Alginate oligosaccharides can maintain activities of lysosomes under low pH condition

The objective of this study was to report that lysosome extracted from egg white could be used as a drug through oral administration for treating diseases by using pH sensitive alginate oligosaccharides. Lysosome-alginate oligosaccharides composite were formulated for oral administration of lysosomes. The dissolution test confirmed the availability of the oral dosage form. When lysosome were used as an independent drug, the activity of protein was lost due to influence of low pH. Its antibacterial activity was also remarkably reduced. However, when lysosome-alginate oligosaccharides composite form was used, antimicrobial activity of lysozyme was maintained. At low pH, a gel-like matrix was formed by alginate oligosaccharides to protect the lysosome. When the pH was increased, alginate oligosaccharides were dissolved and the lysosome was released. SDS–polyacrylamide gel electrophoresis analysis of released lysosomes revealed that alginate oligosaccharide could effectively protect the lysosome from degradation or hydrolysis under acidic conditions for at least 2 h. The results of this study are important for application of lysosomes as therapeutic agents, and also it was confirmed that alginate oligosaccharides have potential as direct delivery system for the oral application of protein derived therapies.

www.nature.com/scientificreports/ In general, AOs has a short chain length, so it has improved water solubility when compared to high molecular weight alginates of the same monomer 12 . Since alginate oligosaccharides (AOs) are considered as non-toxic, non-immunogenic and biodegradable polymers, they are attractive candidates in biomedical applications 13 . AOs are linear polymers of polysaccharides with alternating gel formation properties consisting of β-(1-4)-Dmannosyluronic acid and α-(1-4)-L-glucosyluronic acid. AOs exhibit important biological activities not only in mammals, but also in plants. AOs have additional properties such as antimicrobial effects 14,15 , anti-inflammatory effects 16 , defense-enhancing effects against infection by certain pathogens 17 , and anti-glycating effects 18 . AOs can promote growth and proliferation of human cells, including keratinocytes [19][20][21] and endothelial cells 22 . In addition, they can potentially protect nerve cells from oxidative stress 23 . Finally, AOs have interesting properties for fighting microbial pathogens. AOs can potentiate the action of selected antibiotics by disturbing multidrug-resistant bacteria 24 . Similarly, they can inhibit fungal cell growth and enhance activities of antifungal agents against Candida and Aspergillus species 25 . Here, the purpose of this study was to confirm that the lysosome isolated from egg white can be used as an eco-friendly agent for the treatment of metabolic diseases related to microorganisms living in the intestine through oral drug delivery using pH-sensitive AO.

Results
Formation of lysosome-AOs composite. Three types of alginate oligosaccharides used in this study were depolymerized with alginate by treating sodium alginate at 37 °C for 6 h, 12 h, and 24 h with alginate decomposing enzyme, respectively, to prepare three types of alginate oligosaccharides. The prepared alginate oligosaccharide was analyzed by MALDI-TOF-MS spectroscopy, and each alginate oligosaccharide had an intensity of 379.25 m/z peak corresponding to [M + Na] as 41.96%, 72.03%, and 88.86%. Results of observing the appearance of wild type lysosome, lysosome-2% alginate composite, or lysosome-AOs composite under low pH environment are shown in Fig. 1. Wild type lysosomes without alginate or AOs were used as negative controls. Wild type lysosomes were almost the same before and after the dissolution test. Denaturation of protein was observed in simulated gastric fluid due to its low pH. Lysosome-2% alginate composite was used as a positive control. In acid phase (simulated gastric juice, pH 1.2), alginate gel containing lysosome was formed because pH-sensitive alginate could form spherical lysosome-2% alginate. In the buffer phase (simulated intestinal juice, Figure 1. Appearance of lysosomal suspension immobilized with a pH-sensitive biopolymer in simulated gastric and intestine environment. Type 1 AOs, Type 2 AOs, and Type 3 AOs refer to alginate oligosaccharides made by lyase treatment for 6 h, 12 h, and 24 h, respectively. www.nature.com/scientificreports/ pH 6.8), the alginate gel dissolved to release lysosome. Next, lysosome-AOs (lysosome-Type 1 AOs, lysosome-Type 2 AOs, lysosome-Type 3 AOs) composites formed spherical gel containing lysosome at low pH. In the buffer phase, the gel formed by AOs was dissolved and lysosome was released. Among these three types of AOs, Type 1 AOs behaved like alginate the most.
Antimicrobial activity of the released lysosome. To use lysosomes as a drug, lysosome-2% alginate composite and lysosome-AOs composite were manufactured and applied as oral delivery systems. The antimicrobial activity of wild type lysosome against cell mortality of E. coli was 95% or more ( Fig. 2). Figure 3 shows results of dissolution test, confirming the antimicrobial activity of lysosomes released into the intestine. Wild type lysosomes used for the dissolution test showed approximately 80% decreased antimicrobial activity. However, the antimicrobial activity of the lysosomes used in the dissolution test was maintained after mixing 2%  www.nature.com/scientificreports/ alginate with three types of AOs. The antimicrobial activity of lysosome mixed with 2% alginate was the highest, followed by lysosome mixed with Type 1 AOs.
Protein of the released lysosome. Figure 4 shows SDS-PAGE results. Lane M shows protein marker (DokDo-MARK). Lane 1 shows lysosomal protein extracted from wild type lysosome (without alginate or AOs) as a control. Lanes 2 to 4 schematically describe lysosomal protein patterns extracted from wild type lysosome, lysosome-2% alginate composite, and lysosome-AOs composites formed in simulated gastric fluid (pH 1.2). As a result, the pattern was similar to that shown in lane 1. Lanes 1 and 2 and lanes 3 and 4 showed almost the same pattern. After reaction in simulated gastric fluid, wild type lysosome, lysosome-2% alginate composites, and lysosome-AOs composites were collected and reacted in simulated intestinal fluid. Lanes 5, 6, and 7 show lysosomal protein patterns extracted from wild type lysosomes, lysosome-2% alginate composites, and lysosome-AOs composites dissolved in simulated intestinal fluid (pH 6.8), respectively. As a result, protein pattern extracted from the wild type lysosome (without alginate/AOs) appeared in simulated gastric fluid. This protein pattern did not appear in simulated intestinal fluid. On the other hand, lanes 6 and 7 were lysosomal proteins extracted from lysosome-2% alginate composites and lysosome-AOs composites dissolved and released in simulated intestinal fluid, respectively. Although expression levels and patterns were slightly changed, these results were similar to those under acidic conditions. In particular, lysozyme is another significant protein discovered in egg white. The molecular weight of lysozyme is 14,400 Da. Lysozyme consists of a single polypeptide chain with 129 amino acids 25 . These results confirmed that the pattern of protein extracted from released lysosome appeared below 15 kDa molecular weight (Fig. 4, arrow). In other words, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that mixed alginate and alginate oligosaccharides could effectively protect lysosomes from degradation or hydrolysis under acidic conditions for at least 2 h. Figure 5 shows morphologies of lysosomes observed according to environmental conditions during oral administration. In Fig. 5a, wild type lysosome (unused for experiments) in DW (distilled water) was observed. Figure 5b confirmed the morphology formed by oral administration of lysosomes.

Morphology of complex.
The picture showed lysosomes in pH 1.2 solution (instead of gastric fluid, and lysosomes in pH 6.8 solution (instead of intestinal fluid). As shown in Fig. 5b, the control group (without alginate or AOs) did not have spherical lysosomes at pH 1.2 or pH 6.8. On the other hand, in the case of treatment of 2% alginate with Type 1 AOs, Type 2 AOs, or Type 3 AOs, it was found that spheres seen as lysosomes in pH 1.2 were more clustered by surrounding substrates. Further, at pH 6.8, a spherical lysosome was found independently.

Discussion
Alginate hydrogel exists in the units of D-mannuronic acid and L-guluronic acid, and is a pH-sensitive biopolymer. Alginate has a sol-gel transition characteristic, and Alginate gels tend to be eroded under more neutral and basic pH values than under acid conditions 27 . In other words, alginate hydrogel is an ideal matrix for use in oral sustained release drugs 28 . To overcome the drawback of using the properties of alginate hydrogels but not being degraded in vivo, this study proposes alginate oligosaccharides as a drug matrix in oral sustained release formulations 9 . In this study, we devised a method to utilize low molecular weight AOs to use lysosomes as oral drugs. Lysosome-AOs (lysosome-Type 1 AOs, lysosome-Type 2 AOs, lysosome-Type 3 AOs) composites formed spherical gel containing lysosome at low pH. In the buffer phase, the gel formed by AOs was dissolved and lysosome was released. Of these three types of AO, type 1 AO behaves like the alginate, making gel formation most effective. Alginate has the property of changing to alginate sol form under acidic conditions. Similarly, it was confirmed that AOs changed to sol form under acidic conditions, so that AOs act as a substrate surrounding lysosomes. Dissolution testing is the most common method for evaluating oral modified release delivery systems for colon-specific drug delivery. In-vitro evaluation of oral modified drug delivery systems is generally performed by using standard process procedures or by dissolution testing as appropriate 29 . The dissolution test confirmed that the lysosome activity was maintained up to the intestine when the lysosomes were used for oral administration in the form of AOs complex.
Lysosome activity mediates several processes for cell feeding and antimicrobial defense, including lysosomal fusion with endosomes and phagosomes 2,30 . Thus, lysosomal enzymes are capable of cell digestion 31 . Antimicrobial effects of lysosomes isolated from egg white on different strains including Gram-positive and Gramnegative bacteria have been reported 32 . Wild type lysosomes used for the dissolution test showed remarkably decreased antimicrobial activity than before it was used in the experiment. However, the antimicrobial activity of the lysosomes used in the dissolution test was maintained after mixing 2% alginate or three types of AOs. The antimicrobial activity of lysosome mixed with 2% alginate was the highest, followed by lysosome mixed with Type 1 AOs. Therefore, it can be seen that it is effective to use type 1 AOs together to maintain lysosomal activity up to the intestine.
Lysozyme is another significant protein discovered in egg white. The molecular weight of lysozyme is 14,400 Da. Lysozyme consists of a single polypeptide chain with 129 amino acids 26 . In this study, it was shown that lysosomes mixed with alginate or alginate oligosaccharide were protected so that antibacterial activity could be maintained in acidic conditions (artificial gastric juice) for at least 2 h.

Conclusions
The purpose of this research was to describe that lysosome isolated from egg white could be used as an environmentally friendly antimicrobial agent for treating diseases through oral drug delivery. This study applied sol-gel transition characteristics of biopolymer by pH change. AOs, when processed together with lysosome to use lysosome as a drug, allows drug release in the intestine with a high pH and limits drug exposure in the upper part of the gastrointestinal tract at low pH. Although results described in this study are from preliminary www.nature.com/scientificreports/ experiments for applying lysosomes as therapeutic agents, AOs have possibility as delivery systems for direct application of drugs, proteins, and lysosomes.

Materials and methods
Isolation of lysosome from egg white. Lysosome isolation was performed by homogenizing egg white followed by centrifugation twice under different conditions. First, egg white was homogenized for 30 min, divided into centrifuge bottles, and centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was collected, homogenized for 2 h, and centrifuged at 20,000 × g for 30 min at 4 °C. After the second centrifugation, the supernatant was discarded and only the remaining pellet was collected. The collected pellet was tested for antimicrobial activity and used as lysosomes in all subsequent experiments. The collected pellet was stored at 4 °C in the dark.
AOs production by enzymatic degradation of sodium alginate. To prepare Type 1 AOs, Type 2 AOs, and Type 3 AOs, alginate lyase (Sigma-Aldrich, A1603) was used to catalyze the cleavage of alginate. To make Type 1 AOs, 2% (w/v) of sodium alginate (Sigma-Aldrich, A0682) was prepared by dissolving sodium alginate in 100 ml of distilled water (DW) followed by incubation at 37 °C for 6 h in the presence of 20 U/mg alginate lyase in a rotary shaker at 150 rpm. After the incubation, alginate lyase in the sample was inactivated at 100 °C for 10 min and removed after centrifugation at 12,000×g for 10 min at 4 °C 33 . Type 2 and Type 3 AOs were prepared with the same method except that alginate lyase treatment time was changed from 6 to 12 h for Type 2 AOs and 24 h for Type 3 AOs. Fractions of AOs were collected, freeze-dried, and store at − 4 °C. Confirmation of antimicrobial activity of the released lysosome. To confirm the antimicrobial activity of the lysosome released to the colon, lysosomes isolated from wild type lysosome composite, lysosome-2% alginate composite, and three types of lysosome-AOs composite were used. Escherichia coli BL21 was used for antimicrobial activity tests. For antimicrobial test, after E. coli grew up to OD 600 at 0.7-0.8, cultures were diluted to 10 6 cells/mL with DW. After that, 100 µl of diluted E. coli was mixed with 900 µl lysosome and 100 µl of the mixture was spread onto LB agar plate. The antimicrobial activity of lysosome was measured using colony counts. Results are expressed as the percentage cell mortality (%) according to reference 34 .

Formation of lysosome-
Analysis of proteins extracted from released lysosome. To analyze lysosomal proteins during drug delivery, proteins were extracted from wild type lysosomes, lysosome-2% alginate composite, and lysosome-AOs composite in simulated gastric fluid and intestinal fluid condition followed by SDS-PAGE. Lysosomal proteins were extracted from separated lysosomes. In this experiment, lysis buffer (0.1% NP-40, 0.5 mM DTT, 0.1 mM PMSF) was used. After wild type lysosome and lysis buffer were mixed at a ratio of 1:1, vortexing and cooling were repeated for 20 min and stabilized on ice for 30 min. Finally, we performed centrifugation at 13,000 rpm, 10 min, and 4 °C and collected separated supernatants containing lysosomal proteins extracted from egg white. After filtration of protein through 10 K Amicon Ultra filter, protein concentration was measured with Bradford assay using BSA (bovine serum albumin) as control. Proteins in the gastrointestinal tract were prepared in the same way.
To analyze protein pattern, the amount of lysosomal protein extracted from wild type lysosome, lysosome-2% alginate composite, or lysosome-AOs composite was adjusted 20 µg. Protein was denatured with 5 × SDS-PAGE sample buffer and boiled at 100 °C for 5 min. Then 12.5% separating gel was used to separate proteins by electrophoresis for 3 h. The gel was stained with Coomassie Brilliant Blue overnight and de-stained with de-staining buffer (45% methanol, 10% acetic acid) until Coomassie Brilliant Blue stain was removal.
Observation of the surface by Field emission scanning electron microscope. Surfaces of wild type lysosome, lysosome-2% alginate composite, and lysosome-Types 1, 2, and 3 AOs composite under different pH conditions were observed by Field emission scanning electron microscope (FE-SEM) (Carl Zeiss SUPRA, Germany). Samples were observed after freeze-drying and storage at − 4 °C.