Monophosphoryl Lipid A is a Toll-like receptor 4 agonist. It binds to and activates TLR4 on antigen-presenting cells such as dendritic cells and macrophages. [1]
It may also act directly on T cells, likely through TLR2 and/or TLR4 expressed on these cells. [2]

- MPLA incorporated into liposomes (vacosome) induced bone marrow dendritic cell maturation in a dose-dependent manner. When BMDCs were cocultured with vacosome containing increasing amounts of MPLA (0.1 to 1 μg), the percentage of matured BMDCs increased, reaching a plateau at doses above 1 μg. [1]
- Vacosome containing MPLA and cancer cell membrane significantly promoted BMDC maturation over 72 hours, with the highest maturation percentage (82.4% CD86+, 80.8% CD80+) observed at 72 h, compared to MPLA@Lip or cancer cell membrane alone. [1]
- Vacosome-treated BMDCs and splenocytes showed enhanced antitumor ability against 4T1 cells in vitro, reducing 4T1 cell viability more effectively than immune cells treated with a simple mix of MPLA and cancer cell membrane. [1]
- MPLA (100 μg/mL) induced up-regulation of HLA-DR, CD80, CD86, CD40, and CD83 on human monocyte-derived DCs, though the induction was heterogeneous between donors. Lower doses (5-50 μg/mL) were ineffective. [2]
- MPLA (50-100 μg/mL) induced significant IL-12 p40 production by human DCs, though levels were lower than those induced by LPS (1 μg/mL). IL-12 bioactivity was confirmed by IFN-γ induction in PBMC cultures, which was blocked by anti-IL-12 neutralizing antibodies. [2]
- MPLA (100 μg/mL) induced NF-κB translocation and activation in DCs, similar to LPS. It also modulated TLR expression: TLR2 mRNA was up-regulated, while TLR4 expression was unaffected. [2]
- MPLA (50 μg/mL) induced intracellular calcium mobilization in DCs, whereas LPS at similar doses did not. [2]
- MPLA (100 μg/mL) induced faster ERK1/2 phosphorylation kinetics in DCs compared to LPS (10 μg/mL), while p38 phosphorylation kinetics were similar. Pretreatment with PD98059 (an MEK/ERK pathway inhibitor) enhanced MPLA-induced IL-12 production, suggesting ERK activation negatively regulates IL-12. [2]
- MPLA (10 μg/mL) increased intracellular calcium in CD4 T cell lines and, to a lesser extent, in peripheral blood CD4 T cells. When combined with anti-CD3/anti-CD28 stimulation, MPLA further enhanced calcium mobilization. [2]
- MPLA (10 μg/mL) alone did not induce CD40L expression on resting T cells. However, it significantly increased anti-CD3-induced CD40L expression at both the protein (intracellular staining) and mRNA (RT-PCR) levels. [2]
- MPLA at high doses (100 μg/mL) activated DCs independently of soluble CD14, as anti-CD14 antibodies did not block MPLA-induced IL-12 production, whereas they inhibited LPS (100 ng/mL)-induced IL-12. [2]

- MPLA incorporated into liposomes (vacosome) induced bone marrow dendritic cell maturation in a dose-dependent manner. When BMDCs were cocultured with vacosome containing increasing amounts of MPLA (0.1 to 1 μg), the percentage of matured BMDCs increased, reaching a plateau at doses above 1 μg. [1]
- Vacosome containing MPLA and cancer cell membrane significantly promoted BMDC maturation over 72 hours, with the highest maturation percentage (82.4% CD86+, 80.8% CD80+) observed at 72 h, compared to MPLA@Lip or cancer cell membrane alone. [1]
- Vacosome-treated BMDCs and splenocytes showed enhanced antitumor ability against 4T1 cells in vitro, reducing 4T1 cell viability more effectively than immune cells treated with a simple mix of MPLA and cancer cell membrane. [1]
- MPLA (100 μg/mL) induced up-regulation of HLA-DR, CD80, CD86, CD40, and CD83 on human monocyte-derived DCs, though the induction was heterogeneous between donors. Lower doses (5-50 μg/mL) were ineffective. [2]
- MPLA (50-100 μg/mL) induced significant IL-12 p40 production by human DCs, though levels were lower than those induced by LPS (1 μg/mL). IL-12 bioactivity was confirmed by IFN-γ induction in PBMC cultures, which was blocked by anti-IL-12 neutralizing antibodies. [2]
- MPLA (100 μg/mL) induced NF-κB translocation and activation in DCs, similar to LPS. It also modulated TLR expression: TLR2 mRNA was up-regulated, while TLR4 expression was unaffected. [2]
- MPLA (50 μg/mL) induced intracellular calcium mobilization in DCs, whereas LPS at similar doses did not. [2]
- MPLA (100 μg/mL) induced faster ERK1/2 phosphorylation kinetics in DCs compared to LPS (10 μg/mL), while p38 phosphorylation kinetics were similar. Pretreatment with PD98059 (an MEK/ERK pathway inhibitor) enhanced MPLA-induced IL-12 production, suggesting ERK activation negatively regulates IL-12. [2]
- MPLA (10 μg/mL) increased intracellular calcium in CD4 T cell lines and, to a lesser extent, in peripheral blood CD4 T cells. When combined with anti-CD3/anti-CD28 stimulation, MPLA further enhanced calcium mobilization. [2]
- MPLA (10 μg/mL) alone did not induce CD40L expression on resting T cells. However, it significantly increased anti-CD3-induced CD40L expression at both the protein (intracellular staining) and mRNA (RT-PCR) levels. [2]
- MPLA at high doses (100 μg/mL) activated DCs independently of soluble CD14, as anti-CD14 antibodies did not block MPLA-induced IL-12 production, whereas they inhibited LPS (100 ng/mL)-induced IL-12. [2]

- BMDC Maturation Assay (Vacosome Study): Bone marrow-derived dendritic cells from BALB/c mice were cultured with various formulations (saline, lipid control, MPLA@Lip, cancer cell membrane, vacosome) for 24, 48, or 72 hours. Cells were then stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies and analyzed by flow cytometry. Maturation was defined as the percentage of CD11c+ cells expressing CD80 and CD86. [1]
- In Vitro Killing Assay: BMDCs were first incubated with different formulations for 3 days to induce maturation. These BMDCs were then cocultured with splenocytes for another 3 days. Finally, activated immune cells were cocultured with 4T1 target cells for 24 hours. 4T1 cell viability was assessed using a cell viability assay (likely MTT or similar). [1]
- DC Isolation and Culture (MPLA Mechanism Study): Human monocytes were isolated from PBMCs by adherence and cultured for 6 days with GM-CSF and IL-4 to generate immature DCs. DCs were then stimulated with MPLA (5, 50, or 100 μg/mL), LPS (1 μg/mL), or medium alone for 24 hours. Supernatants were collected for IL-12 p40 ELISA. Cells were harvested for flow cytometry analysis of surface markers (HLA-DR, CD80, CD86, CD40, CD83). [2]
- IL-12 Bioactivity Assay: Supernatants from DCs treated with MPLA or LPS were added to PBMC cultures for 48 hours. IFN-γ in PBMC supernatants was measured by ELISA. To confirm IL-12 dependence, anti-IL-12 neutralizing antibodies or isotype controls (20 μg/mL) were added to parallel cultures. [2]
- Calcium Mobilization Assay: DCs or T cells were loaded with Fluo-3 AM dye in the presence of pluronic acid and sulfinpyrazone. Cells were stimulated with MPLA (50 μg/mL for DCs; 10 μg/mL for T cells), LPS, or anti-CD3/anti-CD28 (for T cells). Fluorescence was monitored by flow cytometry over time. Intracellular calcium concentration was calculated using the formula: [Ca²⁺]i = Kd(F - Fmin)/(Fmax - F), with Kd = 400 nM for Fluo-3. [2]
- CD40L Expression Assay: Purified CD4+ T cells were stimulated with plate-bound anti-CD3 antibody (10 μg/mL) in the presence or absence of MPLA (10 μg/mL) for 16 hours. Cells were fixed, permeabilized, and stained with PE-conjugated anti-CD40L antibody or isotype control, then analyzed by flow cytometry. Parallel samples were processed for RT-PCR analysis of CD40L mRNA. [2]

- BMDC Maturation Assay (Vacosome Study): Bone marrow-derived dendritic cells from BALB/c mice were cultured with various formulations (saline, lipid control, MPLA@Lip, cancer cell membrane, vacosome) for 24, 48, or 72 hours. Cells were then stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies and analyzed by flow cytometry. Maturation was defined as the percentage of CD11c+ cells expressing CD80 and CD86. [1]
- In Vitro Killing Assay: BMDCs were first incubated with different formulations for 3 days to induce maturation. These BMDCs were then cocultured with splenocytes for another 3 days. Finally, activated immune cells were cocultured with 4T1 target cells for 24 hours. 4T1 cell viability was assessed using a cell viability assay (likely MTT or similar). [1]
- DC Isolation and Culture (MPLA Mechanism Study): Human monocytes were isolated from PBMCs by adherence and cultured for 6 days with GM-CSF and IL-4 to generate immature DCs. DCs were then stimulated with MPLA (5, 50, or 100 μg/mL), LPS (1 μg/mL), or medium alone for 24 hours. Supernatants were collected for IL-12 p40 ELISA. Cells were harvested for flow cytometry analysis of surface markers (HLA-DR, CD80, CD86, CD40, CD83). [2]
- IL-12 Bioactivity Assay: Supernatants from DCs treated with MPLA or LPS were added to PBMC cultures for 48 hours. IFN-γ in PBMC supernatants was measured by ELISA. To confirm IL-12 dependence, anti-IL-12 neutralizing antibodies or isotype controls (20 μg/mL) were added to parallel cultures. [2]
- Calcium Mobilization Assay: DCs or T cells were loaded with Fluo-3 AM dye in the presence of pluronic acid and sulfinpyrazone. Cells were stimulated with MPLA (50 μg/mL for DCs; 10 μg/mL for T cells), LPS, or anti-CD3/anti-CD28 (for T cells). Fluorescence was monitored by flow cytometry over time. Intracellular calcium concentration was calculated using the formula: [Ca²⁺]i = Kd(F - Fmin)/(Fmax - F), with Kd = 400 nM for Fluo-3. [2]
- CD40L Expression Assay: Purified CD4+ T cells were stimulated with plate-bound anti-CD3 antibody (10 μg/mL) in the presence or absence of MPLA (10 μg/mL) for 16 hours. Cells were fixed, permeabilized, and stained with PE-conjugated anti-CD40L antibody or isotype control, then analyzed by flow cytometry. Parallel samples were processed for RT-PCR analysis of CD40L mRNA. [2]

- In vitro biocompatibility: Vacosome containing MPLA showed no toxicity to BMDCs when cultured for 1-3 days, with cell viability similar to negative control groups. Only the positive control (1% Triton X-100) significantly reduced viability. [1]
- In vivo safety: Mice immunized with vacosome showed normal body weight gain over 28 days, comparable to saline-treated mice. H&E staining of heart, liver, and kidney at day 28 showed no obvious inflammatory cell infiltration, tissue swelling, adhesion, or hyperplasia, indicating no cardiotoxicity, hepatotoxicity, or nephrotoxicity. [1]

[1]. Recombination Monophosphoryl Lipid A-Derived Vacosome for the Development of Preventive Cancer Vaccines. ACS Appl Mater Interfaces. 2020;12(40):44554-44562.

[2]. Monophosphoryl lipid A activates both human dendritic cells and T cells. J Immunol. 2002;168(2):926-932.

- Monophosphoryl Lipid A is a detoxified derivative of lipopolysaccharide from the cell wall of nonpathogenic Salmonella. It retains immunostimulatory properties but with significantly reduced toxicity compared to LPS. [1][2]
- MPLA has been employed in human vaccine trials for malaria, HIV-1, and meningococcal type B disease due to its ability to bind and activate TLR4, enhancing cell-mediated immunity. [1]
- Due to its hydrophobicity, MPLA requires formulation in delivery systems such as liposomes for effective delivery. In the vacosome study, MPLA was incorporated into liposomes along with cancer cell membrane antigens. [1]
- MPLA acts as an adjuvant by activating APCs, but administration of MPLA alone is insufficient to prime cancer-specific adaptive immunity without co-delivery of tumor antigens. [1]
- The vacosome platform combining MPLA and cancer cell membrane antigens in a liposomal formulation shows promise for clinical translation as a preventive cancer vaccine. The synthesis process is convenient, materials are accessible, and MPLA is FDA-approved as an adjuvant. [1]
- MPLA enhances T cell responses through dual effects: at high doses, it induces DC maturation; at low doses, it acts directly on T cells by increasing calcium mobilization and up-regulating CD40L expression upon TCR engagement, providing co-stimulatory signals back to DCs. [2]
- The mixed Th1/Th2 profile induced by MPLA-treated human DCs may be optimal for humoral responses but may require combination with other adjuvants (e.g., QS21) to enhance Th1 responses and CTL activation for certain applications. [2]

C96H181N2O22P

1746.44

1745.284362

960324-04-3

Monophosphoryl lipid A;1246298-63-4;Monophosphoryl lipid A Triethylamine

131846121

Typically exists as solids at room temperature

28.6

9

22

88

121

2550

14

N([C@H]1[C@@H](O[C@H](CO)[C@@H](OP(O)(O)=O)[C@@H]1OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)OC[C@H]1O[C@H](O)[C@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H]1O)C(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC

JSNQJZJCKAFSHT-OPYGSFAOSA-N

InChI=1S/C96H181N2O22P/c1-7-13-19-25-31-37-39-45-51-57-63-69-85(104)114-79(67-61-55-49-43-35-29-23-17-11-5)73-84(103)98-90-94(119-88(107)74-80(68-62-56-50-44-36-30-24-18-12-6)115-86(105)70-64-58-52-46-40-38-32-26-20-14-8-2)92(120-121(110,111)112)81(75-99)117-96(90)113-76-82-91(108)93(118-87(106)72-78(101)66-60-54-48-42-34-28-22-16-10-4)89(95(109)116-82)97-83(102)71-77(100)65-59-53-47-41-33-27-21-15-9-3/h77-82,89-96,99-101,108-109H,7-76H2,1-6H3,(H,97,102)(H,98,103)(H2,110,111,112)/t77-,78-,79-,80-,81-,82-,89-,90-,91-,92-,93-,94-,95+,96-/m1/s1

[(2S,3R,4R,5S,6R)-2,5-dihydroxy-6-[[(2R,3R,4R,5S,6R)-6-(hydroxymethyl)-5-phosphonooxy-3-[[(3R)-3-tetradecanoyloxytetradecanoyl]amino]-4-[(3R)-3-tetradecanoyloxytetradecanoyl]oxyoxan-2-yl]oxymethyl]-3-[[(3R)-3-hydroxytetradecanoyl]amino]oxan-4-yl] (3R)-3-hydroxytetradecanoate

Glucopyranosyl lipid A free acid; Lapretolimod; Lapretolimod [INN]; 960324-04-3; 6CY69F2AH3; UNII-6CY69F2AH3;

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.)