Development of Dendrimer-5-Flurouracil and Folic acid Conjugation for the Treatment of Colon Cancer via Target Drug Delivery

Introduction

A leading cause of mortality globally, cancer is expected to cause many more deaths in the future, partly due to an ageing world population.¹

Cancer cells are also known as carcinoma, sarcoma, melanoma, lymphoma and leukemia.² The ability of the body's immune cells to recognise and eliminate newly formed cells when there aren't many of them is probably more crucial to the development of cancer than the conversion of a normal cell into a malignant cell.³

Colon cancer ranks second in terms of cancer-related fatalities. The majority of patients with colon cancer will have localised or distant metastases, necessitating either adjuvant chemotherapy after surgery or palliative chemotherapy for metastatic illness. Colonic epithelial cells, which line the lumen of the organ and replace themselves every five days from a stem cell population found at the base of colonic epithelial cell crypts, are the source of colon cancers.⁴

In India, the incidence of cancer increased between 2010 and 2019 at an average yearly rate of 1.2% to 1.4%, according to data from the Cancer Statistics Report 2020. Over the same time period, the average number of deaths from cancer in the nation grew by 0.11 percent. Women had a rate of 103.6 per 100,000 persons compared to 94.1 per 100,000 in men.⁵ Oncological researchers are working hard to develop innovative and effective medicines that help lessen serious adverse effects brought on by traditional medications. Various technologies are already being used in clinical settings or are now being reviewed in clinical studies.

5-fluorouracil (5-FU), a powerful anti-metabolite used to treat a range of solid tumours, is well-established. Bolus 5-FU and leucovorin, the Mayo regimen, are linked to substantial myelosuppression, mucositis, and diarrhoea. Around 35% of patients in one study experienced substantial toxicity, resulting in dose reductions, therapy discontinuations, hospitalizations, and even deaths. Despite making efforts to find clinical markers that can identify individuals at risk of severe toxicity, there remains uncertainty about how to personalize the dosage of 5-FU.⁶

Since some anticancer chemotherapies, like doxorubicin or oxaliplatin, have been demonstrated to depend critically on the host's immune system and anticancer immune responses, it has become obvious that 5FU has the power to have a substantial impact on these systems. It has been demonstrated that 5FU can reduce the frequency of immunosuppressive myeloid-derived suppressor cells (MDSCs) and sensitise cancer cells to death by effector CD8 T cells, suggesting that 5FU could be employed to create effective chemoimmunotherapeutic techniques. However, we have recently demonstrated that 5FU therapy could cause MDSCs to secrete interleukin (IL)1b, favouring the development of tumours.⁷

Dendrimers are linear polymers or tiny molecules that are radially symmetric and nanoscale in size. They also contain symmetric branching units. Dendrimers are artificial macromolecules that are highly defined and have a mix of multiple functional groups. The benefits of this have been clearly defined. They are the newest type of macromolecular nano-scale delivery systems due to their materials. It is amazing how dendritic macromolecules are playing a growing role in cancer treatment and diagnostic imaging.⁸

Conjugates as compared to conventional small molecule treatments; provide a number of important benefits. First, conjugation to a water-soluble polymer can significantly increase a drug's aqueous solubility. As a result of their low water solubility, 40–60% of medicines now under development are thought to have poor bioavailability. Additionally, polymer-drug conjugates have the potential to administer a medicine in a controlled way, with drug release from the conjugate taking place over a predetermined time period. In this method, the delivery's rate and duration may be specifically planned to reach the optimum therapeutic concentration. Thus, it is feasible to prevent the significant variations brought on by periodic delivery, which can result in high systemic drug concentrations that cause toxic effects, organ damage, or other side effects.⁹

Chanphai P et al., (2021) investigated the loading efficiency of folic acid - polyamidoamine (PAMAM)- G3 and folic acid - PAMAM - G4 nanoparticles with Doxorubicin (Dox), Tamoxifen (Tam), and Tetracycline (Tet) in an aqueous solution with a pH of 7.2. The drugfolic acid-PAMAM interactions were characterized by thermodynamic parameters, indicating the involvement of ionic, hydrogen bonding, and van der Waals interactions. Increasing the size of the acid-PAMAM led to enhanced stability and loading efficacy of the drugpolymer conjugates. The order of stability for drugnanoparticles was found to be doxorubicin > tetracycline > tamoxifen. Transmission electron microscopy (TEM) analysis revealed significant morphological changes in the polymer upon drug encapsulation. In vitro experiments demonstrated that folic acid-PAMAM conjugates serve as effective drug delivery tools.¹⁰

In 2016, Bwatanglang IB et al. enhanced the anti-cancer effectiveness of 5-Fluorouracil (5-FU) by utilizing a carrier system with improved targeting capabilities towards folate receptors (FRs) expressed in malignant tissues. They loaded the 5-FU drug onto Mn-ZnS quantum dots (QDs) that were encapsulated with chitosan (CS) biopolymer and conjugated with folic acid (FA) using a simple wet chemical method. To evaluate the in vivo biodistribution and tumor targeting specificity of the 5-FU@FACS-Mn:ZnS in tumor-bearing mice, the Zn(2+) tissue bioaccumulation was analyzed using inductively coupled plasma (ICP) spectroscopy. Furthermore, the in vitro release profile of 5-FU from the conjugates, examined through a diffusion-controlled method, showed a controlled release pattern compared to the release behavior of free 5-FU drug. The newly synthesized 5-FUFACS-Mn:ZnS nanoparticle (NP) system induced a higher level of apoptosis in breast cancer cells in vitro compared to cells treated with the free 5-FU drug. When evaluating the in vivo toxicity of the NPs compared to the control, there was no significant increase in the activities of liver and kidney function biomarkers. However, due to the folate receptor (FA-FRs) chemistry, the NPs specifically accumulated in the tumour of the tumour-bearing mice. These results demonstrated that the 5-FU FACS-Mn:ZnS QDs displayed a selective anti-tumour effect in breast cancer cells in vitro and in vivo, offering a potential strategy to enhance the efficacy of 5-FU and improve tumor targeting specificity with limited systemic toxicity.¹¹

Askarian s et al., (2015) developed polyamidoamine (PAMAM)-pullulan conjugates and investigated their targeting activity in delivering gene into liver cells. The particle size, zeta potential, buffering capacity and ethidium bromide exclusion assays of the conjugates were found in satisfactory range. The final product seems to improve delivery of nucleic acids into the liver cells expressing a glycoprotein receptor with minimal transfection in non-targeted cell.¹²

Materials & Methods

Materials

PAMAM G-5 dendrimer, Dimethyl hydrazine dihydrochloride, folic acid and all other chemicals were procured from Subra Scientific Bengaluru. 5-FU was procured from Yarrow Chem products, Bengaluru. All chemicals used for the study were of analytical grade.

Methods

The formulation of dendrimer of antineoplastic drugs^13-16

Step 1: PAMAM G-5 dendrimer was acylated by the following steps. 50 mg dendrimer was first dissolved in 6 mL methanol in the presence of 27 microliter acetic anhydride.

Another 15 microliter of acetic anhydride was added slowly to the dendrimer solution. The solution was stirred under nitrogen atmosphere at room temperature. After 18 h, Methanol was evaporated from rotary evaporator. The residue was dissolved in water and dialyzed. The obtained sample Ac-G5 was lyophilized and stored in a dry place for further modification and characterization.

Dendrimer Acylated Dendrimer

Step 2: The next step was to develop acylated dendrimer PAMAM-Folic acid -5 fluorouracil acetic acid conjugation. First 6.12 mg of folic acid and 25.45 mg 5 fluorouracil acetic acid were reacted with 29.69 mg of 1- Ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) HCl in a solution of 6 mL dry Dimethylformamide (DMF) and 2 mL dry Dimethyl sulfoxide DMSO for 1h at room temperature. The folic acid and 5 fluorouracil acetic acid solution was added drop wise to 15mL solution of 1.39 micromole acetylated dendrimer solution and vigorously stirred for three days at room temperature. The sample was lyophilized after the dialysis.

Characterization of Compounds

FT-IR Analysis of Drug and conjugate

The structure of the synthesized compounds was elucidated by Fourier transformer – Infrared

Spectrophotometer in KBr – pellet method.

IR values were measured in cm-1 and the results are shown below

Nuclear magnetic resonance (NMR) Spectroscopy

1H-NMR spectra shows the signals at 3.8-4.0 corresponding to the methylene proton of the carboxymethyl proton and the signals at 7.0 and 8.0 ppm related to the NH and CH proton in the conjugate.

In vivo anticancer activity of dendrimer-5-FU conjugate on Ehrlich Ascites Carcinoma (EAC) induced mice model

Animals used

All experiments on animals were conducted by the guidelines of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by the institutional Animal Ethical Committee (IAEC) of Acharya & BM Reddy College of pharmacy with reference number IAEC/ABMRCP/2018-2019/24.

Cancer Cell Count and Induction

The cancer cell count and induction study was carried out by injecting 1 mL of normal saline (0.9%) into donor mouse through intraperitoneal route. After injecting saline, immediately 1 mL of ascites fluid was collected from peritoneal cavity and diluted with normal saline up to 10 ml. From this, 10 µL ascites was taken and placed on Neubauer’s chamber and the number of cells appeared on chamber were calculated and concentration of 1×10⁶ cells were injected to each mouse through intraperitoneal route.

Calculation

Area of one square of WBC chamber (A) = 0.04 mm. Depth of one square of WBC chamber (D) = 0.1 mm. Volume of one square of WBC chamber (V) = Area x Depth = 0.04 mm³ x 0.1 mm = 0.004mm³ . Total volume of 4 squares of WBC chamber = 4 x 0.004 mm³ = 0.016 mm³

Total cancer cells to be induced = (Total number of cancer cells in all 4 squares of WBC chambers(X) divided by total volume of all 4 squares of WBC chambers) multiplied by dilution factor. Dilution factor = 10 . Total number of cancer cells counted in all 4 square = 400 cells. Total volume of 4 chamber = 0.4 mm3 or 0.4µl or 0.0004 ml. That is, 0.0004 mL contains 400 cancer cells. Total cancer cells present per mL = 400 × 10 = 0.0004 = 1× 10⁷ cells/ml. Total cancer cells present in 0.1 mL = 1× 10⁷ × 0.1 = 1× 10⁶ cells. Therefore, 0.1mL of above concentration was injected to each mice intraperitoneal route. (Concentration of 1×10⁶ cells were injected to each mouse intraperitoneal route).

Transplantation of cancer cells^17-19

The EAC cells were maintained in vivo in mice by intraperitoneal transplantation of 1x10⁶ cells suspended in Phosphate Buffered Saline' (PBS) per mouse every 10 days. Ascetic fluid was drawn from EAC cell‑bearing mice at the log phase (days 7-8 of tumor bearing) of the cancer cell growth, and each test animal received 0.1 mL cancer cell suspension containing 1x10⁶ cells intraperitoneal (i.p.). Mice were inoculated with EAC cells and divided into three different groups containing 6 mice in each group.

Treatment schedule

Mice were divided into three groups (n=6) and were provided with food and water ad libitum. All animals in each group received EAC cells (1x10⁶ cells/mouse, i.p.). Group I served as the EAC control. Following a 24‑h period post‑EAC transplantation, group II received Dendrimer-5-FU Conjugate (20 mg/kg., p.o.), and groups III treated with 5FU (20 mg/kg., p.o.), respectively, for fourteen consecutive days. Following 24 h from the last dose, the animals were fasted for 3 h; and the blood was drawn from all the animals in each group by retro-orbital plexus method to estimate haematological parameters.

Determination of haematological parameters

In order to know the effect of the Dendrimer-5-FU Conjugate on haematological parameters in EAC cells bearing mice, a comparison between Group I (Cancer control), Group II (Dendrimer-5-FU Conjugate) & Group III (5FU/ standard) was done.

Blood was drawn from each mice by retro-orbital plexus method and was collected in EDTA vacutainer tube, for the haematological evaluation.

The blood samples were subjected to Animal Blood Counter analyser for RBC, WBC count and the haemoglobin (Hgb) content.^20,21

Statistical analysis

All values were expressed as the mean ± standard error of the mean (n=6). All in vivo data were subjected to ANOVA followed by Dunnett's post hoc test, and P<0.05 was considered to indicate a statistically significant difference upon comparison with cancer control group (EAC Control).

Results

In-vivo anticancer activity of Dendrimer-5-Flurouracil conjugate on EAC induced mice model.

Effect of drugs on WBC (cells 1x10³ /mm³ )

The effect of Dendrimer-5-FU Conjugate (20 mg/kg) and standard 5FU on WBC in EAC induced nude mice are represented in Table 1 and Figure 5.

The result demonstrated that level of WBC in Dendrimer-5-FU Conjugate showed a highly significant (p <0.01) decrease when compared to cancer control. The WBC level in standard 5 FU significantly (p <0.05) decreased compared to cancer control.

Effect of drug on RBCs (cells 1x106 /mm³ )

The effect of Dendrimer-5-FU Conjugate and standard 5 FU on red blood cells in EAC induced nude mice are given in Table 1 and Figure 6.

The result demonstrated that level of RBC in Dendrimer-5-FU Conjugate showed a highly significant (p <0.01) increase when compared to cancer control. The RBC level in standard 5 FU increased and was highly significant (p <0.01) compared to cancer control.

Effect of drugs on Hemoglobin (Hb%)

The effect of Dendrimer-5-FU Conjugate and standard 5 FU on haemoglobin in EAC induced nude mice are given in Table 1 and Figure 7.

The result demonstrated that level of Hb in Dendrimer-5-FU Conjugate showed a highly significant (p <0.01) increase when compared to cancer control. The Hb level in standard 5 FU increased compared to cancer control and was statistically significant (p <0.05).

Effect of drugs on WBC (cells 1x10³ /mm³ )

The effect of Dendrimer-5-FU Conjugate and standard 5FU on WBC in EAC induced nude mice are given in Table 2 and Figure 8

The result shown that level of WBC in Dendrimer-5-FU Conjugate highly significant (P<0.01) decrease when compared to cancer control. The standard 5 FU was decreased in WBC level compared to cancer control and statistically significant (P<0.05).

Effect of drug on RBCs (cells 1x10⁶ /mm³ )

The effect of Dendrimer-5-FU Conjugate and standard 5 FU on RBC in EAC induced mice are given in Table 2 and Figure 9.

The result demonstrated that level of RBC in Dendrimer-5-FU Conjugate showed a highly significant (p <0.01) increase when compared to cancer control. The RBC level in standard 5 FU increased compared to cancer control and was statistically highly significant (p <0.01).

Effect of drugs on Haemoglobin (Hb %)

The effect of Dendrimer-5-FU Conjugate and standard 5-FU on haemoglobin in EAC induced mice are given in Table 2 and Figure 10.

The result demonstrated that level of Hb in Dendrimer-5-FU Conjugate showed a highly significant (p <0.01) increase when compared to cancer control. The Hb level in standard 5 FU increased compared to cancer control which was statistically significant (p <0.005).

Discussion

Conjugates provide a number of important benefits. First, conjugation to a water-soluble polymer can significantly increase a drug’s aqueous solubility. As a result of their low water solubility, 40–60% of medicines now under development are thought to have poor bioavailability. Additionally, polymer-drug conjugates have the potential to administer a medicine in a controlled way, with drug release from the conjugate taking place over a predetermined time period. In this method, the delivery rate and duration may be specifically planned to reach the optimum therapeutic concentration. Thus, it is feasible to prevent the significant variations brought on by periodic delivery, which can result in high systemic drug concentrations that cause toxic effects, organ damage, or other side effects.²²

Dendrimers can be created, producing globular macromolecules that are nearly monodisperse and have a lot of peripheral groups. Therefore, dendrimers are a perfect candidate for a delivery system for the explicit study of the effects of polymer size, charge, composition, and architecture on physiologically relevant features including lipid bilayer interactions, cytotoxicity, internalisation, and so forth. Tumour absorption, bio distribution, and blood plasma retention period. Significant advancements in the delivery of anti-neoplastic and contrast medicines, neutron capture therapy, photodynamic therapy, and photo thermal therapy have all been made in recent years toward the use of dendrimers for therapeutic and diagnostic purposes in the treatment of cancer.²³

Conclusion

Conjugate folic acid and 5-fluorouracil with monodisperse dendrimer molecule was prepared. The structure of the synthesized compounds was elucidated by Fourier transformer – Infrared Spectro - photometer in KBr-pellet method. The structure of the synthesized compounds were predicted by 1H NMR Spectrophotometer. The potency of conjugate as anticancer agent was judged by estimation of haematological parameters in EAC cell bearing mice. The efficiency of the test compound has been compared with the data obtained by standard and control group. From In-vivo studies we understood that, when cancer is induced in mice (Groups I, II, III) there is decrease inRBC, Hb values and an increase in WBC value. The decrease in Hb and RBCs may be due to anaemia and the anaemia encountered in tumour bearing mice is mainly due to reduction in RBC and haemoglobin percentage. With drug treatment, the altered parameters of hemopoietic system changed to normal levels in cancer bearing mice.

Conflict of Interest

The author has no conflict of interest.

Acknowledgement

The authors are grateful to Rajiv Gandhi University of Health Sciences (RGUHS), Bengaluru, Karnatka for providing financial assistance through research grant to carry out the research work.