structure and performance of silk fibroin. It can be degraded by enzymes such as α-chymotrypsin, protease XIV and collagenase IA (Sun et al., 2021). Furthermore, its degradation rate is affected by the material's microstructure, and the degradation rate is the random structure part, silk I structure, and silk II structure in the material from fast to slow (Umuhoza et al., 2020). Lee et al. investigated the degradation behavior of silk fibroin membranes in vitro and in vivo. They found that they were degraded in phosphate-buffered saline, medium and proteinase K solution (Lee et al., 2012). In protease K solution, 80% of this membrane was degraded within 10 days. Meanwhile, another study found also confirmed that, more importantly, its degradation products were also non-toxic and harmless (Lu et al., 2011). Some scholars synthesized silk fibroin hydrogels through a chemical crosslinking reaction of silk fibroin (Wu et al., 2020). By γ-ray irradiation at different doses, the chemically cross-linked silk fibroin exhibited better elasticity and better biodegradability and biocompatibility than the logistically cross-linked hydrogels. In the assay of cell proliferation, the study also evaluated the cell viability of human mesenchymal stem cells on freeze-dried silk fibroin hydrogel scaffolds. No significant cytotoxicity to human mesenchymal stem cells was observed.

Applications in drug delivery system
Due to the unique properties mentioned above, silk fibroin has been widely used as carrier material for various drug delivery systems. In fact, silk fibroin is usually prepared into diverse structural forms with disparate properties to adapt to various drug delivery systems, including hydrogels, porous scaffolds, rods, fibers, and nanoparticles (Zhao et al., 2015). Meanwhile, the drugs loaded could be the first-line drugs used in the clinic such as doxorubicin, and dexamethasone sodium, as well as some Chinese medicine monomers such as quercetin, curcumin (Kim et al., 2014). Since carrier forms of silk fibroin are various, the objects of action and diseases are also different. Therefore, the application of more detailed structural types of silk fibroin is shown in Table 2.
Nanoparticles based on silk fibroin are the most widely used and studied due to the simple and diverse processing technics and the improved in vivo ADME properties steming from the ideal size. The small size usually allows nanoparticles to penetrate tiny capillaries, thereby enhancing the cellular uptake of encapsulated therapeutic molecules (Mathur and Gupta, 2010;Zhao et al., 2015). Moreover, silk fibroin-based nanoparticles may deliver anti-cancer agents to tumor sites and thus have great potential for targeted drug delivery systems. Farokhi et al. also discussed the application of silk fibroin as electrospun nanofibers in the drug delivery system. For example, antibiotics, vitamins, and growth factors have been delivered using silk fibroin electrospun nanofibers as carrier materials and achieved remarkable results (Farokhi et al., 2020). In recent years, silk fibroin as a drug delivery carrier material has made significant development in the field of medicine, including HIV (Crakes et al., 2020), vaccine (Stinson et al., 2020), bone regeneration (Xin et al., 2019), rheumatoid arthritis (Oliveira et al., 2020).
We know that nanoparticles are a common structural form. As a new biomaterial, it is more and more widely used in the medical field. It has significant advantages, such as prolonging the half-life of drugs, reducing the dosage and frequency of administration, enhancing the curative effect, reducing the toxicity of drugs, increasing the accumulation of drugs in tissues, and realizing diversified routes of Administration Hu et al., 2020a;Zhao et al., 2021). The preparation of silk fibroin nanoparticles has become a current hotspot. The desolvation method is the most common method to prepare silk fibroin drug-loaded nanoparticles. It is the process of spontaneously forming insoluble nanoparticles after mixing an aqueous silk fibroin solution with a water-miscible organic solvent. At the same time, in order to meet the requirements of silk fibroin nanoparticles for different purposes, most drugs are connected with silk fibroin through physical adsorption or chemical crosslinking to prepare nanoparticles (Montalbán et al., 2018). However, chemical modification is required to connect silk fibroin with other carrier materials when preparing composite carriers before drug loading. Zhang et al. added 5.0% regenerated silk fibroin solution into water-miscible organic solvents such as acetone and ethanol, the organic solvent volume accounted for at least 70% of the final volume, and silk fibroin was suspended in the mixture of water and organic solvent to form Tiny spherical particles with good crystallinity, nanoparticles spontaneously aggregate to produce slow sedimentation, and finally the sediment can be collected and purified from the mixture by centrifugation and filtration to obtain particles with a particle size ranging from 35 to 125 nm (Zhang et al., 2007). Some scholars used electrospraying to prepare silk fibroin nanoparticles and connected them to the syringe through the nozzle of the high-voltage electrostatic generator, and fixed the whole assembly to the microinjection pump (Qu et al., 2014). Silk fibroin solution was differentiated into droplets under a high-voltage electrostatic field, and then the resulting droplets were continuously collected and frozen in liquid nitrogen. Subsequently, it was freeze-dried in a lyophilizer for 48 h. In order to separate the nano-sized particles, the lyophilized products were suspended in deionized water, then centrifuged, and lyophilized again to obtain silk fibroin nanoparticles (59-75 nm). In addition, we also summarize and describe other standard preparation methods, and their advantages and disadvantages are also shown in Table 3. In these methods, we also find that the size of silk fibroin nanoparticles ranges from 20 nm to 20 μm (Florczak et al., 2021). 4 Drug delivery system for chemotherapy of silk fibroin As mentioned above, the recent main antitumor treatment methods mainly include surgery, radiotherapy, chemotherapy, immunotherapy, etc. Each method has its advantages and disadvantages (Gao et al., 2014;Gao and Lo, 2018;Kaushik et al., 2019;Luo et al., 2019;Boreel et al., 2021). Nevertheless, most patients choose drugs for tumor treatment, such as cytotoxic drugs, but some patients also choose targeted drugs, traditional Chinese medicine decoction, and so on Spiliopoulou et al., 2021;Cortés et al., 2022). However, many anticancer drugs potentially cause toxicity in patients, resulting in high drug resistance and poor prognosis quality of life Bakos et al., 2018;Messina et al., 2019;Abravan et al., 2020). A novel cancer therapy strategy is urgently needed. Drug delivery systems are becoming hotspots since they could reduce adverse reactions, and improve efficacy and safety, Frontiers in Pharmacology frontiersin.org especially in cytotoxic drugs. There seem two primary reasons. First, the advent of albumin-paclitaxel, paclitaxel is a cytotoxic drug, and albumin-paclitaxel as a nano-drug has been successfully marketed and used in clinical practice, which has extensively promoted the development and progress of drug delivery systems (Lv et al., 2021). Secondly, cytotoxic drugs have serious side effects, and some drugs even have problems such as poor water solubility and short half-life, which limit their application (Newell and Sausville, 2016). In order to further solve the use of cytotoxic drugs during chemotherapy, the application of biomaterials in drug delivery systems is an emerging technology (Mancipe Castro et al., 2021). Drug delivery systems prepared by loading cytotoxic drugs with biomaterials can reduce these problems, improve their therapeutic effects, and expand their clinical applications (Al-Mansoori et al., 2021). Therefore, we believe that cytotoxic drugs will gradually become the focus of drug delivery systems. Additionally, drug carrier materials are a determinant in delivery systems. PLA or PLGA was used to build drug delivery systems in many studies and approved by FDA for drug injection. Although various nanocarrier systems are available in the market, a gap exists in drug delivery systems based on different carriers (Aggarwal et al., 2006). Nanotechnology can be applied to the delivery of drugs, providing a new way of cancer treatment. In the past decade, it has been found that materials that can be used as drug carriers include polyurethane functionalized starch (Desai et al., 2021), ferritin (He et al., 2019), gold nanoparticles (AuNPs) , cyclodextrin (Kang et al., 2019), and silk fibroin (Kapoor and Kundu, 2016;Melke et al., 2016). Among them, silk fibroin, a typical natural biomaterial, exhibits versatile properties through tuning the secondary conformations and hierarchical microstructures (Ma et al., 2020). Silk fibroin can be prepared into various drug delivery systems to meet the different requirements of cancer treatment (Wani and Veerabhadrappa, 2018;Xu et al., 2019;Zuluaga-Vélez et al., 2021). In addition, it also has shown promising potential as a carrier in tissue repair (Gholipourmalekabadi et al., 2020), bone defects (Hong et al., 2020a), myocardial infarction (Chen et al., 2018b), nerve repair (Fernández-García et al., 2016), colitis (Diez-Echave et al., 2021), infections (Hassani Besheli et al., 2017), acute pancreatitis (Hassanzadeh et al., 2021), osteoporosis , and cancers (Xie et al., 2016). At present, silk fibroin has been studied and analyzed in the field of tumor as a carrier material for drug delivery.

Breast cancer
Docetaxel can be used to treat breast cancer. However, low water solubility and systemic toxicity seriously hinder its clinical application. Researchers prepared docetaxel-loaded silk fibroin nanoparticles and analyzed their potential antitumor activity against breast cancer cell lines (Al Saqr et al., 2021). The diameter of these nanoparticles was 178-198 nm, with net negative surface charge and encapsulation efficiency ranging from 56% to 72%. In vitro release study showed that the nano-formulation had a biphasic release curve and could release the drug for 72 h. In vitro cell study revealed that compared with free drugs, the nano-formulation significantly increased the cytotoxic potential of breast cancer cell lines and enhanced the uptake of docetaxel by breast cancer cells (Figure 2). Furthermore, the accumulation of cells treated with nanoformulation in the G2/M phase was significantly higher than that of cells treated with free docetaxel. Compared with free docetaxel, the nano-formulation loaded with docetaxel has excellent antitumor activity on breast cancer cells. The results also emphasized the feasibility of using silk fibroin-made nanoparticles as a non-toxic and biocompatible delivery carrier to enhance breast cancer treatment. At the same time, silk fibroin has good biocompatibility and can be used as composite materials with other biological materials for drug delivery systems and targeted formulation development. Arumugam et al. made silk fibroin, cellulose acetate, and gold and silver nanoparticles (CA/SF/Au-Ag) into composite nanofibers. They explored their toxicity to MCF-7 and MDA-MB-231 human breast cancer cells (Arumugam et al., 2021). The study found that CA/SF/Au-Ag nanoparticles have a high inhibitory effect on breast cancer cells, with an IC50 value of 17.54 μg/ml. It also indicated that the targeted nanoparticles were relatively highly inhibitory on breast cancer cells with a lower IC50 than the other three groups. In the activity assay on MCF-7 and MDA-MB-231 cell lines, CA/SF/Au-Ag composite nanofibers exhibited excellent bioactivity compared with other groups. In total, this active targeting nanofiber has a high inhibitory effect on tumor cells with a low IC50. However, due to the lack of in vivo studies, it is difficult to determine the composite nanofibers' active targeting and in vivo antitumor efficacy. Contemporarily, it has been used to build a nano-formulation loading rosmarinic acid, and the activity on breast cancer and cervical cancer cell lines was investigated (Fuster et al., 2021). The results showed that the nano-formulation could inhibit cell proliferation and induce apoptosis of HeLa and MCF-7 cell lines. Consequently, based on the above results, a silk fibroin-based nanoparticle delivery system can enhance the antitumor potential of antibreast cancer drugs.

Liver cancer
Transcatheter arterial chemoembolization is an effective method for the treatment of hepatocellular carcinoma. Some scholars successfully prepared sodium alginate-modified silk fibroin microsphere embolic agent by emulsion crosslinking Frontiers in Pharmacology frontiersin.org method, loaded with adriamycin hydrochloride, and studied its toxicity to liver cancer cells (Chen et al., 2020). The results showed that: firstly, the microspheres had smooth surfaces as well as good sphericity, and the swelling rate of the microspheres could meet the requirements of an arterial embolic agent, also included the sensitivity to pH and Frontiers in Pharmacology frontiersin.org 09 temperature; secondly, the microspheres had good blood compatibility; thirdly, cytotoxicity test showed that microspheres could promote the proliferation of fibroblasts and HUVEC; Finally, the drug loading and release ability of the microspheres were proved by the controlled release curve of adriamycin hydrochloride. This study showed that sodium alginate-modified silk fibroin microspheres could be a potential biodegradable arterial embolization agent to treat liver cancer. Some scholars also successfully prepared the complex of silk fibroin and adenovirus by taking advantage of the excellent biocompatibility of silk fibroin, loaded growth inhibitor 4 and interleukin 24, and analyzed its effect on liver cancer cells (Qu et al., 2019). The preparation and characterization of this complex are shown in Figure 3. The results showed that the constructed complex could effectively mediate the expression of target gene ING4 in SMMC-7721 cells and the secretion of target gene IL-24 in SMMC-7721 cells, thereby inducing apoptosis of hepatoma SMMC-7721 cells. Besides, the viability of SMMC-7721 and L-02 cells infected with the complex was also assessed. It was found that the growth of SMMC-7721 cells was significantly inhibited but that the growth and proliferation of L-02 cells were not affected. The complex constructed in this study showed improved infection efficiency and enhanced suppressive effects on human hepatoma carcinoma SMMC-7721 cells, potentially reducing the dose of adenovirus and maintaining high infection efficiency and tumor inhibition. Currently, most researchers tend to make silk fibroin and other biological materials into a complex and then load drugs or others for anti-liver cancer research. The latest research on Frontiers in Pharmacology frontiersin.org silk fibroin might be a drug delivery carrier material for liver cancer.

Lung cancer
Lung cancer is the most common cancer in terms of morbidity and death, thus choosing the right therapy is crucial (Mottaghitalab et al., 2015). In recent years, silk fibroin as a carrier material to load anti-cancer drugs to treat lung cancer has also become a hot research direction. In 2014, Qu et al. used silk fibroin loaded with cisplatin to prepare a controlled-release preparation to conduct research on lung cancer cell toxicity (Qu et al., 2014). It was found that cisplatin in the nanoparticles could be released slowly and continuously for more than 15 days. Meanwhile, in vitro, anti-cancer experiments showed that silk fibroin-nanoparticles loaded with cisplatin were easily internalized by A549 lung cancer cells, transferred cisplatin to cancer cells, and then triggered apoptosis. However, these particles were not easy to be internalized by L929 mouse fibroblasts, so their inhibitory effect on the growth of normal cells was weak. The silk fibroin nanoparticles loaded with cisplatin showed a sustained and effective killing effect on tumor cells, but the inhibitory effect on normal cells was weak. This research provided a new method for preparing cisplatin-loaded silk fibroin nanoparticles and offered a new carrier system for the clinical treatment of lung cancer and other tumors. Mottaghitalab et al. also used silk fibroin as a drug carrier to load gemcitabine into a targeted nanoformulation. They explored its therapeutic effect on lung cancer in the rat model . Compared with the untargeted silk fibroin nano-formulation and the control group, the gemcitabine-targeted nanoformulation showed higher cytotoxicity, cell uptake and accumulation in lung tissue. Besides, nano-formulation targeting gemcitabine had higher potential in the treatment of induced lung tumors. In histological and radiological analysis, higher survival, lower mortality and no signs of metastasis were  also observed in those animals treated with targeted agents. This study proposes an effective anti-cancer drug delivery system for targeted lung cancer. As its loaded drug, gemcitabine is limited in clinical use because it is cytotoxic. The preparation of nanotargeted formulation with silk fibroin helps to expand its use, improve the therapeutic effect, and reduce its toxic and side effects. The system may be used in the treatment of malignant lung cancer in the future. Moreover, this actively targeted nanoformulation shows a higher therapeutic effect, more aggregation effect and lower mortality in the study. This is a characteristic and advantage that untargeted nano-formulation does not have, and it also shows the advantages and potential of actively targeted nano-formulation. More interestingly, Wang et al. even found that silk fibroin peptide had a significant inhibitory effect on H460 lung cancer cells, induced the cells to arrest in the S phase, and then promoted apoptosis (Wang et al., 2019b). Silk fibroin peptide may become a new type of therapeutic agent for lung cancer treatment.

Gastric cancer
Silk fibroin has also been studied and analyzed in gastric cancer nano-formulations as a drug delivery carrier material. Some researchers prepared nano-formulation from silk fibroin and paclitaxel to explore their effects on human gastric cancer cell lines BGC-823 and SGC-7901 (Wu et al., 2013). In vitro cytotoxicity study, when paclitaxel was incorporated into the nano-formulation, paclitaxel could maintain its pharmacological activity, and silk fibroin had no cytotoxicity to cells. The antitumor effect of paclitaxel silk fibroin nano-formulation in vivo was evaluated on the transplanted tumor model of gastric cancer in nude mice. Compared with systemic administration, local administration of paclitaxel silk fibroin nanoparticles showed excellent antitumor efficacy by delaying tumor growth and reducing tumor weight (Figure 4). In addition, the organs of rats treated with nanoparticles did not show apparent toxicity, indicating that silk fibroin nanoparticles were safe in vivo. Another researcher loaded 5-fluorouracil on the composite made of cyclic pentapeptide and silk fibroin to prepare targeted nanoparticles and investigated its tumor progress on the rat model of gastric cancer (Mao et al., 2018). These results also clarified that silk fibroin-based nano-formulation had ideal active tumor-targeting properties and significantly reduced tumor burden. Crgdfk was conjugated with silk fibroin polypeptide to make the nanoparticles of 5-fluorouracil targeted. In this study, this preparation method makes the prepared nanoparticles have targeted characteristics, and 5fluorouracil can reach more tumor cells to give full play to the antitumor effect. At the same time, the rat organs treated with silk fibroin nano preparation did not show apparent toxicity, which proved that it had good biocompatibility and safety in vivo. In  Moin et al. (2021) Frontiers in Pharmacology frontiersin.org this study, nano preparations with active targeting showed obvious advantages, and the efficacy was more effective when combined with photodynamic therapy. In 2020, Zhang et al. prepared silk fibroin into a hydrogel loaded with gambogic acid to prepare a nano-formulation and studied its antitumor activity in the animal model of gastric cancer . The results showed that the antitumor activity of the hydrogel combined with gambogic acid nano-formulations was higher than that of the hydrogel loaded with gambogic acid and free gambogic acid. The free gambogic acid group had almost no antitumor effect. There was no significant change in body weight in each group, and the white blood cell and hemoglobin counts decreased slightly. The hydrogel delivery system prepared by silk fibroin as a drug carrier showed little systemic toxicity. It was considered a new strategy to improve the efficacy of anti-gastric cancer.

Pancreatic cancer
Pancreatic cancer is a malignant tumor. Its survival rate is extremely low, and its prognosis is still very poor. Hence, novel treatment strategies to combat this deadly disease are urgently needed. Some researchers used silk fibroin as a carrier material to load triptolide and triptorubin to prepare nanoparticle preparations and evaluated its effect on human pancreatic cancer cells . The results showed that the silk fibroin nano-formulation of triptolide could produce a synergistic inhibitory effect on the growth of MIA PaCa-2 and PANC-1 cells, and the growth inhibitory effect of the combination of the two drugs is synergistic rather than additive. It also further suggested that this novel combination may provide a potential treatment for pancreatic cancer. However, there are few studies on silk fibroin related to pancreatic cancer. The effect of silk fibroin as a drug delivery carrier material in the treatment of pancreatic cancer needs further research to confirm. In order to further clarify the application of silk fibroin in drug delivery systems for tumor treatment, we summarized the details and results of silk fibroin as a carrier material for drug delivery systems in different tumor treatment research in the past decade (Table 4).

Prospects and conclusion
Over the past decade, silk fibroin has played an increasingly important role in drug delivery systems as a carrier material, which has aroused the extensive interest of researchers. As described in this article, silk fibroin-based nanocomplexes have been extensively studied in drug delivery and tumor therapy. Furthermore, the researchers have got some positive results in both in vitro and in vivo tests, a breakthrough in nanomedicine. By using cell or animal models, the nanocomposites prepared by silk fibroin as a carrier material have been studied in a variety of tumor diseases, such as breast cancer, liver cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, thyroid cancer, cervical cancer, colorectal cancer, and glioma. The results have found that: 1) the use of fibroin nanoparticles as a non-toxic, biocompatible delivery carrier to enhance the feasibility of tumor treatment effects that have a little inhibitory effect on normal cells; 2) the fibroin can be prepared with other materials into a composite carrier (such as: cellulose acetate, gold and silver nanoparticles, adenovirus, gelatin, etc.), among them, the scaffold of silk fibroin loaded with amino acid modified adenovirus can stimulate the formation of vascular network and thus accelerate the regeneration of dermal tissue, and the composite nanofibers made of silk fibres have a high inhibitory effect on tumor cells (Wang et al., 2019c); 3) the surface of silk fibroin can be properly modified to meet different needs. For example, the sustainedrelease behavior of adriamycin can be enhanced by modifying the surface of silk fibroin with Tween 80 as surfactant; 4) silk fibroin can be prepared into nanocarriers with stimulation response, which can be triggered by the changes of pH value, ultraviolet, near infrared, ultrasonic and magnetic field, to accurately release the drug at the tumor site and enhance the curative effect; 5) the composite nanoparticles prepared from silk fibroin can also significantly improve the tumor inhibition effect through the combination of photothermal therapy (PTT)/tumor-specific photodynamic therapy (PDT)/chemotherapy, with minimal systemic toxicity or adverse reactions; 6) the combination with specifically targeted ligands (such as antibody, growth factor, transferrin, polypeptide, polysaccharide, etc.) helps to enhance the targeting of drug delivery, reduce the toxic and side effects of drugs, and strengthen tumor inhibitory effect in drug delivery system.
The targeted drug delivery system based on silk fibroin will significantly promote the treatment and development of tumors in the future, mainly due to the modifiability of the silk fibroin structure. The structure of it contains active amino and carboxyl groups, which researchers can chemically modify to increase the material's functionality. It has certain biological activity, and SP5-52 peptide sequence (SVSVGMKPSPRP) can target the cell surface of non-small cell lung cancer (NSCLC) . NSCLC cells can increase anticancer accumulation 5.7-fold in tumorigenic lung tissue compared to non-targeted vectors. The SP5-52 peptide was synthesized by solid-phase peptide synthesis on Rink amide MBHA resin. SP5-52 peptide was linked to the surface of silk fibroin nanoparticles with Hatu and diea as coupling reagents to obtain SP5-52 peptide modified nanoparticles. The results indicated that the silk fibroin nanoparticles targeting gemcitabine have a higher potential in treating induced lung tumors than the control group. The results of histopathology and radiological analysis showed that higher survival rate, lower mortality rate, and no signs of metastasis were also observed in those animals treated with SP5-52 peptide Frontiers in Pharmacology frontiersin.org modified silk fibroin@gemcitabine. This study proposed an effective anticancer drug delivery system for specifically targeting induced lung tumors, which can be used to treat malignant lung cancer in the future. In addition to SP5-52 peptide, RGD tumor targeting peptide (Arginine-Glycine-Aspartate) (Rodriguez-Nogales et al., 2016), folic acid (Subia et al., 2014), chondroitin sulfate (Gou et al., 2019b), hyaluronic acid (Ziadlou et al., 2021) can modify silk fibroin for targeted delivery of drugs. Moreover, PEG can also modify silk fibroin, thereby extending the circulation time of drugs in vivo (Zhang et al., 2020b). Therefore, this kind of silk fibroin nano drug delivery system that actively targets tumors is also the focus of current and later research. The application of silk fibroin in disease diagnosis, treatment and prevention has gradually developed, but it loaded anti-cancer drugs to prepare new drug delivery systems is a challenge for clinic use. Most cell and animal studies are short-term observations and lack long-term safety assessment, including survival analysis and toxic and side effects evaluation. In addition, the maximum allowable dose of silk fibroin, drugs, and other materials in animals seems to be an essential reference index for clinical use. Moreover, for the preparation of silk fibroin nanoparticles, whether the size and shape of particles will affect the distribution and accumulation of drugs in tumor tissues to affect the therapeutic effect remains to be further explored and analyzed. Fortunately, silk fibroin-related products such as Seri ® , Surgical Scaffold, Surusil ® , Sofsilk TM and SickVoice ® are on the market, indicating that silk fibroin, as a new medical polymer material that has attracted much attention in recent years, has gone from basic research to clinical application. However, how to transfer the new drug delivery system based on silk fibroin loaded drugs from the laboratory to the clinic for further research and analysis, which first needs to be solved is the mass production of silk fibroin.
Although silk can be obtained through silkworm breeding, this production method has the problems of low yield, long time-consuming and low efficiency. Mehreen et al. believed that the cost of silk obtained from cocoon was low, which could reduce the overall production cost on a larger industrial scale, so as to obtain silk fibroin. However, this does not take into account other factors in the actual upload (Mehreen et al., 2021). At the same time, many reviews did not put forward views on the problems and solutions in the industrial production of silk fibroin, which is a key issue to promote the application of it (Florczak et al., 2014;Wani and Veerbhadrappa, 2018;Florczak et al., 2021;Chis et al., 2022). Based on this, this manuscript puts forward some views on this issue. For production enterprises, their production technology (such as the extraction, preparation, separation and purification of silk fibroin) and the quality control and batch release requirements in GMP mass production have great challenges. At the same time, there are problems such as product quality detection (how to detect the safety, biocompatibility and biodegradability of silk fibroin). These are urgent needs to be solved. Although the laboratory can extract and prepare silk fibroin by itself, few reports on the industrialized production of mature silk fibroin. There is no pharmacopoeia standard or influential industry standard in developed countries to include this variety. In addition, due to the heterogeneity of silk. There are significant differences in amino acid sequence, morphology and manufacturing process between the source materials; therefore, the results of their preclinical characteristics cannot be directly transformed between these studies (Florczak et al., 2020). Consequently, there may be a long time for the transformation, development, and commercialization of the silk fibroin-based drug delivery system. The fact that silk fibroin cannot be mass-produced and quality inspected is also the main reason why it has been difficult to achieve breakthroughs in drug delivery systems since it was approved by the FDA (Florczak et al., 2021). At present, in addition to obtaining silk fibroin from silk, spider silk can also be obtained by breeding spiders and further processed to obtain silk fibroin (Salehi et al., 2020). However, it is also difficult to industrialize and commercialize large-scale production, because spiders are carnivores, putting several spiders together will cause them to bite each other. They have a variety of silk glands, and different glands produce different silk properties (Rising and Johansson, 2015). Hence, it is difficult to collect silk with a single performance. At the same time, the secretion of spider silk from artificially reared spiders has dropped significantly, and the quality has also dropped significantly (Liu et al., 2021). There exist many ways to obtain spider silk, but the problem of quantity and quality is still difficult to solve properly, such as microbial pathway (Wu et al., 2017), animal pathway (Liebsch et al., 2018), plant pathway (Scheller et al., 2001) and electrospinning (Belbéoch et al., 2021). Therefore, the acquisition of silk fibroin is mainly obtained by breeding silkworms and harvesting cocoons.
However, we found that albumin is one of the preferred carrier materials for antitumor drug nanoparticles (Kunde and Wairkar, 2022). Albumin is a natural macromolecular material of protein.
Human serum albumin has high stability, good biocompatibility and biodegradability, no immunogenicity and can be modified structurally (Hong et al., 2020b). It is worth noting that the fundamental properties of silk fibroin are similar to albumin, and it also has the internal stimulation response of pH/lysosomal enzyme/GSH/ROS. It has a wide range of sources, and the acquisition cost is much lower than that of albumin from human blood. Therefore, we believe that the successful experience of albumin as a drug carrier is worthy of reference and learning for silk fibroin. On the other hand, silk fibroin from different sources is different (Lehmann et al., 2022). The slow degradation of silk II crystal structure also makes silk fibroin unsuitable for the situation that silk fibroin needs to be quickly and thoroughly removed from Frontiers in Pharmacology frontiersin.org the body, which may be an obstacle to the application of silk fibroin in drug delivery system (Hu et al., 2020b). Transgenic silk fibroin can achieve quality consistency (Gou et al., 2019a). It can also change the degradation rate of silk fibroin by adjusting the molecular structure. It has also been studied as a drug delivery carrier material, which is a promising and potential solution strategy (Xia et al., 2011;Hofer et al., 2012;Zheng and Zuo, 2021). When a safe and mass production method of silk fibroin is found and applied, the new anti-cancer drug delivery system mediated by silk fibroin will change greatly and be used in clinic.

Author contributions
BY and YL reviewed recent literatures and drafted the present review. HX provided expert analysis and insights into the synthesis of characteristics, application, and the cancers' study of silk fibroin. All authors have read and agreed to the published version of the manuscript.

Funding
The research was supported by the Natural Science