TLR4/toll-like receptor 4
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]

Dendritic cells exposed to 100 μg/mL of monophosphoryl lipid A for two hours detect NF-κB activation and control TLR2 [2]. Human IL-12 production is induced by monophosphoryl lipid A (5~100 μg/mL, 24 hours; dendritic cells)[2]. When monophosphoryl lipid A is increased from 0.1 to 1 μg, the proportion of mature BMDCs likewise rises [1]. Dendritic cell surface markers are upregulated by monophosphoryl lipid A's action. Lipid A monophosphorylation improves T cell responses. Increases in monophosphoryl lipid A (CD4 T cells) stimulate calcium for T cells that have been activated [2].

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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]

It was established that the produced monophosphoester potentiometer A may activate APC and increase the development of Th1 and Th2 iodophor immunological responses in mice [2].

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- BALB/c mice immunized three times with vacosome containing MPLA and 4T1 cancer cell membrane showed significantly higher serum levels of IL-12p70 (33.5 ± 6.5 pg/mL), TNF-α (49.9 ± 7.8 pg/mL), and IFN-γ (55.9 ± 10.3 pg/mL) at day 28 compared to mice treated with saline, cancer cell membrane alone, or MPLA@Lip alone. [1]
- In a 4T1 tumor challenge model, mice pre-immunized with vacosome showed dramatically inhibited tumor growth (76.0 ± 63.9 mm³) compared to saline (1725.3 ± 1158.2 mm³), cancer cell membrane (1488.0 ± 1107.4 mm³), or MPLA@Lip (747.4 ± 454.7 mm³) groups at day 21 post-challenge. [1]
- Vacosome-immunized mice showed prolonged survival, with 50% of mice still alive at 50 days post-challenge, compared to 0% in other groups. [1]
- Flow cytometry analysis of tumors from vacosome-immunized mice showed the highest percentage of CD8+ CTLs (39.4 ± 5.4%) and the lowest percentage of Tregs (38.9 ± 6.7%) compared to other groups. [1]
- Spleens from vacosome-immunized mice showed the highest percentage of effector memory T cells (TEM: 70.4 ± 6.0%) compared to mice treated with cancer cell membrane (38.8 ± 4.4%), MPLA@Lip (49.5 ± 5.4%), or saline (27.8 ± 6.0%). [1]

Western Blot Analysis[2]
Cell Types: dendritic cells
Tested Concentrations: 100 μg/ml
Incubation Duration: 2 hrs (hours)
Experimental Results: Induces NF-κB activation and regulates TLR2.

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- 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]

- Immunization and Tumor Challenge (Vacosome Study): Female BALB/c mice (age not specified) were immunized subcutaneously three times at 7-day intervals with different formulations: saline, cancer cell membrane (1.6 mg), MPLA@Lip (1 μg MPLA in liposomes), or vacosome (containing 1 μg MPLA and 1.6 mg cancer cell membrane in liposomes). Three days after the final immunization (day 0 of tumor stage), mice were challenged subcutaneously with 4T1 cells (number not specified). Tumor growth was monitored every 2-3 days using calipers. Tumor volume was calculated as (length × width²)/2. Mice were euthanized when tumor volume exceeded criteria or for survival analysis up to 50 days. Blood was collected at days 7, 14, and 28 for cytokine analysis (IL-12p70, TNF-α, IFN-γ, IL-6) by cytometric bead array. At day 14 post-challenge, some mice were sacrificed; tumors and spleens were collected for flow cytometry analysis (CTLs, Tregs, TCM, TEM). Major organs (heart, liver, kidney) were collected for H&E staining to assess toxicity. [1]

- 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]

C96H184N3O22P

1763.5008

1762.31

C, 65.38; H, 10.52; N, 2.38; O, 19.96; P, 1.76

1246298-63-4

960324-04-3;

134687617

White to off-white solid powder

9

22

88

122

2550

14

CCCCCCCCCCCCCC(=O)O[C@H](CCCCCCCCCCC)CC(=O)N[C@@H]1[C@H]([C@@H]([C@H](O[C@H]1OC[C@@H]2[C@H]([C@@H]([C@H]([C@H](O2)O)NC(=O)C[C@@H](CCCCCCCCCCC)O)OC(=O)C[C@@H](CCCCCCCCCCC)O)O)CO)OP(=O)(O)[O-])OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC.[NH4+]

UPAZUDUZKTYFBG-HNPUZVNISA-N

InChI=1S/C96H181N2O22P.H3N/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);1H3/t77-,78-,79-,80-,81-,82-,89-,90-,91-,92-,93-,94-,95+,96-;/m1./s1

azanium;[(2R,3S,4R,5R,6R)-6-[[(2R,3S,4R,5R,6S)-3,6-dihydroxy-5-[[(3R)-3-hydroxytetradecanoyl]amino]-4-[(3R)-3-hydroxytetradecanoyl]oxyoxan-2-yl]methoxy]-2-(hydroxymethyl)-5-[[(3R)-3-tetradecanoyloxytetradecanoyl]amino]-4-[(3R)-3-tetradecanoyloxytetradecanoyl]oxyoxan-3-yl] hydrogen phosphate

A1O9D5VN5D; LAPRETOLIMOD AMMONIUM; RefChem:1088361; 1246298-63-4; Monophosphoryl lipid A (synthetic);

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)

DMSO : ~25 mg/mL (~14.18 mM)

Solubility in Formulation 1: 2.5 mg/mL (1.42 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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