TNF-a; bioactive peptide

While it has been demonstrated that the TNFα antagonist, WP9QY, binds to TNFα and inhibits its binding of TNFR-1, it is not clear whether or not WP9QY retains its affinity for binding to TNFα after conjugation to 3D PEG hydrogel networks. To examine the TNFα-binding capability of immobilized WP9QY, we synthesized a crosslinkable and FITC-labeled WP9QY peptide analog, namely acryl-(FITC)WP9QY (Fig. 1a). FRET experiments, an indication of affinity binding, were first carried out in the presence of acryl-(FITC)WP9QY peptide (donor) and AlexaFluor555-labeled TNFα (AF555-TNFα, acceptor) in PBS or PEGDA macromer solutions. As shown in Fig. 2a, an increased normalized FRET was observed as a function of acceptor (AF555-TNFα) concentration at a constant donor (acryl-(FITC)WP9QY) concentration of 500nM. The increased FRET in PBS demonstrates that the synthesized WP9QY analog retains its binding affinity to TNFα. The degree of TNFα-WP9QY binding further increased in PEGDA (both 6kDa and 10kDa) macromer solutions. This was attributed to the high exclusion volume of PEG macromer that increased the local TNFα and WP9QY concentrations, which leads to a movement of the binding equilibrium to TNFα-WP9QY complexes. Note that the increase in the normalized FRET in the presence of PEG macromers cannot be attributed to the auto-fluorescence of PEG molecules, as the FRET signals were normalized to that of PEG/donor solutions without the presence of acceptor. [1]

To examine the binding of TNFα to immobilized peptide antagonist WP9QY, PEGDA solutions were photopolymerized in the presence of acryl-(FITC)WP9QY and soluble AF555-TNFα to form PEG-WP9QY hydrogels. As shown in Figure 1b & 1c, the incorporation of acryl-(FITC)WP9QY in PEG hydrogels can be rigorously controlled by adjusting the concentration of the functionalized peptide in the PEGDA prepolymer solutions. Further, the FRET results (Figure 2b) show that the normalized FRET of TNFα and WP9QY again increases as a function of acceptor concentration, indicating that the immobilized WP9QY retains its binding ability to soluble TNFα. It is worth noting that the extent of TNFα-WP9QY binding in PEGDA hydrogels was lower than the binding in PEGDA solutions, where both TNFα and WP9QY are soluble. In previous work, we demonstrated that the affinity binding of hydrogel network-immobilized peptides to soluble protein is decreased due to the reduced mobility of the immobilized peptide and the “steric hindrance” resulting from the surrounding PEG chains. Nonetheless, the binding of TNFα to immobilized WP9QY in PEG hydrogels is still higher than its binding in PBS (Figure 2b), suggesting a similar, if not higher, TNFα antagonistic effects of PEG-WP9QY hydrogels. The FITC-labeled PEG-WP9QY hydrogels, the presence of AlexaFluor555-TNFα, and the FRET phenomena were all visualized with confocal microscopy (Figure 2c). [1]

Next, the swelling of the PEG-WP9QY hydrogels was characterized as a function of the peptide content. Hydrogel swelling is important in that it dictates the transport properties that affect the time scale of diffusion of important molecules and correlates to the survival of encapsulated cells. To verify that the incorporation of acryl-WP9QY within PEGDA hydrogels does not adversely affect the swelling and diffusional properties of PEG hydrogels, we characterized the equilibrium mass swelling ratio of, as well as insulin (MW: 5.5kDa) diffusion from, PEG-WP9QY hydrogels at physiological pH (7.4). Results indicate that both hydrogel swelling (Figure 1d) and insulin release (data not shown) are unaffected by the incorporation of the acryl-WP9QY peptide at concentrations up to 500μM. [1]

Another factor that would significantly affect the outcome of encapsulated cell fate in the PEG-WP9QY hydrogels is the amount of absorbed TNFα in the gels. In 2D culture, the concentrations of soluble factors can be easily controlled via media supplements. However, when cells are encapsulated in 3D PEG hydrogels, the actual cytokine concentration bound within the gel cannot be assumed equal to that in the media, especially when an affinity peptide is copolymerized within PEG hydrogels to interact with the cytokine. To understand the extent of TNFα penetration/absorption in the affinity PEG-WP9QY hydrogels, we incubated gels in concentrated recombinant TNFα solutions and quantified the concentration of TNFα in the supernatant. As shown in Figure. 3a, the amount of TNFα penetrated/absorbed in PEG-WP9QY hydrogels increases with increasing incubation time, as well as increasing acryl-WP9QY concentrations. However, significant differences were only found at higher WP9QY concentrations (>250μM), indicating that the incorporation of a small quantity of WP9QY peptide (<100μM) in PEG hydrogels does not significantly affect the amount of TNFα absorption, as compared to unmodified PEG hydrogels. Even at high WP9QY peptide incorporation, such as 250μM and 500μM, the absorbed TNFα in the gels are in the bound state. Figure 3b shows the theoretical calculation of the equilibrium concentrations of bound versus free TNFα in PEG-WP9QY hydrogels. While all the TNFα absorbed into the un-modified PEG hydrogels (∼6.8ng/gel after 72hr, Figure 3a) is freely diffusible and able to bind to the TNF-R1 on the encapsulated cells, almost all TNFα is bound to the immobilized WP9QY peptide (when KD<10-5M). It is worth noting that TNFα has a low dissociation constant for TNF-R1 (KD ∼ 1.9 × 10-11 M), implying a strong binding of infiltrated TNFα to the TNF receptor binding loop-derived WP9QY peptide. Although Figure 3b does not take into account the competitive binding of TNFα-TNFR1 nor does it account for TNFα proteolysis/degradation, it should be noted that the incorporated WP9QY peptides are distributed homogenously throughout the PEG gels (Figure 1b) while the encapsulated cells are discretely distributed (Figure 4c & 4d). Further, the WP9QY peptide incorporated in the PEG gels (100 – 500μM) is in large molar excess compared to TNFα level in this study (∼ 50 ng/mL or 2.87 nM) or that found in healing (∼ 0.9 ng/mL or 0.05 nM) or non-healing wound sites (∼ 2.4 ng/mL or 0.14 nM). Therefore, when the infiltrated TNFα is bound to the abundant WP9QY peptide in the gel, it is unable to interact with the encapsulated cells in the WP9QY functionalized gels. [1]

As described above, the pro-inflammatory cytokine TNFα induces apoptosis in a wide variety of cell types. To elucidate the effects of PEG-WP9QY hydrogels in protecting encapsulated cells from TNFα-induced apoptosis, rat pheochromocytoma cells, or PC12 cells, were encapsulated in PEG hydrogels functionalized with the WP9QY peptide. A cell adhesive peptide, CGRGDS, was also immobilized in PEG gels via a thiol-acrylate photopolymerization. The RGD peptide functionality is required to support PC12 survival in a 3D hydrogel. Prior research has revealed that TNFα induces significant apoptosis in differentiated, but not naïve, PC12 cells in 2D culture. We observed a similar phenomenon in 3D hydrogel culture, where un-differentiated PC12 cells in PEG-RGD hydrogels remained viable and nonapoptotic, even with the challenge of TNFα (data not shown). When encapsulated PC12 cells were differentiated with nerve growth factor (2.5S NGF) for 6 days, however, an ∼40% reduction in viability of differentiated PC12 cells was observed in PEG-RGD hydrogels, as compared to the viability of un-challenged cells (Figure 4a). On the other hand, the survival of PC12 cells under TNFα challenge was not affected and maintained a viability comparable to the unchallenged cells in PEG-RGD-WP9QY hydrogels (100 and 250μM WP9QY). In addition, qualitative live/dead staining and confocal imaging results also revealed an increased number of dead cells upon TNFα challenge in un-modified PEG gels, but not in PEG-WP9QY (100μM acryl-WP9QY) gels (Figure 4c & 4d), demonstrating an important role of immobilized WP9QY peptide in protecting encapsulated cells from cytokine-induced cell death. Further examination of caspase 3/7 activation in encapsulated PC12 cells with or without TNFα challenge reveals that the reduced cell survival under TNFα challenge is mediated by an apoptotic pathway (Fig. 4b). [1]

To further explore the efficacy of PEG-WP9QY hydrogels in promoting encapsulated cell function, we photo-encapsulated mouse pancreatic islets within PEG or PEG-WP9QY (100μM) hydrogels and studied the effects of immobilized WP9QY in protecting islet cells from apoptosis and reduced insulin secretion due to the infiltrated TNFα. Pancreatic islets have been shown to undergo apoptotic pathways when challenged with pro-inflammatory cytokines, such as TNFα. Although islet encapsulation and transplantation have been suggested as a solution for curing type 1 diabetes, current encapsulation techniques cannot prevent β-cells damage induced by small pro-inflammatory cytokines. To this end, we successfully fabricated permissive PEG-WP9QY hydrogel environments that support the survival and function of photo-encapsulated pancreatic islets. As shown in Figure S1, although less potent than mouse cytokines, human cytokines do induce significant mouse β-cell apoptosis, suggesting that the PEG-WP9QY hydrogels can be used to prevent hTNFα-induced mouse β-cell apoptosis. Indeed, while mouse islets encapsulated in un-modified PEG hydrogels show an increased caspase 3/7 activity (Figure 5a), impaired cell survival (Figure 5b), and reduced insulin secretion (Figure 5c) under hTNFα challenge, no significant difference was found, with or without hTNFα challenge, when islets were encapsulated within PEG-WP9QY(100μM) functionalized hydrogels. [1]

The use of hydrogels to encapsulate human mesenchymal stem cells (hMSCs) has proven valuable in providing a 3D environment for supporting their osteogenic, chondrogenic, and adipogenic differentiation to restore damaged tissues. However, one question remaining to be answered is that, in the highly inflammatory environments of the wound sites, can the encapsulated hMSCs still undergo their desired differentiation pathways in the presence of pro-inflammatory cytokines? In 2D culture, several publications have revealed the molecular mechanisms of the inhibitory effects of TNFα on osteogenic differentiation of hMSCs. In an attempt to promote 3D hMSCs osteogenic differentiation under TNFα challenge, we photo-encapsulated hMSC in PEG-RGD (3mM CGRGDS) or PEG-RGD-WP9QY (100μM) hydrogels and studied the effects of the infiltrated TNFα on the survival and osteogenic differentiation potential of hMSCs. Our results show that, similar to PC12 (Figure 4b) and islet (Figure 5a) encapsulation, encapsulated hMSCs exhibit higher caspase 3/7 activity under TNFα challenge in PEG-RGD hydrogels, but not in PEG-RGD-WP9QY hydrogels (Figure 6a). Differing from the decreased cell survival in PC12 cells and islets under TNFα challenge, however, is that hMSCs encapsulated in PEG-RGD hydrogels are more proliferative than in PEG-RGD-WP9QY hydrogels in the presence of TNFα. This result is in accordance with a recent report that TNFα induces hMSC proliferation. We verified the proliferative effects of TNFα on hMSC in 2D culture (Figure S3a), as well as in 3D hydrogels (Figure 6b). Since proliferative hMSCs have less differentiation potential and the treatment of TNFα increases proliferation of hMSCs [36], a less osteogenic differentiation might be expected under TNFα challenge. As shown in Figure S3b, when challenged with TNFα in 2D culture, hMSCs show less (on a per cell basis) alkaline phosphatase (ALP) activity, which demonstrates the potential effects of TNFα in inhibiting hMSC osteogenic differentiation. Finally, we show that the use of PEG-RGD-WP9QY functionalized hydrogels successfully prevents the inhibitory effects of TNFα on the osteogenic differentiation of encapsulated hMSCs in a 3D hydrogel culture. As illustrated in Figure 6c, the relative ALP activity of hMSCs encapsulated in PEG-RGD-WP9QY gels is maintained, under the challenge of TNFα, but not in PEG-RGD hydrogels. [1]

We show that PEG-WP9QY hydrogels antagonize TNFα for two weeks (Figure 6b). However, one concern remains regarding the long-term cytokine antagonistic effect of the hydrogels. It should be noted that at sufficiently high peptide concentrations, the release of bound cytokine from immobilized peptide is minimal. It is also likely that the bound cytokine will denature after long-term binding to the immobilized peptide. This study opens an avenue for fabricating functional biomaterials capable of interacting with small cytokines to improve the survival and function of encapsulated cells for in vivo tissue engineering and regenerative medicine applications. The therapeutic efficacy of these cytokine-antagonizing PEG hydrogels can be further expanded through immobilizing other, synergistic peptidomimetic antagonists for additional cytotoxic cytokines, such as interleukin-1β (IL-1β) and interferon γ (IFN- γ). Such peptide-functionalized hydrogel platforms might also serve as an interactive affinity depot, loaded with therapeutic agents, to recruit cells for promoting tissue regeneration.

FRET experiments [1]
To examine TNFα binding to immobilized WP9QY, FRET experiments were designed in PBS, or in the presence of PEGDA macromer solutions or hydrogels. Briefly, TNFα was labeled with alexafluor 555 according to the manufacturer's protocol and used as a FRET acceptor (AF555-TNFα), while acryl-(FITC)WP9QY was used as a FRET donor. Samples for FRET measurements, including acceptor-only samples, donor-only samples, and acceptor and donor samples, were prepared in a 96-well plate. The fluorescence of all the samples was quantified in a microplate reader with three filter sets, including donor (Ex: 485nm, Em: 535nm), acceptor (Ex: 560nm, Em: 595nm), and FRET (Ex: 485nm, Em: 595nm) filters. The obtained fluorescence signals (in PBS or PEGDA solution before and after photopolymerization) were calibrated as described in a previous report. The calibrated FRET for each acceptor concentration was then normalized by the FRET in the absence of acceptor (donor-only) to obtain normalized FRET (see Supporting information).

Cell culture and encapsulation [1]
PC12 cells were maintained in RPMI1640 media containing 15% horse serum, 5% fetal bovine serum, 1% penicillin-streptomycin, and 0.5μg/mL fungizone at 37°C in humid conditions with 5% CO2. For encapsulation, PC12 cells were trypsinized from the flask and mixed with 10wt% PEGDA macromer solution containing 0.025wt% I-2959 and required amount of the acryl-WP9QY peptide. The cell adhesive peptide CGRGDS (2mM) was also incorporated to support cell survival in the PEG gels. Photo-encapsulation was achieved via exposing the cell-containing macromer solutions (40μL/gel, 150,000 cells/gel) under UV similar to the photopolymerization process described above. After encapsulation, the cell-laden gels were maintained in PC12 differentiation media containing 1% horse serum and 50ng/mL 2.5S nerve growth factor for 6 days for neuronal differentiation. Isolated mouse islets were suspended in 10 wt% PEGDA macromer solutions containing the desired amount of acryl-WP9QY and 0.025 wt% I-2959. The islet-macromer solutions containing approximately 20 islets each (30μL) were injected in a perfusion chamber mold (9 mm dia. × 0.5 mm thick, Grace Bio-Labs, Bend, OR) and photopolymerized under UV exposure for 10 minutes. Encapsulated islets were maintained in RMPI 1640 media supplemented with 10 % FBS, 1% penicillin-streptomycin, and 0.5 μg/mL fungizone. Culture media was changed every other day. hMSCs were maintained in growth media (low-glucose DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.5 μg/mL fungizone, and 1ng/mL bFGF) and passage twice every week. All hMSCs used in encapsulations (similar procedure as described above; 150,000 cells/gel) were from passage 3 and maintained in osteogenic differentiation media (high-glucose DMEM containing 10% FBS, 1% penicillin-streptomycin, and 0.5 μg/mL fungizone, 0.01 μM Dexamethasone, 0.2mM ascorbic acid 2-phosphate, 10mM glycerol 2- phosphate).

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TNFα challenge and caspase 3/7 activity measurement [1]
PEG hydrogels and PEG-WP9QY hydrogels containing cells were exposed to recombinant human TNF-α (50 ng/mL) for 24 hours. After which, the cell-laden gels were transferred to 0.6 mL of fresh media containing 10% of AlamarBlue regent and incubated for 3 hours. 200 μL of the solution were transferred to a 96-well clear plate, and the AlamarBlue fluorescence (Ex: 560nm; Em: 590nm) signals were measured to determine the relative cell number in each gel. The gels were then transferred to fresh media containing 50% CaspasGlo 3/7 reagent and incubated for 1 hour at room temperature on an orbital shaker. 200 μL of the solutions were transferred to a white-wall 96-well plate for luminescence measurements in a microplate reader. The obtained luminescence signals were normalized to the previously obtained AlamaBlue fluorescence for the respective gel samples and expressed as relative caspase 3/7 activity. Encapsulated PC12 cells with or without TNFα challenge were stained with a Live/Dead staining kit and imaged with confocal microscopy.

[1]. Functional PEG-peptide hydrogels to modulate local inflammation induced by the pro-inflammatory cytokine TNFalpha. Biomaterials. 2009 Oct;30(28):4907-14.

Hydrogels are an important class of biomaterials used for cell encapsulation and delivery, forming a physical barrier or "immune isolation" between host tissues and encapsulated cells. These semi-permeable gels protect encapsulated cells from recognition by host immune cells and/or antibodies while allowing rapid diffusion of nutrients. However, a previously unresolved issue is that highly permeable hydrogels cannot prevent the infiltration of soluble immune mediators, such as pro-inflammatory cytokines highly expressed in the in vivo wound environment. When encountering pro-inflammatory cytokines, encapsulated cells cannot perform their intended function. This article reports the synthesis, characterization, and application of a peptide-functionalized cytokine antagonist polyethylene glycol (PEG) hydrogel that effectively isolates the pro-inflammatory cytokine tumor necrosis factor-α (TNFα). Results show that, in vitro TNFα treatment significantly inhibited the survival, function, and differentiation of cells encapsulated in unmodified PEG hydrogels (e.g., rat PC12 adrenal pheochromocytoma cells, mouse islet cells, and human bone marrow mesenchymal stem cells or hMSCs). In contrast, cells encapsulated in TNFα-antagonistic hydrogels were unaffected by infiltrating TNFα. This study suggests that controlling the availability of pro-inflammatory cytokines in highly permissible hydrogels is crucial. [1]

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Utilizing the highly specific binding of TNFα-WP9QY, we here report the synthesis, characterization, and application of TNFα-antagonistic PEG hydrogels in enhancing the survival and function of encapsulated cells against TNFα stimulation. We first describe the design and characterization of the affinity binding of human TNFα to WP9QY peptide-functionalized PEG hydrogels using fluorescence resonance energy transfer (FRET), and then characterize the physical properties of the PEG-WP9QY hydrogels, including the degree of swelling, diffusion of small molecules, and uptake of cytokines within the gel. Since TNFα is a potent pro-inflammatory cytokine that can broadly affect various major cell types in vivo, we encapsulated three TNFα-sensitive cell types, including rat adrenal pheochromocytoma cells PC12, mouse islet cells, and human bone marrow mesenchymal stem cells (hMSCs), in a PEG-WP9QY functionalized hydrogel to verify its ability to inhibit TNFα-induced cell fate damage in encapsulated cells. In summary, we synthesized a WP9QY-functionalized TNFα antagonist PEG hydrogel that can chelate cytotoxic TNFα and prolong the survival time and function of encapsulated PC12 cells and mouse islet cells. In addition, the WP9QY-functionalized PEG hydrogel can not only inhibit TNFα-induced hMSC proliferation, but also maintain their osteogenic differentiation potential in 3D hydrogel culture. This study provides a new method for preparing immune-isolating hydrogels for cell therapy, which is expected to be effectively utilized in in vivo tissue engineering applications. [1]

C60H74F3N11O17S2

1342.41828298569

1225.457

199999-60-5

16134442

Tyr-Cys-Trp-Ser-Gln-Tyr-Leu-Cys-Tyr (Disulfide bridge:Cys2-Cys8); L-tyrosyl-L-cysteinyl-L-tryptophyl-L-seryl-L-glutaminyl-L-tyrosyl-L-leucyl-L-cysteinyl-L-tyrosine (2->8)-disulfide; H-Tyr-Cys(1)-Trp-Ser-Gln-Tyr-Leu-Cys(1)-Tyr-OH

YCWSQYLCY (Disulfide bridge:Cys2-Cys8); YCWSQYLCY

White to off-white solid powder

1.5±0.1 g/cm3

1692.1±65.0 °C at 760 mmHg

977.2±34.3 °C

0.0±0.3 mmHg at 25°C

1.701

0.46

16

18

19

86

2300

9

SC[C@@H](C(N[C@H](C(=O)O)CC1C=CC(=CC=1)O)=O)NC([C@H](CC(C)C)NC([C@H](CC1C=CC(=CC=1)O)NC([C@H](CCC(N)=O)NC([C@H](CO)NC([C@H](CC1=CNC2C=CC=CC1=2)NC([C@H](CS)NC([C@H](CC1C=CC(=CC=1)O)N)=O)=O)=O)=O)=O)=O)=O.FC(C(=O)O)(F)F

OXRZFLLXMORPHO-XCLFSWKQSA-N

InChI=1S/C58H71N11O15S2/c1-30(2)21-42-52(77)69-48(57(82)66-45(58(83)84)24-33-11-17-37(73)18-12-33)29-86-85-28-47(68-50(75)39(59)22-31-7-13-35(71)14-8-31)56(81)65-44(25-34-26-61-40-6-4-3-5-38(34)40)54(79)67-46(27-70)55(80)62-41(19-20-49(60)74)51(76)64-43(53(78)63-42)23-32-9-15-36(72)16-10-32/h3-18,26,30,39,41-48,61,70-73H,19-25,27-29,59H2,1-2H3,(H2,60,74)(H,62,80)(H,63,78)(H,64,76)(H,65,81)(H,66,82)(H,67,79)(H,68,75)(H,69,77)(H,83,84)/t39-,41-,42-,43-,44-,45-,46-,47-,48-/m0/s1

(2S)-2-[[(4R,7S,10S,13S,16S,19S,22R)-22-[[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-13-(3-amino-3-oxopropyl)-16-(hydroxymethyl)-10-[(4-hydroxyphenyl)methyl]-19-(1H-indol-3-ylmethyl)-7-(2-methylpropyl)-6,9,12,15,18,21-hexaoxo-1,2-dithia-5,8,11,14,17,20-hexazacyclotricosane-4-carbonyl]amino]-3-(4-hydroxyphenyl)propanoic acid

199999-60-5; H-Tyr-Cys-Trp-Ser-Gln-Tyr-Leu-Cys-Tyr-OH; WP9QY; (2S)-2-[[(4R,7S,10S,13S,16S,19S,22R)-22-[[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-13-(3-amino-3-oxopropyl)-16-(hydroxymethyl)-10-[(4-hydroxyphenyl)methyl]-19-(1H-indol-3-ylmethyl)-7-(2-methylpropyl)-6,9,12,15,18,21-hexaoxo-1,2-dithia-5,8,11,14,17,20-hexazacyclotricosane-4-carbonyl]amino]-3-(4-hydroxyphenyl)propanoic acid; L-Tyrosyl-L-cysteinyl-L-tryptophyl-L-seryl-L-glutaminyl-L-tyrosyl-L-leucyl-L-cysteinyl-L-tyrosine cyclic (2 inverted exclamation marku8)-disulfide; TNF-.alpha. Antagonist; HY-P2612; FT111079;

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.)