TLR4

Microenvironmental priming of MSCs leads to the generation of unique exosomal mRNA content [4]
When examining the type of exosomal mRNAs between unprimed MSCs and CRX-527-primed MSCs, 1915 protein-coding genes exhibited differential expression upon CRX-527 treatment (FDR < 5% and >2 fold-change of normalized read counts). There was significant enrichment of the “inflammatory response” gene ontology (GO) term (GO:0002544) among the CRX-527-induced upregulated exosomal mRNAs (Figure 2; adjusted P-value = 3.8E−4). In addition to the inflammatory response GO term, the upregulated exosomal mRNAs enriched various biological functions like positive regulation of fibroblast migration (GO:0010763; adjusted P-value = 6.2E−12), regulation of intrinsic apoptotic signaling pathways (GO:2001242; adjusted P-value = 1.4E−7), and plasminogen activation (GO:0031639; adjusted P-value = 8.9E−7). These results suggest that CRX-527 priming of MSCs alters exosomal mRNAs content and suggests a role of ex vivo microenvironmental stimuli in modulating inflammation and wound healing.

To determine whether changes in exosomal mRNA content are specific to particular microenvironmental stimuli or represent a general response to MSC priming, we compared the effects of 2 different priming methods of MSCs: TNF-α and CRX-527 on the extent of overlap in exosomal mRNA content changes. We observed a moderate overlap in the differentially expressed exosomal mRNAs between the 2 MSC priming methods (Figure 3). For instance, out of 180 exosomal mRNAs upregulated in response to CRX-527 priming, only 11 (6.1%) were upregulated in response to TNF-α priming. Regarding downregulated genes, 857 out of 1735 (49.4%) exosomal mRNAs affected by CRX-527 priming of MSCs overlapped with those exosomes affected by TNF-α priming of MSCs. Furthermore, the mRNA content changes associated with each MSC priming method were found to be enriched in distinct biological functions (Figure 3). For example, positive regulation of blood vessel endothelial cell migration and fibroblast migration terms were specifically enriched in CRX-527 primed MSC-exosomes upregulated mRNAs but not enriched in TNF-α primed MSC-exosomes upregulated mRNAs. This observation suggests that exosomal mRNA content is governed by the specific microenvironment of MSCs, with differences noted after TNF-α and TLR4 priming.

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Microenvironmental priming of MSCs leads to the modulation of exosomal microRNA content [4]
Recent evidence suggests that microRNA loading of exosomes is a selective and regulated process, rather than a random occurrence. We sought to determine whether the type of MSC microenvironmental priming influences microRNA content of exosomes. To achieve this, we performed small RNA sequencing to profile short RNA fragments in exosomes derived from native MSCs, CRX-527 primed MSCs and TNF-α primed MSCs. We identified 31 differentially expressed exosomal microRNAs in response to CRX-527 treatment (Figure 4A) and 23 differentially expressed exosomal microRNAs in response to TNF-α treatment of MSCs (Figure 4B), respectively. Notably, none of the differentially expressed exosomal microRNAs overlapped between the CRX-527 and TNF-α treatments, suggesting that exosomal microRNA content is influenced by the specific microenvironmental stimuli used to prime MSCs. “Macrophage differentiation” was the top enriched gene ontology (GO) terms based on the enrichment p-value for the microRNA targets of TNF-α primed MSC-exosomes (Figure 4D). TNF-α is a pro-inflammatory cytokine known to induce M1-like polarization of macrophages. Enriching the “macrophage differentiation” GO term suggests that as molecular cargo, exosomes may transport specific microRNAs to initiate monocyte differentiation into macrophages under pro-inflammatory stimuli such as TNF-α perturbation. Figure 4C and D present the top 3 enriched GO terms based on the most significant P-values for CRX-527 and TNF-α priming, respectively.

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Correlations in RNA abundance between parent cells and exosomes [4]
We further conducted mRNA and small RNA sequencing on MSCs to investigate whether RNA abundance is correlated between parental MSCs and their exosomes. We observed moderate correlations between exosomes and their parent cells under native conditions, with Spearman’s Rank correlation coefficients of 0.46 for microRNAs and 0.57 for mRNAs, respectively (Figure 5). This suggests that a subset, but not all, exosomal RNAs indeed recapitulate the RNA abundance of their parental MSCs. These correlations increased when MSCs were exposed to microenvironmental stimuli, such as CRX-527 and TNF-α priming, notably for microRNAs (Figure 5). The Spearman’s rank correlation coefficient (Rho) between exosomal and parental MSC microRNA abundance increased from 0.46 (under native conditions) to 0.78 and 0.76 for CRX-527 and TNF-α primed MSCs, respectively. These data indicate that microenvironmental stimuli of MSCs synchronize microRNA profiles of MSCs with their secreted exosomes. A similar trend was observed for mRNAs, albeit less prominently (Figure 5). Thus, MSC-exosomal RNAs are not simply a random subset of RNAs derived from their parental cells, but are regulated and dependent on the microenvironmental stimuli driving exosome generation.

Morroniside (Mor) is a bioactive compound in Cornus officinalis with anti-inflammatory, neuroprotective and antioxidant properties. Prolonged use of the anesthetic sevoflurane (Sev) has been connected to the development postoperative cognitive dysfunction (POCD). This research aims to elucidate the mechanism of action of Mor to improve cognitive impairment. A model of cognitive dysfunction induced by Sev was established in aged mice and tested for behavioral analysis using the water maze experiment. Histopathological changes and neuronal apoptosis in mouse hippocampus were observed by hematoxylin and eosin (HE) staining, Nissl staining, and TUNEL staining. ELISA and qRT-PCR determined the levels of inflammatory factors. Phenotypic transformation of microglia in hippocampal tissue was assessed by immunofluorescence, flow cytometry, and qRT-PCR. The interaction between Mor and TLR4 was analyzed using molecular docking. Western blot identified the levels of apoptosis-related proteins, synapse-related proteins, and TLR4/NF-κB pathway proteins. Inhalation of Sev caused a notable reduction in learning and spatial memory in old mice, which was dose-dependently ameliorated by Mor. Mor inhibited neuroinflammation, modulated the polarization state of hippocampal microglia, promoted their polarization to M2 type, alleviated Sev-induced hippocampal tissue damage and neuronal apoptosis. Notably, Mor can bind well with TLR4 and reduce TLR4-positive expression. Mor blocked Sev-induced TLR4/NF-κB pathway activation in hippocampal tissues, and the TLR4 agonist CRX-527 attenuated the effect of Mor. In conclusion, Mor blocked the TLR4/NF-κB pathway, reducing hippocampal tissue damage and neuroinflammation caused by Sev, which in turn improving cognitive impairment in aged mice [1].

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Recently, Toll-like receptors (TLRs) have been extensively studied in radiation damage, but the inherent defects of high toxicity and low efficacy of most TLR ligands limit their further clinical transformation. CRX-527, as a TLR4 ligand, has rarely been reported to protect against radiation. We demonstrated that CRX-527 was safer than LPS at the same dose in vivo and had almost no toxic effect in vitro. Administration of CRX-527 improved the survival rate of total body irradiation (TBI) to 100% in wild-type mice but not in TLR4-/- mice. After TBI, hematopoietic system damage was significantly alleviated, and the recovery period was accelerated in CRX-527-treated mice. Moreover, CRX-527 induced differentiation of HSCs and the stimulation of CRX-527 significantly increased the proportion and number of LSK cells and promoted their differentiation into macrophages, activating immune defense. Furthermore, we proposed an immune defense role for hematopoietic differentiation in the protection against intestinal radiation damage, and confirmed that macrophages invaded the intestines through peripheral blood to protect them from radiation damage. Meanwhile, CRX-527 maintained intestinal function and homeostasis, promoted the regeneration of intestinal stem cells, and protected intestinal injury from lethal dose irradiation. Furthermore, After the use of mice, we found that CRX-527 had no significant protective effect on the hematopoietic and intestinal systems of irradiated TLR4-/- mice. in conclusion, CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage.[2]

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TLR4 mediates NTP anti-inflammation effect in the VD mice [3]
Given that the neuroprotective effects of NTP may be mediated at least in part by TLR4, we hypothesized that TLR4 antagonists might have similar effects to NTP in inhibiting inflammation and ameliorating memory, whereas TLR4 agonists might reverse the protective effect of NTP. To further elucidate whether the anti-inflammatory effect of NTP was at least in part mediated by TLR4, the specific TLR4 antagonist TAK242 and the specific agonist CRX-527 were applied. Mice were randomly assigned to the sham group, the BCAS/normal saline (NS) group, the BCAS/NTP group, the BCAS/TAK242 group and the BCAS/NTP/CRX-527 group (each group, n = 12). We found that mRNA levels of IL-1β, IL -6 and TNFα were decreased after NTP or TAK-242 treatment. However, combination of NTP with CRX-527 led to an increase in these mRNA levels (IL-1β: P＜0.001; IL-6:,P＜0.001; TNFα: P＜0.001, compared to the BCAS/NTP group) (Fig. 7A,B and 7C). Similarly, the level of MyD88 and pP65 protein were significantly increased after CRX-527 administration (MyD88: P＜0.001; pP65: P＜0.001, compared to the BCAS/NTP group) (Fig. 7E and F), suggesting that NTP exerted its anti-inflammatory effects at least in part by the TLR4/MyD88/NF-κB pathway in the VD mice. Next, we observed the effects of the two inhibitors on glial cells. More IBA-1 positive cells were observed in the BCAS/NTP/CRX-527 group than the BCAS/NTP group in the CA1 and CA3 regions of the hippocampus (CA1: P＜0.001; CA3: P = 0.004) (Fig. 6A and B). Additionally, GFAP fluorescence staining showed a great increase of the positive cell number in the BCAS/NTP/CRX-527 group than the BCAS/NTP group in CA1 and CA3 regions(CA1: P = 0.0037; CA3: P＜0.001) (Fig. 6C and D). To assess whether improved memory function was associated with TLR4 in the BCAS/NTP group, mice were subjected to the Y-maze test after treatment. In comparison with the sham group, the BCAS/NS group showed a significant decrease in spontaneous alternation rate in Y-maze test (F(4,47) = 27.15,P = 0.000). NTP and TAK242 treatment increased significantly % correct alternation compared to the BCAS/NS group. However, the therapeutic effect of NTP on % correct alternation of the BCAS mice was apparently abolished by CRX-527 administration (P＜0.001, compared to the BCAS/NTP group). Additionally, there was no difference between BCAS/NTP and BCAS/TAK242 group(P＞0.5) (Fig. 8), suggesting that over-activation of TLR4 reverses the effects of NTP on memory impairment, and further TLR4 mediated memory improvement of NTP in the VD mice.

Co-culture assay [2]
In brief, 1×105 RAW264.7 or THP-1 cells were seeded in the Transwell chamber (BIOFIL, 0.4 µm, 6.5 mm diameter), while MODE-K or HIECs were seeded at the bottom of the 12-well plates and then cultured according to the manufacturer’s instructions. RAW264.7 cells or THP-1 cells in the Transwell chamber were treated with CRX-527 12 hours before irradiation. After irradiation, MODE-K cells or HIECs on 12-well plates were used for colony formation, ROS, and Western blot analysis.

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Treatments to the injured ligaments included: (1) PBS (serving as the injured control), (2) 5 × 106 exosomes (exosome), (3) 5 × 106 exosomes from TNF-α-primed MSCs (TNF), or (4) 5 × 106 exosomes from CRX-527-primed MSCs (CRX). Exosome dose was chosen based on prior results showing that delivery of 1 × 106 exosomes reduced the M1/M2 macrophage ratio but did not improve tendon functionality. A dose of 5 × 106 exosomes was the maximum dose and volume that could be added to the torn MCL without excessive leakage. The skin was sutured after treatment administration. A second dose of 5 × 106 exosomes was administered to the contralateral MCL, via needle and syringe, 3 days post-injury (when the presence of macrophages is elevated), to examine if an additional dose of exosomes could provide an additive effect on healing. Fourteen days post-injury, MCLs were collected for mechanical testing. As a time, dependent effect within the mechanical results was not observed, the 2 sides were combined for all data analyses. [4]

Male SPF-grade C57BL/6J mice (20 months old) were kept in a clean-grade animal house. room temperature was 23 °C–28 °C, with humidity levels between 45% and 55%, under a 12-h light–dark cycle, and feeding anddrinking were performed autonomously. Animal experiments followed the 3R principle,changing bedding daily and disinfect-ing facilities, such as food containers, cages, water bottles, anddrinking tubes regularly. After a week of adjustment feeding,mice were randomly divided into control (n=8), Sev (Sev,n=8), Sev-Mor (S-Mor,n=16), and Sev-Mor-TLR4 agonist(S-Mor-CRX-527,n=4) groups. An anesthesia gas monitor was used to monitorthe levels of Sev, carbon dioxide, and oxygen levels. A smallamount of soda lime was spread on the bottom of the inductionbox to prevent carbon dioxide accumulation. Breathable isola-tion pads were laid flat on top of the soda-lime to prevent micefrom inhaling soda-lime dust to burn the mice. A mouse modelof cognitive dysfunction under Sev anesthesia was established according to.

Mice continuously inhaled 2%Sev for 5h, with a total of 1.5L/min airflow achieved using 70% O2 as the carrier gas. After the end of Sev anesthesia, they were returnedto their cages after awakening in a dry and warm environment.Control group: normal inhalation of room air.Control mice were intraperitoneally injected with saline(0.1 mL/100 g). After anesthetization, mice in the Sev groupwere intraperitoneally injected with saline. The mice in theS-Mor group were anesthetized before receiving Mor (30, 60,and 100 mg/kg body weight)at a frequency of every three days over a period of four weeks. Mice in the S-Mor-CRX-527 groupwere anesthetized and injected intraperitoneally with Mor(100 mg/kg bw) every three days for four consecutive weeks,followed by intraperitoneal injection of CRX-527 (0.25 mg/kgbw, V42156, InvivoChem LLC) once a day forthree days during the last three days of Mor treatment. [1]

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Irradiation and treatment [2]
HIECs and MODE-K cells were irradiated with a single dose of 16 Gy X-ray using an irradiator (KUBTEC XCELL 225, 225 KV 13.2 mA 1 Gy/min), while unirradiated control cells were studied in parallel under the same conditions. For mice, 5 Gy total body dose was used to observe the changes of hematopoietic system, 7.5 Gy total body dose was used to observe the changes of intestinal system, and 9 Gy local abdominal irradiation was used to observe the changes of intestinal system. Mice received 0.5 mg/kg CRX-527 by intraperitoneal injection 24 hours and 2 hours before irradiation. Whole body irradiation mice were fixed with a fixed frame and then placed in an irradiator for irradiation. After irradiation, the corresponding tissues were collected for detection.

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Establishment of VD mice model and drug treatment [3]
We established the VD mice model using a bilateral common carotid artery model (BCAS) (Shibata et al., 2004; Shibata et al., 2007; Ihara et al., 2014). After two weeks of adaptation, all mice were randomly divided into five groups: the sham group, the BCAS/normal saline (NS) group, the BCAS/NTP group, the BCAS/TAK242 group and the BCAS/NTP/CRX-527 group (n = 12, each group). Specifically, the mice were anesthetized with 3.0% isoflurane and placed in the supine position. To expose both common carotid arteries, a small incision was made in the midline of the neck. Microcoils of 0.18 (0.18 mm diameter, 2.5 mm length) were placed in both common carotid arteries. Put on one side of the microcoil first, and left the other side about an hour later. The intraoperative and postoperative temperature was kept on a heating pad at 36.5 ± 0.5 °C. When the mice recovered from the anesthesia, they were free to move around the cage and access to water and food. Three days after surgery, both the BCAS/NS group and the BCAS/NTP group were given saline and NTP (50NU/Kg, once per day) for 28 days (Fang et al., 2019), respectively. To observe the effects of NTP on TLR4, intraperitoneal injection of 3 mg/kg TAK-242 or 0.25 mg/kg CRX-527 (tlrl-crx527) was performed according to previous studies and manufacturer instructions (Hua et al., 2015; Yang et al., 2020; Zhang et al., 2022).

[1]. Morroniside ameliorates sevoflurane anesthesia-induced cognitive dysfunction in aged mice through modulating the TLR4/NF-κB pathway. Biomol Biomed. 2024 Dec 6. doi: 10.17305/bb.2024.11433.
[2]. CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage. Front Immunol. 2022 Aug 30:13:927213.
[3]. Neurotropin alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in mice with vascular dementia. Neurochem Int. 2023 Dec:171:105625.
[4]. Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity. Stem Cells. 2024 Jul 8;42(7):636-649.

In summary, our research demonstrates for the first time thatMor mitigates Sev-induced histopathological damage in thehippocampus of elderly mice, promotes the transformation ofmicroglia into the M2 type, and suppresses neuroinflamma-tion. Importantly, Mor blocked the TLR4/NF-κB pathway, sug-gesting that it may alleviate cognitive deficits in aged mice bymodulatingtheTLR4/NF-κBpathway.Thisstudyelucidatesthepotential mechanism of action of Mor in alleviating cognitiveimpairment in aged mice, which provides a new reference forthe clinical treatment of POCD. However, there are still someshortcomings of this research. Owing to time and conditionconstraints, this study had a small sample size. The effect ofMoroncognitiveimpairmentinyoungmiceanditsmechanismof action should be explored further in the future. [1]

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In this study, we demonstrated that CRX-527 protected against irradiation-induced hematopoietic and intestinal injury. Compared with LPS, CRX-527 was less toxic in vivo and in vitro. Mechanistically, the stimulation of CRX-527 significantly increased the proportion and number of HSCs and promoted their differentiation into macrophages, activating immune defense. On this basis, we observed positive changes in intestinal structure and function in mice after ionizing radiation. Activation of the TLR4-related pathway mediated the protective effect of CRX-527 on hematopoietic and intestinal radiation injury. [2]

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In our study, TAK242 inhibited neuroinflammation induced by BCAS, similar to the effects of NTP. Finally, we administered the TLR4-specific agonist CRX-527 to elucidate the underlying mechanisms of NTP effects. CRX-527, a TLR4-specific agonist, induced significant NF-κB promoter activation at 24 h after stimulation (Bowen et al., 2012). CRX-527 induces activation of downstream MyD88 signaling cascades leading to early activation and nuclear translocation of the NF-κB, promoting inflammation (Zhang et al., 2022). We found that CRX-527 abolished the effect of NTP, worsened neuroinflammation, and increased memory impairment, suggesting that TLR4 plays a major role in VD inflammation. The anti-inflammatory effect of NTP in VD mice is through regulation of TLR4. This suggested that NTP blocks the activation of sensor molecules in a neural pathway that could be a treatment for neuroinflammatory nervous system diseases. To sum up, we showed that NTP suppresses inflammation through TLR4/MyD88/NF-κB pathway. [3]

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Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration. [4]

C81N2O19PH151

1488.041

1,487.06

C, 65.38; H, 10.23; N, 1.88; O, 20.43; P, 2.08

216014-14-1

216014-14-1 (CRX-527);216014-05-0 (CRX-547);

Typically exists as solid at room temperature

O=C([C@@H](NC(C[C@@H](CCCCCCCCCCC)OC(CCCCCCCCC)=O)=O)CO[C@H]1[C@@H]([C@H]([C@@H]([C@H](O1)CO)OP(O)(O)=O)OC(C[C@@H](CCCCCCCCCCC)OC(CCCCCCCCC)=O)=O)NC(C[C@@H](CCCCCCCCCCC)OC(CCCCCCCCC)=O)=O)O

REEGNIYAMZUTIO-MGSMBCBTSA-N

InChI=1S/C81H151N2O19P/c1-7-13-19-25-31-34-40-43-49-55-66(97-73(87)58-52-46-37-28-22-16-10-4)61-71(85)82-69(80(91)92)65-96-81-77(83-72(86)62-67(56-50-44-41-35-32-26-20-14-8-2)98-74(88)59-53-47-38-29-23-17-11-5)79(78(70(64-84)100-81)102-103(93,94)95)101-76(90)63-68(57-51-45-42-36-33-27-21-15-9-3)99-75(89)60-54-48-39-30-24-18-12-6/h66-70,77-79,81,84H,7-65H2,1-6H3,(H,82,85)(H,83,86)(H,91,92)(H2,93,94,95)/t66-,67-,68-,69+,70-,77-,78-,79-,81-/m1/s1

O-((2R,3R,4R,5S,6R)-3-((R)-3-(decanoyloxy)tetradecanamido)-4-(((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2-yl)-N-((R)-3-(decanoyloxy)tetradecanoyl)-L-serinate

CRX-527; 216014-14-1; Decanoic acid, (1R)-1-[2-[[(1S)-1-carboxy-2-[[2-deoxy-3-O-[(3R)-1-oxo-3-[(1-oxodecyl)oxy]tetradecyl]-2-[[(3R)-1-oxo-3-[(1-oxodecyl)oxy]tetradecyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]oxy]ethyl]amino]-2-oxoethyl]dodecyl ester

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.)