Design of the New Closo-Dodecarborate-Containing Gemcitabine Analogue for the Albumin-Based Theranostics Composition

Combination therapy is becoming an increasingly important treatment strategy because multi-drugs can maximize therapeutic effect and overcome potential mechanisms of drug resistance. A new albumin-based theranostic containing gemcitabine closo-dodecaborate analogue has been developed for combining boron neutron capture therapy (BNCT) and chemotheraphy. An exo-heterocyclic amino group of gemcitabine was used to introduce closo-dodecaborate, and a 5′-hydroxy group was used to tether maleimide moiety through an acid-labile phosphamide linker. The N-trifluoroacylated homocysteine thiolactone was used to attach the gemcitabine analogue to human serum albumin (HSA) bearing Cy5 or Cy7 fluorescent dyes. The half-maximal inhibitory concentration (IC50) of the designed theranostic relative to T98G cells was 0.47 mM with the correlation coefficient R = 0.82. BNCT experiments resulted in a decrease in the viability of T98G cells, and the survival fraction was ≈ 0.4.


Introduction
Gemcitabine (2 ,2 -difluorodeoxycytidine) has shown to be an active agent against colon, pancreatic, breast, ovary, small cell lung, head and neck, and lung cancers in amalgamation with various anticancer agents. Gemcitabine is considered a gold standard and is the first FDA approved agent used as a monotherapy in the management of advanced pancreatic cancers. However, due to its poor pharmacokinetics, there is need of a newer drug delivery system (DDS) for efficient action [1]. The major goal in DDS is to achieve a higher accumulation of the drug in tumor tissue, and thus to reduce systemic toxicity and side effects of chemotherapeutic agents. The high molecular weight of gemcitabine conjugates may be effective due to their prolonged influence on the tumor. For example, they are represented by polyethylene glycol derivatives bearing gemcitabine and folic acid residues. However, it has been found that even the high molecular weight of polyethylene glycol makes it possible to increase the half-life of conjugates in blood plasma to only two hours [2].
The use of human serum albumin (HSA) as a macromolecular carrier for therapeutic construct creation is a convenient and well-established strategy [3][4][5][6][7][8][9][10]. Albumin is a biocompatible protein and has low toxicity to the organism. Furthermore, HSA contains a number of accessible functional groups for conjugation with low molecular weight compounds, for example, with anticancer drugs. In addition, albumin is able to provide targeted transport of a therapeutic agent and its controlled release [4][5][6][7][8][9]. The reason for this is the presence of specific albumin receptors overexpressed in cancer cells (the glycoproteins Gp18, Gp30 and Gp60 and SPARC) [11][12][13][14]. The size of albumin contributes to its accumulation in the tumor due to an enhanced permeability and retention (EPR) effect.
Gemcitabine showed a weak interaction with HSA, which occurred with cooperative binding in a ratio of 1:1. The gemcitabine binding site is located between the IIA and IIB subdomain interface [15]. Noncovalent binding of gemcitabine to HSA is used in most investigations devoted to therapeutic construct creation, including nanoparticles involving gemcitabine as a therapeutic load [16][17][18][19][20]. There are also a small number of studies in which gemcitabine was covalently attached to HSA. In the study [21], the aim was to create a double-targeted delivery system for gemcitabine. It was conjugated to albumin, and the resulting construct was used to create nanoparticles equipped with folic acid as an additional target molecule. The obtained nanoparticles showed higher toxicity and a cellular uptake in MDA-MB-231 cells overexpressing folic acid receptors. At the same time, the nanoparticles remained stable during blood circulation. Other groups developed an albumin-based enzyme-sensitive (cathepsin B cleavable) nanoplatform for gemcitabine delivery [22]. In vivo NIR imaging of the resulting construct demonstrated its increased accumulation and pronounced retention in the BxPC-3 pancreatic tumors of xenografted mice. Due to reduced deactivation of gemcitabine during circulation, high accumulation and consumption in tumor tissue, and specific intracellular release of gemcitabine, excellent growth inhibition activity without side effects was achieved.
Cancers are complex and involve a variety of pathways, at the same time, tumors often have intrinsic or acquired drug resistance. Therefore, it may be insufficient for regressing tumors to use a single drug to treat cancer. As a result, combination therapy is becoming an increasingly important treatment strategy because multi-drugs can maximize the therapeutic effect and overcome potential mechanisms of drug resistance as they can modulate different signaling pathways in cancer cells. A prospective treatment method is boron neutron capture therapy (BNCT) in which drugs containing enriched boron are accumulated in tumor cells followed by their epithermal neutron beam radiation. This technique offers an advantage over conventional chemo-and radiotherapies as it selectively targets tumor cells without significantly affecting healthy tissues. A combination of gemcitabine and boron-10 for BNCT has not yet been considered by any working group, although there are enough studies in which the therapeutic conjugate contains both gemcitabine and boron atoms [23][24][25][26][27]. In particular, bortezomib, which is a first-generation proteasome inhibitor, is widely used in combination with gemcitabine [28]. In any case, the structures indicated in the abovementioned studies have no structural analogues with our design. The introduction of the boron-containing gemcitabine derivative into the HSA-based theranostic make it possible to combine BNCT and chemotherapy.
In our work, we constructed a new gemcitabine analogue containing a closo-dodecaborate residue (B 12 H 11 ) attached via an N-ethoxy-(2-ethoxy)-N,N-dimethylethan-1-aminium linker to the exocyclic group of the nitrogenous base (compound 1, Figure 1). The 5 -hydroxyl group of gemcitabine was functionalized with a linker containing a maleimide residue at the end for connecting the gemcitabine analogue to HSA. This linker has a phosphamide bond that has been shown to be cleavable under the acidic conditions of cancer tissue [29].
Previously, we reported on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry [29][30][31][32][33][34][35]. Furthermore, we reported on the preparation of the novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells [30]. Herein, we suggest using a boron-containing derivative of gemcitabine (compound 1) as a part of an anticancer theranostic construct. Along with its beneficial properties as a chemotherapeutic agent, boron-containing gemcitabine derivative is a promising agent for boron-neutron capture therapy under imaging control. Previously, we reported on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry [29][30][31][32][33][34][35]. Furthermore, we reported on the preparation of the novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells [30]. Herein, we suggest using a boron-containing derivative of gemcitabine (compound 1) as a part of an anticancer theranostic construct. Along with its beneficial properties as a chemotherapeutic agent, boron-containing gemcitabine derivative is a promising agent for boron-neutron capture therapy under imaging control.

Synthesis of Maleimide Gemcitabine Boron-Containing Derivative 1
Considering gemcitabine, a number of prodrugs have been developed. A significant number of prodrugs are obtained by modifying gemcitabine at the 5'-hydroxyl group. Conjugates with a modification at the 5'-hydroxyl group are much more stable in blood plasma and undergo metabolic transformations mainly inside the cell [36]. However, the peculiarities of the cytosine derivative's metabolism also make it possible to use the heterocyclic base exocyclic amino group for modification [37]. The majority of effective prodrugs with improved pharmacodynamic and kinetic properties have been obtained precisely by modifying the amino group of gemcitabine. They are easy to synthesize, and a wide range of methods is available for incorporating the gemcitabine residue into conjugates with polyethylene glycol, squalene, fatty acids and other molecules. It is known that nucleoside prodrugs containing an amide bond are cleaved by cathepsins B and D, which lead to their inactivation [38,39]. It has been shown that cytosinamides are unstable in plasma, and their half-life time is several hours, which is comparable with that in the presence of cathepsins [40].
Our idea was to use gemcitabine as a multimodal molecule having the exo-heterocyclic amino group and 3′ and 5′ hydroxyl groups of a sugar ring. Since the 3′-OH group is likely to be important for the cytotoxic effect of gemcitabine [41,42], we modified only the 5′-OH group. We used the exo-heterocyclic amino group of gemcitabine to introduce closododecaborate and the 5′-OH group to tether a maleimide moiety through a phosphate and extended hydrophilic diamino linker. The synthesis of this new agent is shown in Scheme 1. It is worth noting that according to the literature [43,44], gemcitabine needs to be phosphorylated for anticancer effects and the phosphamide bond of the final conjugate may be hydrolyzed under acidic conditions in lisosomes after entering cancer cells [29]. Thus, such a modification should not lead to a decrease in the cytostatic properties of gemcitabine.

Synthesis of Maleimide Gemcitabine Boron-Containing Derivative 1
Considering gemcitabine, a number of prodrugs have been developed. A significant number of prodrugs are obtained by modifying gemcitabine at the 5'-hydroxyl group. Conjugates with a modification at the 5'-hydroxyl group are much more stable in blood plasma and undergo metabolic transformations mainly inside the cell [36]. However, the peculiarities of the cytosine derivative's metabolism also make it possible to use the heterocyclic base exocyclic amino group for modification [37]. The majority of effective prodrugs with improved pharmacodynamic and kinetic properties have been obtained precisely by modifying the amino group of gemcitabine. They are easy to synthesize, and a wide range of methods is available for incorporating the gemcitabine residue into conjugates with polyethylene glycol, squalene, fatty acids and other molecules. It is known that nucleoside prodrugs containing an amide bond are cleaved by cathepsins B and D, which lead to their inactivation [38,39]. It has been shown that cytosinamides are unstable in plasma, and their half-life time is several hours, which is comparable with that in the presence of cathepsins [40].
Our idea was to use gemcitabine as a multimodal molecule having the exo-heterocyclic amino group and 3 and 5 hydroxyl groups of a sugar ring. Since the 3 -OH group is likely to be important for the cytotoxic effect of gemcitabine [41,42], we modified only the 5 -OH group. We used the exo-heterocyclic amino group of gemcitabine to introduce closo-dodecaborate and the 5 -OH group to tether a maleimide moiety through a phosphate and extended hydrophilic diamino linker. The synthesis of this new agent is shown in Scheme 1. It is worth noting that according to the literature [43,44], gemcitabine needs to be phosphorylated for anticancer effects and the phosphamide bond of the final conjugate may be hydrolyzed under acidic conditions in lisosomes after entering cancer cells [29]. Thus, such a modification should not lead to a decrease in the cytostatic properties of gemcitabine.
Previously [45], it was found that oxonium derivatives of closo-dodecaborate, such as compound 4 (Scheme 1), are able to react with various amines in high yields. In article [46], a selective alkylation of tertiary amines in the presence of hydroxyl groups using dioxonium derivative 4 was demonstrated. We used this approach to incorporate closo-dodecaborate moiety into gemcitabine's core. For this purpose, we first benzoylated exocyclic amino group of gemcitabine similarly to the benzoylation of cytidine in [47]. Then, benzoyl derivative 2 (Scheme 1) was subjected to the transamination reaction according to the procedure outlined in [48]. Isolated compound 3 (Scheme 1) containing a tertiary amino group was alkylated using dioxonium derivative of closo-dodecaborate 4, similarly to the published procedure in [46]. We slightly modified the protocol and used DMF instead of acetonitrile as a solvent in the reaction for better solubility of the reagents. Thus, Molecules 2023, 28, 2672 4 of 17 compound 5 (Scheme 1), a conjugate of gemcitabine and closo-dodecaborate, was isolated. The structures of conjugate 5 and its precursors were confirmed by 1 H, 13 C, 19 F NMR and ESI mass spectrometry. dioxonium derivative 4 was demonstrated. We used this approach to incorporate closododecaborate moiety into gemcitabine's core. For this purpose, we first benzoylated exocyclic amino group of gemcitabine similarly to the benzoylation of cytidine in [47]. Then, benzoyl derivative 2 (Scheme 1) was subjected to the transamination reaction according to the procedure outlined in [48]. Isolated compound 3 (Scheme 1) containing a tertiary amino group was alkylated using dioxonium derivative of closo-dodecaborate 4, similarly to the published procedure in [46]. We slightly modified the protocol and used DMF instead of acetonitrile as a solvent in the reaction for better solubility of the reagents. Thus, compound 5 (Scheme 1), a conjugate of gemcitabine and closo-dodecaborate, was isolated. The structures of conjugate 5 and its precursors were confirmed by 1 H, 13 C, 19 F NMR and ESI mass spectrometry. In a separate experiment, we found that the reaction of 2 -deoxycytidine 5 -monophosphate with compound 4 readily proceeded with the formation of more lipophilic products with the same UV characteristics. On the other hand, 2-deoxycytidine did not react under the same conditions. It is most likely that phosphate group is the alkylation target in 2 -deoxycytidine 5 -monophosphate. Thus, it was important to carry out phosphorylation after the introduction of closo-dodecaborate to avoid side processes affecting the phos-phate group during alkylation. Compound 5 was selectively 5 -monophosphated to give 6 (Scheme 1) using the protocol from the articles [49][50][51]. Conjugate 6 was isolated as its tris(triethylammonium) salt and characterized by NMR 1 H, 13 C, 19 F and ESI mass.
We then modified the phosphate group in 6 with a diamino linker. During the experiments, we found that the length of the linker between maleimide residue and phosphate is an important point for the stability of such conjugates. Compound 9 ( Figure 2) containing a diaminopropyl linker was found to be unstable in water. We discovered the deficit of maleimide protons according to 1 H NMR of compound 9 (see Supplementary Material). Further, in our attempts to purify 9, it was found that this conjugate converted in several hours at RT into some product 9* (see Supplementary Material) with changed UV characteristics. NMR and mass spectra of this by-product support a hypothesis of hydrolysis of the maleimide core (see Supplementary Material) [52]. There are no maleimide protons in 1 H NMR of 9*, and the main peak in its mass spectra corresponds to [M + H 2 O-1] − of 9. We proposed that a neighboring phosphate group could contribute to the hydrolysis process. Indeed, compound 10 ( Figure 2) containing an extended 2,2 -(ethylenedioxy)bis(ethylamine) linker was found to be stable under the same conditions. So, we used this extended linker for the synthesis of the target compound 1.
In a separate experiment, we found that the reaction of 2′-deoxycytidine 5′-monophosphate with compound 4 readily proceeded with the formation of more lipophilic products with the same UV characteristics. On the other hand, 2-deoxycytidine did not react under the same conditions. It is most likely that phosphate group is the alkylation target in 2′-deoxycytidine 5′-monophosphate. Thus, it was important to carry out phosphorylation after the introduction of closo-dodecaborate to avoid side processes affecting the phosphate group during alkylation. Compound 5 was selectively 5′-monophosphated to give 6 (Scheme 1) using the protocol from the articles [49][50][51]. Conjugate 6 was isolated as its tris(triethylammonium) salt and characterized by NMR 1 H, 13 C, 19 F and ESI mass.
We then modified the phosphate group in 6 with a diamino linker. During the experiments, we found that the length of the linker between maleimide residue and phosphate is an important point for the stability of such conjugates. Compound 9 ( Figure 2) containing a diaminopropyl linker was found to be unstable in water. We discovered the deficit of maleimide protons according to 1 H NMR of compound 9 (see Supplementary Material). Further, in our attempts to purify 9, it was found that this conjugate converted in several hours at RT into some product 9* (see Supplementary Material) with changed UV characteristics. NMR and mass spectra of this by-product support a hypothesis of hydrolysis of the maleimide core (see Supplementary Material) [52]. There are no maleimide protons in 1 H NMR of 9*, and the main peak in its mass spectra corresponds to [M + H2O-1] − of 9. We proposed that a neighboring phosphate group could contribute to the hydrolysis process. Indeed, compound 10 ( Figure 2) containing an extended 2,2′-(ethylenedioxy)bis(ethylamine) linker was found to be stable under the same conditions. So, we used this extended linker for the synthesis of the target compound 1. We relied on the published method in [53] to synthesize compound 7 (Scheme 1), but we changed the process of purification of the target product. The reaction of the maleimide derivative 8 and aliphatic amino group of 7 required completely removing the 2thiopyridine and diamine impurities from 7 to avoid side reactions. We performed threestep purification and isolated the triethylammonium salt of the amine 7. At the last step, activated pentafluorophenyl ether 8 was used for modification of the amine 7 according to the published method in [54]. Maleimide-containing derivative 1 was isolated as its We relied on the published method in [53] to synthesize compound 7 (Scheme 1), but we changed the process of purification of the target product. The reaction of the maleimide derivative 8 and aliphatic amino group of 7 required completely removing the 2-thiopyridine and diamine impurities from 7 to avoid side reactions. We performed threestep purification and isolated the triethylammonium salt of the amine 7. At the last step, activated pentafluorophenyl ether 8 was used for modification of the amine 7 according to the published method in [54]. Maleimide-containing derivative 1 was isolated as its bis(triethylammonium) salt with a high yield. 1 H, 13 C, 19 F NMR and mass spectrometry data confirmed the structure of the target compound.

Bioconjugation
Earlier, our group synthesized fluorophore-labeled homocystamide conjugates of human serum albumin HSA-Cy5-HcyTFAc [30,32]. One-and-a-half copies of trifluoroacetate and a single copy of a fluorophore (Cy5 or Cy7) were covalently attached to a protein via suitable amino acids. This was confirmed by the electron spectroscopy data (Figure 3 Furthermore, it was confirmed by inductively coupled plasma atomic emission spectroscopy. The amount of boron in the sample HSA-Cy5-HcyTFAc-GCB12H11 was 18 times greater than the amount of albumin in it. The amount of boron in the sample HSA-Cy7-HcyTFAc-GCB12H11 was 18.3 times greater than the amount of albumin molecules. Thus, it corresponds to the 1.5 residues of closo-dodecarborate addition per protein molecule.

Cell Viability Assay
The effect of the HSA-Cy5-Hcy-TFAc-GCB12H11 construct on viability of the glioblastoma T98G cell line was determined by standard MTT colorimetric assay [55]. The results are shown in Figure 4. In general, there were no significant differences in the viability of glioblastoma cells incubated with different albumin conjugates; however, the viability of T98G cells decreased in the presence of 0.03 mM or more HSA Cy5-Hcy-TFAc-GCB12H11 (p-value ≤ 0.0001). For the synthesis of the albumin-based theranostic agents HSA-Cy5-HcyTFAc-GCB 12 H 11 and HSA-Cy7-HcyTFAc-GCB 12 H 11 , we used the reactivity of thiolactone (a cyclic thioester) as a latent thiol functionality in thiol-'click' chemistry. The thiol was released by nucleophilic ring opening (aminolysis) in amino groups on the HSA and, subsequently, reacted with a thiol 'scavenger' (a maleimide derivative of the gemcitabine, compound 1 (Scheme 2, path c). Modification of HSA results in the accumulation of oligomeric forms of the protein, which are unstable to the action of dithiothreite (Figure 3, panel A; data for the conjugates with Cy7 are the same as for Cy5 and are not shown here).
Modification of HSA-Cy5-HcyTFAc or HSA-Cy7-HcyTFAc with a threefold excess of the gemcitabine analogue (twofold excess relative to the number of mercapto groups on the conjugate) resulted in the addition of 1.5 residues of the gemcitabine analogue per protein molecule. This was confirmed by MALDI-TOF mass spectroscopy data ( Figure 3C). The mass of HSA differs from the measured mass of HSA-Cy5-HcyTFAc-GCB 12 H 11 by 2474 Da, which corresponds to 1.5 residues of the gemcitabine analogue. The same result was received for HSA-Cy7-HcyTFAc-GCB 12 H 11 ; the mass difference was 2379 Da. Furthermore, it was confirmed by inductively coupled plasma atomic emission spectroscopy. The amount of boron in the sample HSA-Cy5-HcyTFAc-GCB 12 H 11 was 18 times greater than the amount of albumin in it. The amount of boron in the sample HSA-Cy7-HcyTFAc-GCB 12 H 11 was 18.3 times greater than the amount of albumin molecules. Thus, it corresponds to the 1.5 residues of closo-dodecarborate addition per protein molecule.
GCB12H11 and HSA-Cy7-HcyTFAc-GCB12H11, we used the reactivity of thiolactone (a cyclic thioester) as a latent thiol functionality in thiol-'click' chemistry. The thiol was released by nucleophilic ring opening (aminolysis) in amino groups on the HSA and, subsequently, reacted with a thiol 'scavenger' (a maleimide derivative of the gemcitabine, compound 1 (Scheme 2, path c). Modification of HSA results in the accumulation of oligomeric forms of the protein, which are unstable to the action of dithiothreite (Figure 3, panel A; data for the conjugates with Cy7 are the same as for Cy5 and are not shown here).
Modification of HSA-Cy5-HcyTFAc or HSA-Cy7-HcyTFAc with a threefold excess of the gemcitabine analogue (twofold excess relative to the number of mercapto groups on the conjugate) resulted in the addition of 1.5 residues of the gemcitabine analogue per protein molecule. This was confirmed by MALDI-TOF mass spectroscopy data ( Figure  3C). The mass of HSA differs from the measured mass of HSA-Cy5-HcyTFAc-GCB12H11 by 2474 Da, which corresponds to 1.5 residues of the gemcitabine analogue. The same result was received for HSA-Cy7-HcyTFAc-GCB12H11; the mass difference was 2379 Da.

Cell Viability Assay
The effect of the HSA-Cy5-Hcy-TFAc-GCB 12 H 11 construct on viability of the glioblastoma T98G cell line was determined by standard MTT colorimetric assay [55]. The results are shown in Figure 4. In general, there were no significant differences in the viability of glioblastoma cells incubated with different albumin conjugates; however, the viability of T98G cells decreased in the presence of 0.03 mM or more HSA Cy5-Hcy-TFAc-GCB 12 H 11 (p-value ≤ 0.0001).   The increase in cell survival in the presence of HSA (p-value ≤ 0.0001) can be explained by the stimulation of their growth by albumin as a food resource.
Cell survival in the presence of a mixture of HSA and gemcitabine did not decrease within the range of the used gemcitabine concentrations. In the presence of the HSA-Cy5-Hcy-TFAc-GCB 12 H 11 conjugate, the same concentrations relative to gemcitabine resulted in a decrease in the number of surviving cells. The half-maximal inhibitory concentration (IC 50 ) for HSA-Cy5-Hcy-TFAc-GCB 12 H 11 was 0.47 mM with the correlation coefficient R = 0.82. This can be explained by the fact that gemcitabine penetrates into cancer cells better as part of the conjugate than as part of the noncovalent complex with albumin. Another explanation could be the greater efficacy of the gemcitabine analogue compared to gemcitabine itself. In any case, the covalent binding of the gemcitabine analogue to the albumin construct enhances its effectiveness.
Without neutron irradiation, the cell line retained a proliferation rate of over 70% upon treatment with the gemcitabine-containing boronated conjugate HSA-Cy5-Hcy-TFAc-GCB 12 H 11 within its concentration range of 0.03-0.06 mM (Figure 4). Thus, a conjugate concentration of about 0.03 mM can be used to evaluate the effect of the drug on glioma cell colony formation in neutron irradiation experiments.

The Usage of the HSA-Cy7-HcyTFAc-GCB 12 H 11 Conjugate as Boron Delivery Agent in BNCT
We performed the BNCT experiment using the HSA-Cy7-HcyTFAc-GCB 12 H 11 conjugate as a boron delivery agent and 10 B-boronophenylalanine as a positive control for BNCT. We used a clonogenic assay for evaluation of the BNCT effectiveness. The treatment of cells with the boron-modified conjugate and subsequent irradiation was carried out in the same way as in the previous work [30]. Irradiation of cell cultures was carried out at the BINP neutron source [56].
The survival fraction of T98G human glioblastoma cells upon incubation with the conjugate and subsequent irradiation with epithermal neutrons was ≈0.4 ( Figure 5), which differed significantly from the control group (p ≤ 0.0001). Thus, the conjugate HSA-Cy7-HcyTFAc-GCB 12 H 11 shows a synergistic effect between gemcitabine (it has a toxicity to cancer cells without irradiation) and closo-dodecaborate (toxicity to cancer cells is enhanced by neutron irradiation).  In our previous work [30], we described a boron-containing albumin-based conjugate containing thionyl trifluoroacetate (TTFA). TTFA was attached to the protein at arginine residues, followed by treatment with boric acid. The conjugate with TTFA had a similar efficiency under irradiation with epithermal neutrons (p ≤ 0.0001) but at a higher neutron flux (4 × 10 12 cm −2 for the conjugate with TTFA vs. 2.2 × 10 12 cm −2 for the conjugate with gemcitabine).
It is known that TTFA binds to the active site in complex II of the electron transport chain and prevents the reduction in ubiquinone. That inhibits the electron transport chain and can be used to prevent an increase in hyperglycemia-induced mitochondrial reactive oxygen species [57]. Gemcitabine triphosphate has other targets. It competes with deoxycytidine triphosphate in DNA synthesis [58] and inhibits enzyme ribonucleotide reductase, which reduces ribonucleotides to deoxyribonucleotides. The resulting deficiency of In our previous work [30], we described a boron-containing albumin-based conjugate containing thionyl trifluoroacetate (TTFA). TTFA was attached to the protein at arginine residues, followed by treatment with boric acid. The conjugate with TTFA had a similar efficiency under irradiation with epithermal neutrons (p ≤ 0.0001) but at a higher neutron flux (4 × 10 12 cm −2 for the conjugate with TTFA vs. 2.2 × 10 12 cm −2 for the conjugate with gemcitabine).
It is known that TTFA binds to the active site in complex II of the electron transport chain and prevents the reduction in ubiquinone. That inhibits the electron transport chain and can be used to prevent an increase in hyperglycemia-induced mitochondrial reactive oxygen species [57]. Gemcitabine triphosphate has other targets. It competes with deoxycytidine triphosphate in DNA synthesis [58] and inhibits enzyme ribonucleotide reductase, which reduces ribonucleotides to deoxyribonucleotides. The resulting deficiency of cytidine-5'-triphosphate leads to increased incorporation of the triphosphate form of gemcitabine into DNA [59,60]. Different targets of the chemotherapeutic parts of the conjugates can cause a different synergistic effect on any type of cancer cell. It depends on the metabolism of the cancer tissue. Thus, combining a variety of chemotherapeutic agents with boron-containing molecules in a covalent conjugate is promising.
At the same time, the colonogenic assay revealed a decrease in viability with the use of 10 B-boronophenylalanine containing the boron isotope-10 with the lowest surviving fraction compared to the HSA-Cy7-HcyTFAc-GCB 12 H 11 conjugate (p ≤ 0.01). It is worth noting that, here, we used a HSA-Cy7-HcyTFAc-GCB 12 H 11 conjugate made of natural boron which contains 20% 10 B isotope. The BPA control contains solely 10 B. Taking into account the number of boron atoms in one residue and the number of closo-dodecaborate residues attached to albumin, the amount of 10 B in the control is five times greater than in the conjugate. On the one hand, the use of a conjugate containing pure 10 B will lead to greater therapeutic efficiency. On the other hand, since the isolation of pure 10 B isotope is a rather expensive technology, the use of natural boron for BNCT can simplify and reduce the cost of obtaining boron-containing compounds.
The human glioblastoma T98G cell line was received from the Russian cell culture collection (Russian Branch of the ETCS, St. Petersburg, Russia).
Reagents and materials were purchased from Sigma-Aldrich (USA) and Reachem (Moscow, Russia), unless otherwise indicated. Milli-Q water with a conductivity greater than 18 MΩ/cm was used in all experiments. Phosphate buffered saline (PBS) (0.01 M, pH 7.3-7.5, Biolot). Organic solvents were dried and purified using standard procedures.
Mass spectra of proteins and peptides were recorded on a Bruker Autoflex Speed (Bruker Daltonics, Germany) MALDI-TOF mass spectrometer in positive linear mode. A smartbeam-II laser was used. 2,5-Dihydroxyacetophenone (2,5-DHAP) was used as a matrix. Protein samples were desalted by ZipTip C4 pipette tips. An amount of 2 µL of the protein sample solution was mixed with 2 µL of a 2% TFA (trifluoroacetic acid). To the latter solution, 2 µL of the matrix (2,5-DHAP) was added. The mixture was pipetted up and down until crystallization started. Mass spectra were obtained by averaging 3000 laser shots. External calibration was provided by [M + H] + HSA at m/z 66.5 kDa.
ESI mass spectra were registered on Agilent ESI MSD XCT Ion Trap (Agilent Technologies, Santa Clara, CA, USA) in positive or negative mode at The Joint Center for genomic, proteomic and metabolomics studies (IChBFM SB RAN, Novosibirsk, Russia).
The boron content in the resulted protein conjugates was determined by inductively coupled plasma atomic emission spectrometry (ICP AES) on an ICPE-9820 high-resolution spectrometer (Shimadzu, Kyoto, Japan). The samples were not subjected to preliminary incineration and were diluted with deionized water to 7 mL. Calibration dependencies were built using a single-element standard solution Boron Standard (Sigma Aldrich) in the range of 0.01-10 mg/L. The result of the analysis was obtained by averaging over four analytical lines.
SDS-PAGE of human serum albumin conjugates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis using 7% PAAG under Laemmli conditions without the addition of DTT or in the presence of DTT with subsequent Coomassie Brilliant Blue (BioRad) staining.

Cell Viability Assay (MTT Test)
To determine the cytotoxicity of the conjugates, a T98G human glioblastoma cell line was used as well as the previously described protocol with minor modifications [55]. αMEM nutrient medium containing 10% by volume of fetal bovine serum and 1% solution of antimycotic/antibiotics was used. In the wells of a 96-well plate, 100 µL of cell suspension was added in the amount of 5 × 10 3 cells per 1 well in the αMEM nutrient medium containing 1% solution of antimycotic/antibiotics.
The filled plate was incubated for 24 h at (37.0 ± 1.0) • C in an atmosphere of (5.0 ± 0.5)% CO 2 . After 24 h, six dilutions of the conjugates were prepared in the αMEM nutrient medium containing a 1% solution of antimycotic antibiotics with a final conjugate concentration in the well of 0.6, 0.3, 0.02, 0.002, 0.0008, 0.0002 and 0 mM. The obtained dilutions of the test conjugate were added (100 µL of each one) to the wells of the plate. The filled plate was incubated for 24 h at (37.0 ± 1.0) • C in an atmosphere of (5.0 ± 0.5)% CO 2 . After that, the medium was removed, and 200 µL of the appropriate nutrient medium containing 0.25 mg/mL MTT was added to each well. Incubation continued under the same conditions for 4 h. Then, the medium and MTT were removed from the wells, 100 µL of dimethyl sulfoxide was added and the optical density was measured on a multichannel flatbed scanner at a wavelength of λ = 570 nm, ref. 620 nm. The wells without protein samples were used as the zero level for the flatbed scanner.
Then, the mean absorbance and standard deviation were determined in the control wells and for each conjugate dilution concentration. Samples with the conjugate concentration 0 mg/mL were taken as control. Considering the obtained values of the optical density for each concentration of dilution of the conjugate and control samples, the percentage of surviving cells was calculated using Formula (1): Cell viability = AAV sample ./AAV control * (1) * AAV sample. -average absorption value in the wells with the sample; AAV controlaverage absorption value in the wells without the sample.
The calculation of IC50 was carried out according to the protocol presented on the website https://www.sciencegateway.org/protocols/cellbio/drug/hcic50.htm (accessed on 14 October 2022).
Statistical analysis was performed in the following manner. Parametric data are expressed as mean ± standard deviation (SD). Each experiment was repeated at least three times. Statistical analysis was performed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA). To compare more than two datasets, we used two-way ANOVA with multiple comparisons. Differences were considered significant if the p value was <0.05.

Model BNCT Experiments Incubation of the Cell Lines and Irradiation
Human glioblastoma T98G cells were incubated with HSA-Cy5-HcyTFAc-GCB 12 H 11 conjugates (31 µM) then washed by 1 mL of complete culture medium. T98G cells incubated with 10 B-4-borono-L-phenylalanine (50 ppm) for 18h were used as a positive control. Irradiated cells and non-irradiated cells not incubated with any boron compounds served as negative controls.
Neutron irradiation was performed at the accelerator-based epithermal neutron source at the Budker Institute of Nuclear Physics, Novosibirsk, Russia [56]. The layout of the facility is shown in Figure S11 (see Supporting Information).
The neutron source comprises an original design tandem accelerator (vacuum-insulated tandem accelerator VITA), a thin solid lithium target and a neutron beam shaping assembly.
The facility can displace a lithium target in 5 positions; in Figure 1, they are marked as Positions A, B, C, D, E. In these studies, the lithium target was placed in Position A. The PMMA disk with a diameter of 200 mm and a thickness of 72 mm was placed below and close to the target. This disk served as a neutron moderator. The studied cell cultures in test tubes were placed inside a PMMA disk 200 mm in diameter and 40 mm thick at a distance of 25 mm from the center. This disk with test tubes was placed under the moderator with a gap of 10 mm. Cell cultures were irradiated at a proton beam energy of 2.1 MeV and a current of 1.35 mA for 92 min. The neutron flux density at the exit from the moderator was 4 10 8 cm −2 s −1 . Therefore, cell cultures were irradiated with a neutron fluence of 2.2 10 12 cm −2 .

Clonogenic Assay
Irradiated T98G glioblastoma cell lines were seeded in 6-well plates (TPP, Switzerland) at a density of 500 cells per well. The cells were incubated for a week at 37 • C in a humidified incubator under 5% (v/v) CO 2 . Clonogenic analysis was performed according to the method described earlier [63]. The calculation of the plating efficiency and surviving fraction was based on the survival of non-irradiated cells.

Conclusions
The synthesis of a gemcitabine analogue containing closo-dodecaborate can provide a therapeutic agent with a dual nature: it is both a chemotherapeutic agent as well as a tool for BNCT. The covalent attachment of a gemcitabine analogue to a macromolecular carrier such as HSA has multiple goals: increasing the lifetime of the drug in the bloodstream and the specificity of the therapeutic agent to tumor tissue. The albumin core allows the decoration of the final construct with dyes for its detection inside the body: fluorescent dyes (Cy5 or Cy7) or fluorine atoms for MRI. The HSA-Cy5-Hcy-TFAc-GCB 12 H 11 conjugate has a noticeable toxicity against the human glioblastoma T98G cell line at protein concentrations of 0.03-0.06 mM. The half-maximal inhibitory concentration (IC 50 ) for HSA-Cy5-Hcy-TFAc-GCB 12 H 11 is 0.47 mM with correlation coefficient R = 0.82. Experiments with the use of the HSA-Cy7-Hcy-TFAc-GCB 12 H 11 conjugate in BNCT show a decrease in the viability of tumor T98G cells under irradiation with epithermal neutrons with the neutron flux 2.2 × 10 12 cm −2 . Taking into account the toxicity of the conjugate against cancer cells without irradiation, the synergistic effect of gemcitabine and closo-dodecaborate residues takes place. Thus, a design of this type is promising for combination therapy.