Chemotherapeutic boron-containing homocysteinamides of human serum albumin

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Abstract

Combination of boron neutron capture therapy and chemotherapy can provide good efficacy in a cancer treatment. Development of therapeutic constructs that combine these two functions, the possibility of in vitro and in vivo visualization and a convenient platform for selective delivery to the tumor is of great relevance today. In this study, we focused on human serum albumin, a well-known drug delivery platform. We developed constructs based on albumin functionalized with boron clusters, analogues of the chemotherapeutic molecule gemcitabine and signaling molecules. To create the constructs, we developed new analogues of homocysteine thiolactone containing closo-dodecaborate or cobalt bis(dicarbollide) and a gemcitabine analogue containing closo-dodecaborate attached to the C5 carbon atom of the nitrogenous base. We have demonstrated that addition of the gemcitabine analogue to the conjugate structure increases its cytotoxicity towards human glioblastoma cell lines. Among the final conjugates, the highest cytotoxicity is demonstrated by the structure containing cobalt bis(dicarbollide). The final structures accumulate well in the cytoplasm of cancer cells. The albumin conjugate containing cobalt bis(dicarbollide) and a boron-containing gemcitabine analogue is capable of accumulating in the nuclei of T98G cell lines. Thus, both final albumin-based constructs showed sufficient efficacy against the human glioma cell line in vitro. We expect that the therapeutic conjugates we have constructed will significantly increase cytotoxicity against cancer cells when irradiated with epithermal neutrons. Combining a chemotherapeutic residue and a boron-containing group in a single construct provides the potential for more effective glioma therapy.

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M. Wang

Novosibirsk State University

Author for correspondence.
Email: io197724@gmail.com
Russian Federation, Novosibirsk

S. A. Tsyrempilov

Novosibirsk State University

Email: io197724@gmail.com
Russian Federation, Novosibirsk

I. A. Moskalev

Novosibirsk State University

Email: io197724@gmail.com
Russian Federation, Novosibirsk

O. D. Zakharova

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: io197724@gmail.com
Russian Federation, Novosibirsk

A. I. Kasatova

Institute of Nuclear Physics SB RAS

Email: io197724@gmail.com
Russian Federation, Novosibirsk

V. N. Silnikov

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: io197724@gmail.com
Russian Federation, Novosibirsk

T. S. Godovikova

Novosibirsk State University; Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: io197724@gmail.com
Russian Federation, Novosibirsk; Novosibirsk

T. V. Popova

Novosibirsk State University; Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: io197724@gmail.com
Russian Federation, Novosibirsk; Novosibirsk

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Supplementary files

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2. Fig. 1. Scheme of synthesis of homocysteine ​​thiolactone analogs HTL-B12 and HTL-Co(C2B9)2. DIPEA – diisopropylethylamine, DMF – dimethylformamide.

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3. Fig. 2. Characteristics of the homocysteine ​​thiolactone analogue HTL-B12: (a) – 1H-NMR in DMSO-d6, DIPEA – diisopropylethylamine; (b) – IR spectrum in KBr; (c) – 13C-NMR in deuterated acetone; (d) – mass spectrum (electrospray) in CH3CN.

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4. Fig. 3. Characteristics of the homocysteine ​​thiolactone analogue HTL-Co(C2B9)2: (a) – 1H-NMR in deuterated acetone; (b) – 13C-NMR in deuterated acetone; (c) – IR spectrum in KBr; (d) – mass spectrum (electrospray) in CH3CN.

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5. Fig. 4. Scheme of synthesis of human serum albumin conjugates containing a signal label, boron clusters and gemcitabine analogues.

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6. Fig. 5. Characteristics of multifunctional conjugates of human serum albumin: (a) – electronic absorption spectra of HSA and its homocystamides in PBS buffer, pH 7.4; (b) – MALDI-TOF spectra; (c) – SDS-PAGE of homocystamide conjugates of HSA under Laemmli conditions followed by staining with Coomassie blue.

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7. Fig. 6. Characteristics of multifunctional conjugates of human serum albumin: (a) – electronic absorption spectra of HSA and its homocystamides in PBS buffer, pH 7.4; (b) – MALDI-TOF spectra; (c) – SDS-PAGE of homocystamide conjugates of HSA under Laemmli conditions followed by staining with Coomassie blue.

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8. Fig. 7. Viability of T98G cells treated with HSA, conjugates, and HSA + GC mixture for 72 h. Conjugate doses ranged from 0.02 to 60 μM protein equivalent. All data are presented as mean ± SD (n = 3). Two-way ANOVA was used to compare more than two data sets. **** p ≤ 0.0001.

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9. Fig. 8. Results of confocal microscopy of T98G cells treated with HSA-Cy5-HcyB12-GCB12 (a) and HSA-Cy5-HcyCo(C2B9)2-GCB12 (b) conjugates. Red color on slides 2 and 4 is fluorescence of HSA-Cy5-HcyB12-GCB12 and HSA-Cy5-HcyCo(C2B9)2-GCB12 conjugates, blue color on slides 1 and 4 is DAPI fluorescence. Scale bars are 20 μm. Slide 3 is an image of a living cell, 1 is DAPI fluorescence, 2 is conjugate fluorescence, 4 is combined slides 2 and 3.

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10. Fig. 9. Viability of U87 cells treated with HSA, HSA-Cy5-HcyCo(C2B9)2-GCB12, HSA + GC mixture and HSA-Cy5-HcyB12-GCB12 for 72 h. The dose of conjugates was 0.02–60 μM protein equivalent. All data are presented as mean ± SD (n = 3). Two-way ANOVA was used to compare more than two data sets. ** p ≤ 0.01; **** p ≤ 0.0001.

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