Doxorubicin loaded polylactide nanoparticles functionalized histamine promote apoptosis of human gastric cancer cells AGS.

Introduction
Gastric cancer (GC) is one of the most common malignant cancers and the reason for cancer-related death worldwide in the last two decades1,2. Microenvironment of GC reflects the conditions of immune suppression, inflammation, hypoxia, and extracellular matrix remodeling, facilitating migration and metastasis of tumor cells3. Radical surgery followed by chemotherapy administered per os or radiotherapy, also in conjunction with anti-angiogenic therapy and immunotherapy1,4–6, if performed in patients with early gastric cancer, leads to a 90% survival rate in 5 years. In patients with advanced cancer, the prognosis is poor1,4,5. Moreover, oral therapy can be negatively influenced by low gastric pH and ineffective adsorption of the drug in the gut. As a result, therapeutic drug concentration cannot be achieved6,7. Taking into account the benefits of administering drugs per os, there are reasons to improve this method of drug application. Developing proper carriers to deliver and prevent the drug from environmental conditions has been considered. It is a priority due to the liver first-pass effect, poor intestinal permeability, and drug efflux from cancer cells mediated by efflux transporters such as ATP binding cassette transporters (ABC)8,9, as well as the inhibition of the immune system by tumor cells or their adaptation to the niche by various mechanisms. Chemotherapy may also be ineffective due to the development of multi-drug resistance (MDR)7 related to the enhanced production by cancer cells of anti-apoptotic proteins preventing apoptosis, which is the central anti-cancer endogenous mechanism8–10. Considering the above phenomena, developing novel drug delivery systems (DDSs) are designed to target specific tissue and release their cargo in a controlled way in the lesion: passive or active targeting based on functionalization of NPs by proinflammatory agents, may increase vascular permeability, facilitating efficient delivery of NPs and their interaction with cancer cells11,12. To avoid the uncontrolled activity of such molecules, they are attached to the poly(lactic) acid - PLA backbone during the polymerization or post-polymerization process to prepare polymers with active end groups13.
The PLA is frequently used to construct NPs for drug delivery to treat various diseases14–16. Recently, the clinical significance and potential of the poly (lactic acid-glycolic acid) copolymer (PLGA)-based nanostructures have been suggested for their applications in cell transplantation and imaging diagnostics of ischemic stroke17. By utilizing engineered nanoparticles (NPs) that combine polyethylene glycol (PEG) with PLGA, the circulation time and thrombolytic effectiveness of tissue plasminogen activator (tPA) could be extended18. Nevertheless, the influence of PLA end groups on the drug release and anti-cancer efficiency of PLA-based NPs is rather scarce19. One example concerns the functionalization of the PLA backbone with trehalose/dopamine/arginine and the investigation of their interactions with cell surface receptors or proteins20. Our approach with PLA with His end groups is a novel strategy for enhancing the effectiveness of PLA-based NPs loaded with anti-cancer DOX due to the synergistic effect of His and DOX.
It has been shown that His downregulates the proliferation and differentiation of normal and malignant cells21–23. In addition, His increases vascular permeability, potentially facilitating anti-cancer drug delivery to the cancer tissue23–25. In our prior study, the monolayers of AGS gastric cancer cells in in vitro cell cultures were disrupted by DOX-loaded nanoparticles with histamine-modified PLAs (PLA-His), as this led to a reduction in the tight junction protein ZO-124. This study aims to validate the hypothesis that incorporating His residues into NPs DOX (NPs His-DOX) can unseal cancer cell intercellular connections or enhance cell membrane permeability. This alteration would allow particles to enter the cellular environment and subsequently release DOX, thereby achieving optimal therapeutic drug levels. Additionally, encapsulating DOX in His-functionalized PLA NPs also seeks to shield non-cancerous cells from the direct cytotoxic effects of DOX26. In experiments, human gastric cancer cells AGS were exposed in cell cultures in vitro to studied NPs, followed by evaluation of oxidative stress based on lipid oxidation marker—4-Hydroxynonenal (4HNE), DNA damage, and apoptosis in TUNEL assay, as well as detection of caspase 3 (CC3) involved in this process. Furthermore, the cell proliferation and deposition of integrin - intracellular adhesion molecule 1 (ICAM-1) have been determined, which may facilitate the interaction of cytotoxic lymphocytes with cancer cells, thereby driving the elimination of these cells27–29. We expect that the proposed strategy towards AGS cells, based on PLA-His NPs loaded with DOX, will lead to (1) better release of DOX in the cancer milieu, (2) action of the drug in a lower dose due to loosening the cell to cell tight junctions, (3) minimizing damage to non-cancer gastric cells.

Materials and methods

Preparation of NPs by nanoprecipitation
As described previously, the first step in developing NPs was the PLA synthesis with the desired end groups24. The PLAs (Merck KGaA, Darmstadt, Germany) with His (Merck KGaA, Darmstadt, Germany) were prepared in the presence of a catalyst (stannous octoate) or obtained using His as an initiator and polymerization catalyst. Subsequently, we used nanoprecipitation30 method prepared blank or loaded with DOX (Merck KGaA, Darmstadt, Germany) and NPs from the obtained PLAs with His end groups. We used nanoprecipitation methods to obtain these NPs because the NPs loaded with DOX or with His end groups showed stronger cytotoxic properties than NPs obtained by the microfluidic method24. General characteristics of studied NPs have been shown previously24. The size of the resulting NPs ranged from 130 to 250 nm, according to DLS analysis. The schematic procedure for the development of NPs is shown in Fig. 1.

DOX encapsulation efficiency and in vitro drug release
The encapsulation efficiency of DOX was determined after lyophilizing the DOX-loaded NPs and dissolving them in dimethyl sulfoxide (DMSO), as described by Brzezinski et al.24. The fluorescence intensity was measured (λex = 490 nm; λem = 560 nm) to determine the amount of encapsulated drug according to the calibration curve. The encapsulation efficiency (EE) varies between the particle preparation method and the polymeric backbone’s structure, ranging from 49 to 65%. The drug release was investigated in the physiological conditions via dialysis membranes, and from 40 to 50% of DOX was released after 24 h, as described in our previous studies24. Moreover, the results indicated that obtained NPs followed the Korsmeyer-Peppas model, and the Fickian diffusion mechanism could be proposed since n < 0.4524.

Cell culture and conditions of cell exposure to studied formulations
The human AGS (CRL-1739) gastric adenocarcinoma epithelial cell line was obtained from the American Type Culture Collection (ATCC, Rockville, Md., USA). The cells were consistently cultured as a monolayer in complete RPMI-1640 medium (cRPMI; Merck KGaA, Darmstadt, Germany), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; CytoGen, Łódź, Poland) and 1% penicillin/streptomycin (Gibco, Zug, Switzerland). This was done at 37 °C in a humidified environment with 5% CO2. Cells were passaged every seven days using 0.25% trypsin/0.02% EDTA (HyClone, Thermo Fisher Scientific, Waltham, MA, USA), and the medium was refreshed every 3–4 days. All experiments utilized AGS cells at a concentration of 1 × 106 cells/mL.
Different variants of AGS cell cultures were used: in culture medium alone or stimulated for 24 h only with DOX, His, and His-DOX. Furthermore, cells were stimulated with NPs obtained with a catalyst by nanoprecipitation method: empty PLA-OH, PLA-His, PLA-OH loaded with DOX (DOX-loaded PLA-OH), or PLA-His loaded with DOX (NPs PLA-His-DOX). All stimulants were used at a concentration of 1 μg/mL, while DOX was a positive control (PC) (anti-cancer effect) at the concentration of 2 μg/mL, whereas cell culture in culture medium alone was a negative control (NC).

Cell metabolic activity in MTT reduction assay
The influence of empty NPs or NPs containing His, DOX loaded or NPs His-DOX against L929 and AGS cells on metabolic activity of these cells was assessed in the previous cytotoxicity study24 by assessing the cells’ capacity to reduce MTT [(3-(4,5-dimethylthiazol-2-yl)2,5-di-phenyltetrazolium bromide)] as recommended by the Food and Drug Administration and the International Organization for Standardization (ISO) standard 10993-5:2009 Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity.

Preparation of cells for immunohistochemical staining
After stimulation, as described above, cell monolayers were washed three times with phosphate-buffered saline (PBS) (Merck KGaA, Darmstadt, Germany). The cells were fixed with 4% paraformaldehyde solution (Merck KGaA, Darmstadt, Germany) (10 min, room temp.) and washed as described above. Then, cells were permeabilized with 0.02% TritonX100 solution (Merck KGaA, Darmstadt, Germany) (10 min, room temp.) and washed as described above. Free spaces on the plate were blocked with 3% BSA (Merck KGaA, Darmstadt, Germany) in PBS (1 h, room temp.). The cells were then stained with appropriate antibodies according to the methods described below.

Assessment of oxidative stress on the basis of4HNE cell accumulation
Human AGS cells, after exposure to studied NPs (PLA-OH, PLA-His, DOX-loaded PLA-OH or PLA-His), and DOX, His or His-DOX alone, were immunohistochemically stained for detection of 4HNE using rabbit anti-4HNE antibodies fluorescently labeled AlexaFluor560 (Bioss, Woburn, Massachusetts USA) as previously described31. The intensity of fluorescence was measured using a multifunctional spectrophotometer SpectraMax i3 (Molecular Devices, San Jose, CA, USA) at 550 nm (excitation) and 590 nm (emission). Results for 4HNE level were shown in relative fluorescence units (RFU). Three independent experiments were performed in triplicate.

Cell apoptosis and DNA damage
Human AGS cells, after exposure to NPs (PLA-OH, PLA-His, DOX-loaded PLA-OH or PLA-His), DOX, His or His-DOX, were immunohistochemically stained for the presence of pro-apoptotic cleaved caspase 3 (CC3) using mouse anti-CC3 antibodies fluorescently labeled AlexaFluor 790 (Santa Cruz Biotechnology, Dallas, TX, USA) as previously described31. According to the manufacturer’s procedure, apoptosis was also determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Thermo Fisher Scientific, Waltham, MA, USA). DNA damage was determined using primary antibodies against phosphorylated H2AX (Ser139) (Thermo Fisher Scientific, Waltham, MA, USA), which is induced in response to double-strand breaks (DSB), as previously described32. The intensity of fluorescence was measured using a multifunctional spectrophotometer SpectraMax i3 (Molecular Devices, San Jose, CA, USA) at the following wavelengths: 550 nm (excitation) and 590 nm (emission) during the assessment of DNA damage, 782 nm (excitation) and 805 nm (emission) in the TUNEL assay and during CC3 assessment. Results for TUNEL, CC3, and DNA damage were shown in RFU. Three independent experiments were performed in triplicate.

Cell proliferation
Cell proliferation of human AGS cells after stimulation with tested NPs (PLA-OH, PLA-His, DOX-loaded PLA-OH or PLA-His), and DOX, His or His-DOX, as described above, was evaluated using a commercial CyQUANT™ Cell Proliferation Assay (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed with PBS (Merck KGaA, Darmstadt, Germany) and frozen at − 80 °C. Before testing, samples were thawed at room temperature, and cells were lysed in a buffer containing CyQUANT-Red dye prepared according to the manufacturer’s instructions. Fluorescence was measured at 782 nm (emission) and 805 nm (excitation) using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Results were shown in RFU. Three independent experiments were performed in triplicate.

Gastric cancer cell deposition of ICAM-1—ligand for cytotoxic lymphocytes
Human AGS cells, after exposure to studied NPs (PLA-OH, PLA-His NPs, DOX-loaded PLA-OH or PLA-His), DOX, His or His-DOX were immunohistochemically stained using mouse anti-ICAM-1 antibody fluorescently labeled AlexaFluor 647 (Santa Cruz Biotechnology, Dallas, TX, USA) as previously described33. The intensity of fluorescence was measured using a multifunctional spectrophotometer SpectraMax i3 (Molecular Devices, San Jose, CA, USA) at 590 (excitation) and 617 (emission). Results for ICAM-1 were shown in RFU. Three independent experiments were performed in triplicate.

Impact of the studied formulations on AGS cell integrity
The integrity of the AGS cells in cell cultures, in the culture medium alone or after stimulation of cells with tested NPs (PLA-OH, PLA-His, DOX-loaded PLA-OH or PLA-His), DOX, His or His-DOX was determined using a light microscope with an inverted field of view at each time point after 0 h, 24 h, 48 h, and 72 h. The cell integrity was determined as follows: 0—complete cell integrity; 1—low disintegration of cell monolayer (10%), few dead cells; 2—enhanced disintegration of cell monolayer (30%), 20–39% dead cells; 3–50% (medium) disintegration, of cell monolayer, 40–59% dead cells; 4- high disintegration of cell monolayer (70%), 60–79% dead cells; 5- very high disintegration of cell monolayer (90%), 80–99% dead cells; 6-total disintegration of the cell monolayer (100%), only dead cells. The dead cells were assessed on the basis of neutralized metabolic activity in MTT reduction assay as described in paragraph 2.4.
The migration of AGS was evaluated after stimulation of cells with tested NPs (PLA-OH, PLA-His, DOX-loaded PLA-OH or PLA-His), DOX, His or His-DOX, in a “wound healing assay-scratch assay,” which is based on the ability of cells to move into a scratch in the cell monolayer: in the culture medium alone or in the presence of NPs, DOX, His or His-DOX as previously described31,34. The scratched regions were assessed using Image software version 1.48v (National Institute of Health, Bethesda, MD, USA) at 0 h, 24 h, 48 h, and 72 h following cell exposure to the tested probes. The degree of wound healing for each experimental variant was determined by calculating the percentage of cells migrating to the wound area in comparison to untreated cells. Three independent experiments were conducted, each in triplicate.

Statistical analysis
Graphs were generated using GraphPad Prism 10.0 software (https://www.graphpad.com/, GraphPad Software Inc., San Diego, CA, USA). Measurements are presented as median relative fluorescence units (RFU) ± range values. To test differences between groups, we employed the non-parametric U Mann–Whitney test or the Kruskal–Wallis one-way analysis of variance. Statistical analysis was performed using Statistica 13 PL software (https://statistica.software.informer.com/13.3 software, Kraków, Poland). Results were deemed statistically significant at p < 0.05. We assessed normality distribution using the Shapiro–Wilk test (S-W). For data analysis, we applied Dunn’s post-hoc test.

Results and discussion
Nowadays, new DDSs based on NPs that are non-toxic and non-immunogenic are being sought. Modern DDSs should be characterized by the delivery of drugs to pathologically changed tissues, ease of regulation of solubility, bioavailability, and biodistribution of transported biomolecules because most drugs have low molecular weight35,36. Controlled drug delivery can prevent drug-related severe side effects and improve the efficacy of therapy. Delivering a therapeutic dose of the drug is a challenge in the case of GC. These types of tumors are diagnosed at a relatively late stage. To effectively combat gastric cancer, the drug must maintain its activity in the acidic pH of gastric juice. Conventional anti-cancer chemotherapy is often limited due to low water solubility and permeability, limited circulation time, poor drug targeting, low biodistribution, and toxic effects, local or systemic37. It has been shown that NPs, e.g., liposomes, micelles, and dendrimers, can be used in GC treatment to achieve enhanced efficiency, high specificity, reduced toxicity, and excellent stability37. Moreover, biologically active substances can be added to NPs in addition to the drug to increase the anti-GC activity. The effects of biocomponents may be linked with breaking the tight intercellular junctions, ensuring better drug delivery. Shahzad Lodhi et al. formulated purified transferrin using gold NPs capped with cysteine, targeted against GC cells. The cysteine-gold NP-conjugated transferrin demonstrated efficient binding with the cell receptor, suggesting that it could be a novel diagnostic probe for targeting tumor sites38. Yang et al. revealed that lipid-based NPs, such as hyaluronic acid combined with sorafenib (SRF) and cisplatin (CDDP), can be used to treat GC39. Another example is NPs co-loaded with 5-fluorouracil (FU) and paclitaxel coated with a monoclonal antibody targeting sialyl-Lewis A, which mediates GC metastasis40. Studies by Zang et al. have shown that mPEG-PCL (methoxypoly(ethylene glycol)-polycaprolactone) NPs significantly increased cell death in GC malignancies41. Nanotechnology can substantially improve the pharmacokinetic properties of drugs.
In this study, we used DOX-loaded NPs containing PLAs with histamine residues (NPs His-DOX). We employed nanoprecipitation to prepare NPs from the obtained PLAs since the encapsulation efficiency was higher than that of microfluidics. In this study, we used PLAs with hydroxyl (PLA-OH) or histamine (PLA-His) end groups obtained with or without a catalyst24. The functionalization of the polymeric backbone is very important in terms of the activity of the resulting NPs. Previously, we showed the highest cytotoxic activity of DOX-loaded NPs containing PLAs with histamine residues compared to non-functionalized ones, such as PLA-OH loaded with DOX24. Furthermore, using a confocal laser scanning microscope, we showed the intracellular accumulation of DOX in the AGS cells treated with the prepared NPs. It has been demonstrated that DOX-loaded NPs can penetrate the cell membrane24. The current study has been dedicated to determining the effectiveness and potential mechanism of anti-cancer activity of the developed NPs (PLA-OH, PLA-His, DOX-loaded PLA-OH, or PLA-His).
In this study for demonstration and discussion, the results for NPs obtained with a catalyst are provided due to the lack of differences for the NPs variant obtained without a catalyst. In the first stage of the study, the influence of DOX, His, His-DOX, and NPs: PLA-OH, PLA-His, DOX-loaded PLA-OH, PLA-His-DOX on the integrity of the AGS cell monolayer after 24 h, 48 h, and 72 h of cell exposure was assessed versus the integrity of cell monolayer in culture medium alone or integrity of cells treated with DOX at 2 µg/mL, which consisted a positive control for cell monolayer disintegration. The images of the cell monolayers are shown in (Fig. 2). In our previous study24, to determine the cytotoxicity of tested NPs against the reference non-cancer mouse fibroblasts or AGS cancer cells, the following concentrations of DOX were tested: 2.5, 1.25, 1.0, 0.5 and 0.25 µg/mL. At 2.5 µg/mL of DOX, the studied NPs showed high cytotoxicity towards fibroblasts (10% live cells versus 90% dead cells) or AGS cells (15% live cells versus 85% dead cells). At 1 µg/mL of DOX, NPs were not cytotoxic to fibroblasts (29% dead cells versus 71% live cells)—meeting in vitro biosafety conditions, according to ISO norm (cell viability minimum 70%), and showed slight cytotoxicity towards AGS cells (60% live cells versus 40% dead cells). In the present study a concentration of DOX 2 µg/mL, which was low cytotoxic against cancer cells, was selected to see whether His may improve this effect of DOX. The cell integrity was assessed according to the scale, which facilitated grading of the area of cell detachment, which was demonstrated as a heat map (Fig. 3). The following grading of cell disintegration was developed: (0)—no cell disintegration; (1)—10% (low); (2)—30% (enhanced); (3)—50% (medium); (4)—70% (high); (5)—90% (very high), and (6)—100% (total) disintegration of the cell monolayer.

No influence on the integrity of AGS cell monolayer was observed in the culture medium with un-loaded PLA-OH compared to cell monolayers in the culture medium alone. All other stimulants: DOX, His, His-DOX, or PLA-OH, PLA-His NPs, DOX-loaded PLA-OH or PLA-His NPs caused the disintegration of the AGS cell monolayer after 24 h, 48 h and 72 h of culture (Figs. 2 and 3). DOX caused the disintegration of the cellular monolayer by 50%, 70%, and 90% after 24 h, 48 h, and 72 h, respectively (Figs. 2 and 3). In cell cultures with His, after 24 h and 48 h, only 10% disintegration of the cell monolayer was observed. In 72 h cultures, the cell damage increased to 30% (Figs. 2 and 3). The His-DOX induced 30%, 50%, or 70% disintegration of the cellular monolayer after 24 h, 48 h, and 72 h, respectively (Figs. 2 and 3). In the presence of DOX-loaded PLA-OH, after 24 h, the cell disintegration reached 30%, whereas after 48 h and 72 h, it was 50% (Figs. 2 and 3). Treatment of cells with PLA-His resulted in 10%, 50%, and 70% disintegration of the cellular monolayer after 24 h, 48 h, and 72 h, respectively.
Most importantly, stimulation of cells with PLA-His-DOX resulted in 30%, 50%, and 90% disintegration of the cellular monolayer after 24 h, 48 h, and 72 h, respectively (Figs. 2 and 3). From studied NPs variants, those containing His (PLA-His, PLA-His-DOX) induced the most potent cytotoxic effect, causing cell disintegration, and His enhanced the cytotoxic effect of DOX. Increased disintegration of AGS cell monolayer by PLA-His-DOX may be caused by the presence of histamine in the NPs. Several studies revealed that His could increase microvascular endothelial paracellular permeability42–44. Majno and Palade (1961) first suggested that histamine works by opening the inter-endothelial-cell tight junctions, facilitating the penetration of DOX-loaded NPs into the cancer milieu. Previously, we showed that histamine attached to NPs DOX-loaded reduces the amount of tight junction protein ZO-1 in AGS cells24.
His acts through its four G-protein-coupled receptors (GPCRs)—histamine receptors 1 to 4 (H1R to H4R) by inducing the cascade of inflammation and increasing vascular permeability in the gastrointestinal tract, endothelial cells, and lymphocytes45. Zabner et al. showed that His alters E-cadherin mediated cell-to-cell adhesion of airway epithelial cells46. Komarova and Malik revealed that His may regulate endothelial cell permeability via paracellular and transcellular transport pathways47. Permeability mediators, including His and H1R, lead to the rapid mobilization and intracellular secretion of Ca2+, and the Rho/ROCK activation pathway is critical for His-induced cell barrier disruption48–50. The above-suggested mechanisms of His related enhancement of cell barrier permeability may also improve the delivery of DOX encapsulated in the studied NPs His-DOX.
The disintegration of the AGS cell monolayer in this study was combined with the inhibition of cell migration in the presence of NPs containing DOX, His, or His-DOX, confirming a downregulation of cell functions. In the wound healing assay, the cells migrated only in the culture medium alone or in the presence of PLA-OH NPs, and the wound gradually healed over time. The remaining stimulants did not promote AGS cell migration and wound healing over time and caused detachment of cells from the adhesive surface (Fig. 4). The strongest inhibition of cell migration and wound healing was shown in the cell cultures with NPs PLA His-DOX.

During carcinogenesis and cancer progression, cancer cells undergo metabolic changes resulting in the accumulation of different metabolites51–53, including 4-HNE—a marker of lipid oxidation and therefore the increased oxidative stress, in conjunction with DNA damage and chromosomal aberrations54, inhibition of DNA repair processes55,56 followed by cell apoptosis57,58.
AGS cells cultured in culture medium alone or treated with PLA-OH do not stimulate 4HNE production over 24 h, 48 h, and 72 h. DOX, His, or His-DOX alone significantly enhanced (p < 0.05) 4HNE production over time, similarly to NPs DOX-loaded PLA-OH, PLA-His, and PLA-His-DOX. Adding DOX to NPs with His (PLA-His-DOX) significantly enhanced (p < 0.05) oxidative stress compared to PLA-His alone over 24 h, 48 h, and 72 h (Fig. 5). Interestingly, PLA-His-DOX induced the highest 4-HNE production after 24 h of cell exposure to this NPs formulation (Fig. 5A).

DNA was not damaged after 24 h, 48 h, and 72 h in cell cultures of AGS cells in culture medium alone or treated with His alone, PLA-OH or PLA-His. Attachment of DOX to His (His-DOX) or PLA-His-DOX variant resulted in significantly increased (p < 0.05) DNA damage compared to His alone or PLA-His after 24 h, 48 h, and 72 h of cell exposure. Introduction of His, DOX, or His-DOX into NPs maintained the effect of DNA damage by DOX, His, or His-DOX alone (Fig. 6). Although His or PLA-His increased oxidative stress, the results show no correlation with DNA damage. These may indicate that in the milieu of His/PLA-His, the cellular anti-oxidative mechanisms may play a role. By comparison, His-DOX or PLA-His-DOX formulations significantly increased (p < 0.05) oxidative stress related to DNA damage. The effect of PLA-His-DOX may be due to DOX-driven oxidative stress and/or the direct interaction of DOX with DNA.

Furthermore, we hypothesized whether the DNA damage is followed by cell apoptosis as an expected anti-cancer effect. The presence of PLA-OH in cell culture did not induce a proapoptotic CC3 and signs of apoptosis after 24 h, 48 h, and 72 h of cell exposure, which was consistent with no DNA damage. After 24 h, only in the DOX environment, the number of AGS cells undergoing apoptosis was increased, as shown by the TUNEL assay. Regarding the production of CC3, only an increasing trend was demonstrated. After 48 h or 72 h, DOX, His, His-DOX, DOX-loaded PLA-OH, and PLA-His-DOX significantly (p < 0.05) enhanced apoptosis of AGS cells. The results in Figs. 7 and 8 show that the presence of His, DOX, or His-DOX in the NPs interior facilitates the maintenance of the effect of DNA damage and the enhancement of apoptosis by DOX, His, or His-DOX alone (Figs. 7 and 8). Under these conditions, the number of cells undergoing apoptosis was related to increased oxidative stress, accumulation of 4 HNE in cells, and cell proliferation inhibition (Fig. 5)56–58. A reduction of AGS cell proliferation was demonstrated after 24 h, 48 h, and 72 h coincubation of cells with tested soluble substances and NPs. However, the statistically significant (p < 0.05) inhibition of cell growth was noted after 24 h and 48 h in the milieu of DOX, PLA-His-DOX, and after 72 h in the milieu of DOX, His-DOX, DOX-loaded PLA-OH, PLA-His as well as PLA-His-DOX (Fig. 9). However, it has been shown that His can downregulate cell proliferation and differentiation of normal and malignant cells, and a suggested mechanism depends on histamine receptors—H2R antagonists21–23. In addition, His promotes increased vascular permeability and potentially may help to deliver DOX23,25. In vivo histamine dihydrochloride is used in numerous clinical trials as an adjuvant to IL-2-driven immunotherapy involving T lymphocytes and natural killer (NK) cells59.

However, the role of ICAM-1 in anti-cancer response related to His cannot be excluded. This integrin is expressed in various cell types (epithelial cells, keratinocytes, fibroblasts, and immune cells), playing an essential role in cell–cell, cell-extracellular matrix interaction, cell signaling, and immune process. It facilitates the transmigration of inflammatory cells60,61. Although intense expression of ICAM-1 may facilitate the spreading of cancer cells, on the other hand, it may also reflect the elevated immunity against tumor cells by rendering tumor cells more sensitive to adhesion of CD8 + T lymphocytes and lymphocyte-mediated cytotoxicity62–66. Scholer et al. showed that ICAM-1 blockade decreased infiltration of cytolytic T lymphocytes to the tumor microenvironment (TME)67. The increased ICAM-1 expression on other cells in the TME also enhances the tumor infiltration by CTLs and activation of these cells68–70.
We showed diminished ICAM-1 deposition on AGS cells cultured in the presence of DOX, which was increased on AGS exposed to His, His-DOX, or NPs PLA-His and PLA-His-DOX, and this effect was related to the presence of His (Fig. 10).

Results of this study reveal that NPs containing His and DOX-loaded showed better cytotoxic effect than DOX-loaded NPs against gastric cancer cells AGS in vitro due to induction of elevated oxidative stress in conjunction with an increased DNA damage and apoptosis while diminished cell proliferation. The increased deposition of ICAM-1 on AGS cells exposed to NPs His-DOX was shown in cell cultures. It may facilitate the elimination of cancer cells by cytotoxic lymphocytes in vivo. DOX-loaded NPs composed of PLA-His, due to the presence of His, disintegrate the AGS cell monolayer better than DOX-loaded PLA-OH NPs, which may facilitate penetration of the neoplastic lesion by NPs and effective drug delivery to the tumor cells. However, the cytotoxicity of His-DOX was higher than that of PLA-His-DOX. The potential reason could be PLA. The effect of PLA-His-DOX is lower, maybe due to the slow release of loaded DOX. This may facilitate a prolonged effect of drug delivered in the site of cancer tissue.
Further studies are needed to determine whether histamine-enriched and DOX-loaded NPs may contribute to tumor control in animal models of gastric tumors. The results of this study demonstrated that NPs obtained with a catalyst were similar to NPs developed without a catalyst, suggesting that both procedures for preparing NPs are helpful.
To sum up, nowadays GC it is one of the most common cancers worldwide, particularly prevalent in Asia, especially in China, where both its incidence and mortality rates are significantly high. Common clinical treatments for GC show different efficacy and limitations. Based on NPs, DDS are being used to treat GC to achieve greater anti-cancer efficiency, high specificity, and stability while lower toxicity towards non-cancer cells. In this study, we used PLA NPs containing His as end groups of PLA and loaded with DOX. These NPs are characterized by biocapacity, controlled drug release, and better drug penetration due to the addition of His. This is of particular importance due to the coverage of cancer cells with the extracellular matrix, which hinders the penetration of the drug into the deeper layers of the tumor and the remodeling of the extracellular matrix.
The studied NPs PLA-His-DOX potentially may offer several advantages in the treatment of GC:Targeted drug delivery per os, selectively transporting the drug cargo to the tumor milieu while minimizing damage to non-cancer tissues.

Prolonged systemic drug circulation due to its sequential release from NPs.

Overcoming multidrug resistance by adjusting drug release rates.

Increased immune response against GC by increasing ICAM-1 deposition, which makes tumor cells more sensitive to CD8 + T lymphocytes and induction of lymphocyte-mediated cytotoxicity.

An in vivo mouse model of GC—FVB/N-Tg (Ins1-GAS)1Sbr/J will be introduced in a future research for preclinical evaluation of the studied NPs. We expect that these new NPs will improve the efficacy of GC treatment, facilitate overcoming the limitations of classical therapies due to drug resistance of cancer cells, and minimize side effects towards non-cancer cells.

Conclusions
The rationale behind using histamine-functionalized PLAs combined with DOX towards gastric cancer cells was to achieve the combined effect of His, which influences the integrity of cell membranes, and the classical anticancer drug DOX. The present study demonstrates that the NPs, DOX-loaded and composed of PLA with histamine end groups, exhibit better anti-cancer activity against AGS cells in cell cultures in vitro, comparable with DOX-loaded NPs composed of PLA with hydroxyl end groups.
DOX-loaded NPs composed of PLA-His facilitated the AGS cell monolayer disintegration and delayed monolayer reconstruction in response to mechanical damage in a "wound healing test—scratch assay." These effects were linked with higher oxidative stress, increased DNA damage, elevated apoptosis, and inhibition of cell proliferation. It was also shown that NPs His-DOX significantly increased the production of the cell adhesion molecule ICAM-1, which in vivo can facilitate the interaction of immunocompetent cells with cancer cells. The mobilization of such cells may increase the effectiveness of the anti-cancer immune response.
The results presented herein reveal the increased potential of obtained NPs His-DOX towards gastric cancer cells in vitro. Further study using this novel NPs formulation and advanced cancer cell models in vitro, facilitating the assessment of NPs cell interactions, will confirm the anti-cancer potential of studied NPs before using dedicated animal models.