1. Introduction
Natural alkaloid colchicine (
1) (
Figure 1) is a mitotic poison that binds to intracellular protein tubulin and prevents mitotic spindle formation. This leads to a block in cell proliferation and reduced cell motility [
1,
2]. Orally administrated colchicine has an elimination half-life of 20–40 h [
3]. It binds to neutrophils and serum albumin and may cause the body to produce fewer blood cells of different types [
4]. Colchicine is metabolized by intestinal and hepatic cytochrome CYP3A4 and excreted via the hepatobiliary and renal routes. The alkaloid significantly affects the gastrointestinal tract inducing nausea, vomiting, and diarrhea in 5–10% of patients even at approved doses [
5], which prevents its usage in cancer treatment and other diseases. The low therapeutic index of colchicine, like many other tubulin-binging agents, is caused by its inability to yield concentrations high enough at the target site to trigger apoptosis, associated with the non-specific cytotoxicity towards normal tissues and organs. Numerous attempts have been made to modify the molecule in order to reduce its inherent toxic side effects [
6].
Colchicine biodistribution studies demonstrated its bioaccumulation in liver, intestine, kidney, and heart at organ/muscle ratios 11, 5, 7, and 6, respectively [
8]. Unspecific tissue partition of colchicine may be reduced by decorating it with hydrophilic groups such as polyethylene glycol (PEG) [
9,
10]. Another way to sequester colchicine from non-target organs is to incorporate it into a drug carrier which can improve the therapeutic antitumor index via “enhanced permeability and retention effect” [
11] due to an increased uptake of macromolecules by tumors.
Among various polymers used to develop drug carriers, chitosan (
3,
Figure 1) has significant advantages over analogues due to its biodegradability, low toxicity, and multiple amino groups used to obtain derivatives with desired properties [
12]. Chitosan was used to develop various delivery systems for insulin, morphine, DNA, siRNA, proteins and peptides [
13,
14,
15]. Chitosan based nanoparticles (NPs) can be developed as theranostic agents for both drug delivery and diagnostics [
16,
17,
18].
The pharmacokinetics of nanosized carriers in living organisms is quite complex and depends on a number of factors, e.g., physical and chemical properties of NPs, surface functionalization, and permeability of various tissue membranes [
19]. For the biodistribution analysis, it is essential to incorporate a reliable label into a drug or a delivery vehicle to monitor its accumulation in tissues and body fluids. In many cases experimental data are obtained by different poorly compatible methods, some of which have low sensitivity; and many studies provide limited information on ADME (absorption, distribution, metabolism, and excretion). For example, after investigation of biodistribution and pharmacokinetics of
125I-labeled PLGA NPs in mice, Panagi et al. [
20] found that the accumulation of NPs mostly occurs in liver with minor amounts in lungs, intestine, and muscles, while kidney and gall inputs were not estimated. Flaten et al. [
21] studied the biodistribution of
3H-labeled camptothecin and its liposome formulation in HT-29 mouse tumor model. Although all major organs were collected and studied, the analysis was performed 20 h post injection which made it impossible to analyze primary biodistribution and excretion. Body distribution of chitosan NPs loaded with a fluorescent dye and siRNA/cisplatin was studied using bioimaging for qualitative and PCR method for quantitative analysis in lung cancer nude mice model. Analysis was conducted from 12 to 120 h and demonstrated blood clearance and a major accumulation of NPs in liver, kidneys, and tumor [
22].
Recently, our group synthesized a number of heterocyclic allocolchicinoids [
23,
24,
25] possessing similar to colchicine antitumor activity but lower systemic toxicity. It was found that the conjugate (
4) of colchicinoid
2 with chitosan
3 (40 kDa) (
Figure 1) is more effective in tumor growth inhibition in mice compared to intact colchicinoid
2, which was associated with a better entry of nanoparticles into tumor tissues and less systemic toxicity [
7]. The goal of this work was to synthesize fluorescent furano-allocolchicinoid derivative (
AC,
6), fluorescent furano-allocolchicinoid—chitosan conjugate (
AC-Chi,
8) and to develop AC-chitosan nanoparticles (
AC-NPs), and analyze their biodistribution in mice using fluorescent signal in tissues and body liquids as a quantitative label.
2. Materials and Methods
2.1. Materials
Commercially available reagents («Sigma-Aldrich», Steinheim, Germany; «Alfa Aesar», Kandel, Germany; «ACROS ORGANICS», Geel, Belgium) were used without additional purification. Column chromatography was performed using Macherey–Nagel Kieselgel 60 (70–230 mesh). 1H and 13C NMR spectra of 6 were recorded at room temperature in CD3OD on Agilent DD2 400 instruments. Chemical shifts (δ) are reported in parts per million (ppm) from tetramethylsilane (TMS) using the residual solvent resonance (CD3OD: 3.31 ppm for 1H NMR, 49.00 ppm for 13C NMR). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; dd = doublet of doublets; dt = doublet of triplets; td = triplet of doublets). 1H-NMR spectra of the initial chitosan and chitosan derivatives were obtained on a Bruker DRX 500 spectrometer in 0.01 M DCl/D2O at 30 °C. EI mass spectra (70 eV) were obtained on a DSQ II mass-spectrometer (Thermo Electron Corporation, Austin, TX, USA) with a quadrupole mass-analyzer. MALDI mass spectra were obtained on a MALDI-TOF mass-spectrometer Bruker Microflex LT.
Medium molecular weight (MW) chitosan (≈40 kDa) with deacetylation degree 0.94 (Aladdin Chemistry Co., Ltd., Shanghai, China); Chitosan with MW ≈ 200 kDa and deacetylation degree 0.85 was purchased from ZAO «Bioprogress» (Moscow, Russia). Samples were purified by sequential precipitation with 30% CH3COOH and 12% NH4OH. Caproic anhydride (Fluka, Germany), sodium tripolyphosphate (Sigma-Aldrich, St. Louis, MO, USA), calcium chloride (Pacreac, Barcelona, Spain), acetic acid, ammonium hydroxide (Chimmed, Moscow, Russia), ethylenediaminetetraacetic acid (EDTA), (Sigma-Aldrich, St. Louis, MO, USA), Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, phosphate-buffered saline (PBS), L-glutamine, ampicillin, Rodamine B (Sigma-Aldrich, St. Louis, MO, USA) were used as received.
2.2. Synthesis of Fluorescently Labeled Furano-Allocolchicinoid (6)
Compound
2 was prepared according to the previously proposed procedure from commercial colchicine [
23]. Compound
5 (see
Scheme 1) was synthesized from commercial rhodamine B according to the method proposed by Nguyen and Francis [
26].
A Schlenk flask was degassed, filled with argon, and charged with EDC·HCl (28 mg, 0.146 mmol) and compound 5 (45 mg, 0.073 mmol). Anhydrous CH2Cl2 (0.5 mL) was added, and the mixture was stirred for 30 min at 0 °C. A second Schlenk flask was charged with furano-allocolchicine 2 (30 mg, 0.073 mmol) and DMAP (5 mg, 0.036 mmol). The flask was then filled with argon and dry CH2Cl2 (0.5 mL) was added. The mixture from the first flask was transferred into the second flask by using a syringe. The resulting mixture was stirred at 0 °C for 1 h and then at room temperature for 3 h. After the solvent removal under reduced pressure the residue was dissolved in H2O (30 mL), saturated with NaCl and then extracted with multiple portions of 2:1 iPrOH/CH2Cl2 until a faint pink color persisted. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The product was purified by column chromatography, eluent: petroleum ether/ethyl acetate/ethanol (1:1:1) to give 6 as a dark purple solid (45 mg, 0.045 mmol, 61%). 1H NMR (400 MHz, CD3OD) δ 8.09 (d, J = 6.6 Hz, 2H), 7.76 (d, J = 5.9 Hz, 2H), 7.68–7.65 (m, 1H), 7.59 (s, 1H), 7.53–7.50 (m, 1H), 7.43 (s, 1H), 7.28 (d, J = 9.5 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.95 (s, 2H), 6.82 (s, 1H), 6.75 (s, 1H), 5.22 (s, 2H), 4.74 (dd, J = 11.9, 6.8 Hz, 1H), 3.89 (d, J = 8.2 Hz, 6H), 3.70–3.66 (m, 8H), 3.46 (s, 3H), 3.40 (s, 8H), 2.66 (s, 3H), 2.53 (dd, J = 12.7, 5.8 Hz, 2H), 2.25 (m, 4H), 2.03 (s, 3H), 1.29 (t, J = 6.8 Hz, 12H). 13C NMR (101 MHz, CD3OD) δ 173.91, 172.47, 159.28, 159.27, 157.21, 157.01, 156.21, 154.16, 153.81, 152.14, 143.03, 142.44, 139.19, 136.60, 133.18, 132.27, 131.76, 131.29, 130.88, 128.94, 127.76, 126.38, 123.82, 115.42, 114.87, 109.05, 108.11, 107.77, 106.55, 97.36, 61.62, 61.41, 59.66, 56.62, 50.80, 46.91, 39.87, 39.78, 31.42, 30.68, 30.08, 28.63, 22.68, 12.84. MS (MALDI-TOF) = 1003.9 [M]+. Preparation 6 is designated as AC in the text.
2.3. Synthesis of Fluorescently Labeled Furano-Allocolchicinoid-Chitosan Conjugate (8)
Rhodamine B derivative
5 was conjugated with
4 according to the published procedure [
7]. Furanoallocolchicinoid-chitosan conjugate
4 (≈40 kDa, 50 mg, 0.001 mmol) was dissolved in 6 mL of distilled water, acidified with acetic acid to pH = 6 and diluted with 15 mL of methanol, after which
5 (23 mg, 0.037 mmol), EDC·HCl (28 mg, 0.148 mmol) and NHS (17 mg, 0.148 mmol) were added and the mixture was stirred for 24 h at room temperature. The resulting solution was dried under reduced pressure, washed with toluene, CH
2Cl
2 (5 × 50 mL) and dried in vacuo. The product
8 was obtained as a bright-pink solid mass (69 mg). For the in vivo experiments 10 mg of product
8 was dissolved in acidic 50% ethanol overnight at stirring. Final concentration of furano-allocolchicinoid was estimated by MTT assay using free furano-allocolchicinoid as a reference and was 0.7 mM [
7]. Preparation
8 is designated as
AC-Chi in the text.
2.4. Synthesis of N-Laurylchitosan (Kashirina et al., 2018)
Modification of chitosan with lauric and succinic residues is necessary for the reduction of the charge of preparations as negative
ζ-potential of the drug used for IV injections is preferential [
7]. Chitosan (Heppe Medical Chitosan GmbH, MW ≈ 40 kDa, deacetylation degree 0.86) 100 mg, was dissolved in 10 mL of 2% acetic acid and 30 mL of methanol, a solution of
N-hydroxysuccinimide ether of lauric acid with different molar ratios (0.1–1 mmol) in 10 mL of methanol was added, and the reaction mixture was stirred for 12 h at rt. Methanol was evaporated, the aqueous solution was dialyzed against 0.1% acetic acid and freeze-dried.
N-laurylchitosan was obtained in the form of a light white powder with 85% yield [
27].
2.5. Synthesis of N-Laurylsuccinoylchitosan (Kashirina et al., 2018)
Succinic anhydride (50–500 mmol) was added to the
N-laurylchitosan solution (80 mg, 2 mmol) in 8 mL of 2% acetic acid and 32 mL of methanol, and the reaction mixture was stirred for 12 h at rt. Methanol was evaporated, the aqueous solution was dialyzed against 0.1% acetic acid and freeze-dried.
N-laurylsuccinoylchitosan was obtained in the form of a white powder (65 mg, 81%).
1H NMR (500 MHz, DCl/D
2O) δ 2.04 (
N-acetyl), 2.54 ((CH
2)
2, succinoyl); 1.21 (CH
2, lauryl). The degree of substitution (succinoyl/lauryl) was calculated from the ratio of the integral intensity of the
N-succinoyl- and
N-lauryl-radical proton signals to the proton signal at 2C (δ 4.56).
1H NMR spectra are shown in
Figure S1.
2.6. Preparation of N-laurylsuccinoylchitosan Nanoparticles Containing Furano-Allocolchicinoid
Product 6 dissolved in DMSO (20 mM) was added drop-wise (0.02%) to N-laurylsuccinoylchitosan 30% alcohol solution (1 mg, 0.17 mmol) under magnetic stirring at 30 rpm until opalescence occurred, which was estimated by a Specol 11 spectrophotometer (Carl Zeiss Jena, Jena, Germany) at 590 nm. The pH of the reaction mixture was maintaining 7.4 with a 5M solution of Na2CO3. The reaction mixture was stirred for 1 h at rt. The solution was dialyzed against saline overnight. NPs were formed during dialysis. The particle diameter was determined by dynamic light scattering (DLS). N-Laurysuccinoylchitosan nanoparticles containing furano-allocolchicinoid (6) are designated as AC-NPs in the text.
2.7. Standardization of AC Formulations
All furano-allocolchicinoid preparations contained rhodamine B bound to furano-allocolchicinoid. MFI was used as the main label in biodistribution studies. To obtain a comparable MFI injected, formulations were dissolved in saline or acidic ethanol to calibrate the quantity of MFI/mL.
2.8. Cell Cultures
Human pancreatic PANC-1, ovarian HeLa tumor cell lines, immortalized embryonic kidney HEK293 cells and murine colon CT26 cell line were used in the study. HEK293 were grown in DMEM supplemented with 10% fetal calf serum (FCS), pen-strep-glut (all from PanEco, Moscow, Russia). All other cell lines were grown in RPMI-1640 with the same supplements. Cells were passaged by trypsinization using Trypsin/EDTA solution (PanEco, Moscow, Russia) twice a week. Twenty-four hours before the assays, cells were seeded in 96 well plates at 104 cells/well and incubated overnight to achieve standardized growth conditions.
2.9. MTT-Assay
Cytotoxic effect of different formulations was estimated by a standard 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2
H-tetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) test as described earlier [
28]. In short, different dilutions of the formulations using series of dilutions were prepared on a separate plate and then transferred in 100 µL to the plates with the cells. Non-treated cells served as controls. Plates were incubated for 72 h. For the last 6 h 250 μg/mL of MTT was added in 10 μL/well. After this, the incubation culture medium was removed and 100 μL of DMSO was added to each well. Plates were incubated at shaking for 15 min to dissolve formazan. Optical density was read on spectrophotometer Titertek (UK) at 540 nm. Results were analyzed by Excel package (Microsoft). Cytotoxic concentration giving 50% of dead cells (IC
50) varied between cells lines and is shown on
Figure 2. The inhibition of proliferation (inhibition index, II) was calculated as [1 − (OD
experiment/OD
control)], where OD was MTT optical density.
2.10. Dynamic Light Scattering (DLS)
The average liposome and HC NP diameters were determined using 90 Plus Particle Size Analyzer (Brookhaven, NY, USA) in water (25.0 ± 0.1 °C) at a scattering angle of 90° and wavelength of 661 nm using Big Particle Sizing Software. Zeta potential of NPs was determined in 10 mM KCl solution using identical Big Pal Zeta-Potential analyzer hard-ware and software.
2.11. Confocal Microscopy
AC-NPs were overloaded onto a microscopic glass slide, air dried, covered with a cover glass in a polymerizing medium Mowiol 4.88 (Calbiochem), and analyzed using Eclipse TE2000 confocal microscope (Nikon).
For the analysis of intestinal pathology fractions of small intestine were excised 5 h post injection of the furano-allocolchicinoid preparations, washed out from feces, fixed with 4% paraformaldehyde, frozen in Tissue-Tek (Sakura, Alphen aan den Rijn, The Netherlands), and cryosectioned (ThermoScientific, Waltham, MA, USA). Control sections were stained with DAPI to visualize nuclei; experimental sections were additionally stained with phallaidin-Alexa488 (Life technologies, Waltham, MA, USA) staining actin microfilaments. Analysis was conducted using confocal microscope Nikon E2000 (Tokyo, Japan).
2.12. Flow Cytometry
To study leukocyte binding of furano-allocolchicinoid formulations were mixed with 1 mL of fresh mice blood, incubated 1 h at 37 °C in 5% CO2 conditions. Red blood cells were removed by lysing solution (BD, Franklin Lakes, NJ, USA). Samples were analyzed using gating to macrophages, neutrophils and lymphocytes on FACSCalibur device (BD, Franklin Lakes, NJ, USA). Total 20,000 events were collected. The results were analyzed using WinMDI 2.8 software. Total mean fluorescence intensity was summarized and percentages of fluorescence were calculated for each cell population.
2.13. Extraction Efficiency
To estimate extraction efficacy in in vivo experiments 1 mL of tissue homogenates of liver, kidney, spleen or blood obtained from intact mice were prepared in saline. Equal fixed amount (20 μL) of fluorescent AC formulations were added to the homogenate samples and incubated overnight at +4 °C at shaking. After that 1 mL of 0.3N HCl in 70% ethanol extraction buffer [
29] was added to each sample and again incubated overnight at +4 °C at shaking. The mixtures were then transferred to Eppendorf microcentrifuge tubes and centrifuged at 10,000 rpm for 20 min. Samples (200 µL) in triplets were transferred to black plates and the fluorescence was measured at 490 nm using Glomax Multi spectrofluorimeter (Promega, Madison, WI, USA). Control amount of furano-allocolchicinoid preparations were diluted in the same manner. Extraction efficacy was calculated using the following equation: EE = (mean fluorescence intensity (MFI) in samples)/(MFI in controls)·100.
2.14. Biodistribution Experiments
2.14.1. Mice
C57BL/6 mice were purchased from Pushchino Affiliation of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow. All mice were 6–8 weeks old and maintained in minimal pathogen animal facility at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow.
Mice were allocated in three groups and were injected IV in the tail vein with rhodamine labeled AC, AC-Chi, or AC-NPs. Experiments were repeated 4 times and pooled results are presented. Totally 12 mice per time point were used in each group.
2.14.2. Ethical Approval
All studies were conducted in an AAALAC accredited facility in compliance with the PHS Guidelines for the Care and Use of Animals in Research, protocol #232 from 24 May 2018.
2.14.3. Rhodamine Extraction from Organs
At different time intervals blood was collected from the orbital sinus of anaesthetized (Isoflurane, Baxter Healthcare, San Juan, Puerto-Rico) mice and coagulated. Serum was used directly without extraction. Mice were sacrificed by cervical dislocation at different times post injection (0.5, 2, 5, 10, 20 h). Residual blood in organs was removed by transcardial perfusion by heparinized saline solution. Thigh muscle piece, spleen, kidneys, liver, and lungs were collected, weighed, and homogenized using steel strainer in 1 mL saline solution per 0.3 g of tissue weight. Rhodamine extraction from different tissues was conducted by acidic ethanol buffer [
29]. To this end 1 mL of tissue homogenate was mixed with 1 mL 0.3N HCl in 70% ethanol and incubated overnight at +4 °C at shaking. The mixtures were then transferred to Eppendorf microcentrifuge tubes and centrifuged at 10,000 rpm for 20 min. Cleared samples (200 µL) in triplets were transferred to black plates and the fluorescence was measured at 490 nm using Glomax Multi spectrofluorimeter (Promega). Organs and serum from intact mice (
n = 4) were used to estimate a cut-off limit of autofluorescence. The cut-off limit was subtracted from the mean fluorescence intensity (MFI) measured in 200 µL of cleared extracts. Biodistribution was analyzed among serum, muscles, leukocytes, spleen, kidney, liver, and lungs. In some experiments leukocyte binding was also included in the analysis. For this, blood was collected in heparinized tubes; red blood cells were lysed, and rhodamine form leukocytes was extracted as described.
2.14.4. Statistical Analysis
Statistical analysis was performed using Excel software and Student’s t-test. Comparison values of p < 0.05 were considered statistically significant.
4. Discussion
Colchicine is an old drug with a good antimitotic activity and unique combination of properties; it has the potential to be competitive with modern drugs used in the medical practice. Recent studies show that colchicine modifications, such as its conjugation with isotope binding sites DOTA or NOTA, and radionuclide or incorporation into polymeric nano/microparticles such as mesoporous silica nanoparticles or glycopeptide dendrimers, can improve its antitumor activity [
30,
31,
32,
33]. The common features of these modifications are an increase in the size of colchicine molecule and a slower release from the drag carrier. To verify the role of a molecular size of colchicine delivery system on biodistribution and toxicity, two new imaging agents of different sizes were synthesized:
AC with MW 1 kDa, and
AC-Chi of 41 kDa.
AC-NPs nanoparticles with 450 nm in diameter were developed by incorporation of
AC into
N-laurysuccinoylchitosan.
AC-Chi preparation is also likely to form nanoparticles due to low solubility of chitosan at neutral pH.
Our results on biodistribution demonstrated a decreased excretion of nano/micro sized formulation via hepatobiliary root, low accumulation in kidney and lungs, and increased retention in spleen. The same longer retention of colchicine conjugates in non-excretory organs was demonstrated earlier [
8,
34]. However, we found an increased quantity of
AC-Chi and
AC-NPs in spleen only. In any case, longer retention in non-excretory organs can be translated into a better accumulation of such drugs in tumors, as has been shown by Korde et al. [
34], and Erfani et al. [
8]. In this work we have compared furano-allocolchicinoid preparations of three different sizes. As a result, biodistribution of medium size
AC-Chi demonstrated an intermediate pattern between
AC and
AC-NPs showing a role of size of delivery system. Chitosan can also modify the effect on biodistribution. Earlier we have shown a better antitumor effect of AC-Chi which depended on the amount of AC in the preparations [
7].
Decoration of toxic preparations can decrease its side effects. The major side effects of colchicine (nausea, vomiting, diarrhea) result from the block of gastrointestinal cell division by colchicine. It appeared that both AC-Chi and AC-NPs are less toxic to the intestinal cells possibly due to a partial degradation of chitosan in liver.
Our results demonstrated that an increase in the size of the preparation results in the increase in the biodistribution. Which size of colchicine derivative carrier will provide a better effect when used as antitumor therapy should be further studied.