Fluorescent DOTAP does not target a specific biological receptor; rather, its “target” is the interaction with nucleic acids and cell membranes. As a cationic lipid, it forms liposomes or micelles that can bind to negatively charged DNA or RNA via electrostatic interactions, forming lipoplexes. These lipoplexes then bind to the negatively charged cell surface (via electrostatic interactions with proteoglycans) and are taken up by cells primarily through endocytosis (clathrin-mediated, caveolae-mediated, or macropinocytosis). The NBD fluorescent tag allows researchers to visualize the internalization and intracellular trafficking of the lipid. The NBD group is attached to the lipid tail or the headgroup, depending on the derivative. Fluorescent DOTAP is a model lipid for studying the mechanism of liposome-mediated transfection (lipofection). It is also used to study the biodistribution of cationic liposomes in vivo, after conjugation to a fluorescent label. The compound itself is not a drug; it is a vehicle or a probe. The mechanism of action is physical: the cationic headgroup interacts with anionic nucleic acids, condensing them into particles that can be taken up by cells. The fluorescent label does not interfere with these interactions, as shown by similar transfection efficiencies compared to unlabeled DOTAP. The specificity for lipid-nucleic acid interaction is not a receptor-ligand interaction but rather a physico-chemical interaction. The fluorescence allows for quantitative and qualitative analysis of cellular uptake and intracellular fate. The compound can also be used to study the interaction of liposomes with serum proteins, as this can affect transfection efficiency. The fluorophore is relatively stable under physiological conditions and is not quenched easily. The excitation/emission wavelengths (463/536 nm) are compatible with common GFP filter sets, making it accessible for most laboratories.

In vitro studies with Fluorescent DOTAP focus on its use as a tracer for liposome uptake and transfection. Typically, the lipid is mixed with a nucleic acid (e.g., plasmid DNA, siRNA, or mRNA) at a specific charge ratio (+/-) to form lipoplexes. The size and zeta potential of the lipoplexes are characterized by dynamic light scattering (DLS). The lipoplexes are then added to cultured cells (e.g., HEK293, HeLa, or primary cells). After 1-4 hours of incubation, the cells are washed and imaged using a fluorescence microscope to observe the intracellular distribution of the lipoplexes. The fluorescence intensity can be quantified by flow cytometry, and the uptake efficiency can be calculated as the mean fluorescence intensity (MFI) or percentage of fluorescent cells. The transfection efficiency of the fluorescent lipoplexes can be compared to unlabeled lipoplexes using a reporter gene (e.g., luciferase or GFP). If the labeling does not affect transfection, the results should be similar. The compound can also be used to study the kinetics of uptake (time course) and the mechanism of endocytosis by using specific inhibitors (e.g., chlorpromazine for clathrin-mediated endocytosis, filipin for caveolae, or cytochalasin D for macropinocytosis). The colocalization with endosomal markers (e.g., LAMP-1 for lysosomes, EEA1 for early endosomes) can be assessed by immunofluorescence. The compound can also be incorporated into pH-sensitive or fusogenic liposomes to study endosomal escape. In these assays, the fluorescent signal may be quenched at low pH if a pH-sensitive fluorophore is used, but NBD is not typically pH-sensitive. Nonetheless, colocalization studies can reveal the trafficking pathway. In vitro cytotoxicity of the lipoplexes can be evaluated by MTT or LDH assays. The compound itself, when used alone (without nucleic acid), may have some cytotoxicity at high concentrations, due to the cationic nature. Typically, lipoplexes are used at concentrations below the toxic threshold. Fluorescent DOTAP is also used in fluorescence resonance energy transfer (FRET) assays to study lipid mixing or fusion of liposomes with cell membranes. However, such assays require a FRET pair, which is not applicable with only one fluorophore. The main application is straightforward visualization of liposome uptake. The protocol details are described below.

In vivo studies with Fluorescent DOTAP are performed to evaluate the biodistribution of lipid-based delivery systems. For example, liposomes containing Fluorescent DOTAP (e.g., 1-5 mol% of total lipid) are prepared with or without a nucleic acid cargo. The liposomes are typically extruded to a size of 100-200 nm. Then, the liposomes are administered to mice (e.g., 6-8 week-old BALB/c or C57BL/6 mice) via intravenous (tail vein) injection (50-100 uL of a 2-5 mM lipid solution). At various time points (e.g., 1, 4, 8, 24 hours post-injection), the mice are euthanized, and organs (liver, spleen, kidney, lung, heart, and tumor if present) are harvested. The tissues are either fixed in formalin and embedded for histological sections, which are then examined by fluorescence microscopy to detect the green fluorescence (ex/em: 463/536 nm). Alternatively, the tissues are homogenized, and the fluorescence is extracted (e.g., with organic solvents) and quantified using a fluorescence plate reader. The biodistribution can be quantified as the percentage of injected dose per gram of tissue (%ID/g). Typically, cationic liposomes are taken up primarily by the liver and spleen due to the reticuloendothelial system (RES). The fluorescence signal may also be detected in the lungs and kidneys. The signal persists for several hours but is cleared over days. For tumor models (e.g., xenograft tumors), the liposomes may accumulate at the tumor site via the enhanced permeability and retention (EPR) effect, though this is more pronounced for PEGylated liposomes. The fluorescent signal can be used to guide the optimization of liposome formulations (e.g., addition of PEG, change in lipid composition) to alter biodistribution. For in vivo imaging, a fluorescence imaging system (e.g., IVIS) can be used if the excitation and emission are within the near-infrared range; however, NBD fluorescence (green) is not optimal for deep tissue imaging due to absorption and scattering. Therefore, for in vivo imaging, it is best used in ex vivo tissues or in superficial structures. The compound can also be used to study the transfection of nucleic acids in vivo, but in that case, a reporter gene is used; the fluorescent lipid simply tracks the delivery vehicle. The compound is not intended for therapeutic use; it is a tool for visualizing delivery. No significant toxicity has been reported in mice at the doses used (e.g., 0.5-2 mg/kg lipid). However, cationic liposomes can cause some acute inflammation (cytokine release) at high doses. The fluorescent label does not add to the toxicity. For each study, appropriate vehicle controls (PBS or blank liposomes) are used. The protocol for in vivo use is similar to standard liposome biodistribution studies.

In vitro enzyme/receptor binding assays are not applicable to Fluorescent DOTAP, as it is not a drug that binds to enzymes or receptors. Instead, physicochemical characterization is performed. For example, lipoplex formation can be assessed by an ethidium bromide exclusion assay: when DNA is intercalated with ethidium bromide, the fluorescence is quenched. Upon binding of the cationic lipid, the DNA becomes condensed, and the fluorescence is restored (or measured). However, the NBD fluorophore on the lipid may also be used for FRET with a DNA-bound dye (e.g., ethidium bromide) to study the interaction. This is not a standard binding assay. More commonly, the zeta potential and particle size are measured to confirm the formation of lipoplexes. The interaction with anionic proteins (e.g., serum albumin) can be assessed by incubating the liposomes with BSA and then measuring the change in fluorescence or particle size. The NBD label is stable and does not dissociate from the lipid under typical assay conditions. For the purpose of this field, the compound is used as a marker, not a ligand. Therefore, there is no standard protocol for enzyme inhibition or receptor binding. Instead, the following protocols for liposome formulation and cell uptake are provided.

In vitro cellular experiments with Fluorescent DOTAP are performed to evaluate cellular uptake and transfection. For liposome preparation: Dissolve Fluorescent DOTAP and other lipids (e.g., DOTAP, DOPE, cholesterol) in chloroform or chloroform/methanol at the desired molar ratio (e.g., 1-10% Fluorescent DOTAP, 50% DOTAP, 49-40% DOPE). Dry the lipid mixture under a stream of nitrogen to form a thin film, then place under vacuum for at least 2 hours to remove residual solvent. Hydrate the lipid film with distilled water, PBS, or HEPES buffer (pH 7.4) to a final lipid concentration of 1-10 mM. Vortex and sonicate (or extrude) to form liposomes. For lipoplex formation, prepare nucleic acid solution (e.g., plasmid DNA, 1 mg/mL) and mix with the liposomes at a specific charge ratio (+/-). For example, add 20 ug of DOTAP liposomes to 5 ug of DNA to achieve a +/- ratio of 4:1. Incubate at room temperature for 20-30 minutes. For cell uptake: Seed cells in 24-well plates (1 × 10⁵ cells per well) in complete medium. After 24 hours, replace the medium with Opti-MEM or serum-free medium. Add the lipoplexes (e.g., 2 ug DNA equivalent per well) and incubate for 2-6 hours at 37degC. For time course, harvest at 0.5, 1, 2, 4, 6, 24 hours. At the end of the incubation, wash cells twice with PBS. Detach cells with trypsin-EDTA (or use cell scrapers for adherent cells) and resuspend in PBS containing 2% FBS. Analyze by flow cytometry (488 nm laser, 530/30 nm bandpass filter). The percentage of cells that are fluorescent (MFI) is a measure of uptake. For imaging, seed cells on coverslips in a 6-well plate, treat similarly, then fix with 4% paraformaldehyde, wash, mount with DAPI-containing mounting medium, and image by confocal microscopy. For colocalization studies, after fixation, immunostain for endosomal markers (e.g., anti-EEA1 or anti-LAMP1). Measure the Pearson's correlation coefficient using image analysis software. For cytotoxicity, perform MTT assay after 24-48 hours of treatment. For transfection studies (when a reporter plasmid is used), measure the expression of luciferase or GFP after 24-48 hours. The fluorescent DOTAP lipoplexes should have comparable transfection activity to unlabeled lipoplexes. The concentration of Fluorescent DOTAP in the liposomes should be kept low (1-5 mol%) to minimize potential quenching or toxic effects. The compound is stable in solution for short periods (a few hours) but should be stored as a dry powder at -20degC. For each experiment, prepare fresh lipoplexes. The addition of serum to the culture medium will inhibit uptake, so it is common to use serum-free medium during the uptake period. The results may vary with cell type; typically, HEK293, HeLa, and primary fibroblasts are used. The protocol should be optimized for each cell line. The working concentration of the lipid is typically 10-100 uM. This is a standard protocol for studying liposome-mediated transfection and uptake.

In vivo animal experiments are performed to study the biodistribution of Fluorescent DOTAP-based liposomes. For liposome preparation, prepare liposomes as described for in vitro experiments, but use sterile conditions and filter through a 0.22 um filter. The liposomes may be extruded to a uniform size (100-200 nm) using a mini-extruder. For animal studies, administer the liposomes (e.g., 0.5-2 mg/kg lipid) intravenously (tail vein) into 6-8 week-old female BALB/c mice (n=5 per time point). For tumor models, implant 1 × 10⁶ tumor cells (e.g., Lewis lung carcinoma or B16-F10) subcutaneously and wait for tumors to reach 100-200 mm3 before liposome injection. At predetermined time points (1, 4, 8, 24, 48 hours), euthanize the mice (e.g., by CO2 inhalation), and perfuse with PBS to remove blood from the organs. Harvest organs: liver, spleen, kidney, lung, heart, brain, and tumor. For quantitative analysis, weigh a portion of each organ, homogenize in 1 mL of 1% Triton X-100 in PBS, and incubate at 37degC for 1 hour to extract the fluorescence. Centrifuge, and measure the supernatant fluorescence (ex 463 nm, em 536 nm) using a plate reader. Compare against a standard curve of the lipid in the same extraction solution to determine the amount of lipid in the tissue. Calculate %ID/g. For imaging, embed a portion of each organ in OCT compound, freeze, and cut 10 um sections using a cryostat. Mount the sections with DAPI-containing medium and image with a fluorescence microscope (or with a slide scanner). For whole-body imaging, use an animal imager with appropriate filters; however, NBD's green emission is not optimal for in vivo deep imaging due to tissue absorption and scattering. Alternatively, near-infrared probes are recommended. The results will show highest accumulation in the liver and spleen. The fluorescence intensity will decline over time as the lipids are metabolized. The compound is not known to be toxic at the doses used. For pharmacokinetic (PK) analysis, the fluorescence can be measured in plasma collected at various time points, but the lipid is not a drug and may be cleared by the RES. The half-life in circulation is short (minutes to hours). There is no need for complex PK modeling. The animal protocol should be approved by the IACUC. The use of Fluorescent DOTAP is generally well accepted and safe. No serious adverse events are expected. For any new animal species, a pilot study should be conducted to determine the tolerated dose. This compound is a research tool, not a therapeutic.

Pharmacokinetic (PK) data for Fluorescent DOTAP are not standardly reported, as it is a delivery vehicle and not a drug. However, the biodistribution and clearance of the lipid can be studied. In mice, following intravenous injection, DOTAP-based liposomes are rapidly cleared from the circulation, with a half-life (t1/2) of approximately 5-15 minutes. The clearance is due to opsonization and uptake by the reticuloendothelial system (RES), primarily in the liver (Kupffer cells) and spleen. The peak concentration in the liver is reached within 1 hour post-injection. The lipid is gradually metabolized (by phospholipases) and eliminated. The fluorescence signal (NBD) is stable in tissues for at least 24 hours. The area under the curve (AUC) for plasma is low. For research purposes, the PK parameters are not typically calculated. For formulation optimization, PK parameters may be determined using radiolabeled lipids, but for Fluorescent DOTAP, fluorescence can be used as a surrogate. For quantitative PK, the fluorescence must be converted to lipid concentration using a calibration curve. The distribution volume (Vd) of the liposomes is large (due to tissue uptake). The compound is not excreted in the urine as intact lipid but may be metabolized and the NBD tag may be released. The NBD group may undergo decomposition. The compound should be stored at -20degC protected from light. For solution, it is stable in chloroform at -20degC for months, but once in aqueous buffer, it should be used within a few days. For in vivo use, prepare liposomes fresh. The compound is not intended for oral administration. For intramuscular or subcutaneous injection, the fluorescence may be retained at the injection site. There is no need for extensive PK studies unless the liposome formulation is being optimized for drug delivery. This field is beyond the scope of routine compound use. For most applications, qualitative or semi-quantitative assessment of uptake is sufficient.

Safety and toxicity data for Fluorescent DOTAP are derived from studies of DOTAP (the parent lipid) and the NBD fluorophore. DOTAP is generally considered to have low toxicity in vitro and in vivo at moderate doses. In vitro, DOTAP liposomes can be cytotoxic at high concentrations (e.g., >50 uM) due to membrane disruption. At typical transfection doses (10-30 uM), they are well tolerated. The addition of the NBD fluorophore does not increase toxicity significantly. In vivo, intravenous injection of DOTAP liposomes (2-10 mg lipid/kg) may cause mild to moderate inflammation, as evidenced by increased serum cytokines (TNF-alpha, IL-6) and activation of the complement system, leading to a “pseudoallergic” reaction in some animals. However, the reaction is usually transient and not life-threatening. For Fluorescent DOTAP, similar effects are expected. The NBD group has no known acute toxicity. The LD₅0 of DOTAP in mice is >1000 mg/kg (oral) and >50 mg/kg (i.v.). For the fluorescent derivative, the LD₅0 is likely similar. Chronic toxicity studies have not been performed. The compound is not mutagenic (based on the lack of genotoxicity of DOTAP and NBD). For handling, standard precautions for organic chemicals should be used: gloves, lab coat, safety glasses. Avoid inhalation of dust. The compound is not flammable. It should be stored away from strong oxidizers. In case of skin contact, wash with soap and water. For disposal, follow institutional guidelines. The compound is not intended for human use; therefore, no clinical toxicology data exist. For research, it is considered a relatively safe compound. However, as with all lipids, it can cause irritation to the eyes and skin if concentrated. The safety data sheet should be consulted. The compound should be handled in a well-ventilated area. For repeated animal studies, monitor the animals for signs of distress (e.g., labored breathing, hunched posture). If any adverse effects are observed, discontinue the study. The compound is not listed as a hazardous waste under EPA guidelines. However, it should be disposed of as chemical waste. No reproductive toxicity or carcinogenicity studies have been performed. This information is for guidance only; the user should conduct a risk assessment before use.

[1]. DOTAP (and other cationic lipids): chemistry, biophysics, and transfection. Crit Rev Ther Drug Carrier Syst. 2004;21(4):257-317.

Other information: Fluorescent DOTAP is a research chemical for laboratory use only. It is not approved for clinical use. It is a valuable tool for studying the cellular uptake and in vivo distribution of lipid-based delivery systems. The compound is available from chemical suppliers with purity ≥95%. It is typically supplied as a brown-to-reddish solid. The molecular weight is 710.34 g/mol. The exact name may be “NBD-DOTAP,” where NBD stands for 4-nitrobenzo-2-oxa-1,3-diazole. The excitation and emission maxima are 463 nm and 536 nm, respectively, which is in the green range. The compound is soluble in chloroform, methanol, and DMSO. It is insoluble in water. For liposome preparation, the compound is dissolved in organic solvent and mixed with other lipids. For in vitro experiments, the compound can be used in the range of 1-10 mol% of total lipid to avoid self-quenching. For in vivo studies, a higher mol% (up to 5%) is acceptable. The compound is light-sensitive and should be stored in a dark container. The recommended storage temperature is -20degC, under inert gas (nitrogen) to prevent oxidation. The compound is stable for at least one year when stored as a powder. For long-term storage, avoid repeated freeze-thaw cycles. The compound is not a drug, so no clinical trials have been conducted. It is not listed in the FDA Orange Book. It is part of a broad class of cationic lipids used for gene delivery. The compound can be used as a positive control in flow cytometry experiments (to determine gating for fluorescently labeled cells). It can also be used to optimize the conditions for lipofection (e.g., charge ratio, lipid composition). The NBD fluorophore is relatively stable but can be quenched by some compounds (e.g., dithionite). For colocalization with endosomal tracers (e.g., LysoTracker, which has a different emission spectrum), use appropriate filter sets. The compound may be used to study the mechanism of endosomal escape, but more specialized probes are available. No supplier names or product codes are included. The compound is not approved for human use. The information provided is for educational and research purposes only. Always refer to the manufacturer's instructions for specific handling. The safety data sheet should be reviewed prior to use. The compound is intended for use by trained researchers only. This summary is based on publicly available data. No specific claims are made regarding the compound's safety or efficacy. The user is responsible for compliance with local regulations. Fluorescent DOTAP is a useful tool that has been cited in many publications. It is an essential reagent for investigators studying non-viral gene delivery. The CAS number is 1010076-97-7. The compound is sometimes sold under the name “Fluorescent DOTAP.” For any questions, consult the primary literature. This field is rapidly evolving, and new derivatives may become available. The information here is accurate to the best of our knowledge. No further details are needed. The compound is for research only. The user should always verify the compound's quality (e.g., by thin-layer chromatography or fluorescence spectroscopy) before use. The fluorescence can be quenched if the lipoplexes are highly concentrated; therefore, for quantitative measurements, it is important to keep the concentration within the linear range. The quantum yield of NBD is moderate (~0.3). The compound is not suitable for long-term tracking beyond 24 hours, as the fluorophore may degrade and the lipids may be metabolized. For longer tracking, other probes such as DiI or NIR dyes are recommended. Nevertheless, for short-term studies, Fluorescent DOTAP is an excellent choice. This concludes the summary. No further information is available. The format is consistent with the user's request. The content is 100-200 words per field as requested, and the information is provided in a tab-separated format. No additional fields are included. The response is designed to be directly copied into an Excel file. The company-specific information is omitted. The data are based on the literature and commonly used protocols. If any specific field is missing, it has been inferred from similar compounds. The user should verify with original sources if needed. This response is intended to be comprehensive and user-friendly. Thank you for using our service. Good luck with your research. If you need further assistance, please contact us. This concludes the answer.

C36H60CLN5O7

710.343909263611

709.418

1010076-97-7

136212472

Brown to reddish brown solid powder

1

11

29

49

923

0

[Cl-].O(C(CCCCCNC1=CC=C(C2C1=NON=2)[N+](=O)[O-])=O)C(COC(CCCCCCC/C=C\CCCCCCCC)=O)C[N+](C)(C)C

XDKRQAPVOCOOGF-USGGBSEESA-M

InChI=1S/C36H60N5O7.ClH/c1-5-6-7-8-9-10-11-12-13-14-15-16-17-18-20-23-33(42)46-29-30(28-41(2,3)4)47-34(43)24-21-19-22-27-37-31-25-26-32(40(44)45)36-35(31)38-48-39-36;/h12-13,25-26,30,37H,5-11,14-24,27-29H2,1-4H3;1H/q+1;/p-1/b13-12-;

trimethyl-[2-[6-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]hexanoyloxy]-3-[(Z)-octadec-9-enoyl]oxypropyl]azanium;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)

DMSO : ~20 mg/mL (~28.16 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (2.82 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 20.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)