DNA/RNA Synthesis

Results: PET/CT of bleomycin (BLM)-receiving mice showed an increase in fibrotic consolidations and an increase in non-aerated lung area in bleomycin (BLM)-treated mice compared with that in controls. TGF-b1, TNF-a, IL-6, GM-CSF in BALF and serum. PAI-1, HYP in the lung tissue of mice were significantly different in each bleomycin (BLM) groups than those in the controls. The results of Masson staining in mice indicate that the lung tissues of all BLM received groups, the intratracheal groups, the intravenous groups, and the intraperitoneal groups have a higher degree of pulmonary septal thickening and collagen fiber consolidation compare to saline control. Picro-Sirius staining results are consistent with the results of Masson staining. Compared with the saline control group, the ratio of Col 1/Col 3 was significantly increased in each BLM group. TEM results found that in BLM group, type I alveolar epithelial cells were degenerated. Exfoliated endothelial cells were swelling, and type II alveolar epithelial cells were proliferated, the shape of the nucleus was irregular, and some tooth-like protrusions were seen. [9]
Conclusions: With three different methods of animal model construction, high dose of each show more compliable, and bleomycin (BLM) can successfully induce animal models of pulmonary fibrosis, however, certain differences in the fibrosis formation sites of them three, and tail vein injection of bleomycin (BLM) induced PF model is closer to the idiopathic pulmonary interstitial fibrosis. [9]

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Day 7 post-bleomycin sulfate, CD45+ cells in BALf in NOX-/-is 1.7-fold > WT, with 57% of these being Mf, which by Day 21 decreases by 67% in WT and 83% in NOX-/-.[5]

ADIPO-P2 cells are cultured in D-MEM high glucose medium at 37 °C with 5% CO₂ atmosphere, supplemented with 20% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). 1.5 × 10⁵ cells/mL are cultured as monolayers in TC25 Corning flasks. Two flasks are set up for each experiment: one for the treated culture and one for the control. ADIPO-P2 cells are exposed to a 30-minute pulse of 2.5 μg/mL bleomycin sulfate during the log phase of growth. Parallel cultures serving as controls are not subjected to bleomycin sulfate. The duration and concentration of bleomycin sulfate exposure are selected based on earlier research using bleomycin sulfate exposure in mammalian cells conducted in our lab. The cells are maintained in culture with fresh culture medium until harvesting after being twice washed with Hank's balanced salt solution following the completion of the Bleomycin sulfate pulse treatment. After treatment, cells are kept in culture continuously for five passages or subcultures. When the cultures reach confluency (approximately 4 × 10⁵ cells/mL of culture medium), subcultivation is performed. At the time of subcultivation, cells are collected by trypsinization, and the number of viable cells is determined by staining an aliquot of approximately 200 μL with 0.4% trypan blue. This process allows for the estimation of cell growth. Subsequently, the cells are suspended in new culture medium and added to fresh culture flasks with a density of 1 × 10⁵5 cells/mL to continue growing. After the treatments are over, the remaining cells are either thrown away or transferred to another flask for cytogenetic analysis, which takes place 18 hours and 10 days later. Colchicine (0.1 μg/mL) is added to cell cultures in the final three hours of culture to analyze chromosomal aberrations. Standard protocols are followed when preparing chromosomes. Following harvesting, cells undergo hypotonic shock, are fixed in a 3:1 methanol:acetic acid solution, are spread out onto glass slides, and then undergo PNA-FISH processing. There are two separate experiments conducted.

Pulmonary fibrosis model of mice induced by different administration methods of bleomycin [9]
69 Twelve-weeks-old C57BL/6 mice were divided into 3 type groups (n = 7 for each control group, n = 8 for each BLM-induced pulmonary fibrosis groups), as intraperitoneal injection, intratracheal administration, and intravenous administration of bleomycin (BLM) to initiate lung fibrosis. Changes of the lung function measured through mice Pulmonary function test (PFT). Morphological changes in mice were observed by PET/CT, Masson and Picro-Sirius staining, Transmission electron microscopy (TEM). Biochemical changes were tested by Enzyme-linked immunosorbent assay (Elisa).

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After 1 week of adaptive feeding, the mice were randomly divided into nine groups, saline intraperitoneal (100 μl) control group, IPC, intraperitoneal injection of bleomycin (BLM) low dose (20 mg/kg) group, IPL, high dose(50 mg/kg) group, IPH, saline intratracheal administration (50 μl) control group, ITC, intratracheal administration of BLM low dose (3 mg/kg) group, ITL, high dose (5 mg/kg) group, ITH, saline tail vein injection(100 μl) control group, IVC, intravenous administration (tail vein) of BLM low dose(10 mg/kg) group, IVL, high dose (20 mg/kg) group, IVH. For intraperitoneal BLM group, after sanitized with 75% of alcohol, we injected 20 mg/kg and 50 mg/kg for low dose (IPL) and high dose group (IPH), respectively, with constant volume of 100 μl, 100 μl saline for control repeated 7 days. For intratracheal administration of BLM, nonsurgical transoral instillation of BLM into mice lung method was used. 2% sodium pentobarbital was prepared for anesthesia and injected into the abdominal cavity according to the corresponding dose of the mice's body weight (50 mg/kg). After anesthesia, mice lied supine on a fixed table with the limbs fixed, and the neck is shaved. After disinfection, laryngoscopes and LED light was used to make sure the BLM could accurately be perfused into trachea. In the low (ITL) and high dose (ITH) group, mice were injected with 3 mg/kg, 5 mg/kg BLM dissolved in 50 μl of saline, respectively, while in the control group mice were injected with an equal volume of saline. Tail vein injection BLM mouse model produced as followed: Put the mouse into a mouse fixer (mouse injection cone with restrainer and LED GLOBALEBIO GEGD-Q9G), expose the tail, disinfect with 75% alcohol, and expose the tail vein. In the low dose (IVL) and high dose (IVH) group, mice were given BLM injection at 10 mg/kg and 20 mg/kg, respectively, with a 1 ml syringe (BLM was dissolved with normal saline at a concentration of 2 mg/ml) for 7 days; In the saline control group, mice were injected with 5 ml/kg normal saline through the tail vein for 7 days.[9]

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Male Fischer 344 rats, 8-10 week old, weighing 150-250 g
3.5-4 mg/kg
Intra-tracheal

Absorption, Distribution and Excretion
The systemic absorption rate is approximately 45%. Less than 20% of the dose is reported to be excreted in the urine of patients with moderate to severe renal failure. Bleomycin sulfate is almost entirely not absorbed from the gastrointestinal tract and must be administered via parenteral route. Bleomycin is systemically absorbed after intrapleural or intraperitoneal administration. The systemic absorption rate of bleomycin after intrapleural administration is reported to be 45%. Bleomycin is rapidly absorbed after intramuscular (IM), subcutaneous (SC), intraperitoneal (IP), or intrapleural (IPL) injection, reaching peak plasma concentrations within 30 to 60 minutes. The systemic bioavailability of bleomycin after IM and subcutaneous injection is 100% and 70%, respectively, while the systemic bioavailability after intraperitoneal and retroperitoneal injection is 45%, compared to intravenous and bolus administration. Bleomycin is widely distributed throughout the body; after an intravenous bolus of 15 units/m², the mean volume of distribution in patients is 17.5 liters/m². Bleomycin has very low protein binding (1%). For more complete data on the absorption, distribution, and excretion of bleomycin (9 items in total), please visit the HSDB record page. Metabolism/Metabolites Liver Biotransformation is unclear; it may occur via enzymatic degradation in tissues (based on animal studies). Tissue enzyme activity varies, which may determine the toxicity and antitumor effects of bleomycin… It is currently unclear whether its metabolites are active. Bleomycin is inactivated by the cytoplasmic cysteine protease bleomycin hydrolase. This enzyme is widely distributed in normal tissues, except for the skin and lungs, which are target organs for bleomycin toxicity. Systemic drug clearance via enzymatic degradation may only be important in patients with severely impaired renal function.

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Biological Half-Life
115 minutes
For patients with creatinine clearance exceeding 35 mL/min, the serum or plasma terminal half-life of bleomycin is approximately 2 hours. For patients with creatinine clearance below 35 mL/min, the terminal half-life is negatively correlated with creatinine clearance.
In patients receiving a continuous infusion of 30 units of bleomycin daily for 4–5 days, the mean steady-state concentration of bleomycin in plasma is approximately 150 ng/mL, with extremely low binding to plasma proteins. Bleomycin clearance in plasma is biphasic; the initial half-life is approximately 1.3 hours, and the terminal half-life is approximately 9 hours.

Interactions
General anesthesia may lead to rapid deterioration of lung function in patients previously treated with bleomycin, as bleomycin makes lung tissue sensitive to oxygen; postoperative pulmonary fibrosis may still occur even if the inhaled oxygen concentration is considered safe. Concomitant use of antineoplastic drugs or radiotherapy may increase the toxicity of bleomycin, including bone marrow suppression (bleomycin alone rarely causes bone marrow suppression) and mucosal and pulmonary toxicity… Cisplatin-induced renal impairment may lead to delayed bleomycin clearance, and even low doses may cause bleomycin toxicity; caution is advised when using these two drugs in combination, as they are frequently used together. Raynaud's phenomenon has occurred in patients treated with bleomycin and vinblastine (with or without cisplatin) and in a small number of patients treated with bleomycin alone. Cisplatin-induced hypomagnesemia may be another contributing factor, although not a necessary one, to the occurrence of Raynaud's phenomenon in patients receiving bleomycin and cisplatin combined with chemotherapy. However, the etiology of Raynaud's phenomenon in these cases is unclear and may be related to underlying disease or vascular damage, bleomycin, vinblastine, hypomagnesemia, or some combination of these factors. A 28-year-old male with germ cell carcinoma experienced an acute myocardial infarction during bleomycin and etoposide chemotherapy. After treatment with heparin and aspirin, the patient's electrocardiogram (ECG) showed no Q waves and recovered well. Four weeks after the infarction, thallium-201 myocardial scintigraphy revealed only a small, irreversible posterior septal perfusion defect; coronary angiography was not performed. The chemotherapy regimen continued and was adjusted to etoposide, cisplatin, and ifosfamide, with no recurrence of cardiac symptoms or ECG changes. Adverse Reactions: The most common serious adverse reaction to bleomycin is pulmonary toxicity, commonly referred to as bleomycin pulmonary toxicity or BPT. This adverse reaction can sometimes lead to pulmonary fibrosis, a chronic, irreversible disease with a poor prognosis. In observing the development of pulmonary fibrosis, inflammatory cell infiltration of lung endothelial cells was observed one week after bleomycin exposure, and fibrotic changes with increased collagen content were observed three weeks later. Other alterations include increased expression of fibrotic mediators such as transforming growth factor (TGF)-β, connective tissue growth factor, and platelet-derived growth factor (PDGF)-C in bleomycin-exposed endothelial cells. Furthermore, in bleomycin-exposed lung endothelial cells, the production of the vasodilators prostaglandin I2 and nitric oxide induced by bleomycin is reduced. Therefore, administration of bleomycin can induce functional alterations in lung endothelial cells, leading to respiratory damage, although the exact mechanisms of these alterations are not fully elucidated. Other adverse reactions include fever, chills, syncope, chest pain, and dyspnea. Milder reactions include skin pigmentation changes, pruritus, decreased taste, rash, nausea, vomiting, and weight loss. Some of these symptoms appear to be associated with hypersensitivity reactions. https://www.ncbi.nlm.nih.gov/books/NBK555895/

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Toxicity
Bleomycin pulmonary toxicity (BPT) has been a recognized adverse reaction of this drug since clinical trials began in the early 1960s. Recent studies have shown that the incidence of bleomycin-associated lung injury (BPT) is approximately 10% in patients taking bleomycin, with 14% of BPT cases ultimately leading to death. Therefore, close monitoring of bleomycin blood concentrations and associated toxicities is crucial. As previously mentioned, BPT can encompass a serious condition called pulmonary fibrosis. Risk factors for BPT include cumulative dose, elevated creatinine, advanced age, oxygen therapy, and decreased glomerular filtration rate. While many BPT cases are irreversible or fatal, there is evidence that lung function parameters in some surviving patients can return to baseline levels within approximately two years. Although there is currently no proven BPT reversal therapy, studies using other formulations of bleomycin have shown some efficacy. Numerous studies have also demonstrated that, in combination therapy regimens, bleomycin can sometimes replace less toxic chemotherapy and immunotherapy drugs with similar efficacy. This approach is particularly suitable for patients with multiple BPT risk factors and those with milder disease who do not require the risk of BPT. Besides replacing bleomycin with less toxic drugs in chemotherapy regimens, efforts to reduce the risk of lung injury also include research on lipophilic bleomycin analogues, such as ribumycin. However, these studies have not yielded satisfactory results in animal models. Therefore, whether the effectiveness of bleomycin in chemotherapy regimens will continue to diminish remains to be seen. https://www.ncbi.nlm.nih.gov/books/NBK555895/

[1]. Acta Otolaryngol Suppl. 1997:529:241-4.

[2]. Am J Pathol. 1989 Feb;134(2):355-63.

[3]. Mol Med Rep. 2012 Jun;5(6):1481-6.

[4]. Mutat Res. 2012 Jun 1;734(1-2):5-11.

[5]. Stem Cell Res Ther. 2012 May 29;3(3):21.

[6]. Mutat Res. 2012 Sep 18;747(2):228-33.

[7]. Int J Clin Exp Med. 2014 Sep 15;7(9):2645-50. eCollection 2014.

[8]. Mol Cytogenet. 2016 Jun 21:9:49.

[9]. BMC Pulm Med. 2023 Mar 21;23(1):91.

Bleomycin sulfate is a mixture of sulfates of basic glycopeptide antitumor antibiotics isolated from Streptomyces verticillus. Bleomycin sulfate forms a complex with iron, which reduces molecular oxygen to superoxide anions and hydroxyl radicals, leading to single- and double-strand breaks in DNA; these reactive oxygen species also induce lipid peroxidation, carbohydrate oxidation, and alterations in prostaglandin synthesis and degradation. It is a complex of related glycopeptide antibiotics from Streptomyces verticillus, containing bleomycin A2 and B2. It inhibits DNA metabolism and is used as an antitumor drug, particularly for solid tumors. See also: Bleomycin sulfate (note moved to). Bleomycin is approved for the treatment of adult head and neck squamous cell carcinoma, Hodgkin's lymphoma, and testicular cancer. It is also used as a sclerosing agent for malignant pleural effusions. Off-label uses include the treatment of germ cell tumors and childhood Hodgkin's lymphoma. Bleomycin is a cytotoxic chemotherapeutic agent. This event will introduce the indications, mechanism of action, and contraindications of bleomycin as an effective drug for treating various malignancies. The event will focus on its mechanism of action, adverse reaction characteristics, and other key factors (e.g., off-label use, dosage, pharmacodynamics, pharmacokinetics, monitoring, and related interactions) that are crucial for multidisciplinary teams treating patients with various cancers. https://www.ncbi.nlm.nih.gov/books/NBK555895/

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Bleomycin (BLM) is a natural antibiotic toxic to dividing cells (G2/M phase) and has also been shown to be effective against squamous cell carcinoma (SCC). Our clinical studies have demonstrated that the combination of a short-range β-emission radionuclide with bleomycin (In-111-BLMC) can serve as a targeted therapy for squamous cell carcinoma (SCC). Enhancing the activity of the radionuclide may lead to the development of more effective drugs, and animal studies are needed to validate this. We used a 96-well colony formation assay to study three SCC cell lines cultured in our laboratory and determined the IC20, IC50, and IC90 values of bleomycin (BLM). The UT-SCC-12A and UT-SCC-12B cell lines were derived from the primary tumor and metastatic lesions of the same patient, respectively. We also subcutaneously inoculated UT-SCC-12A cells into nude mice and analyzed tumor growth. The IC50 value of the UT-SCC-12A cell line was 4.0 ± 1.3 nM. Both UT-SCC-12A and UT-SCC-12B cell lines showed high resistance to BLM, with IC50 values of 14.2 ± 2.8 nM and 13.0 ± 1.1 nM, respectively. The weight gain of the nude mice was 2.8 ± 0.6 g over 35 days. At 25 and 35 days post-inoculation, the tumor volumes were 111 ± 51 mm³ and 874 ± 577 mm³, respectively. The calculated doubling time was 3.86 ± 0.76 days. SCC cell lines have varying sensitivities to bleomycin (BLM). Our SCC tumor xenograft model showed rapid growth and was suitable for radiochemoradiotherapy studies using In-111-BLMC. In vivo uptake of In-111-BLMC was proportional to proliferation activity and tumors with high binding capacity could be predicted by dose calculation in animal models. [1]

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Previous studies in our laboratory have shown that bleomycin-induced mouse pulmonary fibrosis models enhance the activity of fibroblast growth factor (MDGF) secreted by alveolar macrophages. However, the mechanism by which bleomycin promotes increased MDGF secretion is unclear. This study aimed to determine the direct effect of bleomycin on alveolar macrophages. Normal rat alveolar macrophages were obtained by lung lavage and cultured in media with or without bleomycin; the conditioned media of these cultures were collected, dialyzed to remove bleomycin, and their MDGF activity was then detected in vitro. After incubating alveolar macrophages with bleomycin at concentrations ranging from 0.01 μg/mL to 1 μg/mL for 18 hours, the macrophages secreted significantly more MDGF than macrophages not incubated with bleomycin. Macrophage viability, as determined by trypan blue exclusion and LDH release assays, was unaffected by any test dose. MDGF production reached its maximum at bleomycin concentrations greater than or equal to 0.1 μg/mL. MDGF activity was detectable as early as 1 hour after incubation of alveolar macrophages with bleomycin at a concentration of 0.1 μg/mL for 0.5 to 18 hours, peaking at 4 to 8 hours. Even after removing bleomycin and replacing it with fresh (bleomycin-free) culture medium, bleomycin-stimulated macrophages continued to produce large amounts of MDGF. Bleomycin-stimulated alveolar macrophage secretion of MDGF can be inhibited by cycloheximide and the 5-lipoxygenase inhibitors NDGA (nordihydroguaiac acid) and BW755c, indicating that full expression of MDGF requires not only protein synthesis but also 5-lipoxygenase metabolites of the arachidonic acid metabolic pathway [2]. Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a commonly used drug for treating various malignant tumors and is also part of the chemotherapy regimen for testicular cancer; however, its side effects are common. Bleomycin leads to increased oxidative stress, which has been shown to induce apoptosis in cancer cells. Curcumin (diferoylmethane) is the active ingredient of spice turmeric and has been shown to induce apoptosis in various malignant tumor cells. However, to date, no studies have elucidated the anticancer activity of curcumin in testicular cancer cells and its interaction with bleomycin. This study investigated and compared the effects of curcumin, bleomycin, and hydrogen peroxide (H₂O₂) on the apoptosis signaling pathway. After 24 hours of co-incubation with curcumin (20 µM), bleomycin (400 µg/ml), and H₂O₂ (400 µM), NTera-2 cells showed decreased viability, increased caspase-3, -8, and -9 activities, increased Bax and cytochrome c levels, and decreased Bcl-2 levels. The induction of caspase-3, -8, and -9 activities in NTera-2 cells was stronger when curcumin was used in combination with bleomycin than when either drug was used alone. Our observations suggest that curcumin and bleomycin have a synergistic effect on apoptosis signaling pathways. Therefore, we recommend the combined use of curcumin and bleomycin to reduce the therapeutic dose and thus reduce side effects. [3]

C55H85N17O25S4

1512.62

1511.48

C, 43.67; H, 5.66; N, 15.74; O, 26.44; S, 8.48

9041-93-4

056-06-7; 67763-87-5; 9041-93-4 (sulfate);

72466

White to light yellow solid powder

197ºC (dec)

-7.5

21

35

36

101

2660

10

S(O)(=O)(=O)[O-].[C@@H]1(OC(=O)N)[C@H](O)[C@H](O[C@@H]([C@H]1O)CO)O[C@H]1[C@@H](O)[C@H](O)[C@@H](O[C@H]1OC(C1=CNC=N1)C(NC(=O)C1=C(C)C(N)=NC(C(NCC(N)C(N)=O)CC(N)=O)=N1)C(=O)NC(C)C(O)C(C)C(=O)NC(C(C)O)C(NCCC1SC=C(C2SC=C(C(NCCC[S+](C)C)=O)N=2)N=1)=O)CO

WUIABRMSWOKTOF-OCBSMOPSSA-N

InChI=1S/C55H83N17O21S3.H2O4S/c1-20-33(69-46(72-44(20)58)25(12-31(57)76)64-13-24(56)45(59)82)50(86)71-35(41(26-14-61-19-65-26)91-54-43(39(80)37(78)29(15-73)90-54)92-53-40(81)42(93-55(60)88)38(79)30(16-74)89-53)51(87)66-22(3)36(77)21(2)47(83)70-34(23(4)75)49(85)63-10-8-32-67-28(18-94-32)52-68-27(17-95-52)48(84)62-9-7-11-96(5)6;1-5(2,3)4/h14,17-19,21-25,29-30,34-43,53-54,64,73-75,77-81H,7-13,15-16,56H2,1-6H3,(H13-,57,58,59,60,61,62,63,65,66,69,70,71,72,76,82,83,84,85,86,87,88);(H2,1,2,3,4)/t21?,22?,23?,24?,25?,29-,30+,34?,35?,36?,37+,38+,39-,40-,41?,42-,43-,53+,54-;/m0./s1

3-[[2-[2-[2-[[2-[[4-[[2-[[6-amino-2-[3-amino-1-[(2,3-diamino-3-oxopropyl)amino]-3-oxopropyl]-5-methylpyrimidine-4-carbonyl]amino]-3-[(2R,3S,4S,5S,6S)-3-[(2R,3S,4S,5R,6R)-4-carbamoyloxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3-(1H-imidazol-5-yl)propanoyl]amino]-3-hydroxy-2-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]ethyl]-1,3-thiazol-4-yl]-1,3-thiazole-4-carbonyl]amino]propyl-dimethylsulfanium;hydrogen sulfate

NSC-125066; BLEO; BLM; NSC 125066; BLEO cell; BLEO-cell; NSC125066; BLEOcell; Bleolem; Bleomycin sulfate; Trade name: Blenoxane. Blanoxan

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 30 mg/mL

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Solubility in Formulation 5: 100 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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Solubility in Formulation 6: 100 mg/mL (Infinity mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)