Doxorubicin (DOX) is a common chemotherapeutic drug for treating a number of cancers and widely used as the first-line in breast cancer treatment [1-4]. Over time, cancer cells develop resistance to chemotherapy, which is a significant contributor to chemotherapy failure and disease recurrence [5,6]. The drug resistance is one of the challenges that need to be solved urgently in clinical approaches [7]. The resistance of the cancer to the chemotherapy is a complex phenomenon involving multiple mechanisms such as DNA damage repair ability, cell cycle change, apoptosis retardation, epigenetic modifications and abnormal activation of multiple signaling pathways [8,9]. In this regard, it is necessary to reduce chemotherapy DOX doses and modify its bio-distribution in order to reduce its side effects, improve its concentration in tumors and decrease its drug resistance [10-12].

Nanoparticles (NP)-based drug delivery has been widely investigated for reducing the chemotherapeutic agents’ diverse side effects and improving their anti-tumor activity by specifically targeting the cancer cells owing to impaired lymphatic drainage and the distinctively disorganized vasculature of cancer cells with many pores, leading to an enhanced permeability and retention effect which can be utilized by nano-drug delivery system as it augments the accumulation of the chemotherapeutic drugs in tumors and minimizes their uptake by healthy tissue [13]. With their selective targeting property and usefulness as a carrier agent, zinc oxide nanoparticles (ZnONP) can be good substitutes for traditional cancer therapy [14].

ZnONPs have potent inhibitory effects against tumor cells due to their inherent toxicity, which they achieve by generating intracellular reactive oxygen species (ROS) production and triggering the apoptotic signaling pathway, making them a good candidate as anti-cancer drugs [15,16]. ZnONP could exhibit high cancer cell selectivity, retention and regulated release of ligated as well as loaded drugs [17,18].

Folic acid (FA) is absolutely essential for DNA methylation, repair and synthesis, as well as the metabolism of amino acids and RNA. Thus, FA plays a crucial role in cell growth, proliferation, and survival, emphasizing its special significance in the establishment and maintenance of cancer cells [19]. Cancer cells strongly express FA receptor or folate receptor (FR) comparing to normal cells that are poorly expresses or absent, and this is associated with advanced stage disease and is a negative breast cancers prognostic factor. Consequently, different tumor cells have been reported to express FR more frequently and it was found to be associated with poor chemotherapy response and tumor survival outcome [20]. The location of folic acid receptor (FAR), density, distribution pattern, higher FA absorbing capacity, and specificity towards FA make it a potential target for nanotherapeutics [21]. Addition of molecular targeting agents such as FA to nanosized delivery systems, such as ZnONP, to provide selective delivery of DOX, increased drug accumulation in the tumor area and controlled release of the drug, thereby reducing toxicity and thus improving the benefit/risk profile for patients [22].

Tumors treatment with NP loaded with conventional chemotherapeutics and other adjuvants such as folic acid resulted in the induction of anti-tumor immunity, thereby inhibiting primary tumor growth without causing side effects. Most significantly, it prevented primary tumor metastasis and recurrence. NP combined with conventional chemotherapeutics and other adjuvants act as an excellent nano-drug delivery system (DDS) in cancer therapy [23] and will improve the safety and efficacy of cancer immunotherapies that provide durable therapeutic responses [24-26].

The composition of tumor immune infiltration changes in response to anti-neoplastic drugs, which is decisive for the success of treatment. Therefore, increased numbers of intra-tumoral T lymphocytes and increased proportion of cytotoxic CD₈⁺-T lymphocytes (CTLs) after chemotherapy predict a favorable therapeutic response in human breast cancer treated with NP containing the systemic chemotherapeutic drug DOX and other adjuvants such as FA (NP /DOX/FA) [27,28]. Several clinical researches have shown that severe lymphopenia can adversely affect the response to chemotherapy in several types of cancer [29]. Thus, mouse tumors responded more efficiently to NP- conjugated DOX and other adjuvants when they grow in syngeneically immunocompetent mice than when they grow in immunodeficient hosts [30,31].

Notably, the essential contribution of the immune system to the success of systemic chemotherapy has been validated in several transplantable mouse models of carcinoma, sarcoma, and lymphoma [32,33]. Both transplantable neoplasms and tumors brought on by the chemical methylcholanthrene (MCA) are susceptible to immune surveillance, which causes them to progress slowly in immunocompetent. As an alternative, oncogene-driven cancers might learn how to activate immune escape mechanisms and/or fail to activate a natural immune surveillance [34]. Numerous efficient therapeutic anti-cancer drugs, such as chemotherapy and chemotherapeutic-loaded nanoparticles (NP) conjugated with DOX and/or FA, can result in the characteristics of cancer immunogenic cell death (ICD). To stimulate a protective anti-cancer immune reaction, ICD-susceptible cells can be used as vaccines. Additionally, ICD-inducing anti-cancer treatments like chemotherapeutic-loaded NP combined with FA lose their effectiveness when cancer cells' ICD signal emission is suppressed, when the immune system's ability to perceive these signals is hampered, and when vital immune effectors, like lymphocytes CD₄⁺-T cells and CD₈⁺-T cells, are depleted [34].

In view of these considerations, the current study was designated to investigate the anti-tumor immune response and anti-oxidant effects of doxorubicin (DOX)-conjugated ZnONP (ZnONP/DOX), folic acid-conjugated ZnONP (ZnONP/FA) and DOX and folic acid-conjugated ZnONP (ZnONP/DOX/FA) in breast cancer model.

Reagents

Zinc oxide nanoparticles (ZnONP) were manufactured in Nanotech. Lab. (Inc., Cairo, Egypt). Doxorubicin (DOX) was dissolved in phosphate-buffered saline (PBS: Lonza, Bio Whittaker, USA) and frozen at -80°C until use. Folic acid (FA: Sigma-Aldrich Co., USA. Ammonium chloride potassium (ACK) buffer (Lonza, Bio Whittaker, USA). Monoclonal antibodies: CD₄, CD₈, CD₃₃₅ purchased from Pharmingen (San Diego, CA, USA).

Synthesis of ZnONP Composites

ZnONP has been prepared as previously described [35] by hydrolysis and condensation of zinc acetate dihydrate using potassium hydroxide at low temperature. ZnONP precipitate washed with methanol and then dispersed in methanol/chloroform mixture. ZnONP/DOX conjugate were prepared by coating DOX onto ZnONP outer surface. ZnO nanopowder dispersion (5 mg/mL) in distilled water (DW) and a DOX solution (5 mg/ml) in DW were mixed at a 1:1 volume ratio and incubated for 1 h at room temperature. After centrifugation at 8000 g for 15 min, the ZnONP/DOX conjugates pellet was resuspended in distilled water and stored at - 80 °C. ZnONP/FA conjugates were prepared by coating FA onto ZnONP outer surface. ZnO nano powder dispersion (5 mg/mL) in distilled water (DW) and a FA solution (5 mg/ml) in DW were mixed at a 1:1 volume ratio and incubated for 1 h. After centrifugation at 8000 rpm for 15 min, the ZnONP/FA conjugates precipitate was resuspended in distilled water and keep at -80°C. To prepare ZnONP/FA/DOX conjugates, 0.2 g ZnONP/FA conjugates was suspended in 100 ml of PBS (pH 7.4) then sonicated for 1h. 10 ml of DOX (50 mg/25ml), was added to ZnONP/FA and stirred at dark condition overnight then centrifuged at 1500 rpm for 10 min. After loading, UV spectrum of supernatant observed at 480 nm was 0.460 cm^-1. To determine loading capacity, the precipitate was collected and dried in vacuum dictator.

Mice

Seventy female Swiss albino mice (6-8 weeks old, weighing 25±2g) purchased from National Research Centre Animal House (Dokki, Giza, Egypt) were divided into 7 groups (n=10) and given a standard pellet diet and tap water ad libitum under hygienic conditions. The experimental protocol was conducted according to the principles for the use and care of research animals National Institutes of Health (NIH) guidelines, as well as the recommendations of the Institutional Animal Ethical Committee (EAC), Faculty of Science, Tanta University, Tanta, Egypt (approval #: IACUC-SCI-TU-0062).

Tumor Cell Line and Tumor Model Preparation

EAC tumor cell line (Pharmacology and Experimental Oncology Unit, National Cancer Institute, Cairo University, Cairo, Egypt) retained in ascitic tumor form in naïve female Swiss albino mice by weekly IP transplantation of 1×10⁶ cells/mouse [36]. The ascitic fluid of EAC tumor cells was collected from the mice peritoneal cavity and diluted with PBS and counted using Trypan Blue dye exclusion assay in a Neubauer hemocytometer (cell viability was > 95%). To prepare the tumor model, 0.25 × 10⁵ EAC tumor cells were IP inoculated into the naïve female Swiss albino mice.

Tumor Implantation and Experimental Design

Tumor implantation and in vivo experimental design is illustrated in Figure 1. Sixty female Swiss albino mice were IP implanted with 2.5 × 10⁵ EAC cells suspended in 100 µl PBS/mouse and divided into six groups (n=10). On day 7 after EAC tumor transplantation, group 1 (EAC group) IP administered with PBS, group 2 (DOX group) IP injected with DOX (0.4 mg/mouse), group 3 (ZnONP group) IP received ZnONP (0.5 mg/mouse), group 4 (ZnONP/FA group) IP inoculated with ZnONP/FA (0.5 mg/mouse), group 5 (ZnONPs/DOX group) IP injected with ZnONP/DOX (0.7 mg/mouse) and group 6 (ZnONP/DOX/FA group) IP administrated with ZnONP/DOX/FA (0.8 mg/mouse) once/day for six days. On day 15 post EAC inoculations, all groups of mice were sacrificed, spleen and peripheral blood (PB) were harvested for cellularity and anti-tumor immune responses investigations and sera were collected for biochemical analyses. Depend upon our previous unpublished data, the nominated in vivo intraperitoneal (IP) doses of ZnONP nanocomposites used here were 0.4 mg/mouse, 0.5 mg/mouse, 0.4 mg/mouse, 0.6 mg/mouse and 0.8 mg/mouse for DOX, ZnONP, ZnONP/FA, ZnONP/DOX and ZnONP/DOX/FA, respectively.

Figure 1: Tumor challenge and in vivo study design.

Tumor Cells Collection and Quantifying

Ascitic EAC tumor cells were collected from treated and nontreated-EAC mice using a 5 ml-syringe containing PBS, after they being anesthetize/analgesized. The EAC tumor cells were washed twice and resuspended in PBS. ACK buffer was added to lyse red blood cells. The suspensions of EAC cells were centrifuged (3000 g, 5 min, 4 °C). The EAC cell pellets washed and resuspended in PBS. EAC cells counts and viability were investigated by a Trypan blue dye exclusion assay (cell viability was > 95%).

Assessment of Total Apoptosis Rate of EAC Tumor Cells by Flow Cytometry

Ascitic EAC tumor cells harvested from cancer-bearing mice treated with PBS, DOX, ZnONP, ZnONPs/DOX, ZnONPs/FA and ZnONPs/DOX/FA. EAC tumor cells washed by ice-cold PBS and the cell density was calculated. EAC tumor cells resuspended in 1X annexin-binding buffer to obtain a final density of 1 × 10⁶ cells/ml. 100 μl of the cell suspension was placed into 1.5-ml eppendorf tubes and 5-μl annexin V-fluoresceinisothiocyanate (FITC), and 1 μl PI (100 μg/ml) working solution was added. EAC tumor cells incubated for 15 min at room temperature and resuspended in 400 μl of 1X annexin-binding buffer, gently mixed, and the samples were then stored on ice. The cells were subsequently subjected to a flow cytometric analysis.

Splenocytes Collection and Counting

Single-cell suspension and count of splenocytes were prepared as previously described [37,38]. In brief, spleens harvested from the treated and nontreated-EAC mice were washed with PBS. Splenocyte suspensions were prepared by dissociating spleen tissues on 60 μm mesh sieves screens (Sigma, St. Louis, MO) and lysing of RBC was carried out with ACK buffer. Splenocytes counts and viability were investigated by a Trypan blue dye exclusion assay (cell viability was > 95%). Splenocytes then washed in PBS and diluted in RPMI 1640 provided with 5% fetal calf serum (FCS).

Flow Cytometric Analysis of CD4+-T, CD8+-T and CD335 Cell Subsets

Fresh single-cell suspensions of splenocytes were prepared from spleen of testing mice. 1 × 10⁶ splenocytes were stained with Anti-mouse CD₄ (clone: RM4-5; BD biosciences, San Diego, CA, USA), anti-mouse CD₈ (clone: YTS-169; BD biosciences, San Diego, CA, USA) and anti-mouse CD₃₃₅ (NKp46) (clone: 29A1.4; BD biosciences, San Diego, CA, USA) then left for 30 min at room temperature in the dark before being cooled on ice for 1 min. The stained splenocytes were washed twice and resuspended in 0.3 ml of 0.5% bovine serum albumin and 0.02% sodium azide solution. Flow cytometry was used to detect the CD₄⁺-T, CD₈⁺-T cell subsets, and CD₃₃₅ cell subsets using a FACS Calibur system (BD Biosciences, San Jose, CA). All data analysis was done with Cell-Quest software (Becton Dickinson, San Jose, CA).

Sera Collection

Blood was gathered from the retro-orbital sinus of the mice into test tubes and left to stand for 3 hours to ensure complete coagulation. After centrifugation (3000 g, 10 min), the sera samples were sucked out and stored at - 80°C for biochemical analysis.

Analysis of the Peripheral Blood Immune Cells Profile

Mice were anesthetize/analgesized and blood was gathered from retro-orbital sinus in heparinized microhematocrit tubes. A Nihon Kohden automated haematology analyzer (model MEK-6318K, Japan) was used to assess blood parameters, including the counts leucocytes (WBC) (10³/cmm) and relative percentages of their differentials (neutrophils, lymphocytes, basophils, and monocytes) in peripheral blood (PB).

Evaluation of Oxidative Stress Markers

The concentration of superoxide dismutase (SOD) in serum was determined spectrophotometrically based on the superoxide radicals generation produced by xanthine and xanthine oxidase, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazon dye. Briefly, mixed substrate (300 μL) was added to diluted hemolysates (200 μl). The samples were mixed well and 75 μl xanthine oxidase was added to reactions. The absorbance was monitored at 505 nm and the level of SOD was then evaluated according to the instruction of the manufacturer (Ransod®-Randox Lab, Antrim, UK).

The concentration of catalase (CA) in serum was measured spectrophotometrically as previously described [39]. Briefly, 10 μl of sample was incubated with 100 μmol/ml of H₂O₂ in 0.05 mmol/l Tris-HCl buffer for 10 min. The reaction was terminated by rapidly adding 50 μl of 4% ammonium molybdate. Yellow complex of ammonium molybdate and H₂O₂ was measured at 410 nm.

The level of malondialdehyde (MD) in serum was measured using the thiobarbituric acid reaction method [40]. Quantification of the reactive substances of thiobarbituric acid was determined at 532 nm by comparing the absorption to the standard curve of MDA equivalents generated by acid-catalyzed hydrolysis of 1,1,3,3-tetramethoxypropane. To measure the serum level of MDA, a working solution containing 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.25 N hydrochloric acid was prepared. For each sample, 250 μl serum and 500 μl working solution were mixed and placed in boiling water for 10 min. The samples then centrifuged (5000 g, 10 min) after getting cool. Finally 200 μl of each supernatant was transferred to microplates and the optical densities at were measured 535 nm.

Statistical Analysis

The numerical data was reported as a mean ± SD. Statistical analysis was carried out by a one-way analysis of variance (ANOVA). Statistical significance was evaluated using a Tukey and a post-hoc test followed by Dunnett’s multiple comparison tests. All p values were two sided, with p < 0.05 considered significant.

In Vivo Anti-Tumor Activity of Znonp Conjugates

The results in Figure 2 revealed that treatment of cancer-bearing mice with DOX, ZnONP, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA resulted in significant decrease in the fold change of EAC tumor cell counts comparing to that in cancer-bearing mice received PBS (0.39±0.03, 0.67±0.12, 0.23±0.04, 0.36±0.05, 0.21±0.02, respectively versus 1.00±0.00) (Figure 2). Additionally, comparing to their values in cancer-bearing mice received DOX, fold change of EAC tumor cell counts recorded remarkable decrease in cancer-bearing mice inoculated with ZnONP/DOX, ZnONPs/FA and ZnONPs/DOX/FA conjugates (0.39±0.03 versus 0.23±0.04, 0.36±0.05 and 0.21±0.02, respectively) (Figure 2). Importantly, the treatment of cancer-bearing mice with ZnONP recorded significant elevation in the fold change of EAC tumor cell counts comparing to that in cancer-bearing mice received DOX alone (0.67± versus 0.39±0.03) (Figure 2). Fold changes of EAC tumor cell counts in cancer-bearing mice received ZnONP/DOX, ZnONPs/FA and ZnONPs/DOX/FA recorded significant decreases comparing to their values in cancer-bearing mice received ZnONP alone (0.23±0.04, 0.36±0.05 and 0.21±0.02, respectively versus 0.67±0.13 ) (Figure 2).

Figure 2: In vivo anti-tumor potentials of ZnONP conjugates on EAC tumor-bearing mice. EAC-bearing mice IP injected with PBS, DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP /DOX/FA. Ascitic EAC cells were harvested from peritoneal cavity and washed twice with PBS and their viability was determined using trypan blue assay. Data were represented as mean ± SE (n= 10). Difference between groups was considered statistically significant at P < 0.05. Note: ^a,b Statistically significant difference as compared to the corresponding means of EAC tumor-bearing mice received PBS(a) and EAC tumor-bearing mice received DOX (b) within each column.

Assessment of Total Apoptosis Rate Of EAC Tumor Cells By Flow Cytometry

The data herein revealed that the treatment of cancer-bearing mice with DOX, ZnONP/DOX and ZnONP/FA remarkably increased the total apoptosis rate of EAC tumor cells comparing to that in cancer-bearing mice received PBS alone ( 15.05±0.05, 19.30±1.55 and 9.45±0.45, respectively versus 6.65±0.37) ( Figure 3). Injection of cancer-bearing mice with ZnONP/DOX resulted in obvious increase in the total apoptosis rate of EAC tumor cells comparing to that in cancer-bearing mice treated with DOX alone (19.30±1.55 versus 15.05±0.05) (Figure 3). Similarly, inoculation of cancer-bearing mice with ZnONP/FA led to remarkable increase in the total apoptosis rate of EAC tumor cells comparing to that in cancer-bearing mice inoculated with ZnONP alone (9.45±0.45 versus 5.85±0.04) (Figure 3).

Figure 3: Potentials of ZnONP conjugates on the total apoptosis rate of EAC tumor cells in EAC-bearing mice treated with ZnONP conjugates. EAC-bearing mice IP injected with PBS, DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA. Mice were sacrificed on day 15 post tumor challenge and ascitic EAC tumor cells were harvested from peritoneal cavity and washed twice with PBS. Tumor cells were stained with Propidium Iodide (PI) and annexin V then analyzed by flow cytometry.

Phenotypic Expression of Splenic Lymphocyte CD4+-T and CD8+-T Cells and NK (CD335) Cells Subsets

Phenotypic expression of splenic lymphocytes CD₄⁺-T and CD₈⁺-T cells of cancer-bearing mice treated with DOX, ZnONP, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA composites were done using flow cytometry (Figures 4 and 5). The results presented here showed that the treatment of cancer-bearing mice with DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA composites markedly induced the expression of splenic CD₄⁺-T cells compared to that of EAC group received PBS alone (25.80%, 26.70%, 22.10%, 29.60%, , 39.10%, respectively versus 12.20%) (Figure 4). Comparing to their proliferation rate value in cancer-bearing mice treated with DOX, splenic lymphocyte CD₄⁺-T cells in Cancer-bearing mice treated with ZnONP, ZnONP/FA or ZnONP/DOX/FA recorded high proliferation rate (25.80% versus 26.70%, 29.6% and 39.10%, respectively) (Figure 4).

Similarly, treatment of cancer-bearing mice with DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA resulted in obvious induction in the proliferation rate of CD₈⁺-T cells comparing to that of EAC group received PBS (22.90%, 20.80%, 25.60%, 23.50%, 38.80% respectively versus 11.10%) and induced the expression of CD₈⁺-T cells in spleen comparable to that in EAC-group received PBS alone (Figure 5). Comparing to their proliferation rate value in cancer-bearing mice treated with DOX, splenic lymphocyte CD8⁺-T cells in cancer-bearing mice treated with ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA recorded high proliferation rate value (22.90% versus 23.50%, 25.60% and 38.80%, respectively) (Figure 5). Interestingly, treatment of cancer-bearing mice with ZnONP/DOX/FA highly increased the phenotypic expression of splenic lymphocyte CD₄⁺-T and CD₈⁺-T cells comparing to their values in cancer-bearing mice received PBS (Figures 4 and 5).

Figure 4: Phenotypic analysis of splenic lymphocytes CD4⁺T cells in EAC tumor-bearing mice. EAC-bearing mice IP implanted with PBS, DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA. Mice were sacrificed on day 15 post tumor implantation and splenocytes were harvested, incubated with antimouse-CD4 mAbs then analyzed by flow cytometry for the markers indicated on the representative histogram. (A) Representative flow cytometry analyses of splenic lymphocytes CD4⁺T cells gated from splenic lymphocyte CD4⁺T cells of treated EAC tumor-bearing mice. (B) Absolute numbers of CD4⁺T cells of treated EAC tumor-bearing mice. Note: ^a,b Statistically significant difference as compared to the corresponding means of EAC tumor-bearing mice received PBS(a) and EAC tumor-bearing mice received DOX (b) within each column.

Figure 5: Phenotypic analysis of splenic lymphocytes CD8⁺T cells in EAC tumor-bearing mice. EAC-bearing mice IP implanted with PBS, DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA. Mice were sacrificed on day 15 post tumor implantation and splenocytes were harvested, incubated with antimouse-CD8 mAbs then analyzed by flow cytometry for the markers indicated on the representative histogram. (A) Representative flow cytometry analyses of splenic lymphocytes CD4⁺T cells gated from splenic lymphocyte CD4⁺T cells of treated EAC tumor-bearing mice. (B) Absolute numbers of CD8⁺T cells of treated EAC tumor-bearing mice. Note: ^a,b Statistically significant difference as compared to the corresponding means of EAC tumor-bearing mice received PBS(a) and EAC tumor-bearing mice received DOX (b) within each column

Phenotypic analysis of splenic NK (CD335⁺) cells in cancer-bearing mice treated with DOX, ZnONP, ZnONP/FA, ZnONP/DOX or ZnONP/DOX/FA conjugates were determined by flow cytometry. The data herein indicated that the treatment of cancer-bearing mice with DOX, ZnONP, ZnONP/FA, ZnONP/DOX or ZnONP/DOX/FA composites markedly induced the expression of splenic NK (CD335⁺) cells comparing to that in EAC group received PBS (34.20%, 29.30%, 31.90%, 54.10%, 58.50%, respectively versus 10.20%) (Figure 6). Importantly, treatment of cancer-bearing mice with ZnONP/DOX or ZnONP/DOX/FA highly increased expression of NK (CD335⁺) cells comparing to that in cancer-bearing mice received PBS (54.10% and 58.50%, respectively versus 10.20%) (Figure 6). Comparing to their proliferation rate value in cancer-bearing mice injected with DOX, splenic NK (CD335⁺) cells in cancer-bearing mice inoculated with ZnONP/DOX and ZnONP/DOX/FA revealed high proliferation rate (34.20% versus 54.10% and 58.50, respectively) (Figure 6).

Figure 6: Phenotypic analysis of splenic NK (CD₃₃₅⁺) cells of EAC tumor-bearing mice. EAC-bearing mice IP implanted with PBS, DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA. Mice were sacrificed on day 15 post tumor implantation and splenocytes were harvested, incubated with antimouse-CD335 mAbs then analyzed by flow cytometry for the markers indicated on the representative histogram. (A) Representative flow cytometry analyses of splenic lymphocytes CD4⁺T cells gated from splenic lymphocyte NK (CD₃₃₅⁺) cells of treated EAC tumor-bearing mice. (B) Absolute numbers of NK (CD₃₃₅⁺) cells of treated EAC tumor-bearing mice. Note: ^a,b Statistically significant difference as compared to the corresponding means of EAC tumor-bearing mice received PBS(a) and EAC tumor-bearing mice received DOX (b) within each column

Analysis of the Peripheral Blood Immune Cells Profile

The results of the total count of leucocytes post treatment of cancer-bearing mice with DOX, ZnONP, ZnONP/FA and ZnONP/DOX/FA indicated a marked increase in the total count of WBC (7.20±1.42 × 10³, 25.26±8.41 × 10³, 20.06±6.78 × 10³, 10.16±0.17 × 10³, respectively), and a slight increase in ZnONP/DOX-treated cancer-bearing mice (5.40±1.37 × 10³) comparing to cancer-bearing mice received PBS (5.33±2.19 × 10³) and naïve mice received PBS (4.62±0.89 × 10³) (Table 1). Treatment of cancer-bearing mice with DOX, ZnONP or ZnONP/FA significantly increased the total count of leucocytes comparing to naïve mice received PBS alone (7.20±1.42× 10³, 25.26±8.41× 10³ or 20.06±6.78× 10³, respectively versus 4.62±0.89 × 10³) (Table 1). Additionally, injection of cancer-bearing mice with ZnONP resulted in significant increase in total WBC count comparing to that of cancer-bearing mice received PBS (25.26± 8.41× 10³ versus 5.33± 2.19× 10³ (Table 1). Comparing to cancer-bearing mice received DOX, the treatment of cancer-bearing mice received ZnONP led to a significant elevation in the WBC count (7.20±1.42 versus 25.26±8.41× 103) (Table 1).

The treatment of cancer-bearing mice with DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA markedly decreased the relative number of lymphocytes comparing to naïve mice receive PBS (69.33±6.48 × 10³, 61.33±8.68 × 10³, 69.66±6.35 × 10³, 52.33±3.28 × 10³, 59.66±9.66 × 10³ and 62.66±6.74 × 10³, respectively versus 87.33±0.66 × 10³) (Table 1). The injection of cancer-bearing mice with DOX, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA resulted in marked decrease in the relative number of lymphocytes comparing to that in cancer-bearing mice receive PBS (61.33±8.681 × 10³, 52.33±3.28 × 10³, 59.66±9.66 × 10³ and 62.66±6.74 × 10³, respectively versus 69.33±6.48 × 10³) (Table 1). Comparing to their values in cancer-bearing mice received DOX, the relative number of lymphocytes obviously decreased in cancer-bearing mice treated with ZnONP/DOX or ZnONP/FA (61.33±8.68× 10³ versus 52.33±3.28× 10³ and 59.66±9.66× 10³) (Table 1).

Table 1: Alternations in peripheral blood immune cells profile of EAC-bearing mice treated with PBS, DOX, ZnONP, ZnONP/DOX, ZnONP/FA or ZnONP/DOX/FA.

Despite the advancements in cancer treatment made over the last decades, resistance to traditional chemotherapeutics and/or new targeted drugs remains a significant hurdle for cancer therapies. Drug resistance, one of the main reasons of death, is the primary cause of most cancer relapses. Drug resistance can be acquired during therapy or intrinsically preexists before treatment [41]. Drug resistance may be successfully overcome by correctly identifying drug-resistance targets following chemotherapy and implementing sequential therapy. Consequently, the combination treatment based on chemotherapy enhanced by nanotechnology was the current paradigm in clinical research, which has the potential to significantly improve therapeutic efficacy with few adverse effects on normal tissues [42]. By directing drugs to specifically target cancer cells, nanotechnology-based drug improves chemotherapy and lessens its harmful side effects [423]. ZnONP demonstrated cancer cell-specific toxicity through the production of ROS and destruction of mitochondrial membrane potential, which activates caspase cascades and causes cancerous cells apoptosis [44].

Clinically, it is still difficult to create an effective and tumor-specific therapeutic approach that can both attack the primary tumor and train the host immune system to eliminate other distant tumors [45]. ZnONP-associated drug delivery looks promising for enhancing therapeutic efficiency by delivering immune-stimulating agents to the site of interest, such as leucocytes and lymphoid organs initiating anti-tumor immunity. Additionally, ZnONP nanocomposites with innately immunogenic characteristics can be specifically created to heighten the immune response in the cancer microenvironment [46]. ZnONP nanocomposites have the capacity to trigger a specific anti-tumor immune response [47]. Due to their ability to stimulate the production of immunoregulatory cytokines in cancer patients, ZnONP nanocomposites have been used as an effective immunoadjuvant that increases the anti-tumor immunity while decreasing the power and exposure time of DOX-based chemotherapy needed to eradicate the primary tumor and inhibit distant metastases. It is advised to assess the immunomodulatory effects of ZnONP nanocomposites on immune cells in order to completely comprehend how they affect the anti-tumor immunity [48].

Data presented here demonstrated that the combinations of the effective penetration properties of ZnONP nanocomposites: ZnONP, ZnONPs/DOX, ZnONPs/FA and ZnONPs/DOX/FA resulted in a potent ability to suppress the fold change of EAC tumor cells count in cancer-bearing mice with low cytotoxicity on healthy cells compared to naïve and cancer-bearing mice received PBS, possibly due to improved their uptake. This combination significantly augmented anti-proliferative potentials against EAC tumor cells in cancer-bearing mice increasing the total apoptosis rate of EAC tumor cells. These data are in line with the findings of [47] that ZnONP conjugates selectively cause significant change in the fold change of EAC tumor cells count and the total apoptosis rate of EAC tumor cells, cytotoxicity and autophagy in cancer cells such as human ovarian cells and gingival cancer cells, which is likely to be mediated by ROS and oxidative stress assembly via p53 pathway and superoxide formation via the mitochondrial intrinsic pathway. Anti-proliferative capability of ZnONP conjugates to cancerous cells was found due to the apoptosis induction and destruction of mitochondrial membrane potential leading to caspase cascades activation followed by apoptosis of cancerous cells [44].

Additionally, the ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA conjugate regimens allows for targeted chemo-photothermal treatment and controlled drug release in a single system [44]. Carrying heat and drug expressively to cancerous cells along with its targeted synergistic effect of chemo-photothermal therapy enhancing their uptake and cytotoxicity against breast cancer cells facilitating controlled drug release and targeted chemo-photothermal therapy in a single system. The efficacy of ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA in breast cancer (MCF-7) and colon carcinoma (HT-29) cells affirming their efficiency in drug delivery to cancerous cells with minimum toxicity to heath cells and high therapeutic efficacy [49].

To account for the concern that ZnONP composites may cause a reduction or increasing in the responsiveness in the immune system through the immune-related cells subsets such as lymphocytes; CD₄⁺-T and CD₈⁺-T cells and NK cells, we performed phenotypic analysis by flow cytometry of CD₄⁺-T and CD₈⁺-T cells and NK cells in mice treated with ZnONP-based therapeutic composites; ZnONP, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA.

Tumor treatment with NP loadedwithconventional chemotherapeutics andotheradjuvantssuch as FA resulted in induction of anti-tumor immunity, therebysuppressing the growth of the primarytumorwithoutcausing side effects. Significantly, it suppressedprimarytumorrecurrenceandmetastasis [50]. NP combinedwithconventional chemotherapeutics andotheradjuvantsact as excellentdrugdeliverysystems (DDS) in cancertherapy [23] andwillimprovetheefficiency of cancer immunotherapies thatprovidedurabletherapeuticresponses [24-26].

The data herein reported that the treatment of cancer-bearing mice with ZnONP, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA showed putative immunomodulatory effects on the phenotypic expression and proliferative response of splenic subsets of lymphocyte; CD₄⁺-T and CD₈⁺- T cells and NK cells. Interestingly, ZnONP/DOX nanocomposite significantly increased the phenotypic expression and proliferative immune response of lymphocytes CD₄⁺-T and CD₈⁺- T cells and NK cells. ZnONP-based therapeutic composites promotes innate immunity-type responses from CD₄⁺-T and CD₈⁺-T cells and NK cells and may affect different human immune cells and their production of exosomes that have a role in cell to cell communication through an effect on FcγR-mediated anti-tumor immune responses in cancer host. Additionally, ZnONP-based therapeutic interventions may be successful in elevating a group of cytokines important for eliciting a Th₁-mediated anti-tumor immune response with essential anti-tumor immunity [51]. ZnONP-based therapeutic composites may function via a two-fold mechanism to eliminate cancer cells by direct and preferential cytotoxic actions, and by enhancing the type of immunity most effective at eliciting an in vivo anti-tumor response and ability of ZnONP-based therapeutic composites to increase the expression of IFN-γ and IL-12 in primary immune human cells causing appreciable cancer cell death. The ability of ZnONP-based therapeutic composites to induce TNF-α may also help to promote anti-tumor immunity in the tumor host by induce the differentiation of immune lymphocytes cells; CD₄⁺-T and CD₈⁺-T cells [52].

Our results come to agree with findings of [51] who observed anti-tumor immunity through a greater resistance of naïve T cells to ZnONP nanocomposites -associated toxicity compared to memory T cells, indicating that the toxic potential of ZnONP-based therapeutic nanocomposites is dependent on the activation of the cells and lymphocytes being the most resistant to ZnONP in the tumor tissues. Besides, ZnONP nanocomposites induced anti-tumor immunity through a significant release of the pro-inflammatory cytokines IL-12, Interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in human primary immune cells at concentrations below those causing relevant cancer cell death. The tuning of NK cell activation resulting in release of immune activating cytokines, such as IFN-γ, and TNF-α, which can act on other innate immune cells, including dendritic cells (DC) and epithelial cells and on adaptive immune cells, such as lymphocytes; CD₄⁺-T and CD₈⁺-T cells leading to anti-tumor immunity [53]. Also, pectin-guar gum-zinc oxide nanoconjugate was employed as an immunomodulator to particularly attack cancer cells, which showed enhanced anti-cancerous effects [54].

 Notably, several transplantable models of carcinoma, sarcoma, and lymphoma have demonstrated the crucial role the immune system plays in the effectiveness of systemic chemotherapy [30-33]. Both transplantable neoplasms and tumors brought on by the chemical methylcholanthrene (MCA) are susceptible to immune surveillance, which causes them to progress slowly in immunocompetent. As an alternative, oncogene-driven cancers might learn how to activate immune escape mechanisms and/or fail to activate a natural immune surveillance [34].

Tumor-infiltrating, CD₄⁺-T and CD₈⁺-T cells and NK cells are known to exert an anti-tumor effect [55]. The NP-based therapeutic nanocomposites achieved powerful cytosolic delivery of membrane impermeable molecules resulting in efficient CD₄⁺-T and CD₈⁺-T cell priming [56]. Further, because of the liposomes' fast aggregation in tumors, tumor infiltration was triggered. by CD₄⁺-T and CD₈⁺-T cells, cytokine and granzyme secretion and elicited effective anti-tumor action without causing systemic toxicity [57]. Moreover, NP nanocomposites exerted significant innate and acquired immunity for the treatment and prevention of tumors by promoting the activation of NK cells, CD₄⁺-T and CD₈⁺-T cells, and their infiltration into tumor tissues. Synergistically, the CD₄⁺-T and CD₈⁺-T cells are enhanced by treatment with ZnONP composites to eradicate tumor cells by antigen cross presentation promoting the activation of NK cells and their infiltration into tumor tissue. Besides, ZnONP composites treatment resulted in a significant secretions of TNF-α and IFN-γ from the infiltration of CD₈⁺-T cells in tumor tissue contributing to the anti-tumor immunity capability [58].

The composition of tumor immune infiltration changes in response to antineoplastic drugs, which is decisive for the success of treatment. Therefore, increased numbers of intra-tumoral T lymphocytes and increased proportion of cytotoxic CD₈⁺-T lymphocytes (CTLs) after chemotherapy predict a favorable therapeutic response in human breast cancer treated with NP containing the systemic chemotherapeutic drug DOX and other adjuvants such as FA (NP/DOX/FA) [27,28]. Several clinical researches have shown that severe lymphopenia can adversely affect the response to chemotherapy in several different types of cancer [29]. Thus, mouse tumors responded more efficiently to NPs conjugated to DOX and another adjuvant when they grow in syngeneically immunocompetent mice than when they grow in immunodeficient hosts [59,60]. Synthesized NP nanocomposites were also shown to induce activation of tumor cell killing processes and improve the tumor cell killing capacities of lymphocytes increasing the production of anti-tumor cytokines IFN-γ, IL-2 and TNF-α which further led to the killing of tumor cells and inhibition of tumor growth and enhanced expression of CD₄⁺-T and CD₈⁺-T cells and NK cells [54,61].

The increase in the differentiated bone marrow progenitor cells or the decrease in progenitor cell frequency could result in reduction or increasing in the count of WBC and absolute number of neutrophils, lymphocytes and monocytes in peripheral blood and therefore a responsive anti-tumor immune approaches, we performed WBC, neutrophils, lymphocytes and monocytes counts in mice injected with ZnONP, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA nanocomposites. The present results indicated that there were no many significant differences in the counts of peripheral WBC, neutrophils, lymphocytes and monocytes, increasingly or decreasingly after treatment of cancer-bearing mice with ZnONP nanocomposites comparing to those of cancer-bearing-mice received PBS. Thus, ZnONP nanocomposites induce a homeostatic condition of decreased progenitor cell frequency in the myeloid compartment of the bone marrow without adverse effects on WBC, neutrophils, lymphocytes and monocytes productions.

The most abundant WBC in the body is the neutrophil. The majority of mature neutrophils are found in the blood, bone marrow, spleen, and liver, as well as other marginal pools. Only a tiny number are found in tissue [63]. Neutrophils are the first cells to be recruited to the site of inflammation and other malignancies having an important role in the clearance of pathogens. However, neutrophils can also interact with the adaptive immune system in malignant host by promoting naïve lymphocytes T cells to transition into T helper 1 cells and can present antigens to B-cells [63]. The current results also come to agree with findings of [64]. who indicated that macrophages and granulocytes have been regarded as mediators of acute inflammation following single exposure to ZnONP nanocomposites resulting in a dynamic immune response involving neutrophils, accompanied by peripheral leukocyte depletion. ZnONP are activators of several human neutrophil functions and that they inhibit their apoptosis by a de novo protein synthesis-dependent and ROS-independent mechanism [65,66]. In cancer-bearing host treated with ZnONP, the homeostasis of neutrophils is disturbed temporarily, or even for a long term, which leads to the variation of neutrophil numbers. However, higher levels of neutrophils are found in the blood of patients with advanced cancer, and this might be due to the up-regulation of Granulocyte colony stimulating factor (G-CSF) in multiple cancer types [67]. However, repeated exposures of ZnONP nanocomposites caused mild lymphophilia and eosinophilia in blood. It is also possible that increased exposure to ZnONP-based therapeutic nanocomposites may cause immunomodulation in cancerous host [65]. Numerous therapeutically effective anti-cancer medications, including chemotherapy and chemotherapeutic-loaded NP conjugated with doxorubicin (DOX) and/or FA, can cause the hallmarks of cancer immunogenic cell death (ICD). To stimulate a protective anti-cancer immune reaction, ICD-susceptible cells can be used as vaccines. Additionally, ICD-inducing anti-cancer treatments like chemotherapeutic-loaded nanoparticles combined with folic acid lose their effectiveness when cancer cells' ICD signal emission is suppressed, when the immune system's ability to perceive these signals is hampered, and when vital immune effectors, like lymphocytes CD₄⁺-T and CD₈⁺-T cells, are depleted [34].

The significant decreases in lymphocytes and the significant increases in WBC and neutrophils in tumor-bearing host were closely related to the administration of ZnONP-based therapeutic nanocomposites because these changes were remarkable and showed a clear-cut dose-anti-tumor immune responses [69]. In tumor-bearing host, ZnONP nanocomposites enhance the phagocytosis of human neutrophils [70] and positively modulate the degranulation process in human neutrophils demonstrating the pro-inflammatory effect of ZnONP on the host immunity [71]. A restricted group of chemotherapeutic nanoparticles in combination with adjuvants, such as folic acid, can cause immunogenic stress and death in tumor cells. As a result, the patient's dying cancer cells act as a vaccine to trigger an immune reaction specific to the tumor, which in turn can control (and occasionally even eradicate) remaining cancer (stem) cells [72].

ZnONP exhibits a detrimental impact on the lipid, protein, and nucleic acid of the tumor tissues at high levels of oxidative stress. It has been suggested that oxidant generation and antioxidant depletion are the common pathways through which anticancer drugs trigger apoptosis in cancer cells [73]. Therefore, in our results the levels of MDA, along with the activities of antioxidant enzymes SOD and CAT were determined. The findings of the current study demonstrated that a significant increase in MDA levels were observed in EAC tumor-bearing female mice when compared to naïve group. Our data are coincide with previous findings that reported an elevated levels of MDA in breast cancer. Also, Ehrlich tumors exhibited significant increases in MDA and considerable decreases in SOD and CAT MDA content manifests the level of lipid peroxidation and indirectly represents the level of damage to the tissue [74].

A decrease in endogenous antioxidant enzymes SOD and CAT with enhanced free radical generation and MDA is well documented in carcinogenesis [75]. Several reports documented that MDA, the end product of lipid peroxidation, are seen to be higher in cancer tissues than in normal tissues [76]. The findings of the current study demonstrated that dosing of Cancer-bearing mice with ZnONP nanocomposites resulted in decease of serum level of MDA. The inhibition of SOD and CAT activities as a result of tumor growth was also reporte [77] These out comes are agreed with our study that decrease in the level of SOD and CAT. Antioxidant enzymes act as the primary line of defense against ROS which suggest their usefulness in estimating the risk of oxidative damage induced during carcinogenesis [78].

Treatment of tumor-bearing host with ZnONP nanocomposites and cyclophothamide (CP) together attenuated the oxidative damage and disturbance in the antioxidant defense system induced by cancer conventional therapeutic cyclophosphamide (CP). So, cancer patients treated with CP advised to take ZnONP to prevent the side effects of chemotherapy [79]. Co-administration of ZnONP plus DOX induced a significant induction in SOD and CAT activities and significantly decrease in the testicular MDA concentration as compared with the cancer-bearing mice received DOX alone. ZnONP nanocomposites protect the cell membranes against oxidative damage, decrease free radicals and MDA levels, and increase the antioxidant enzyme SOD and CAT levels [80-82], Thalidoamide-induced reductions in CAT and SOD were alleviated by ZnONP nanocomposites, which also decreased tissue MDA. Hence, it can be concluded that the ZnONP nanocomposites (ZnONP, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA) could be the most efficient anti-cancerous agents due to their strong therapeutic potential and low systemic toxicity [83].

Taken together, ZnONP with its effective penetration properties and DOX resulted in a potent ability to suppress the growth of EAC tumor cells, but ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA significantly augmented anti-proliferative rate of EAC tumor cells overcoming the resistance to chemotherapeutics and reduce their side effects with low cytotoxicity on healthy cells. Furthermore, ZnONP/DOX, ZnONP/FA and ZnONP/DOX/FA have putative immune-modulatory effects on T-lymphocytes subsets CD₄⁺-T and CD₈⁺-T cells, stimulating the cytokine expressions and inducing NK cytotoxic effects. As a result, this research offers a hopeful combinatorial approach to induce anti-tumor immunity in malignancies, overcoming chemotherapeutics resistance and decreasing their side effects and more studies are required to connect the biomedical application of this regimen to diagnostic and therapeutic approaches.

Funding Information

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Conflict Of Interest Statement

The authors declare that they have no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics Statement

This study was approved by the ethics committee, ethics number: IACUC-SCI-TU-0062.

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