Doxorubicin prodrug; Topoisomerase

In this study, researchers synthesized alloc-DOX as a bioorthogonal prodrug (Supplementary Figs. 23–26). Doxorubicin (DOX) is clinically used for cancer treatment; it acts by binding DNA and eliciting enzyme-mediated strand breakage. By caging the primary amine with the allylcarbamate group, alloc-DOX (Kalloc-DOX = 7.96 × 104 M−1) showed a much lower binding affinity towards calf thymus DNA than did DOX (KDOX = 9.36 × 105 M−1), as revealed by a fluorescence titration method (Fig. 4a,b and Supplementary Fig. 27, Kalloc-DOX and KDOX represent the binding constants for the interactions of alloc-DOX and DOX with ctDNA, respectively.). This weaker DNA binding ability of the alloc-DOX was because the reduction in positive charge of the daunosamine moiety made the DNA–drug intercalating complex less stable. Meanwhile, the decaging of alloc-DOX catalysed by Pd-TNSs in solution was proved by high performance liquid chromatography (HPLC) analysis (Supplementary Fig. 28). Such a masking strategy could afford a less active prodrug while allowing restoration by bioorthogonal catalysis.[2]

Meanwhile, the in vivo toxicity of alloc-DOX was determined by measuring the weight changes of mice and comparing with those of mice treated with DOX (Supplementary Fig. 36). Intraperitoneal (i.p.) injection of alloc-DOX up to 150 mg kg–1 every three days led to weight loss of ~7.7%; meanwhile, serious weight loss and death were caused by DOX when its dose was over 10 mg kg–1, suggesting the low toxicity of the prodrug in vivo. Based on the toxicity tolerance data, to underline the less toxic feature of prodrugs while avoiding unexpected side effects at too high doses, equitoxic doses of alloc-DOX (100 mg kg–1) and DOX (5 mg kg–1), with a 20 times difference, were applied in the following antitumour studies.[2]

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Next, B16-F10 tumour-bearing mice were randomly divided into five groups and treated with the alloc-DOX/PT-MN combination, DOX, alloc-DOX, PT-MNs or PBS once every three days. DOX, alloc-DOX and PBS were administered by i.p. injection. Before injection of prodrugs, PT-MNs were inserted into the tumour site from the surface skin and fixed there for one hour to allow the needles to swell thoroughly. Bioluminescence imaging and tumour volumes were recorded to visualize tumour growth and evaluate the therapy efficiency (Fig. 5a). As shown in Fig. 5a–c, like the control group treated with PBS, no delayed tumour growth was observed for groups treated with alloc-DOX or PT-MNs alone, indicating that neither the prodrug nor the catalytic device alone had an antitumour effect. A moderate inhibitory outcome was observed in the DOX-treated group, but the tumours grew quickly after DOX administration was stopped. By contrast, the alloc-DOX/PT-MN combination suppressed tumour growth much better, leading to the smallest tumours in mice after four times of treatment. In addition, no obvious body weight fluctuation was observed for the mice in all groups (Fig. 5d), indicating the low side effects of the prodrug and the PT-MN device. Importantly, TUNEL assay verified apoptotic signals only in the groups treated with alloc-DOX/PT-MNs and DOX, implying a similar tumour cell killing mechanism (Fig. 5e).[2]

Interactions between DOX or alloc-DOX and ctDNA[2]
Fluorescence titrations were performed to study the interactions between ctDNA and DOX or alloc-DOX. The solutions were excited at a wavelength, λEx = 490 nm, and spectra were collected from 520 to 720 nm at 25 °C. The titrations were performed by adding increasing amounts of ctDNA directly into the cuvette containing 2 µM DOX or alloc-DOX solution. During the equilibration, illumination was avoided. All the samples were prepared in pH 7.5, 10 mM Tris buffer containing 1 mM ethylenediaminetetraacetic acid disodium.[2]

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Prodrug activation by Pd-TNSs[2]
Similar to the process of alloc-RH 110 activation, Pd-TNSs (1 mg) were first dispersed in 0.9 ml PBS buffer and then mixed with 100 µl alloc-DOX stock solution prepared in DMSO (1 mM). The mixture was stirred at 37 °C in the dark. At indicated time points, 5 µl of the reaction medium was taken out and mixed with 50 µl methanol. After centrifugation at 21,100g for 5 min, the upper solution was analysed by HPLC. The HPLC analysis was performed on a Waters Alliance e2695 system with a Kromasil C18 HPLC column (particle size, 5 μm; pore size, 300 Å; length by inner diameter, 250 mm × 4.6 mm) at a flow rate of 1.0 ml min–1 and a column temperature of 40 °C. The mobile phase consisted of water/acetonitrile (68:32) with 0.1% trifluoroacetic acid. All chromatograms were recorded by measuring the ultraviolet absorption at wavelength λ = 254 nm.

Cytotoxicity assays[2]
Three cell lines, namely B16-F10, 4T1 and Hep G2 cells, were seeded in 96-well plates at a density of 10,000 cells in 200 µl culture medium per well. After 24 h incubation, cells were treated with different concentrations of DOX and alloc-DOX in triplicate. For the alloc-DOX/PT-MN combination group, PT-MNs with arrays were cut into smaller patches that were large enough to cover one well, and about 58 needles of each patch were immersed in the culture medium. The volume of culture medium was about 350 µl per well. After 48 h incubation, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, and the plate was incubated for an additional 4 h. After that, the cells were lysed by addition of 100 µl DMSO. Optical absorbance was measured in a microplate reader at 570 nm with 660 nm set as the reference wavelength. For apoptosis studies by flow cytometry, Annexin V-APC (allophycocyanin) and SYTOX Green double staining was performed with an Apoptosis Detection Kit (Invitrogen, Thermal Fisher Scientific) according to the manufacturer’s instructions. For laser scanning confocal microscopy analysis of DNA fragmentation after different treatments, a TUNEL assay was conducted according to the manufacturer’s instructions; meanwhile, the cell nuclei were stained with Hoechst 33342 for colocalization. To demonstrate the generation of DOX in the extracellular space, the culture medium was withdrawn and mixed thoroughly with methanol/chloroform (3:2). After centrifugation at 21,100g for 30 min, the organic phase was isolated and dried under nitrogen. The residue was redissolved in the acetonitrile and analysed by HPLC. To analyse the potential catalyst leakage, the upper culture medium was withdrawn to a tube, while the cells at the bottom were digested with 150 µl concentrated nitric acid. All the solutions of each well were collected, mixed with 1 ml aqua regia and further digested. After centrifugation at 21,100g for 30 min, the supernatants were diluted and analysed by inductively coupled plasma mass spectrometry.

Toxicity of DOX and alloc-DOX in vivo[2]
The solutions of alloc-DOX and DOX were prepared by dissolving alloc-DOX in PBS buffer with the assistance of Tween 80 (2%). To decide the dose that could be used for antitumour studies, the toxicity of alloc-DOX was determined by measuring the weight loss of mice after i.p. injection every three days. The body changes of mice treated with DOX were also measured for comparison. Reversible weight change within 10% was considered safe.[2]

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Antitumour activity mediated by the bioorthogonal catalysis in vivo[2]
The 1 × 106 luciferase-tagged B16-F10 melanoma tumour cells were subcutaneously injected into the right flank of C57BL/6J mice. When the tumour sizes reached about 50–100 mm3, the mice were randomly divided into different groups, and respectively treated with the alloc-DOX (100 mg kg–1)/PT-MN combination, DOX (5 mg kg–1), alloc-DOX (100 mg kg–1), PT-MNs or PBS, once every three days, for four times total. The drug, prodrug and PBS were administered by i.p. injection. For groups treated with PT-MNs, the size of PT-MNs was adjusted to cover the tumour area, and the PT-MNs were inserted into the tumour sites one hour before prodrug administration and fixed with Liquivet Rapid Tissue Adhesive to allow the thorough swelling of the needles. Tumour growth was monitored by bioluminescence signals after i.p. injection of luciferin (150 mg kg–1, catalogue no. LUCK-100, Gold Biotechnology), and images were obtained using an IVIS Lumina imaging system. The changes in tumour size were measured by a digital caliper, and the tumour volume (mm3) was calculated by the following equation: long diameter × short diameter2 × 0.5. If the mice showed signs of impaired health, or once the volume of the tumour exceeded 1,500 mm3, the mice were euthanized with CO2. To assess the potential toxicities, changes in mice body weight were measured, and histological analysis using hematoxylin and eosin staining was conducted on the tumour and major organs (that is, heart, liver, spleen, lung and kidney) at the end of the treatment. For apoptosis analysis, the fixed tumour sections were stained by TUNEL Assay Kit BrdU-Red according to the manufacturer’s protocol and the cell nuclei were stained with Hoechst 33342. The stained tumour slides were then imaged by a Zeiss LSM 880 confocal microscope. For the groups treated with DOX-MNs or intratumourally injected DOX, the doses of DOX given were similar to the average amount of DOX generated in the group treated with alloc-DOX/PT-MNs at each corresponding treatment time point, which was determined by collecting all the DOX measured in the tumour, blood and major organs.[2]

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Analysis of DOX, alloc-DOX and Pd distribution in plasma and tissues[2]
After i.p. injection of DOX or alloc-DOX into mice bearing tumours with a size around 200 mm3, blood was collected by retro-orbital puncture using a BD microtainer capillary blood collector and BD microgard closure. Plasma was isolated by centrifugation at 1,000g for 10 min. The 50 µl plasma was withdrawn and mixed with 150 µl acetonitrile to extract DOX and alloc-DOX. All tissues (heart, liver, spleen, lung, kidney and tumour) were weighed after drying the surfaces with wipers and homogenized in 200 µl acetonitrile thoroughly with Cole-Parmer Tissue Tearor 985370-07 homogenizer. After shaking all the samples for 24 h at room temperature in the dark, the tubes containing plasma or homogenized tissues were centrifugated at 21,100g for 30 min. Then 30 µl of the upper solution was taken out, mixed with a known amount of DOX and alloc-DOX internal standard and then analysed by HPLC using the same protocol described above. A similar procedure was conducted for groups treated with DOX-MNs or intratumour injection of DOX. To analyse the Pd content in plasma and tissues, plasma (50 µl) and homogenized tissues were first dissolved in 500 µl concentrated nitric acid for 1 day. Then, 500 µl aqua regia was added to each sample, and the mixtures were incubated for another day. After removing the tissue debris by centrifugation (21,100g, 30 min), the supernatants were withdrawn, and the Pd contents were measured by inductively coupled plasma mass spectrometry.

[1]. Nano-palladium is a cellular catalyst for in vivo chemistry. Nat Commun. 2017 Jul 12;8:15906.

[2]. Bioorthogonal catalytic patch. Nat Nanotechnol. 2021 Aug;16(8):933-941.

Palladium catalysts have been widely adopted for organic synthesis and diverse industrial applications given their efficacy and safety, yet their biological in vivo use has been limited to date. Here we show that nanoencapsulated palladium is an effective means to target and treat disease through in vivo catalysis. Palladium nanoparticles (Pd-NPs) were created by screening different Pd compounds and then encapsulating bis[tri(2-furyl)phosphine]palladium(II) dichloride in a biocompatible poly(lactic-co-glycolic acid)-b-polyethyleneglycol platform. Using mouse models of cancer, the NPs efficiently accumulated in tumours, where the Pd-NP activated different model prodrugs. Longitudinal studies confirmed that prodrug activation by Pd-NP inhibits tumour growth, extends survival in tumour-bearing mice and mitigates toxicity compared to standard doxorubicin formulations. Thus, here we demonstrate safe and efficacious in vivo catalytic activity of a Pd compound in mammals.[1]

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Bioorthogonal catalysis mediated by transition metals has inspired a new subfield of artificial chemistry complementary to enzymatic reactions, enabling the selective labelling of biomolecules or in situ synthesis of bioactive agents via non-natural processes. However, the effective deployment of bioorthogonal catalysis in vivo remains challenging, mired by the safety concerns of metal toxicity or complicated procedures to administer catalysts. Here, we describe a bioorthogonal catalytic device comprising a microneedle array patch integrated with Pd nanoparticles deposited on TiO2 nanosheets. This device is robust and removable, and can mediate the local conversion of caged substrates into their active states in high-level living systems. In particular, we show that such a patch can promote the activation of a prodrug at subcutaneous tumour sites, restoring its parent drug's therapeutic anticancer properties. This in situ applied device potentiates local treatment efficacy and eliminates off-target prodrug activation and dose-dependent side effects in healthy organs or distant tissues.[2]

C31H33NO13

627.59

627.195

561022-65-9

171714282

Typically exists as solid at room temperature

2.6

6

13

9

45

1160

6

OC1=C2C(C3=CC=CC(OC)=C3C(=O)C2=C(O)C2[C@H](C[C@](O)(C(=O)CO)CC1=2)O[C@]1([H])O[C@@H](C)[C@@H](O)[C@@H](NC(=O)OCC=C)C1)=O

MYTMRYOJAKUVBH-BPZXBQJLSA-N

InChI=1S/C31H33NO13/c1-4-8-43-30(40)32-16-9-20(44-13(2)25(16)35)45-18-11-31(41,19(34)12-33)10-15-22(18)29(39)24-23(27(15)37)26(36)14-6-5-7-17(42-3)21(14)28(24)38/h4-7,13,16,18,20,25,33,35,37,39,41H,1,8-12H2,2-3H3,(H,32,40)/t13-,16-,18-,20-,25+,31-/m0/s1

prop-2-enyl N-[(2S,3S,4S,6R)-3-hydroxy-2-methyl-6-[[(1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1H-tetracen-1-yl]oxy]oxan-4-yl]carbamate

N-Alloc doxorubicin

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.)