Molecular farming expression of recombinant fusion proteins applied to skincare strategies

Molecular farming for skincare research

Molecular farming is an interdisciplinary subject of agronomy, engineering, and medicine, as it can be widely used in industry, environmental protection, and health industries. Molecular farming, also known as a plant bioreactor, utilizes gene editing technology to enable the expression of pharmacologically important proteins in plant models (Kowalczyk et al., 2022). There are three types of transformation. The target gene in the plant expression vector is transformed into the plant genome by Agrobacterium tumefaciens to yield a stable heredity, called stable nuclear transformation. Alternatively, transient expressions based on agroinfiltration or virus-based vectors have been developed to complement transgenic plants that offer rapid and high-level protein expression within a few days. Alternately, the target gene was inserted into the chloroplast genome, and the foreign protein was expressed in the chloroplast (Krenek et al., 2015; Liu et al., 2023; Shanmugaraj, Bulaon & Phoolcharoen, 2020). Owing to its advantages and rapid development, molecular farming has attracted global attention from researchers and industries, including skincare research and industry (Shanmugaraj, Bulaon & Phoolcharoen, 2020). Advances in biotechnology have led to the establishment of efficient plant expression systems, most of which have been extensively studied. Currently, plants such as Nicotiana tabacum L, Brassica napus, Solanum tuberosum L, Solanum lycopersicum L, Glycine max, Arabidopsis thaliana L, Zea mays L, and Oryza sativa seeds have been used to express foreign recombinant proteins, thus earning plant bioreactors the name “chemical factories.” Recent use of plant bioreactors hinges on the expression of biological agents with potential commercial value, such as vaccine antigens, antibodies, nutritional supplements, and industrial enzymes (Kumar, Singh & Singh, 2022; Yang et al., 2021). With the commercialization of the recombinant glucosidase drug, the first commercialized bioactive agent expressed in plant cells for the treatment of Gaucher’s disease, it is safe to say that the use of plant bioreactors has entered a new era (Kowalczyk et al., 2022). Other proteins that have been expressed in plant bioreactors include human serum albumin (HSA), expressed in O. sativa. Using crystallography, the plant bioreactor-expressed HSA was confirmed to be structurally similar to the plasma HSA, suggesting that they share the same biological activity (Kuo et al., 2013). In veterinary medicine, the subunit vaccine of bovine viral diarrhea virus (BVDV), a serious cause of global economic losses in farm animals, has been expressed in modified alfalfa crops and is shown to be highly effective (Aguirreburualde et al., 2013). The human insulin-like growth factor binding protein-3 (Higfbp-3), a multifunctional protein, is another protein that has been expressed in plants, specifically N. tabacum. Interestingly, the protein was expressed in N. tabacum seeds using the signal peptide (SP) and c-terminal tetrapeptide AFVY of phaseolin. Analysis of expression showed that recombinant Higfp-3 accumulated in N. tabacum seed with a high yield (Cheung et al., 2009). Multiple therapeutic proteins synthesized in plants are either at the preclinical or clinical trials stage with the potential of achieving commercialization. For example, in the area of skin health, multiple studies have shown the applicability of molecular farming in skin health research and skin care product development. Nazeri, Aram & Niazi (2023) used N. tabacum to express hyaluronic acid (HA) and found that plants can produce the precursor HA but not the mature HA as they lack hyaluronic acid synthase. To overcome this limitation, these authors successfully transfected the human hyaluronate synthase two gene into N. tabacum and confirmed its retention of function, thus enabling the production of mature HA in plants (Nazeri, Aram & Niazi, 2023). Other genes relevant to skincare that have been expressed in plants include superoxide dismutase (SOD) and basic fibroblast growth factor (bFGF). Previously, SOD was mainly derived from animal tissues. However, in the past decade, functional SOD with potential application across various biological fields has been obtained from plant-based expression systems (Che et al., 2020; Ren et al., 2018). Ding et al. (2006) expressed bioactive bFGF in soybeans and showed that the recombinant protein can retain its proliferative function. These findings confirm the feasibility of plants as natural bioreactors for the production of cytokines and growth factors relevant to skincare.

Review methodology

The data presented herein were acquired following a rigorous search from several well-established academic search engines and specific journal databases. These included Google eScholar, PubMed, Web of Science, Baidu Academic, and Microsoft Academic Search. Google eScholar and PubMed served as the main search tools, accounting for more than half of the used literature. To optimize and find the relevant literature, we used the boolean search method with the following keywords: Arabidopsis and transgenic, molecular agriculture, molecular farming, plant bioreactor, transdermal peptide, cytokine and transdermal peptide, molecular agriculture and pharmaceutical protein, cosmetics and plant bioreactor, and molecular agriculture and cytokines. Thereafter, relevant reports were acquired, studied, and assessed for suitability to the topic and content of the review, thus ensuring the quality and accuracy of interpretations of the findings and conclusions presented herein.

Commercial strategies for expression of foreign proteins using molecular agriculture

Application of cytokine expression in molecular agriculture

Molecular farming can be applied to the study and application of recombinant protein expression, and it has been demonstrated for proteins such as cytokines and growth factors. Molecular farming can also be used to assess the applicability of various expression methods. Plants have several advantages that make them efficient bioreactors. First, their offspring can stably inherit the transfected genes to ensure the continuous production of the recombinant proteins. This also allows the acquisition of plants already expressing the desired protein, thus reducing the production cost and providing the prerequisite basis for commercialization (Ma, Drake & Christou, 2003). Second, plants have functional, post-translational modification machinery (Huang & McDonald, 2012), which enables the processing of recombinant proteins to ensure the retention of structure and function. Third, unlike other expression systems, plant expression systems do not require heat sources, and the purified proteins are free from endotoxins, or other sensitizing substances. As such, these expression systems reduce the potential damage of the recombinant proteins and are likely to cause less sensitization and adverse reactions in people who use the final products (Moustafa, Makhzoum & Trémouillaux-Guiller, 2016). Fourth, except for foreign proteins, the extraction of the whole plant contains a lot of antioxidants, vitamins, and antibacterial substances, which can be easily separated from the plant (Sitarek et al., 2020; Hoang, Moon & Lee, 2021; Marchev & Georgiev, 2020). The proper mix of exogenous protein and plant derivatives plays a synergistic role in skincare (Miliauskas, Venskutonis & Van Beek, 2004). Not only can it promote the full utilization of raw materials, but it is also in line with the current demand for skincare raw materials from the pure natural concept (Hoang, Moon & Lee, 2021).

To date, multiple studies have shown the feasibility of expressing skincare-relevant proteins in plant bioreactors. Proteins that have been successfully expressed in plants include aFGF, bFGF, FGF21, KGF-2, and EGF and the actual plants used to express these proteins include A. thaliana, S. lycopersicum, N. tabacum, and Carthamus tinctorius Linn. aFGF, bFGF, KGF-2, EGF, and among others, are proven to delay aging (Mehta & Fitzpatrick, 2007; Hou et al., 2020), promote whitening (Smit, Vicanova & Pavel, 2009), remove acne and other spots, moisturize, as well as eliminate wrinkles, and brighten the skin. Additionally, some of these growth factors have been demonstrated to enhance skin regeneration and reduce scar formation (Bloemen et al., 2009; Dolivo, 2022). These studies illustrated the potential prospect of the use of plants as bioreactors in the field of skincare. Thus, how to make plant-derived proteins reflect higher application value in skin care has become an urgent problem to be solved. Studies have shown that the incorporation of the expression of cytokines and growth factors with beneficial effects on skin health in different plant parts may confer synergetic effects. Specific plant parts, such as roots, leaves, and fruits/seeds, are rich in anthocyanins, vitamin E, and antioxidants, and thus they have been utilized in multiple skincare products (Jo et al., 2020; Michalak, 2022). The expression of recombinant proteins in plant parts containing high levels of antioxidants can help increase skincare efficacy when applying into products. Another approach is the fusion protein strategy to enhance their transdermal properties (Bolhassani, Jafarzade & Mardani, 2017). Oil-body cytokine fusion can also significantly improve the transdermal rate of the cytokine. The permeability mechanism of oil body may be similar to that of liposomes. The drugs carried by liposomes show a strong ability to penetrate through the skin. Qiang et al. (2018) studied the oil body carrying oleosin-hEGF-hEGF and showed that it can rapidly penetrate the skin tissue and the amount of absorption is higher than EGF not fused to oil body. Zhao et al. (2015) used A. thaliana to express aFGF fused with oil body, which were specifically stored in the seeds, and subsequent toxicological tests showed that the fusion protein has no obvious toxicity and would not cause any irritation or skin allergy. Suggestively, this could allow the expression of pharmacologically beneficial proteins in the desirable plant parts or the avoidance of the expression of these proteins in certain plant parts. This can lead to increased reusability and value of plant bioreactors. Coupled with the general preference for naturally derived skincare products, molecular farming undoubtedly holds unique advantages over conventional methods used to synthesize proteins in the skincare industry.

Feasibility of molecular farming synthesis of tdp1 fusion egf protein

Epidermal growth factor (EGF) is a small-molecule polypeptide composed of 53 amino acid residues with multiple functions (Boonstra et al., 1995). EGF has several beneficial effects on the skin and has been used for various purposes in skincare. For example, recombinant human epidermal growth factor (rhEGF) is used in anti-acne creams. Using split-face randomized double-blind trials, rhEGF has been demonstrated to significantly improve acne clearance with little to no side effects (Kim et al., 2014a). Aside from treating acne, EGF has also been used to treat skin melasma and is known to be non-invasive (Lyons, Stoll & Moy, 2018). Additionally, EGF is known to be an anti-aging factor, and its topical application reduces wrinkles while increasing dermal collagen in multiple clinical studies (Ha et al., 2017; Yu & Driscoll, 2011; Kim et al., 2014b). EGF is also known to accelerate wound healing and reduce scarring, and as such, it is widely used post-surgery, especially in reconstruction and minimally invasive facial plastic surgery (Ratanapokasatit & Sirithanabadeekul, 2022). Evidently, EGF has a wide range of applications in skincare. However, the lack of effective transdermal properties of EGF reduces the efficacy of the EGF products. Thus, the development of improved transdermal EGF delivery methods remains a major challenge for its future application in skin health (Shin et al., 2023). To resolve this, recent studies have fused EGF with TD1 (a CPP), conferring improved dermal penetration and enhanced efficacy for the TD1-EGF product (Ruan et al., 2013). However, TD1-EGF has been largely expressed in yeast or other prokaryotic systems (Ruan et al., 2014). The expression of TD1-EGF in plants may confer additional benefits mentioned above, especially when expressed in plants used as raw materials for skincare products.

A potential model plant for the expression of fused EGF isA. thaliana, as it has a high infection rate compared to other plants. Additionally, A. thaliana has a strong adaptability and fast growth rate, a large biomass, and a high content of anthocyanins, which may have synergistic effects on the skin (Zheng et al., 2021). Furthermore, A. thaliana extracts are used as raw materials or ingredients in multiple cosmetics. EGF has been expressed in various plant systems, including A. thaliana, S. tuberosum, N. tabacum and O. sativa (Thomas & Walmsley, 2014), but there has been no investigation into the expression of TD1-EGF. A study by Qiang et al. (2020) expressed double-EGF fused with olesion in A. thaliana and explored the quantity and quality of the resultant olesion-hegF-Hegf protein. The recombinant protein was found to retain the bioactivity of EGF and was more permeable compared to the control. The replication of a similar recombinant gene design may yield a superior-performing TD1-EGF. Jin et al. (2014) explored the transdermal efficacy of hEGF fused with either a single- or double-TD1 and showed that the double-TD1 fused EGF is at least five times more efficient than the control compared with just 3 $\times$ more efficient in the case of the single-TD1-fused EGF. This suggests that the higher transdermal permeability of double-TD1-fused EGF may improve the therapeutic outcome and lead to wider medical applications.

In order to ensure the stable expression of TD1 in plants, we will optimize the preference of the TD1 codon and change part of its amino acid residues through point mutation to obtain the TD1 mutant transdermal peptide TDP1, which is fused to the EGF gene that is also subjected to codon optimization. In the preliminary design, a flexible linker is inserted between the two protein molecules to prevent the structure of the two protein molecules from affecting the function of the fusion protein (Chen, Zaro & Shen, 2013). In future studies, the feasibility of TDP1-EGF expression in a whole plant system will be tested in A. thaliana. The transdermal ability and biological activity of TDP1-EGF will also be evaluated with references to skincare, and hopefully, this will provide a new application model of plant bioreactor in the field of skin healthcare products for exploring new ideas for the development of natural skincare raw materials.

Outlook

Transdermal delivery is an attractive route for the administration of drugs because of its high acceptance among patients and avoidance of the first-pass hepatic metabolism. In the route of transdermal drug delivery, the availability of CPPs has led to improved efficacy of the drugs and broaden the selectivity of the drugs that can be used. In the field of skincare, epidermal growth factor (EGF) is widely recognized for its various functions, which include whitening and anti-aging effects, accelerating wound healing, and minimizing scar formation. To enhance the transdermal absorption of EGF, a transdermal peptide called TDP1 was fused to the C-terminus of EGF to produce a fusion protein known as TDP1-EGF, which can then be expressed in a plant bioreactor.

Research on plant bioreactors has been going on for over four decades. Initially, plants were used to express antibodies, vaccines, and medicinal proteins. Over time, the advantages and disadvantages of using plants as bioreactors have become clear. More and more growth factors are produced by plants and used in the field of skin care. Today, we understand that it is not necessary to express foreign proteins in specific plant tissues to improve yield. The selection of a suitable plant expression vector and transformation mode can significantly improve the bioaccumulation of the fusion protein TDP1-EGF.

For example, inflorescence transformation can integrate the target gene into the plant genome and stabilize inheritance and whole-plant expression. In addition, selecting appropriate plant expression vectors that contain an enhanced 35S promoter and 35S terminator will increase the accumulation of the fusion protein TDP1-EGF. Furthermore, it has become clear that plants are not suitable bioreactors for expressing proteins that require high purity, such as antibodies, due to the potential difficulty of separation and purification. Despite this, plants are still being explored for the expression of vaccine antigens. Indeed, plant bioreactors have a great potential for applications in various fields of medicine, and with further development, the challenge of purification will be overcome. Here, we selected two fusion protein tags, His-tag and TDP1, which not only have overcome the difficulties of downstream purification but also the difficulties of transdermal drug delivery. Advancements in the use of plant bioreactors have enabled their use in skincare product research and development. The use of plant bioreactors in skincare product development fulfills the need for effective products as well as the desire for plant-derived products. In addition, plants are considered an environmentally friendly source of raw materials, and this will ease concerns about the sources of the raw materials used in industry and research. Thus, plant bioreactors are more suitable for direct application of the expressed proteins. In order to improve the safety of the fusion protein TDP1-EGF, we expressed TDP1-EGF in Arabidopsis, because Arabidopsis has a simple biomass and a short growth cycle, and it contains anthocyanins and other by-metabolites that are conducive to skincare. Moreover, there are presently few skincare products on the market that exploit the synergetic effects of recombinant proteins and active substances in plants. Therefore, TDP1-EGF is bound to become more competitive and popular, especially in the cosmetic industry. While molecular farming has a broad prospect in the field of skincare products, there is a need to further our current understanding of the use of plants as bioreactors and how to scale up their production for the skincare market.