Xanthan gum/oil body-microgel emulsions with enhanced transdermal absorption for accelerating wound healing.

The oil body comprises lipid droplets surrounded by a surface embedded with oil body-related proteins. To form a drug delivery system, an oleosin can be fused with foreign proteins and bound to the oil body surface. Here, safflower oil bodies carrying oleosin-human epidermal growth factor (hEGF) were mixed with xanthan gum to form self-assembled polymers, referred as an oil body microgel emulsion (OBEME) without any chemical crosslinking agent. The physicochemical properties of OBEME were evaluated and compared with those of natural lipid droplets. The electrostatic interaction between xanthan gum and oil bodies prevents excessive cross-linking and forms a uniform network structure. The basic properties of OBEME were characterized by scanning electron microscopy, cryo-scanning electron microscopy, rheology, and thermogravimetric analysis. The OBEME is an interconnected network and presents a smooth surface without any pores; it remains stable at room temperature for 90 days, and is not affected by low-speed centrifugation and repeated freeze-thaw cycles as indicated by particle size, potential, and fluorescence microscopy analyses. The OBEME enlarges the skin tissue gap, enhances skin permeability, and shows a good slow-release effect in the transdermal absorption test in vivo. It demonstrates a wound healing effect; further, it regulates the inflammatory response of full-layer skin wounds in rats, as well as accelerate angiogenesis, and promote re-epithelialization and remodeling. The OBEME as a bioactive molecule-carbohydrate complex can effectively accelerate skin regeneration and has great translational potential to provide low-cost alternative wound care treatments.

Safflower oil body nanoparticles deliver hFGF10 to hair follicles and reduce microinflammation to accelerate hair regeneration in androgenetic alopecia.

Androgenetic alopecia (AGA) affects physical and mental health with limited therapeutic options. Novel materials and delivery methods have considerable potential to improve the current paradigm of treatment. In this study, we used a novel plant nanoparticle of safflower oil body (SOB) loaded with human fibroblast growth factor 10 (hFGF10) to target hair follicles and accelerate hair regeneration in AGA mice with few adverse effects. Our data revealed that the average particle size of SOB-hFGF10 was 226.73 ± 9.98 nm, with a spherical and uniform structure, and that SOB-hFGF10 was quicker to preferentially penetrate into hair follicles than hFGF2 alone. Using a mouse model of AGA, SOB-hFGF10 was found to significantly improve hair regeneration without any significant toxicity. Furthermore, SOB-hFGF10 inhibited dihydrotestosterone (DHT)-induced TNF-α, IL-1ß, and IL-6 overproduction in macrophages in relation to hair follicle microinflammation, thereby enhancing the proliferation of dermal papilla cells. Overall, this study provides an applicable therapeutic method through targeting hair follicles and reducing microinflammation to accelerate hair regeneration in AGA.

Optimization of the extraction conditions and dermal toxicity of oil body fused with acidic fibroblast growth factor (OLAF).

INTRODUCTION: Oil body (OB), a subcellular organelle that stores oil in plant seeds, is considered a new transdermal drug delivery system. With the increasing understanding of the OB and its main protein (oleosin), numerous studies have been conducted on OB as "carrier" for the expression of exogenous proteins. In our previous study, oil body fused with aFGF (OLAF) was obtained using a plant oil body expression system that had been preliminarily proven to be effective in accelerating the healing of skin wounds. However, no dermal toxicological information on OLAF is available. OBJECTIVE: To ensure the dermal safety of OLAF, a series of tests (the acute dermal toxicity test, 21-day repeat dermal toxicity test, dermal irritation test and skin sensitisation test) were conducted after optimising the extraction protocol of OLAF. MATERIALS AND METHODS: To improve the extraction rate of OLAF, response surface methodology (RSM) was first employed to optimise the extraction conditions. Then, Wistar rats were exposed to OLAF (400 mg·kg-1 body weight) in two different ways (6 hours/time for 24 hours and 1 time/day for 21 days) to evaluate the acute dermal toxicity and 21-day repeated dermal toxicity of OLAF. In the acute dermal toxicity test, clinical observations were conducted to evaluate the toxicity, behaviour, and health of the animals for 14 consecutive days. Similarly, the clinical signs, body weight, haematological and biochemical parameters, histopathological changes and other indicators were also detected during the 21 days administration. For the dermal irritation test, single and multiple doses of OLAF (125 mg·kg-1 body weight) were administered to albino rabbits for 14 days (1 time/day). The irritation reaction on the skin of each albino rabbit was recorded and scored. Meanwhile, skin sensitisation to OLAF was conducted using guinea pigs for a period of 28 days. RESULTS: Suitable extraction conditions for OLAF (PBS concentration 0.01, pH of PBS 8.6, solid-liquid ratio 1:385 g·mL-1) were obtained using RSM. Under these conditions, the extraction rate and particle size of OLAF were 7.29% and 1290 nm, respectively. In the tests of acute dermal toxicity and 21-day repeated dermal toxicity, no mortality or significant differences were observed in terms of clinical signs, body weight, haematological parameters, biochemical parameters and anatomopathological analysis. With respect to the dermal irritation test and skin sensitisation test, no differences in erythema, oedema or other abnormalities were observed between treatment and control groups on gross and histopathological examinations. CONCLUSIONS: The results of this study suggest that OLAF does not cause obvious toxicity, skin sensitisation or irritation in animals.

Dermal toxicity, dermal irritation, and delayed contact sensitization evaluation of oil body linked oleosin-hEGF microgel emulsion via transdermal drug delivery for wound healing.

Objective: The expression of therapeutic proteins in plant oil body bioreactors has attracted much attention. But its safety is not yet clear. This article determines the risk of safety after using the drug. Methods: The oil body-linked oleosin-hEGF microgel emulsion (OBEME) was prepared by mixing the xanthan gum with suitable concentrations in an appropriate proportion. Skin irritation and sensitization reaction were investigated in rats and guinea pigs using OBEME as test article.Results: The OBEME did not produce dermal erythema/eschar or oedema responses. The dermal subacute and subchronic toxicity of OBEME were evaluated in accordance with OECD guidelines. Compared with the control group, the basic physical signs, such as weight, feed, drinking, excretion, and behaviour of experimental animals, were not abnormal. In addition, no abnormality was found in haematological parameters, biochemical indexes, relative organ weight, and histopathological observation of organs, and there was no significant difference compared with normal saline treatment group. Therefore, we conclude that OBEME has no toxic effects and is safe and reliable to be used for topical application.

Molecular Pharming of the Recombinant Protein hEGF-hEGF Concatenated with Oleosin Using Transgenic Arabidopsis.

We set out to assess the NIH/3T3 cell proliferation activity of Arabidopsis oil body-expressed recombinant oleosin-hEGF-hEGF protein. Normally, human epidermal growth factor (hEGF) is purified through complex process, however, oleosin fusion technology provides an inexpensive and scalable platform for its purification. Under a phaseolin promoter, we concatenated oleosin gene to double hEGF (hEGF-hEGF) with plant-preferred codons in the expression vectors and the construct was transformed into Arabidopsis thaliana (Arabidopsis). The transgenic Arabidopsis was validated by RT-PCR and the content of recombinant protein oleosin-hEGF-hEGF was quantified by western blot. Subsequently, the proliferation assay and transdermal absorption were determined by MTT method and immunohistochemical staining, respectively. First, the expression level of hEGF was recorded to be 14.83-ng/µL oil body and due to smaller size transgenic oil bodies expressing the recombinant oleosin-hEGF-hEGF, they were more skin permeable than those of control. Second, via the staining intensity of transgenic oil bodies was greater than EGF at all time points via immunohistochemical staining in transdermal absorption process. Lastly, activity assays of oil bodies expressed oleosin-hEGF-hEGF indicated that they stimulated the NIH/3T3 cell proliferation activity. Our results revealed oil-body-expressed oleosin-hEGF-hEGF was potential new material having implications in the field of medicine.

Genome-Wide Identification, Expression Profiling, and Functional Validation of Oleosin Gene Family in Carthamus tinctorius L.

Carthamus tinctorius L., commonly known as safflower, is an important oilseed crop containing oil bodies. Oil bodies are intracellular organelles in plant cells for storing triacylglycerols (TAGs) and sterol esters. Oleosins are the most important surface proteins of the oil bodies. We predicted and retrieved the sequences of eight putative C. tinctorius oleosin (Ctoleosin) genes from the genome database of safflower. The bioinformatics analyses revealed the size of their open reading frames ranging from 414 to 675 bp, encoding 137 to 224 aa polypeptides with predicted molecular weights of 14.812 to 22.155 kDa, all containing the typical "proline knot" motif. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) determined the spatiotemporal expression pattern of Ctoleosin genes, which gradually increased and peaked during flowering and seed ripening, and decreased thereafter. To validate their role in plant development, we transformed and overexpressed these eight putative Ctoleosin genes in Arabidopsis. Overexpressing Ctoleosins did not affect leaf size, although silique length was altered. Arabidopsis transformed with Ctoleosin3, 4, and 5 grew longer siliques than did the wild-type plants, without altering seed quantity. The 100-grain weight of the transgenic Arabidopsis seeds was slightly more than that of the wild-type seeds. The seed germination rates of the plants overexpressing Ctoleosin4 and 6 were slightly lower as compared with that of the wild-type Arabidopsis, whereas that in the other transgenic lines were higher than that in the wild-type plants. The overexpression of Ctoleosin genes elevated the oil content in the seeds of transgenic Arabidopsis. Our findings not only provide an approach for increasing the oil content, but also for elucidating the intricate mechanisms of oil body synthesis.

A new nanoscale transdermal drug delivery system: oil body-linked oleosin-hEGF improves skin regeneration to accelerate wound healing.

BACKGROUND: Epidermal growth factor (EGF) can promote cell proliferation as well as migration, which is feasible in tissue wound healing. Oil bodies have been exploited as an important platform to produce exogenous proteins. The exogenous proteins were expressed in oil bodies from plant seeds. The process can reduce purification steps, thereby significantly reducing the purification cost. Mostly, the diameter of oil body particle ranges between 1.0 and 1.5 µm in the safflower seeds, however, it reduces to 700-1000 nm in the transgenic safflower seeds. The significant reduction of particle size in transgenic seeds is extremely beneficial to skin absorption. RESULTS: The diameter of oil body in the transgenic safflower seeds was recorded in the range of 700-1000 nm. The smaller particle size improved their skin absorption. The expression level of oleosin-hEGF-hEGF in T3 transgenic seeds was highest at 69.32 mg/g of seeds. The oil body expressing oleosin-hEGF-hEGF had significant proliferative activity on NIH/3T3 cells and improved skin regeneration thereby accelerating wound healing in rats. The wound coverage rate exceeded 98% after treatment for 14 days with oil body expressing oleosin-hEGF-hEGF, while the saline without EGF group and wild type oil body group both showed less than 80%. The neonatal fibroblast and collagen were found to be increased in the safflower oil body expressing oleosin-hEGF-hEGF treatment group. TGF-ß1, bFGF and VEGF were noted as important growth factors in the repair of cutaneous wounds. Their expression level increased after 4 and 7 day treatment, but decreased after 14 days. Therefore, it can promote skin regeneration to accelerate wounds healing. CONCLUSIONS: The expression of oleosin-hEGF-hEGF in T3 transgenic seeds was 80.43 ng/µL oil body. It had significant proliferative activity on NIH/3T3 cells and improved skin regeneration to accelerate wound healing in rats. The expression process of TGF-ß1, bFGF and VEGF increased at first and then gradually declined.

High-efficiency production of bioactive oleosin-basic fibroblast growth factor in A. thaliana and evaluation of wound healing.

Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factors family. It is a highly specific mitogenic factor for many cell types, as though it be involved in wound repair, angiogenesis, nerve nutrition and embryonic development etc. Oil bodies have been applied for medicine, foodstuff and industry field. The heterogonous proteins expressed in oil bodies have distinct advantages, such as less purification steps and low costs. In this study, bFGF was expressed in A. thaliana seeds using oleosin fusion technology. The pOTB-bFGF vector contained an oleosin-bFGF fusion gene and a glufosinate resistance gene for selection. Transgenic A. thaliana lines were obtained by the floral dip method and protein expression was identified by SDS-PAGE and western blotting in transgenic A. thaliana lines. Moreover, MTT assays showed that the oil bodies expressed oleosin-bFGF fusion protein had a remarkable proliferation effect on NIH/3T3 cells and animal experiments showed that it could effectively decrease wound size and accelerate granulation tissue maturation. In conclusion, this may be a better method of producing oleosin-bFGF fusion protein to meet the increasing demand in its pharmacological application.

Oil Body-Bound Oleosin-rhFGF-10: A Novel Drug Delivery System that Improves Skin Penetration to Accelerate Wound Healing and Hair Growth in Mice.

Recombinant human fibroblast growth factor 10 (rhFGF-10) is frequently used to treat patients with skin injuries. It can also promote hair growth. However, the effective application of rhFGF-10 is limited because of its poor stability and transdermal absorption. In this study, polymerase chain reaction (PCR) and Southern blotting were used to identify transgenic safflowers carrying a gene encoding an oleosin-rhFGF-10 fusion protein. The size and structural integrity of oleosin-rhFGF-10 in oil bodies extracted from transgenic safflower seeds was characterized by polyacrylamide gel electrophoresis and western blotting. Oil body extracts containing oleosin-rhFGF-10 were topically applied to mouse skin. The absorption of oleosin-rhFGF-10 was studied by immunohistochemistry. Its efficiency in promoting wound healing and hair regeneration were evaluated in full thickness wounds and hair growth assays. We identified a safflower line that carried the transgene and expressed a 45 kDa oleosin-rhFGF-10 protein. Oil body-bound oleosin-rhFGF-10 was absorbed by the skin with higher efficiency and speed compared with prokaryotically-expressed rhFGF-10. Oleosin-rhFGF-10 also enhanced wound closure and promoted hair growth better than rhFGF-10. The application of oleosin-rhFGF-10 in oil bodies promoted its delivery through the skin, providing a basis for improved therapeutic effects in enhancing wound healing and hair growth.