Cyanocobalamin-Modified Colistin–Hyaluronan Conjugates: Synthesis and Bioactivity

Polymeric drug delivery systems enhance the biopharmaceutical properties of antibiotics by increasing their bioavailability, providing programmable and controlled-release properties, and reducing toxicity. In addition, drug delivery systems are a promising strategy to improve the intestinal permeability of various antimicrobial agents, including colistin (CT). This study describes the modification of conjugates based on CT and hyaluronic acid (HA) with cyanocobalamin (vitamin B12). Vitamin B12 was chosen as a targeting ligand because it has its own absorption pathway in the small intestine. The resulting polysaccharide conjugates contained 95 μg/mg vitamin B12 and the CT content was 335 μg/mg; they consisted of particles of two sizes, 98 and 702 nm, with a ζ-potential of approximately −25 mV. An in vitro release test at pH 7.4 and pH 5.2 showed an ultra-slow release of colistin of approximately 1% after 10 h. The modified B12 conjugates retained their antimicrobial activity at the level of pure CT (minimum inhibitory concentration was 2 μg/mL). The resulting delivery systems also reduced the nephrotoxicity of CT by 30–40% (HEK 293 cell line). In addition, the modification of B12 improved the intestinal permeability of CT, and the apparent permeability coefficient of HA–CT–B12 conjugates was 3.5 × 10−6 cm/s, corresponding to an in vivo intestinal absorption of 50–100%. Thus, vitamin-B12-modified conjugates based on CT and HA may be promising oral delivery systems with improved biopharmaceutical properties.


Introduction
Modification of known drugs is a simple and cost-effective strategy for pharmaceutical research and development, especially for antimicrobial drugs [1,2]. Bacterial resistance to modern antibiotics is increasing rapidly, and the development of new-generation antimicrobial agents is a long and expensive process; moreover, most synthesized molecules fail in the preclinical and clinical stages due to their poor biopharmaceutical properties [3]. Multidrugresistant bacteria, including multidrug-resistant Gram-negative ESKAPE pathogens, exhibit resistance to three or more classes of antibiotics (e.g., carbapenems and third-generation cephalosporins) and are a major cause of mortality [4]. One potential antimicrobial drug that could benefit from various modifications of a known molecule is the peptide antibiotic colistin (CT) [5][6][7]. The disadvantages of CT therapy are its severe side effects such as nephrotoxicity and neurotoxicity [8][9][10]; moreover, CT is practically not absorbed in the The aim of the present study was to obtain vitamin-B12-modified conjugates of CT with HA and to investigate their potential in terms of the oral delivery of CT. It was hypothesized that functionalization with vitamin B12 would enhance intestinal permeability and absorption of the polymeric conjugates through receptor-mediated endocytosis. The concept of conjugating CT to polymers to improve its biopharmaceutical properties has been used previously [25][26][27][28]. However, to the best of our knowledge, this study was the first attempt to develop an oral form of CT that can be absorbed in the GI tract by grafting vitamin B12 as a targeting ligand.

Synthesis of the Succinyl Cyanocobalamin (Suc-B12)
Analysis of the literature showed that, for the synthesis of succinyl derivatives of cyanocobalamin, the OH group of the ribose (substitution occurs first, resulting in monosubstituted succinyl-B12) or the less reactive capable secondary hydroxyl group (a significant excess of reagent results in disubstituted succinyl-B12) can be used (Figure 1a) [29]. The reaction is typically performed in dry DMSO under anhydrous conditions in the presence of DMAP or pyridine as electron donors [23,[29][30][31]. This type of conjugation does not interfere with the recognition of vitamin B12 by the transfer proteins involved in its absorption [15]. h). Furthermore, the degree of substitution with CT fragments of 8-10% provided antim crobial activity at the level of pure CT, as well as reduced cytotoxicity [7,24].
The aim of the present study was to obtain vitamin-B12-modified conjugates of with HA and to investigate their potential in terms of the oral delivery of CT. It was h pothesized that functionalization with vitamin B12 would enhance intestinal permeabil and absorption of the polymeric conjugates through receptor-mediated endocytosis. T concept of conjugating CT to polymers to improve its biopharmaceutical properties h been used previously [25][26][27][28]. However, to the best of our knowledge, this study was first attempt to develop an oral form of CT that can be absorbed in the GI tract by graft vitamin B12 as a targeting ligand.
To obtain modified HA-CT-B12 conjugates, we exploited the carbodiimide activation of the carboxyl groups of Suc-B12 and their further coupling to the amino groups of the conjugated CT (Figure 1b). We used Suc-B12 in an amount of 10 mol% (30% by mass) relative to HA-CT, because this amount is able to provide an acceptable level of intestinal permeability of nanoformulations according to [23]. According to spectrophotometry, the B12 content in the HA-CT-B12 conjugate was 95 µg/mg ( Table 2). The coupling of vitamin B12 to the HA-CT conjugate was demonstrated by Fourier transform infrared (FTIR) spectroscopy ( Figure 2). In the FTIR spectrum of the HA-CT-B12 conjugate, a characteristic band was observed at 1730 cm −1 , corresponding to the C=O vibrations of the ester bond of the succinyl linker of vitamin B12.

Modification of the HA-CT Conjugate with Suc-B12
For B12 modification, we used previously synthesized HA-CT conjuga tent was 367 ± 1 μg/mg, the apparent hydrodynamic diameter (Dh) was 50 and the ζ-potential was −20 mV) [7]. A study of HA-CT conjugates showed sulting compound was sufficiently stable, with less than 3% CT released w pH 7.4 [7].
To obtain modified HA-CT-B12 conjugates, we exploited the carbodii tion of the carboxyl groups of Suc-B12 and their further coupling to the ami the conjugated CT ( Figure 1b). We used Suc-B12 in an amount of 10 mol% (3 relative to HA-CT, because this amount is able to provide an acceptable leve permeability of nanoformulations according to [23]. According to spectropho B12 content in the HA-CT-B12 conjugate was 95 μg/mg ( Table 2).
The coupling of vitamin B12 to the HA-CT conjugate was demonstrate transform infrared (FTIR) spectroscopy ( Figure 2). In the FTIR spectrum of B12 conjugate, a characteristic band was observed at 1730 cm −1 , correspondin vibrations of the ester bond of the succinyl linker of vitamin B12. According to the dynamic light scattering (DLS) data, Dh of the HA-C gate was 98 ± 22 nm, with the presence of some aggregates of 702 ± 248 nm (T pattern was in good agreement with our previous DLS studies, in which the the conjugates belonged to individual macromolecules (unimers) and the sl longed to their aggregates. The ζ-potential of the conjugates was −25.4 ± 0.3 m their colloidal stability ( Table 2). Table 2. Characterization of the HA-CT-B12 conjugate. According to the dynamic light scattering (DLS) data, D h of the HA-CT-B12 conjugate was 98 ± 22 nm, with the presence of some aggregates of 702 ± 248 nm ( Table 2). This pattern was in good agreement with our previous DLS studies, in which the fast mode of the conjugates belonged to individual macromolecules (unimers) and the slow mode belonged to their aggregates. The ζ-potential of the conjugates was −25.4 ± 0.3 mV, indicating their colloidal stability (Table 2).
Scanning electron microscopy (SEM) confirmed the presence of spherical particles less than 100 nm in size, which was in good agreement with the DLS data ( Figure 3).
Scanning electron microscopy (SEM) confirmed the presence of sph less than 100 nm in size, which was in good agreement with the DLS data (

The pH Stability Studies and In Vitro CT-Release Profile
Vitamin B12 is absorbed by active transport in the small intestine (i.e with an average pH of 7.4-7.5) [32]. At the same time, the transit of the different parts of the gastrointestinal tract with different pH levels and enz tions affects its effectiveness; for successful absorption in the small intes must be stable at the pH of the stomach (pH in the fasted/fed state) and re absorption as quickly as possible (to transit from the stomach to the intesti time in the stomach is typically from 5 min to 2 h; usually a meal prolongs dosage form in the stomach. To accelerate the transit of the drug from the small intestine, it is recommended to take it with water and on an empty st case the drug almost immediately reaches the small intestine). The drug small intestine for an average of 3-4 h, independent of meals [14]. We seque the stability of the HA-CT-B12 conjugate at pH 1, 6.8, and 7.4 and showe thesized compounds remained stable during transit through different part intestinal tract and were able to reach the target site (ileum) unchanged (Ta the stability at the pH of the ileum provided a good potential for the success of the conjugates into the blood.

The pH Stability Studies and In Vitro CT-Release Profile
Vitamin B12 is absorbed by active transport in the small intestine (i.e., in the ileum, with an average pH of 7.4-7.5) [32]. At the same time, the transit of the drug through different parts of the gastrointestinal tract with different pH levels and enzyme compositions affects its effectiveness; for successful absorption in the small intestine, the drug must be stable at the pH of the stomach (pH in the fasted/fed state) and reach the site of absorption as quickly as possible (to transit from the stomach to the intestine). Residence time in the stomach is typically from 5 min to 2 h; usually a meal prolongs the stay of the dosage form in the stomach. To accelerate the transit of the drug from the stomach to the small intestine, it is recommended to take it with water and on an empty stomach (in this case the drug almost immediately reaches the small intestine). The drug remains in the small intestine for an average of 3-4 h, independent of meals [14]. We sequentially studied the stability of the HA-CT-B12 conjugate at pH 1, 6.8, and 7.4 and showed that the synthesized compounds remained stable during transit through different parts of the gastrointestinal tract and were able to reach the target site (ileum) unchanged (Table 3). In turn, the stability at the pH of the ileum provided a good potential for the successful absorption of the conjugates into the blood. In vitro release kinetics studies of the designed conjugates at the inflammatory site pH (5.2) showed an ultra-slow release of CT via the hydrolysis of amide bonds (Figure 4), which was in agreement with our previous studies [7,24]. In summary, approximately 1% of CT was released in 10 h, and these slow kinetics require confirmation of the antimicrobial activity of CT in vitamin-B12-modified conjugates.
In vitro release kinetics studies of the designed conjugates at the inflammatory site pH (5.2) showed an ultra-slow release of CT via the hydrolysis of amide bonds (Figure 4), which was in agreement with our previous studies [7,24]. In summary, approximately 1% of CT was released in 10 h, and these slow kinetics require confirmation of the antimicrobial activity of CT in vitamin-B12-modified conjugates.

Antimicrobial Activity
Previously, we observed that conjugation of CT with HA at a DS of 8% did not reduce its potency compared to pure CT [7].
A study of antimicrobial activity against Pseudomonas aeruginosa (1 × 10 7 CFU/mL) showed that the HA-CT-B12 conjugate also had antimicrobial activity comparable to that of pure CT. Both minimum inhibitory concentrations (MICs) were 2 μg/mL, indicating that the antibiotic activity was maintained despite the modification with vitamin B12. At the same time, a mixture of HA with vitamin B12 at an equivalent concentration had no significant effect on visible bacterial growth ( Figure 5).

Antimicrobial Activity
Previously, we observed that conjugation of CT with HA at a DS of 8% did not reduce its potency compared to pure CT [7].
A study of antimicrobial activity against Pseudomonas aeruginosa (1 × 10 7 CFU/mL) showed that the HA-CT-B12 conjugate also had antimicrobial activity comparable to that of pure CT. Both minimum inhibitory concentrations (MICs) were 2 µg/mL, indicating that the antibiotic activity was maintained despite the modification with vitamin B12. At the same time, a mixture of HA with vitamin B12 at an equivalent concentration had no significant effect on visible bacterial growth ( Figure 5).

Cytotoxicity Study
We investigated the potential nephro-and neurotoxicity of the new HA-CT-B12 conjugates on kidney (HEK 293) and brain (T 98G) cell lines. Equal amounts of HA+B12 mixture and free CT were used as controls. In vitro cytotoxicity experiments showed that the

Cytotoxicity Study
We investigated the potential nephro-and neurotoxicity of the new HA-CT-B12 conjugates on kidney (HEK 293) and brain (T 98G) cell lines. Equal amounts of HA+B12 mixture and free CT were used as controls. In vitro cytotoxicity experiments showed that the HA-CT-B12 conjugate increased cell viability by 1.3-and 1.4-fold compared to native CT at CT concentrations of 0.5 and 1.0 mg/mL, respectively (Figure 6a). At the same time, the concentrations of free CT tested had no toxic effect on glioblastoma cells; however, even in this experiment, the viability of T 98G cells in the presence of the HA-CT-B12 conjugate was approximately 10-15% higher than in the presence of native CT (Figure 6b). Thus, HA-CT-B12 conjugates reduced the toxicity of CT against kidney (HEK 293) and brain (T 98G) cells.

Cytotoxicity Study
We investigated the potential nephro-and neurotoxicity of the new HA-CT-B1 jugates on kidney (HEK 293) and brain (T 98G) cell lines. Equal amounts of HA+B1 ture and free CT were used as controls. In vitro cytotoxicity experiments showed th HA-CT-B12 conjugate increased cell viability by 1.3-and 1.4-fold compared to nat at CT concentrations of 0.5 and 1.0 mg/mL, respectively (Figure 6a). At the same tim concentrations of free CT tested had no toxic effect on glioblastoma cells; however in this experiment, the viability of T 98G cells in the presence of the HA-CT-B12 con was approximately 10-15% higher than in the presence of native CT (Figure 6b). HA-CT-B12 conjugates reduced the toxicity of CT against kidney (HEK 293) and b 98G) cells.

Caco-2 Cell Permeability Assay
The Caco-2 cell line (human colon adenocarcinoma) has enterocyte characte After monolayer formation, the cells form junctional complexes and microvilli on t ical surface. It is known that the permeability of the Cao-2 cell monolayer is highly

Caco-2 Cell Permeability Assay
The Caco-2 cell line (human colon adenocarcinoma) has enterocyte characteristics. After monolayer formation, the cells form junctional complexes and microvilli on the apical surface. It is known that the permeability of the Cao-2 cell monolayer is highly correlated with the processes of drug absorption in the intestine. The disadvantage of the Caco-2 cell line is its cancer origin and phenotypic instability; however, Caco-2 cells are widely used in pharmacological studies because they allow robust results to be obtained [29]. The apparent permeability coefficient in vitro (Papp) for substances absorbed through the intestinal wall by active transport had lower values compared to in vivo experiments because of the lower degree of expression of ionic and peptide transport proteins in Caсo-2 cells. Therefore, the main area of application of Caсo-2 cell culture is the qualitative assessment of intestinal permeability [33].
In general, absorption in the human GIT is 50 to 100% for compounds with an in vitro Papp greater than 1 × 10 −6 cm/s. These compounds are promising for the development of oral dosage forms [34,35]. Intestinal permeability testing in the Caco-2 cell model showed that the B12-modified conjugates based on CT and HA had an acceptable Papp to allow potential absorption in the gastrointestinal tract (3.5 × 10 −6 cm/s). In comparison, the Papp of free CT and free vitamin B12 in this experiment were 0.04 × 10 −6 cm/s and 5.4 × 10 −6 cm/s, respectively (Figure 7).

Materials and Reagents
In this study, we used previously synthesized CT conjugates based on sodium hyaluronate (HA) with a DS for CT moieties of 8 mol% (HA-CT). The CT content in the HA-CT conjugates was 367 ± 1 μg/mg. In phosphate-buffered saline (PBS, pH 7.4), HA-CT conjugates were present as unimers and nanoparticles with hydrodynamic diameters (Dh) of 50 and 560 nm, respectively; the ζ-potential was −20 mV. The HA (MW of 1.8 × 10 5 ) was obtained from Shandong Focuschem Biotech (Qufu, Shandong, China); the Dh and ζ-potential in the PBS were 34 nm and −26 mV, respectively [7].

Materials and Reagents
In this study, we used previously synthesized CT conjugates based on sodium hyaluronate (HA) with a DS for CT moieties of 8 mol% (HA-CT). The CT content in the HA-CT conjugates was 367 ± 1 µg/mg. In phosphate-buffered saline (PBS, pH 7.4), HA-CT conjugates were present as unimers and nanoparticles with hydrodynamic diameters (D h ) of 50 and 560 nm, respectively; the ζ-potential was −20 mV. The HA (MW of 1.8 × 10 5 ) was obtained from Shandong Focuschem Biotech (Qufu, Shandong, China); the D h and ζ-potential in the PBS were 34 nm and −26 mV, respectively [7].

Synthesis of Succinyl Cyanocobalamin (Suc-B12)
Suc-B12 was prepared according to the method described in [23] with certain modifications: B12 (0.05 g, 0.037 mM) was dissolved in 5 mL of dry DMSO, then an equimolar amount of DMAP and a 10-or 100-fold molar excess of succinic anhydride were added. The reaction mixtures were stirred for 24 h and the resulting products (Suc-B12-10 and Suc-B12-100, respectively) were precipitated with acetone and dried at 40 • C for one day.
The products were characterized by mass spectrometry using a maXis impact Q-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with an electrospray ionization (ESI) source (Bruker Daltonics GmbH, Bremen, Germany) operated in the positive ionization mode. Mass calibration was performed with sodium formate solution (calibration mode HPC, standard deviation (SD) 0.308 ppm). Flow injection mode was used for analysis: mass range from 50 to 1300 m/z, spectra rate 1 Hz, and line and profile spectra stored. The acquisition parameters were as follows: Source: end plate offset 500 V, capillary 4500 V, nebulizer 0.4 bar, dry gas 4.0 L/min, and dry temperature 180 • C. Collision cell: collision energy 7.0 eV, transfer time 90.0 µs, and pre-pulse storage 10.0 µs. Mass spectra were analyzed and deconvoluted using DataAnalysis ® 5.0 software (Bruker Daltonics GmbH, Bremen, Germany).

Modification of the HA-CT Conjugates with Suc-B12
Suc-B12 (25 mg, 0.018 mM) was dissolved in 2.5 mL DMSO, then 1.5-times molar amounts of EDC and NHS were added and stirred for 30 min for carbodiimide activation of the COOH groups. A 10 mL aqueous solution of HA-CT (55 mg, 0.18 mM) was added to the mixture and stirred overnight. The resulting product (HA-CT-B12) was dialyzed against distilled water for 5 days (until the pink color of the dialysis medium disappeared), and was then lyophilized.
The presence of vitamin B12 in the HA-CT-B12 conjugate was confirmed by FTIR using a Vertex 70 IR Fourier spectrometer (Bruker Optics, Ettlingen, Germany) equipped with a ZnSe-attenuated total reflectance accessory (PIKE Technologies, Fitchburg, WI, USA). A correction was applied to the attenuated reflectance spectra to account for the depth of penetration of the irradiation as a function of wavelength.
The conjugation efficiency was determined spectrophotometrically with a UV-1700 PharmaSpec spectrophotometer (Shimadzu, Kyoto, Japan) at 360 nm using a calibration with B12.
The CT content (µg/mg) in the HA-CT-B12 conjugate was calculated from the CT content in the HA-CT conjugate (367 µg/mg) determined by 1 H NMR spectroscopy [7], and the B12 content in the HA-CT-B12 conjugate (95 µg/mg) was determined by UV-VIS spectrophotometry, as described above.

Size and Surface Morphology of the HA-CT-B12 Conjugates
The hydrodynamic radii and ζ-potential of HA-CT-B12 were measured using a Photocor Compact-Z device (Photocor Ltd., Moscow, Russia) with a 659.7 nm He-Ne laser at 25 mV power and a detection angle of 90 • .
The morphology of HA-CT-B12 conjugates was analyzed by SEM using a Tescan Mira 3 instrument (Tescan, Brno, Czech Republic). For this purpose, the nanosuspension of HA-CT-B12 conjugates was centrifuged (15 min, 5000 rpm) to separate aggregates, and the obtained sample was placed on double-sided carbon tape and dried in a vacuum for 48 h; then, SEM images were obtained in the secondary electron mode at an accelerating voltage of 20 kV and an operating electric current of 542 pA; the distance between the sample and the detector was 6-7 mm.

The pH Stability Studies and In Vitro CT-Release Profile
To study the stability of the conjugates at the pHs of different parts of the GIT (stomach, duodenum, and ileum), 1 mg of the sample was placed in a Vivaspin ® Turbo 4 centrifugal concentrator (MWCO 10,000), and 1 mL of 0.1 M HCl at pH 1 (gastric conditions) was added. The sample was incubated at 37 • C, and after 2 h, the medium was completely ultracentrifuged at 4500 rpm and replaced with 1 mL phosphate buffer at pH 6.8 (pH of duodenal medium). The sample was incubated at 37 • C, and after 2 h, the medium was completely ultracentrifuged at 4500 rpm and replaced with 1 mL of PBS at pH 7.4 (pH of the ileum, the main site of vitamin B12 absorption, as well as blood pH). The sample was incubated for 10 h. At regular intervals, the medium was completely centrifuged at 4500 rpm and replaced with 1 mL of fresh PBS [7,24].
To determine the release profile of CT under conditions simulating the inflammatory site environment, 1 mg of the conjugate was dissolved in 1 mL of phosphate buffer at pH 5.2 (inflammatory site pH) and was incubated at 37 • C. At regular intervals, 1 mL of the medium was ultracentrifuged at 4500 rpm using a Vivaspin ® Turbo 4 centrifugal concentrator (10,000 MWCO). The CT content in the filtrates was determined by LC-MS, as previously reported [7,24].

Antimicrobial Activity
The antimicrobial activity of HA-CT-B12 was tested using the microtiter broth dilution method, as previously reported [7,24]. P. aeruginosa ATCC 27853 (Museum of Microbiological Cultures, State Research Institute of Highly Pure Biopreparations, St. Petersburg, Russia) was used as the experimental microorganism.
Briefly, HA-CT-B12 conjugate stock solutions were prepared by diluting samples in Mueller-Hinton broth to a maximum 2-fold concentration equivalent to CT. Serial dilutions of CT (64 to 0.25 µg/mL) were then plated on Luria-Bertani agar culture plates. The antimicrobial efficacy of the HA-CT-B12 conjugates was confirmed by the absence of P. aeruginosa growth.
P. aeruginosa suspension was serially diluted 1:100 in Müller-Hinton broth to obtain a concentration of approximately 1 × 10 7 CFU/mL. Then, 125 µL of the inoculum was added to the wells of the culture plate containing HA-CT-B12 solutions in Müller-Hinton broth. The plate also contained positive controls-100% growth (bacteria only), sterility control (Mueller-Hinton broth only), and HA+B12 at equivalent concentrations (blank). The plate was incubated for 24 h at 37 • C and the optical density (OD) was measured at 630 nm using an ELx808™ Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA). Relative bacterial growth (%) was calculated as the ratio of OD630 at each concentration of the test samples to OD630 in the control (0 µg/mL). Each sample was tested in triplicate in three independent replicates (n = 9).

Caco-2 Cell Permeability Assay
The human colon adenocarcinoma cell line Caco-2 was obtained from the Russian Cell Culture Collection (Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia). Caco-2 cells were cultured in DMEM medium supplemented with 10% FBS, Lglutamine, and penicillin/streptomycin in 75 cm 2 cell culture flasks (Jet Biofil, Guangzhou, China) at 37 • C in a humidified atmosphere containing 5% CO 2 in a CO 2 incubator. When the cells reached 60-70% confluence, the cells were sub-cultured into new flasks (1:6 ratio).
Caco-2 cell permeability protocol: For the experiment to study permeability and changes in the barrier properties of the intestinal epithelium, cells (0.5 mL, 100,000 cells per well) were seeded on the apical side of the 1.0 µm pore diameter membrane (Corning Incorporated, Corning, NY, USA) in cell culture inserts for 24-well plates. A total of 1 mL of complete DMEM medium was added to the basolateral chamber. The medium in the apical and basolateral chambers was changed every other day. Within 7 days, the cells formed a confluent monolayer and then polarized over the next 14 days, with tight junctions and microvilli formation, i.e., the cells acquired the properties of enterocytes. On day 21 of the cultivation, the medium was removed and the cell monolayer was washed three times with PBS. The studied samples (1 mg CT, 28.5 mg vitamin B12, and 300 mg HA-CT-B12 conjugate) were dissolved in 5 mL PBS, and 0.5 mL of the obtained solutions were added to the apical chamber. A total of 1 mL of PBS was added to the basolateral chamber, and 1 mL of PBS was removed from the basolateral chamber every 30 min for 2 h. This was followed by the addition of 1 mL of fresh PBS to maintain the same volume. The integrity of the Caco-2 monolayer was checked by leakage of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran, Sigma Aldrich (St. Louis, MO, USA)). A freshly prepared solution containing 5 mg/mL 4-kDa FITC-dextran dissolved in PBS was added to the apical chamber for incubation at 37 • C for 2 h. Samples were collected from the bottom chamber and fluorescence intensity was measured using a fluorescence plate reader (excitation 492 nm; emission 520 nm). If the FITC-dextran apparent permeability (Papp) was less than 1 × 10 −6 cm/s, cells were considered acceptable for further experiments.
Each sample was assayed in three independent series (n = 3). The content of vitamin B12 and the content of the HA-CT-B12 conjugate equivalent to vitamin B12 were determined spectrophotometrically at 360 nm using a calibration curve. In addition, it was confirmed that the HA-CT-B12 conjugates were chemically stable under the conditions of this experiment and did not release free vitamin B12 within 2 h (as determined by spectrophotometric analysis).
The Papp was calculated using the following equation [33]: where Papp is the apparent permeability coefficient (cm/s); dQ dt -is the permeation rate (µg/s); A is the monolayer area (0.3 sm 2 ); and C 0 is the concentration in the apical chamber at the initial moment of time (µg/mL).

Conclusions
Oral drug delivery is characterized by simplicity and patient convenience as well as improved safety compared to intravascular injection. Increasing the intestinal permeability of peptide antibiotics is a current and challenging task. This study included several key aspects to address this challenge. First, we developed a convenient synthetic method to modify CT-and HA-based conjugates with a specific targeting ligand (vitamin B12) to improve intestinal permeability. Second, we demonstrated the stability of the obtained conjugates under gastrointestinal conditions (at pH 1, 6.8, and 7.4), as well as the preservation of antimicrobial activity at the level of free CT (2 µg/mL), with a decrease in nephrotoxicity (by 30-40%). Third, we demonstrated that modification with B12 actually improved the intestinal permeability of CT; the Papp of HA-CT-B12 conjugates was 3.5 × 10 −6 cm/s, which is significantly higher than the Papp of pure CT (0.04 × 10 −6 ). Furthermore, the Papp value of 3.5 × 10 −6 cm/s corresponds to an in vivo intestinal absorption of 50-100%. Thus, B12 modification of CT-and HA-based conjugates may be an effective strategy for the development of oral delivery systems with improved bioavailability and a reduced toxicity profile of the peptide antibiotic CT.