Editorial For reprint orders, please contact: reprints@futuremedicine.com Genetically modified silk fibroin nanoparticles for drug delivery: preparation strategies and application prospects Dingpei Long*,1,2 , Bo Xiao1,2 & Didier Merlin1,3 1 Institute for Biomedical Sciences, Center for Diagnostics & Therapeutics, Digestive Disease Research Group, Georgia State University, Atlanta, GA 30302, USA 2 State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics & Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, PR China 3 Atlanta Veterans Affairs Medical Center, Decatur, GA 30033, USA *Author for correspondence: dlong26@gsu.edu “SF molecules can be designed and modified through genetic modification, and subsequently processed to drug-loading GMSF-NPs with excellent new characteristics.” First draft submitted: 28 April 2020; Accepted for publication: 29 May 2020; Published online: 17 July 2020 Keywords: drug delivery • genetic modification • nanoparticle • silk fibroin Silk fibroin (SF), a natural protein extracted from the cocoons of the silkworm (Bombyx mori), is a US FDAapproved natural polymer. It has been used in silk-based medical devices for sutures and as a support structure during reconstructive surgery [1]. Compared with synthetic polymers such as poly(lactic-co-glycolic acid), poly(lactic acid) and poly(glycolic acid), SF has high biocompatibility, tunable biodegradability and low immunogenicity. In comparison with natural polymers (e.g., chitosan, collagen and gelatin), SF has excellent mechanical properties, unique self-assembling ability and versatile processability in an aqueous environment [1,2]. Recently, SF has been utilized as a drug-delivery material; notably, SF-based nanoplatforms exhibit intrinsic anti-inflammatory activity, wound-healing capacity, high drug encapsulation efficiency, and the ability to undergo lysosomal environmentresponsive drug release [2]. In recent years, researchers have sought to use physical, chemical and even genetic modification methods to prepare modified SF-based biomaterials with new functions for biomedical applications [1]. The improvement of SF by genetic modification techniques has shown great potential to endow SF-nanoparticles (NPs) with new characteristics, including high-level cell-targeting and cell-entry efficiency that could effectively promote the application of SF-NPs for drug delivery in the field of nanomedicine. In this editorial, we will discuss the advantages of SF-NPs and the specific genetic modification methods that have been used to improve SF for NP drug delivery applications. We will also highlight the strategies that can be applied to achieve precision medicine using genetically modified SF-NPs (GMSF-NPs) with different properties. Possible challenges and future research directions are also presented. Why should we use genetic modification to improve SF? To date, SF-based biomaterials have been modified by various methods, ranging from the molecular level to the macroscopic level; these include chemical conjugation-based composite modification, direct feeding, genetic modification, mesoscopic assembly and macroscopic mixing [3]. Genetic modification has been used to regulate the insertion or replacement of exogenous DNA in the B. mori genome, or to knock out the endogenous silk protein genes of B. mori by means of silkworm genetic improvement technologies, such as transgenics [4] and genome editing [5]. Using overexpression of exogenous proteins or downregulation/knockout of endogenous silk proteins, researchers have obtained functional genetically modified SF (GMSF) from the cocoons of genetically modified (GM) silkworms [4,5]. Of the modification methods mentioned above, only genetic modification can change the composition of silk protein itself. Once a GM silkworm strain is established, the silkworms can be easily proliferated and retained. The inserted transgene can be carefully chosen to modify the SF for desirable characteristics. C 2020 Future Medicine Ltd 10.2217/nnm-2020-0182 Nanomedicine (Lond.) (Epub ahead of print) ISSN 1743-5889 Editorial Long, Xiao & Merlin How can we use genetic modification to improve SF? SF is synthesized and secreted by the silk gland of B. mori, and mainly contains three proteins: fibroin heavy chain (Fib-H; Mw = 391.6 kDa), fibroin light chain (Fib-L; Mw = 27.7 kDa) and fibrohexamarin (also called P25; Mw = 25.2 kDa) [6]. Fib-H is the major component (92%, w/w) of the SF; it is amphiphilic in nature and contains hydrophobic crystalline regions and hydrophilic noncrystalline regions in an interval arrangement. SFNPs accordingly have the characteristics of internal hydrophobicity and external hydrophilicity, and thus can load both hydrophilic and hydrophobic drugs. The hydrophobic crystalline regions of Fib-H have repeating sequences of (Gly-Ala-Gly-Ala-Gly-Ser)n , which tend to self-assemble into antiparallel β-sheet structures [7]. The β-sheet structures determine the physical properties and stability of the SF-NPs, and controlled release of the drugs can be achieved by adjusting the crystallinity and β-sheet content of SF-NPs. In order to create functional GMSF-NPs, the SF must be improved via direct genetic modification, wherein the gene sequences that encode the component proteins of natural SF are modified so as to obtain GMSF containing different component proteins that can be functionalized in SF-NPs. This is generally achieved in two ways: • Transgenic technology can be used to regulate the expression of a fusion gene composed of an exogenous protein/polypeptide coding sequence and the Fib-H or Fib-L sequence. Such silkworm transgenic technology has been used to generate GMSF containing various exogenous proteins or polypeptides, such as fluorescent proteins, spider dragline silk proteins and various growth factors, and functional polypeptides. These GMSF have great potential to create new functional GMSF-NPs; • Some functional small molecule protein/polypeptide coding sequences have been selectively inserted into the endogenous Fib-H or Fib-L genes by using genome-editing tools (e.g., ZFN, TALEN and CRISPR/Cas systems) combined with homology-directed repair. This method does not introduce any exogenous protein or polypeptide into the natural SF, which to a certain extent ensures the stable molecular assembly of the SF. Moreover, GM Fib-H or GM Fib-L can be artificially designed and modified as needed. Preparation & application strategies of GMSF-NPs for drug delivery Due to the good chemical versatility and miscibility of SF, SF-NPs are traditionally loaded with various materials (e.g., metal particles, proteins, polypeptides and polysaccharides) to enhance the performance of micro or nano structures, physicochemical properties and biological response. These materials may be included during particle preparation or loaded to prepared particles by direct mixing, surface adsorption or chemical coupling, so as to create the functionalized SF-NPs [8]. In contrast, exogenous fusion proteins/polypeptides with different functions introduced by genetic modification will bind with the endogenous component proteins of SF through disulfide bond formation and participate in the self-assembly of SF in the silk glands of transgenic silkworms [6]. The stability of GMSF-NPs loaded with exogenous proteins/polypeptides is better than that of functional SF-NPs prepared by direct mixing or surface adsorption, and this strategy can significantly reduce the influence of chemical cross-linking on drug encapsulation [9]. Functional GMSF-NPs may then be prepared with high-level cell-targeting and/or cell-entry properties, highly controlled drug release ability or other characteristics, as discussed in the following. GMSF-NPs with cell-targeting properties In active targeting, a high-level affinity between engineered ligands and receptors overexpressed on the target cell surface is used to selectively transport drugs to specific sites and enable delivery of the NPs and/or the loaded drug [10]. For tumor cells, a variety of target ligand proteins (e.g., HER-2, EGFR, TNF-α, VEGF and VEGFR) and peptides (e.g., CTX, cRGD, iRGD and OCT) have been widely modified for use as active targeting components in nano-targeted drug delivery systems (DDSs). As natural SF-NPs are not targeted, the preparation of drug-loaded GMSF-NPs with surface expression of a relevant ligand or receptor can be an important strategy for creating functional GMSF-NPs that can achieve high-level cell-targeting. GMSF-NPs with cell-entry efficiency As the main site of cell metabolism and physiological signal transduction, the cytoplasm is an important target of anticancer drug delivery. However, the NP-mediated delivery of a drug to the cytoplasm requires that it cross the cell membrane. Cell-penetrating peptides (CPPs), such as Pep-1, pVEC and polyhistidine, are 5 to 30 amino-acid peptides that can directly carry active substances across the cell membrane, thereby skipping the conventional 10.2217/nnm-2020-0182 Nanomedicine (Lond.) (Epub ahead of print) future science group Genetically modified silk fibroin nanoparticles for drug delivery: preparation strategies & application prospects Editorial endocytosis pathway [11]. GMSF-NPs with high cell-entry efficiency can be created by expressing CPP fusion proteins in SF or integrating CPPs into the endogenous Fib-H or Fib-L molecules via genetic modification [12]. GMSF-NPs with improved controlled drug release capacity The β-sheet is the most basic secondary structure in silk-based biomaterials, including SF and spider silk. It not only critically stabilizes the particle structure; it also plays a leading role in controlled drug release. Studies have shown that the controlled drug release ability of SF-NPs can be regulated by modulating the β-sheet content [13,14]. Our group recently created GMSF-NPs containing the synthetic spider dragline silk protein, MaSp1 (data not published). The proportion of β-sheet structure content was significantly higher in the generated GMSF-NPs compared with SF-NPs prepared by the same process, and the GMSF-NPs exhibited significantly more controlled drug release than SF-NPs. These findings indicate that the introduction of spider-like structural proteins to improve the β-sheet content and self-assembly process of GMSF-NPs could effectively improve their controlled drug release ability. GMSF-NPs with other excellent characteristics Recently, Teramoto et al. created transgenic silkworm strains that expressed B. mori phenylalanyl tRNA synthetase variants in silk glands, and found that the synthetase variants supported the efficient in vivo incorporation of synthetic non-natural azidophenylalanine into SF molecules [15]. If this GMSF can be made into drug-loading GMSF-NPs, its non-natural amino acids could effectively reduce the recognition and degradation of SF proteins by various digestive enzymes. When used in a DDS, this could improve drug stability and alleviate drug toxicity toward normal cells. In another set of studies, GMSF containing temperature-responsive elastin-like proteins were used to develop temperature-sensitive GMSF-NPs [16]. Other work showed that enzyme-sensitive GMSF-NPs targeting tumor tissues could be prepared by introducing substrate peptides of enzymes present at high concentrations in tumor tissues (e.g., the MMP site specifically recognized by matrix metalloproteinases) into GMSF [17]. GMSF containing various fluorescent proteins (e.g., EGFP, mKate2 and EYFP) has been used to develop fluorescent GMSF-NPs with drug-tracer abilities [18,19]. Finally, studies have indicated that GMSF containing bioactive proteins/peptides (e.g., EGF, VEGF or hbFGF) can be directly used to prepare bioactive drug-loaded GMSF-NPs [20] that are expected to have a better drug-loading rate and improved drug-loading stability than drug-loaded SF-NPs prepared by mixing a natural SF solution with the drug. Conclusion & future perspective The improvement of SF by genetic modification techniques has shown great potential to endow SF-NPs with new characteristics (e.g., high-level cell-targeting and cell-entry efficiency) that could effectively promote the application of SF-NPs for drug delivery in the field of nanomedicine. To date, SF-NPs have been developed to deliver proteins, small molecules and anticancer drugs. SF molecules can be designed and modified through genetic modification, and subsequently processed to drug-loading GMSF-NPs with excellent new characteristics. However, several challenges must be overcome in the preparation and future application of GMSF-NPs: first, the processes used to prepare GMSF-NPs should be optimized according to the types of exogenous proteins/polypeptides to be introduced. Second, the introduced exogenous proteins/peptides should not affect the synthesis, secretion or self-assembly of SF in vivo and must not destroy the desirable characteristics of natural SF, such as its biocompatibility and versatile processability. Third, functional GMSF-NPs must not have any toxic effect on normal cells or overall individuals. Finally, the therapeutic effect of GMSF-NPs in vivo needs to be studied in more detail. In the future, we believe the development of new GMSF-NP-based DDSs and formulations will form the basis for increasing the accuracy of targeted therapy and reducing the side effects of conventional delivery methods. future science group 10.2217/nnm-2020-0182 Editorial Long, Xiao & Merlin Financial & competing interest disclosure This work was supported by the National Institutes of Health of Diabetes and Digestive and Kidney (RO1-DK-107739 & RO1DK-116306 to D Merlin), the Department of Veterans Affairs (Merit Award BX002526 to D Merlin). D Merlin is a recipient of a Senior Research Career Scientist Award from the Department of Veterans Affairs (BX004476). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. References 1. Holland C, Numata K, Rnjak-Kovacina J, Seib FP. The biomedical use of silk: past, present, future. Adv. Healthc. Mater. 8(1), 1800465 (2019). 2. Mottaghitalab F, Farokhi M, Shokrgozar MA, Atyabi F, Hosseinkhani H. Silk fibroin nanoparticle as a novel drug delivery system. J. Control. Rel. 206, 161–176 (2015). 3. Zhou Z, Zhang S, Cao Y et al. Engineering the future of silk materials through advanced manufacturing. Adv. Mater. 30(33), 1706983 (2018). 4. Tamura T, Thibert C, Royer C et al. 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