{"id":58473,"date":"2024-06-25T11:36:51","date_gmt":"2024-06-25T11:36:51","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=58473"},"modified":"2024-07-11T11:57:47","modified_gmt":"2024-07-11T11:57:47","slug":"formulation-and-characterization-of-nanoparticle-protein-sirtuin-1-nps1-by-nanoprecipitation-technique","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/formulation-and-characterization-of-nanoparticle-protein-sirtuin-1-nps1-by-nanoprecipitation-technique\/","title":{"rendered":"Formulation and Characterization of Nanoparticle-Protein Sirtuin 1 (NPS1) by Nanoprecipitation Technique"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Atherosclerosis is a chronic inflammatory disease of the\narteries that alters the structure and function of the three layers of the\ncoronary artery wall1,2<\/sup>. According to current theories, endothelial\ncell dysfunction is one of the first steps in forming atherosclerosis2<\/sup>.\n The process of atherosclerosis is caused\nby several stages: the formation of fat lines, the construction of atheroma,\nand the formation of atherosclerotic plaques. Various risk factors cause\nendothelial cell damage, one of which is inflammation. Acute inflammation has two impacts on EPC\nmobilisation and recruitment: the temporary restricted inflammatory response and\nthe prolonged or exaggerated inflammatory stimulation. EPC mobilisation may be\ntriggered by the transient restricted inflammatory response3<\/sup>. After\nvascular trauma, EPCs are immediately mobilised and help wounded arteries\nrevascularize in response to increased proinflammatory cytokine levels4<\/sup>.  IL-1\u03b2 upregulates the expression of VEGF,\nVEGFR-2, and adhesion molecules in endothelial cells and is implicated in EPC\nrecruitment and transit to ischemic tissue in a VEGF-dependent manner5<\/sup>. <\/p>\n\n\n\n

SIRT1 is a histone deacetylase that relies on nicotinamide\nadenine dinucleotide and helps regulate the cell cycle and apoptosis6<\/sup>.\nSirtuin protein 1 (SIRT1) is the human sirtuin closely related to the yeast\nprotein Sir2 sequence. SIRT1 is widely expressed in EPC cells and is a\nlongevity gene that plays an essential role in its protective function7,8<\/sup>.\nAn age-dependent decrease in SIRT1 levels was observed in arteries, indicating\nits role in the aging of the cardiovascular system. SIRT1 delays replicative\nsenescence and premature senescence in stem cells and differentiated cells\nexposed to oxidative stress9<\/sup>.<\/p>\n\n\n\n

Nanoparticles are one of the technologies that develop the\ndevelopment of cardiovascular drugs. In nanotechnology, a particle is defined\nas an object that is small in size, has a role, and behaves as a unit\nconcerning its properties and transport. Nanoparticles have many advantages so\nthat they are considered capable of delivering drugs: easy to absorb, easy to\ndissolve, and bioavailability and safety in target tissues and organs10<\/sup>.\nOne of the most popular nanoparticles used is chitosan. Chitosan is a valuable\nbiomaterial generated from the deacetylation of chitin\n(poly(\u03b2-1,4-N-acetyl-d-glucose-2-amine)), which is one of the most prevalent\nnatural polysaccharides found in the cell walls of microorganisms such as\nyeast, fungus, and the exoskeletons of crustaceans and insects. The molecular\nweight and degree of deacetylation primarily determine chitosan’s physical and\nchemical characteristics. Chitosan is a biocompatible, biodegradable,\nbioadhesive, and nontoxic substance. Chitosan, a naturally occurring\nbiocompatible polymer, has been widely studied in pharmaceutical and industrial\nresearch for drug delivery and biological applications.<\/p>\n\n\n\n

Chitosan can control the rate of medication release, extend\ntherapeutic efficacy, and deliver pharmaceuticals to the proper spot in\nthe body. Chitosan may be dissolved in water under acidic circumstances to\nyield a high positive charge density. Chitosan’s positive charge allows it to\ninteract with polyanions and create complexes, then develop a gel and adhere to\nmucous membranes, increasing drug absorption across cellular barriers11<\/sup>.\nHowever, there are currently no studies that explain further the specific\npackaging of chitosan and SIRT1 protein. Therefore, this study aims to report the formulation\nand characterization stages of nanoparticles carrying SIRT1 (NPS1) with\ndifferent solvents, to meet the optimal solvent used in drug design\ndevelopment. <\/p>\n\n\n\n

Materials And Methods:\n<\/strong><\/strong><\/p>\n\n\n\n

Nanoparticle formulation-SIRT1 (NPS1)<\/strong><\/p>\n\n\n\n

This study uses four groups of formulas: K1, K2, F1, and F2.\n<\/p>\n\n\n\n

K1 is a control NPS1 with a solvent ratio of acetone:methanol\n(3:2) without protein SIRT-1 (Cat. Number ab54334, ABCAM, USA); <\/p>\n\n\n\n

K2 is control of NPS1 with aquadest as a solvent without\nprotein SIRT-1; <\/p>\n\n\n\n

F1 is a formulation of NPS1 with a solvent ratio of acetone:methanol\n(3:2) with SIRT-1 protein 7.5 \u03bcL;<\/p>\n\n\n\n

F2 is the formulation of NPS1 with aquadest as a solvent\nwith SIRT-1 protein 7.5 \u03bcL. <\/p>\n\n\n\n

Aquadest used in this research is water for injection (WFI).<\/p>\n\n\n\n

Preparation of Nanoparticle-SIRT1 (NPS1) by the\nnanoprecipitation method <\/strong><\/p>\n\n\n\n

NPS1 with ethanol or aquadest solvent was prepared by the\nnanoprecipitation method. The manufacture is done by preparing an organic\nsolvent (acetone methanol = 3:2). Then, the organic phase (FO) consists of\nchitosan and phosphatidylcholine mixed with 5 mL of organic solvent until\ndissolved. Dissolution can be assisted by using a sonicator or stirring on a\nhotplate stirrer for 1-2 hours. After everything was dissolved, 7.5 L of SIRT-1\nrecombinant protein was added. Afterward, prepare an aqueous phase (FA)\nconsisting of 1% Tween 80 (1 gram of tween 80 dissolved in 100 mL WFI). Then\nperform FA quantification. Comparison FA:FO = 15:1 (FA 15 times of FO). If the\nFO is dissolved in 5 mL, prepare the FA 15 times (75 mL), then add the FO into\nthe FA slowly while stirring, followed by ultraturax at a speed of 8000 rpm for\n5 minutes. Stirring was carried out for 18 hours. In this stirring, overheating\nis not carried out to prevent the protein from being degraded. After 18 hours\nof stirring, the nanoparticles were ready to be harvested. The procedure was\ncarried out twice for K1 and F1. The difference from this operation is that no\nprotein is added to K1.<\/p>\n\n\n\n

NPS1 with aquadest solvent was made by preparing the organic\nsolvent (chitosan dissolved in WFI and HCl 0.2 N) and then melting phosphatidic\ncholine at 80o<\/sup>C. Temperature lowered, and the organic solvent was\nadded in the previous step. After the temperature is confirmed to drop and the\nFO is at room temperature, 7.5\u00b5L of SIRT-1 recombinant protein is added.\nComparison FA: FO = 15:1, followed by ultraturax at a speed of 8000 rpm for 5\nminutes. Stirring was carried out for 18 hours. In this stirring time,\noverheating is not carried out to prevent the protein from being degraded.\nAfter 18 hours of stirring, the nanoparticles were ready to be harvested. This\nprocedure was repeated twice for K2 and F2. The difference from this operation\nis that no protein is added to K2.<\/p>\n\n\n\n

Characterization of NPS1 by pH Test, Organoleptic Test, and\nIdentification of Protein in NPS1 by Nanodrop and SDS-PAGE<\/strong><\/p>\n\n\n\n

The pH test was carried out using a pH meter, and in the\norganoleptic test, the color and odor of the preparation were observed. The\nformulation results were then observed based on pH 4. Protein levels contained\nin SIRT1 were identified using Nanodrops in 2 stages. Identification of protein\nin the supernatant was made by centrifuging  the formulation that had been stirred for 18\nhours at a speed of 5000 rpm for 5 minutes (LW Scientific EZ Swing 5K (C5)\nCentrifuge, Hettich Mikro 22R Refrigerated Centrifuge). Then, the pellets\nseparated from the supernatant (containing the harvested nanoparticles). <\/p>\n\n\n\n

Identification of proteins using Nanodrop at an absorbance\nof 280, both in the pellet and in the supernatant. Protein is expected to be\npresent in the supernatant. <\/strong><\/p>\n\n\n\n

The concentration value indicates that protein is present in\nthe solution. The blank used for both supernatant and pellet was WFI. For the\nstep 2 procedure, 10 mL of each supernatant was centrifuged at 10,000 rpm for\n15 minutes. All ten samples were divided into eight Eppendorf tubes and\ncentrifuged for 15 minutes. The supernatant and pellets were collected\naccording to each sample to obtain four pellet and four supernatant samples.\nPellets were dissolved with Tris-HCl pH 8.0 100 \u03bcL per tube, pipetting was\ncarried out slowly to mix everything and then collected into new tubes for each\npellet sample in all formulations. The samples identified proteins using\nNanodrop at an absorbance of 280 nm, both in the pellet and the supernatant. At\nthis stage, it is expected that the protein will be present in the pellet. If\nthe reading is positive, the concentration value indicates that protein is\npresent in the solution. The blank used for pellets was Tris-HCl pH 8.0, while\nfor the supernatant using WFI. <\/p>\n\n\n\n

Result<\/strong><\/p>\n\n\n\n

NPS1 Formulation<\/strong><\/p>\n\n\n\n

The results showed that in the first confirmation stage (Stage I) there was a protein concentration of 0.28 mg\/mL and in the second stage there was protein in the supernatant at 0.09 mg\/mL. The results of the nanodrops are presented in Table 1.<\/p>\n\n\n\n

Table 1: Results of SIRT1 Protein Identification on NPS1<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Nanodrop<\/strong><\/p>\n

Identification<\/strong><\/p>\n<\/td>\n

\n

Formulation<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

K1<\/strong><\/p>\n<\/td>\n

\n

K2<\/strong><\/p>\n<\/td>\n

\n

F1<\/strong><\/p>\n<\/td>\n

\n

F2<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Step I<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Pellet<\/p>\n<\/td>\n

\n

-0.14<\/p>\n<\/td>\n

\n

0.02<\/p>\n<\/td>\n

\n

-0.00<\/p>\n<\/td>\n

\n

0.03<\/p>\n<\/td>\n<\/tr>\n

\n

Supernatant<\/p>\n<\/td>\n

\n

0.30<\/p>\n<\/td>\n

\n

0.20<\/p>\n<\/td>\n

\n

0.28<\/p>\n<\/td>\n

\n

0.04<\/p>\n<\/td>\n<\/tr>\n

\n

Step II<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Pellet<\/p>\n<\/td>\n

\n

-0.01<\/p>\n<\/td>\n

\n

0.36<\/p>\n<\/td>\n

\n

0.09<\/p>\n<\/td>\n

\n

0.19<\/p>\n<\/td>\n<\/tr>\n

\n

Supernatant<\/p>\n<\/td>\n

\n

0.07<\/p>\n<\/td>\n

\n

-0.05<\/p>\n<\/td>\n

\n

-0.16<\/p>\n<\/td>\n

\n

0.05<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: Stage I (5000 rpm centrifugation); Phase II (10,000 rpm centrifugation); negative, and a value of 0 indicates no undetectable protein in NPS1. A positive value indicates the presence of a detected protein. In this study, the protein is expected to be present in the supernatant in step I and the pellet at stage II.<\/p>\n\n\n

 <\/strong>NPS1\ncharacterization (pH and organoleptic)<\/strong><\/p>\n\n\n\n

The pH observations were repeated to show that the pH of the\nSIRT1 nanoparticles was acidic in each formulation (Table 2).<\/p>\n\n\n\n

Table 2: Observations of pH and organoleptic NPS1<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Group<\/strong><\/p>\n<\/td>\n

\n

pH<\/strong><\/p>\n<\/td>\n

\n

Organoleptic<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

K1<\/p>\n<\/td>\n

\n

5.412 \u00b1 0.73<\/p>\n<\/td>\n

\n

Clear yellow; smells like egg yolk (phosphatidylcholine-like)<\/p>\n<\/td>\n<\/tr>\n

\n

K2<\/p>\n<\/td>\n

\n

3.624 \u00b1 0.45<\/p>\n<\/td>\n

\n

Deep yellow; smells like egg yolk (phosphatidylcholine-like)<\/p>\n<\/td>\n<\/tr>\n

\n

F1<\/p>\n<\/td>\n

\n

5.418 \u00b1 0.55<\/p>\n<\/td>\n

\n

Deep yellow; smells like egg yolk(phosphatidylcholine-like)<\/p>\n<\/td>\n<\/tr>\n

\n

F2<\/p>\n<\/td>\n

\n

4.182 \u00b1 0.07<\/p>\n<\/td>\n

\n

Clear yellow; smells like egg yolk (phosphatidylcholine-like)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: Each formulation has an acidic pH range (each N=5). The organoleptic test showed that the formulation’s color and odor were yellow, a mixture of chitosan and phosphatidylcholine.<\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

SIRT1\nmodulates various molecular signaling pathways important for vascular function\nin various types of vascular cells11<\/sup>. SIRT1 inhibits the expression\nof endothelial adhesion molecules such as intracellular adhesion\nmolecule-1 and vascular cell adhesion molecule-1 by suppressing nuclear factor\nNFKB, where SIRT1 deacetylates and inhibits the RelA\/p65 subunit of NF-\u03baB12<\/sup>.\nAs a result, SIRT1 inhibits monocyte-to-endothelial cell binding, as well as\nmonocyte transmigration to the arterial wall, indicating its anti-inflammatory\neffect on endothelial cells13<\/sup>. For this reason, in an in vitro<\/em> study in this study, SIRT1\npackaging used a drug carrier method with the nanoprecipitation method as a\ncarrier for SIRT1 protein to penetrate the EPC cell membrane.<\/p>\n\n\n\n

The\nnanoprecipitation method refers to the technique carried out by Luque-Alcaraz\net al.10<\/sup> with several adjustments to protein conditions that do not\nrequire heating and normal room temperature while manufacturing nanoparticles14<\/sup>.\nThe purpose of not heating is to keep the protein from being degraded during\nthe stirring process for 18 hours. Normal room temperature refers to previous\nresearch which states that nanoparticles will be stable at room temperature15<\/sup>.\nFurther confirmation was carried out using Nanodrop to determine whether there\nwas a SIRT1 protein in the previously prepared nanoparticles. As in one of the\nformulations, F1, the results showed that in the confirmation of the first\nstage (Stage I), there was a protein concentration of 0.28 mg\/mL, and in the\nsecond stage, there was protein in the supernatant at 0.09 mg\/mL. The results\nof the nanodrops are presented in Fig. Table 1. Thus, the results show that\nthere is the protein in F1.<\/p>\n\n\n\n

The\nyellow color in NPS1 is the color of phosphatidylcholine mixed with chitosan as\na polymer carrier. This study also found that the odor produced from NPS1 is a\ndistinctive odor of phosphatidylcholine. The average pH value in each\nformulation is at an acidic pH. The size of the nanoparticles is highly\ndependent on pH, as shown in previous studies, which showed that the measure\nwas due to the nucleation process in the slow formation of silver nanocrystals,\nso the particles formed were quite large16-19<\/sup>. On the other hand, at\na high pH, \u200b\u200bit is easier to form small sizes20-23<\/sup>.Thus it can be\nseen that NPS1 may have a relatively large particle size when viewed from the\npH point of view. F1, a formulation made with acetone:methanol solvent, has a\nlow pH, but in the process of checking nanoparticles with nanodrops, F1 tends\nto adsorbed proteins. Therefore, in this study, F1 has the potential to be\nfurther developed.<\/p>\n\n\n\n

The\nnanoparticles in this study are in line for use in drug delivery, and have been\npublished in several reports. The use of nanoparticles in delivering SIRT1, for\nexample, is to influence transcription factors and mitochondrial biogenesis24<\/sup>,\ndeliver siRNA to target cells25<\/sup>, and reduce the side effects of\nusing a drug26<\/sup>. In this study, SIRT1 packaging used a drug carrier\nmethod with the nanoprecipitation method as a carrier for SIRT1 protein to\npenetrate the EPC cell membrane. In the manufacture of nanoparticles, the\nchosen polymer is chitosan. Previous research revealed that chitosan could\nprotect grape seed polyphenols from degradation and EPCs from oxidative stress\ncaused by H2<\/sub>O2<\/sub>27-29<\/sup>. The protection of\nchitosan makes a valid reason for choosing the polymer as a nanoparticle drug\ncarrier. In addition, chitosan is also a carrier with good biodegradability,\nlow toxicity, hemostatic, and good biocompatibility. Therefore, efforts to\npackage SIRT1 protein using chitosan as a polymer are significant to study.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

This research is an early stage research that uses\nNPS, which is a nanoparticle, as an external carrier for the SIRT1 molecule.\nThis research observes solvents that can be used so that NPS can work well in\ncarrying SIRT1. Based on the pH and organoleptic test showed that the NPS1\nformulation was acidic, yellow in color, and had a characteristic odor. The\nlimitations of this study were that SEM, particle size, and nanoparticle\nvalidation were not performed according to NPS1. However, from this study, it\nis possible to be developed further because nanoparticles are considered a good\nalternative as a technology to add exogenous SIRT1 protein.<\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

We thanked to Ministry of Research, Technology and Higher Education of the Republic of Indonesia for funding the experiment. We gratefully acknowledge all participants of this study.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors have no conflicts of interest regarding this investigation.<\/strong> <\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The experiment is funded by Ministry\nof Research, Technology and Higher Education of the Republic of Indonesia\n(Grant number: 1\/UN10.F17\/PT.01.03.2\/2022). <\/p>\n\n\n\n

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