The Influence of Protein Loading in Emptied Yeast on its Bactericidal and Anticancer Effectiveness Volume 55- Issue 1

Nawal Abd El-Baky, Mona M Sharaf and Amro A Amara*

• Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), Egypt

Received: January 25, 2024; Published: February 09, 2024

*Corresponding author: Amro A Amara, Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, P.O. Box 21934 Alexandria, Egypt

DOI: 10.26717/BJSTR.2024.55.008635

Abstract PDF

While yeasts were employed for delivery of drugs, active RNA or DNA, they were in minor cases utilized in delivery of certain antigenic proteins. The loading of bioeffective proteins inside emptied cells can preserve their structure and bioefficiency. In the present work, loaded lactoferrin inside emptied yeast was assayed for bactericidal and anticancer effectiveness after loading and compared with free protein. Its bactericidal efficiency was assayed against S. aureus, S. typhi, K. pneumonia, S. sonnei, P. vulgaris, S. marcescens, and E. coli. Its cytotoxicity was checked on human skin fibroblast (HSF) and potential anticancer impact on epidermoid skin carcinoma of human (A-431). S. aureus, S. sonnei, and E. coli were the most susceptible to loaded protein (concentration of minimum inhibition (MIC) of 0.65 mg/ml), but K. pneumonia and P. vulgaris were the least susceptible ones having MIC equal to 2.6 mg/ml. The MIC for loaded protein remained the same against K. pneumonia compared to free protein. Conversely, MIC for loaded protein amplified twice against S. aureus, S. typhi, S. sonnei, S. marcescens, and E. coli, and amplified four times in case of P. vulgaris. IC50 of loaded protein on A-431 was >1.25 mg/ml, and on HSF was 2.77 mg/ml. These outcomes pointed out the drop in bactericidal potency for loaded protein inside emptied yeast against six of seven tested pathogens related to free protein. Moreover, the toxicity was comparable on both normal (HSF) and anticancer cells (A-431).

Keywords: Loaded Protein; Emptied Yeast; Bactericidal; Anticancer; Bioeffective Proteins

Abbreviations: A-431: Human Epidermoid Skin Carcinoma; FBS: Fetal Bovine Serum; HSF: Human Skin Fibroblast; MIC: Concentration of Minimum Inhibition

Since the eighties of 19 century, proteins have gained extensive acceptance as drugs, with insulin as a unique model [1,2]. Protein therapies or therapeutic candidates include either purified proteins from natural sources or recombinant ones for instance hormones, enzymes, antibodies, cytokines, vaccines from protein subunits, etc. [3-5]. However, their delivery remains a hot topic for research [6]. Lactoferrin as therapeutic candidate was earlier verified [7]. Its bactericidal potency was showed to be based on binding to various sites on cell surfaces of bacteria [8-12]. Its anticancer effectiveness was also verified [7,13,14]. Lactoferrin does its anticancer potential via provoking caspase-1 as well as IL-18, triggering CD8+ and CD4+, activating natural killer and IFN-γ T cells, hindering angiogenesis, and inducing apoptosis [15]. Lactoferrin had the capacity for constraining or motivating division of cells, reliant on whether its impact intended for healthy or cancer cells [16,17]. It was also found to affect cells that produce melanin resulting in approximately twenty reduction percent in pigmentation. It was immersed transdermally conquering production of melanin [18]. Its recombinant form could trigger propagation and migration of fibroblasts, and keep their survival [19]. Yeasts (S. cerevisiae) are profitable for carrying drugs owing to their safety and cost effectiveness. Furthermore, they are cultivable lacking whichever extra costs. Also, phospholipids in their membranes behave in a similar way to liposomes and thus could encapsulate various molecules [20-23].

Their thick wall containing glucan, mannoprotein layer, and chitin (only minor quantity) made them a type of continuous discharge system for delivery of drugs [24]. Yeast was formerly chemically emptied from all of its contents [25]. Drugs like berberine and gossypol acetic acid were introduced into yeast cells for their delivery [26,27]. While, delivery of certain antigenic proteins was also reported for yeast [28]. In an earlier study (under publication), lactoferrin derived from milk of camel was introduced into emptied yeast. The present work was conducted to examine the influence of protein loading inside emptied yeast on its bactericidal and anticancer effectiveness.

Introduction of Chloramphenicol into Emptied Yeast

Chloramphenicol (Bioshop, Canada) was dissolved at concentration 50 μg/ml in absolute ethanol, and filter sterilized. Emptied yeast was added to 2 ml of chloramphenicol, let at room temperature for half hour, followed by evaporating ethanol. Chloramphenicol in emptied yeast was used as bactericidal standard. Protein (lactoferrin derived from milk of camel) previously loaded inside emptied yeast (under publication) was involved in the coming assays.

Assessment of Chloramphenicol into Emptied Yeast

To conclude the quantity of taken chloramphenicol dissolved in ethanol by emptied yeast, a microscopic glass slide was weighted and emptied yeast was prepared as a slide smear without heating during fixation. As a substitute, cells were left to dry at 37 °C. After drying, slide was weighted again to calculate the smear weight. About 500 µl of chloramphenicol (50 μg/ml dissolved in ethanol) was added on top of the smear. The slide was left to enable the cells to take the drug for 20 min, and then the slide was dried again. After washing the slide with distilled water to get rid of any excess drug (outside emptied yeast), the slide was dried again at 37 °C and weighed. The amount of chloramphenicol contained within the cells was calculated from the difference in smear weight before and after drug addition.

Bactericidal Assessment for Loaded Lactoferrin Inside Emptied Yeast

The utilized bacterial pathogens in broth microdilution check to value bactericidal efficiency of loaded lactoferrin/chloramphenicol inside emptied yeast include Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 19430, Klebsiella pneumonia, Shigella sonnei ATCC 25931, Proteus vulgaris, Serratia marcescens, and Escherichia coli ATCC 25922. All of which incubated overnight at 37 °C in LB broth. To measure bactericidal efficiency of loaded lactoferrin/chloramphenicol inside emptied yeast and assess their MIC, broth microdilution was done. Dilutions (serial) were done at two-fold from loaded protein (starting with 5.2, and reaching 0.325 mg/ml) and added to plates of bacteria. Additionally, the same was done for loaded chloramphenicol (starting 20, and reaching 1.25 μg/ml). After 12 h incubation at 37 °C, growth was assessed. Test was carried out in triplicates [29].

Cultures of Skin Cells

HSF and A-431 have been gotten from Nawah Scientific Inc. (Cairo, Egypt). Cells maintenance was carried out at 37°C in DMEM supplemented media (10% of heat inactivated fetal bovine serum; FBS) in humidified, 5% (v/v) CO₂ atmosphere.

Cytotoxicity and Anti-Carcinogenicity Assays

Standard MTT test has been conducted for cell viability estimation for HSF and anti-carcinogenicity for A-431 [30,31]. Aliquots of cells suspension (100 µL at 5x 10³ cells) were loaded in plates and incubated in complete DMEM media for 24 h. Cells were treated with different concentrations of Cisplatin (standard drug) and loaded protein inside emptied yeast. Following 48 h of exposure, medium was removed and MTT introduced. The released formazan was detected with DMSO. The absorbance was valued at ʎmax 570 nm.

Bioeffective proteins are interesting candidates that can be applied in different applications [3-5]. Some of them even enclose short peptide(s) that add to their functionality in a similar pathway or different one. Lactoferrin is one of those proteins that is extensively consumed for different purposes. This protein is a scavenger for so many and variable activities beneficial for us [7-15]. The fascinating thing is that lactoferrin is already naturally produced within our secretions. In the present work, lactoferrin derived from milk of camel previously introduced into emptied yeast (under publication) was analyzed to examine the influence of protein loading inside emptied yeast on its bactericidal and anticancer effectiveness. Bactericidal efficiency of loaded protein was checked against seven bacterial pathogens. Its cytotoxicity on HSF and anticancer efficacy on A-431 were also assessed.

Assessment of Chloramphenicol into Emptied Yeast

The amount of chloramphenicol contained within emptied yeast was about 20 μg.

Bactericidal Assessment for Loaded Lactoferrin Inside Emptied Yeast

In view of Table 1 and presented MIC, S. aureus, S. sonnei, and E. coli were the most susceptible to loaded protein (MIC of 0.65 mg/ml), but K. pneumonia and P. vulgaris were the least susceptible ones having MIC equal to 2.6 mg/ml. The MIC for loaded protein remained the same against K. pneumonia compared to free protein. Conversely, MIC for loaded protein amplified twice against S. aureus, S. typhi, S. sonnei, S. marcescens, and E. coli, and amplified four times in case of P. vulgaris. This drop in bactericidal potency may be caused by slow freeing of the loaded protein or may be its binding to yeast surface.

Table 1: Bactericidal efficacy for loaded lactoferrin inside emptied yeast.

Cytotoxicity and Anti-Carcinogenicity Assays

A normal cell line (HSF) was used to investigate the toxicity of loaded protein and A-431 cancer cells were for anticancer evaluation. Both cell types were treated using Cisplatin/standard anticancer drug (0.03-300 μg/ml) and loaded protein inside emptied yeast (0.004-1.25 mg/ml). The aim was to investigate the impact of protein loading on its anticancer efficacy on skin cancer cells. Figure 1, plates showed clearly that Cisplatin behave differently on normal than cancer cells, yet loaded protein behaved nearly the same way especially at concentration of 1.25 mg/ml it even caused more toxicity to healthy cells. The MTT data for HSF are in Tables 2 & 3. While, those for A-431 are showed in Tables 4 & 5. The LC₅₀ details on both cell lines were calculated as in Figures 2 & 3. The findings displayed that the cytotoxicity against the normal cell line under the experimental condition for Cisplatein was 4 µg/ml, while for loaded protein it was 2.77 mg/ml. The anti-carcinogenicity for Cisplatin on cancer cells was 5.24 µg /ml, while for the loaded protein it was >1.25 mg/ml. The above-mentioned result indicated that the loaded protein under the experimental conditions is highly toxic for cancer as well as healthy cells. In contrast, Cisplatin exhibited the standard criteria of the antitumor compound and proved to be selective. These outcomes differ significantly from a former study that applied free lactoferrin (non-loaded).

Table 2: The viability % of HSF after treatment with various Cisplatin concentrations.

Note: Control: untreated cells, Blank: DMSO only

Table 3: The viability % of HSF after treatment with various loaded protein concentrations.

Note: Control: untreated cells, Blank: DMSO only

Table 4: The viability % of A-431 after treatment with various Cisplatin concentrations.

Note: Control: untreated cells, Blank: DMSO only

Table 5: The viability % of A-431 after treatment with various loaded protein concentrations.

Note: Control: untreated cells, Blank: DMSO only

Figure 1

Figure 2

Figure 3

The obtained data of the free protein demonstrated that the concentration of free protein that spared the life of all WI-38 cells (Human normal cells) was 425.2 μg/ml while the concentration that could kill 50% of cancer cells; HepG-2, Caco-2, MCF-7 and Hela were 1011, 2127, 1229, 1352 μg/ml respectively, which means that free protein could selectively kill cancer cells [14]. One could also conclude that the loaded protein in emptied yeast apparently enables a higher concentration around the yeast cells before full dissociation in the surrounding medium. That might explain its high toxicity on both of normal and cancer cells. More investigations are needed to adjust the release of the loaded protein by yeast and the best conditions that can readjust its toxicity based on its concentration in certain volume during the start point of the release till the full re-evacuation of the loaded protein from emptied yeast.

The influence of protein loading in emptied yeast was unfortunately negative on its bactericidal and anticancer effectiveness. That might indicate a controlled freeing of the protein from emptied yeast in case of bacterial control. The high toxicity in case of cell lines treatment can be linked to a higher concentration around the yeast cells before full dissociation in the surrounding medium. More investigations are needed.

The authors declare no conflict of interest.

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