## 2 Materials and Methods  
_Chemicals_: Chitosan (medium molecular weight, CAS No. : 9012-76-4), lactic acid (CH\({}_{3}\)CH(OH)COOH) (80%, Chimtex, Dimitrovgrad, Bulgaria), Tween(r) 80 (CAS No. :9005-65-6, Sigma-Aldrich, Darmstadt, Germany), sodium tripolyphosphate (TPP) (CAS No. :7758-29-4, Acros Organics, Geel, Belgium), phosphate-buffered saline (PBS, P-3813, Sigma Aldrich), and hydrochloric acid (CAS No. : 7647-01-0, Sigma-Aldrich, Darmstadt, Germany) were used in the experiments. _Chitosan particle synthesis_: The chitosan particles were synthesized by the ion gelation method [41] with modifications. A 0.5 % chitosan solution (40 mL) was prepared in 1% lactic acid under magnetic stirring. Then, 1% Tween 80 (20 mL) was added to the transparent acidic chitosan solution and 2% sodium tripolyphosphate (TPP) (32 mL) was gradually added drop by drop to the mixture, which was agitated for 40 min under magnetic stirring at 500 rpm. The obtained suspension was sonicated for 4 min in an ultrasonic homogenizer (SONOPULS HD 2070.2, BANDELIN electronic GmbH & Co. KG, Wertheim Germany) and subsequently centrifuged for 15 min at 5300\(\times\)\(g\) in a Heraeus Labofuge 200 (Thermo Electron Corporation, Waltham, Massachusetts, USA). The obtained chitosan particles were washed with milli-Q water three times, freeze-dried in a laboratory freeze dryer (Biobase, BK-FD10P, Jinan City, Shandong Province, China), and stored in a desiccator for further use. The synthesized biopolymer particles were eco-friendly because they were made from natural products that are ecological and have no toxic side effects. The chitosan microparticles were stable after storage for 6 months, which was proven by periodic measurements of their size and an exploration of their morphology under a digital microscope. _Cells and treatment protocol_: The experiments were performed on a colon cancer epithelial cell line (Colon 26) kindly provided by Prof. [PERSON] of the Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences. The cells were cultured in Dulbecco's Modified Eagle's medium (DMEM, Sigma-Aldrich, Germany) with a high concentration of glucose (4500 mg/L) and supplemented with 10 % heat-inactivated fetal bovine serum (FSB, Sigma-Aldrich, Germany) and antibiotics (100 U/mol penicillin and 100 ug/mol streptomycin) in a humidified atmosphere at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). Twenty-four hours before the experiments were conducted, adhesive cells were trypsinized, centrifuged (800\(\times\)\(g\)/5 min), counted (using the trypan blue method), and re-suspended in fresh medium without antibiotics but with specific pH values obtained previously. The re-suspension of the cancer cells in the medium without antibiotics was included in the treatment protocol in order to eliminate their potential to have an influence on the biochemistry of the cultured cells and an impact on the experimental endpoint [42]. Colon 26 epithelial cells were seeded in 12-well plates at a density of 5 \(\times\) 10\({}^{4}\) cells per 1 mL of cultured medium and incubated in a humidified atmosphere at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). Untreated cells (cells incubated only in fresh medium without antibiotics and with a pH = 8.0) were used as controls for all experiments. The cells were divided into three groups. To simulate different acidosis conditions, cells were exposed to a medium with a corrected pH. Two types of acids were used in the experiments: 1% lactic acid (group 1) and 0.1 mol/L hydrochloric acid (group 2). Different volumes from both types of acids were added drop by drop into a cell medium (pH = 8.0) under permanent mixing to obtain six different specific pH values (pH = 6.0, pH = 6.6, pH = 7.0, pH = 7.4, pH = 7.8, and pH = 7.9). Group 3 contained cells exposed to normal and pH-corrected mediums and treated with the newly synthesized chitosan microparticles applied at concentrations of 1000 mg/mL, 500 mg/mL, 200 mg/mL, 100 mg/mL, 50 mg/mL, 25 mg/mL, and 10 mg/mL.