Fluorescent dye

Stock Solution Preparation
1. Protein Preparation
To achieve optimal labeling efficiency, prepare the protein (antibody) at a concentration of 2 mg/mL.
(1) The pH of the protein solution should be 8.5±0.5. If the pH is below 8.0, adjust it using 1 M sodium bicarbonate.
(2) If the protein concentration is below 2 mg/mL, the labeling efficiency will significantly decrease. For optimal labeling efficiency, the final protein concentration should range between 2-10 mg/mL.
(3) The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions, as these can interfere with labeling efficiency.
2. Dye Preparation
Dilute the anhydrous DMSO with CY dye to prepare a 10 mM stock solution. Mix thoroughly using a glass tube or vortex.
Note: It is recommended to aliquot the CY stock solution and store it at -20°C or -80°C, protected from light.
Before use, activate the dye with a condensation solution (500 μg/mL) before proceeding with the labeling experiment.
3. Calculation of Dye Working Solution Volume
The amount of CY dye required for the labeling reaction depends on the amount of protein to be labeled. The optimal molar ratio of CY dye to protein is approximately 10.
Example: If the protein to be labeled is 500 μL of 2 mg/mL IgG (MW=150,000), and 1 mg of CY dye is dissolved in 100 μL of DMSO, the detailed calculation for the required volume of CY dye (using CY3-NHS ester as an example) is as follows:
(1) mmol (IgG) = mg/mL (IgG) × mL (IgG) / MW (IgG) = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7×10^-6 mmol
(2) mmol (CY3-NHS ester) = mmol (IgG) × 10 = 6.7×10^-6 mmol × 10 = 6.7×10^-5 mmol
(3) μL (CY3-NHS ester) = mmol (CY3-NHS ester) × MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7×10^-5 mmol × 917.05 mg/mmol / 0.01 mg/μL

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Instruction for Use
1. Labeling Reaction
(1) Take the calculated amount of freshly prepared 10 mM CY dye and activate it (approximately 10 μL of stock solution mixed with 50 μL of 500 μg/mL condensation solution). Slowly add the activated dye to 0.5 mL of the protein sample solution. Gently mix by shaking, then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent denaturation or inactivation of the protein. (2) Place the reaction tube in a light-protected area and incubate at room temperature with gentle shaking for 60 minutes. Every 10–15 minutes, gently invert the tube several times to ensure thorough mixing of the reactants and to enhance labeling efficiency.
2. Protein Purification and Desalting
The following protocol uses a Sephadex G-25 column for purifying the dye-protein conjugate as an example.
(1) Prepare the Sephadex G-25 column according to the manufacturer’s instructions.
(2) Load the reaction mixture onto the top of the Sephadex G-25 column.
(3) When the sample runs just below the surface of the resin, immediately add PBS (pH 7.2-7.4).
(4) Continue adding PBS (pH 7.2-7.4) to the column to complete the purification. Collect the fractions containing the desired dye-protein conjugate.

Factor VIIa, Cy5.5-labeled, was created for tumor imaging. While unbound Cy5.5 did not locate to any xenografts or organs, Cy5.5 tagged with these targeting proteins specifically localized to tumor xenografts for at least 14 days. Anti-tissue factors in tumor VECs can be seen using this technique, which can be utilized to track treatment responses in vivo and identify primary tumors and metastases [1]. For preoperative MRI and intraoperative fluorescence imaging of gliomas, a pH/temperature-sensitive magnetic nanogel conjugated with Cy5.5-labeled lactoferrin (Cy5.5-Lf-MPNA nanogel) was created as a promising contrast agent[2].

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Glioma is the most common primary brain tumor and causes a disproportionate level of morbidity and mortality across a wide range of individuals. From previous clinical practices, definition of glioma margin is the key point for surgical resection. In order to outline the exact margin of glioma and provide a guide effect for the physicians both at pre-surgical planning stage and surgical resection stage, pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin (Cy5.5-Lf-MPNA nanogels) were developed as a promising contrast agent. Due to its pH/te mperature sensitivity, Cy5.5-Lf-MPNA nanogels could change in its hydrophilic/hydrophobic properties and size at different pH and temperatures. Under physiological conditions (pH 7.4, 37 °C), Cy5.5-Lf-MPNA nanogels were hydrophilic and swollen, which could prolong the blood circulation time. In the acidic environment of tumor tissues (pH 6.8, 37 °C), Cy5.5-Lf-MPNA nanogels became hydrophobic and shrunken, which could be more easily accumulated in tumor tissue and internalized by tumor cells. In addition, lactoferrin, an effective targeting ligand for glioma, provides active tumor targeting ability. In vivo studies on rats bearing in situ glioma indicated that the MR/fluorescence imaging with high sensitivity and specificity could be acquired using Cy5.5-Lf-MPNA nanogels due to active targeting function of the Lf and enhancement of cellular uptake by tailoring the hydrophilic/hydrophobic properties of the nanogels. With good biocompatibility shown by cytotoxicity assay and histopathological analysis, Cy5.5-Lf-MPNA nanogels are hopeful to be developed as a specific and high-sensitive contrast agent for preoperative MRI and intraoperative fluorescence imaging of glioma [4].

Subcutaneous inoculation of cancer cells [2]
U87EGFRviii glioma cells, MiaPaCa and ASPC-1 pancreatic cancer cells at 106 cells/0.1 mL, and KB-V1 SCC cells at 3 × 106 cells/0.1 mL, were inoculated subcutaneously suspended in PBS. An aliquot of Cy5.5-FFRck-fVIIa or unconjugated Cy5.5 containing approximately 0.03 mg of Cy5.5/0.1 mL/mouse was injected intravenously into the lateral tail vein of athymic nude mice when all tumors reached 0.5-1.0 cm in diameter.

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Conjugation of Cy5.5 with factor VIIa, anti-TF antibody, FFRck-fVIIa and paclitaxel-FFRck-fVIIa [2]
Factor VIIa (5 mg/mL), FFRck-fVIIa (ASIS, Batch NLDP013: 7 mg/mL), and anti-TF antibody (1 mg/mL) were dissolved in distilled water and dialyzed in 2 liters of 0.1 M Na-carbonate buffer (pH8.8) for 48 hours. Cy5.5 (10 mg) was dissolved in 3 mL of 100% DMSO. An aliquot of Cy5.5 was added to the following proteins in approximately the indicated Cy5.5 : protein ratios: fVIIa (1.5 : 1), FFRck-fVIIa (2 : 1), paclitaxel-FFRck-fVIIa (2 : 1) and anti-TF antibody (2 : 1), based on calculations following the manufacturer’s instruction. The mixtures were stirred gently for 1-1.5 hours at room temperature. The resulting Cy5.5-protein conjugates were separated from unconjugated Cy5.5 by a Sephadex G25-150 column previously equilibrated with 0.1 M Na-carbonate buffer (pH 8.8). In a typical experiment, 1.8 mg of fVIIa in 0.6 ml in 0.1M sodium-bicarbonate buffer, pH8.8 was incubated with 1 mg of Cy5.5 mono-NHS ester in DMSO in 0.3 ml at room temperature for 1 h. Cy5.5-fVIIa and free Cy5.5 dye were separated using the Sephadex G25-150 column (8 ml). 0.3 ml (0.324 mL =6 drops)/fraction was collected (1 drop = 54 μL) for fractions 2-6, containing Cy5.5-fVIIa. Then fractions 7-14 with no color were eluted at 1ml/fraction. Free Cy5.5 dye was eluted from fractions 15-21 and thereafter. Absorbance reading at A280 and A678 identified fractions containing Cy5.5-fVIIa (protein) and free CY5.5 dye (no protein). Fractions with higher protein were determined using a Micro BCA protein assay kit and pooled. The protein concentration of the pooled fraction (1 mL total volume) typically was 0.7 mg/mL. The Cy5.5 to fVIIa ratio was calculated as 1.24:1, using extinction coefficients for fVIIa and Cy5.5 dye, 1.39 × 10 5 M-1cm-1 and 2.5 × 105 M-1cm-1, respectively. The ratio of Cy5.5 to anti-TF antibody was calculated as 1.86:1, using the extinction coefficient 1.7 × 105 M-1cm-1 for the antibody, as determined by following the manufacturer’s manual.

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In vitro cytotoxicity [4]
C6 cells and NIH/3T3 mouse embryo fibroblast cell line (NIH/3T3) were used for cell viability studies according to a previous report [14]. The medium containing Cy5.5-Lf-MPNA nanogels or MPNA nanogels was added in a dilution series (cell medium containing 0, 25, 50, 75 and 100 µg/mL Fe). The control was the culture medium without the nanogels. After 24, 48 and 72 h of incubation, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to wells. After incubation for 4 h, formazan crystals were solubilized by 100 μL of isobutanol in the incubator overnight. The absorbance of each well was read on a microplate reader (1420 multilabel counter) at 560 nm. The relative cell viability (%) related to the control wells containing cell culture medium without nanoparticles was calculated by [A]test/[A]control × 100%, where [A]test is an absorbance value of the tested cell, and [A]control is an absorbance value of the control group. The average result was calculated from 6 samples.

In vivo biocompatibility [4]
The normal rats were randomly divided into four groups (n = 9). The rats in one group were left without any treatment as the control. The rats in other three groups were injected with saline, Cy5.5-Lf-MPNA nanogels and MPNA nanogels (12 mg Fe/kg body weight) via the tail vein, respectively. At the time of the 21st day post-injection, rats were euthanized. 4 mL of blood was collected from femoral artery and sent to the clinical laboratory of Huazhong University of Science and Technology Hospital for the important biological function analysis immediately. Meanwhile, rats were perfused with sodium chloride (250 mL), and various tissues (heart, liver, spleen, lung, kidney and brain) were collected for histological examination. All the tissues were stained with H&E according to the standard clinical laboratory protocol and reviewed by a pathologist with expertise in veterinary pathology.

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Imaging Cy5.5 near infrared in vivo [2]
Imaging of Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa and anti-TF antibody was monitored over time by detecting Cy5.5 in the whole animal according to the instructions of the IVIS Lumina Imaging System 100 Series (Xenogen). Standard filter set pairs for Cy5.5 were selected in the Filter Lock box and ensured that the excitation (615-665 nm) and emission (695-770 nm) filters were properly paired for Cy5.5. Imaging was carried out daily for up to 26 days after the injection (Figures 2-5). Mice were anesthetized by an intraperitoneal injection of the mixture of ketamine (50 mg/mL), xylazine (20 mg/mL) and sterile distilled water mixed at a ratio of 8, 1 and 9 volumes according to the IACUC approved protocol at Emory University. In Figure 5, tumors and normal organs were individually dissected and imaged. Cy5.5 was imaged at 2 days after the i.v. injection using the IVIS Imaging System 100 Series located in the Department of Animal Facility according to the manufacturer’s instructions.

[1]. Ptaszek M. Rational design of fluorophores for in vivo applications. Prog Mol Biol Transl Sci. 2013;113:59-108.

[2]. Visualizing cancer and response to therapy in vivo using Cy5.5-labeled factor VIIa and anti-tissue factor antibody. J Drug Target. 2015 Apr;23(3):257-65.

[3]. Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513. doi:10.1016/j.dyepig.2017.06.029.

[4]. pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin for MR and fluorescence imaging of glioma in rats. Biomaterials. 2013 Oct;34(30):7418-28.

[5]. A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology. Theranostics. 2017 Feb 26;7(5):1047-1061.

Several classes of small organic molecules exhibit properties that make them suitable for fluorescence in vivo imaging. The most promising candidates are cyanines, squaraines, boron dipyrromethenes, porphyrin derivatives, hydroporphyrins, and phthalocyanines. The recent designing and synthetic efforts have been dedicated to improving their optical properties (shift the absorption and emission maxima toward longer wavelengths and increase the brightness) as well as increasing their stability and water solubility. The most notable advances include development of encapsulated cyanine dyes with increased stability and water solubility, squaraine rotaxanes with increased stability, long-wavelength-absorbing boron dipyrromethenes, long-wavelength-absorbing porphyrin and hydroporphyrin derivatives, and water-soluble phthalocyanines. Recent advances in luminescence and bioluminescence have made self-illuminating fluorophores available for in vivo applications. Development of new types of hydroporphyrin energy-transfer dyads gives the promise for further advances in in vivo multicolor imaging. [1]

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We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses. [2]

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In this review paper, some of the important fundamentals in the chemistry of cyanine dyes were explained. This include topics like structure and resonance forms of cyanine dyes, naturally occurring cyanine dyes, different classes of cyanine dyes and formation mechanisms of cyanine dyes. This covers methine cyanine dyes, apocyanine dyes, styryl cyanine dyes (hemicyanine dyes), aza-styryl cyanine dyes)aza-hemicyanine dyes(, merocyanine dyes (acyclic merocyanine dyes and cyclic merocyanine dyes) squarylium cyanine dyes (aromatic squarylium cyanine dyes and heterocyclic squarylium cyanine dyes), spectral sensitization evaluation of cyanine dyes, solvatochromic evaluation of cyanine dyes, halochromic evaluation of cyanine dyes, cyanine dyes for CD-R and DVD-R, cyanine dyes as fluorescent labels for nucleic acid research, mechanisms of dimethine cyanine dyes and mechanisms of apocyanine dyes. In addition, in the introduction section of this review paper some light is focussed on some important uses and applications of cyanine dyes. This special and/or specific type of collective review in the fundamentals, principles, knowledge and/or the understanding of cyanine dyes chemistry has been paid little attention and is lacking in the chemistry literature. [3]

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Progress in pharmaceutical development is highly-dependent on preclinical in vivo animal studies. Small animal imaging is invaluable for the identification of new disease markers and the evaluation of drug efficacy. Here, we report for the first time the use of a three-dimensional fluorescence bioimager called FLuorescence Emission Computed Tomography (FLECT) for the detection of a novel recombinant fluoroprobe that is safe, easily prepared on a large scale and stably stored prior to scan. This novel fluoroprobe (Targ-Cy7) comprises a single-chain antibody-fragment (scFvTarg), which binds exclusively to activated-platelets, conjugated to a near-infrared (NIR) dye, Cy7, for detection. Upon mouse carotid artery injury, the injected fluoroprobe circulates and binds within the platelet-rich thrombus. This specific in vivo binding of the fluoroprobe to the thrombus, compared to its non-targeting control-fluoroprobe, is detected by the FLECT imager. The analyzed FLECT image quantifies the NIR signal and localizes it to the site of vascular injury. The detected fluorescence is further verified using a two-dimensional IVIS® Lumina scanner, where significant NIR fluorescence is detected in vivo at the thrombotic site, and ex vivo, at the injured carotid artery. Furthermore, fluorescence levels in various organs have also been quantified for biodistribution, with the highest fluoroprobe uptake shown to be in the injured artery. Subsequently, this live animal imaging technique is successfully employed to monitor the response of the induced thrombus to treatment over time. This demonstrates the potential of using longitudinal FLECT scanning to examine the efficacy of candidate drugs in preclinical settings. Besides intravascular thrombosis, we have shown that this non-invasive FLECT-imaging can also detect in vivo pulmonary embolism. Overall, this report describes a novel fluorescence-based preclinical imaging modality that uses an easy-to-prepare and non-radioactive recombinant fluoroprobe. This represents a unique tool to study mechanisms of thromboembolic diseases and it will strongly facilitate the in vivo testing of antithrombotic drugs. Furthermore, the non-radiation nature, low-cost, high sensitivity, and the rapid advancement of optical scanning technologies make this fluorescence imaging an attractive development for future clinical applications. [5]

C47H59N3O14S4

1018.24

Cy5.5;210892-23-2;Cy5.5 acetate

Typically exists as solid at room temperature

4

9

25

89

2280

0

CCN\1C2=C(C3=C(C=C2)C(=CC(=C3)S(=O)(=O)O)S(=O)(=O)O)C(/C1=C/C=C/C=C/C4=[N+](C5=C(C4(C)C)C6=C(C=C5)C(=CC(=C6)S(=O)(=O)O)S(=O)(=O)O)CCCCCC(=O)[O-])(C)C.CCN(CC)CC.CCN(CC)CC.CCN(CC)CC.CCN(CC)CC

QPRAQNURYXILTG-UHFFFAOYSA-N

InChI=1S/C41H44N2O14S4.4C6H15N/c1-6-42-31-18-16-27-29(21-25(58(46,47)48)23-33(27)60(52,53)54)38(31)40(2,3)35(42)13-9-7-10-14-36-41(4,5)39-30-22-26(59(49,50)51)24-34(61(55,56)57)28(30)17-19-32(39)43(36)20-12-8-11-15-37(44)45;4*1-4-7(5-2)6-3/h7,9-10,13-14,16-19,21-24H,6,8,11-12,15,20H2,1-5H3,(H4-,44,45,46,47,48,49,50,51,52,53,54,55,56,57);4*4-6H2,1-3H3

N,N-diethylethanamine;6-[2-[(1E,3E,5Z)-5-(3-ethyl-1,1-dimethyl-6,8-disulfobenzo[e]indol-2-ylidene)penta-1,3-dienyl]-1,1-dimethyl-6,8-disulfobenzo[e]indol-3-ium-3-yl]hexanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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