Introduction

Immunotherapy of diseases ranging from autoimmune disorders to different types of cancer as well as infectious diseases, require proper control of immune system cells against the danger of interest [1,2,3,4,5,6,7,8,9,10,11,12,13]. Immune system responds back to the danger signals coming from pathogens, tumor cells, and damaged cells during autoimmune reactions or tissue injury [1,2,3,4,5,6,7,8,9,10,11,12,13]. This response is the secretion of signaling molecules, growth, differentiation, polarization, and migration factors for the immune system cells [1,2,3,4,5,6,7,8,9,10,11,12,13]. The small molecules that turn on or off these signaling pathways in the immune cells are known as cytokines and chemokines (migratory factors) [1,2,3,4,5,6,7,8,9,10,11,12,13].

Macrophages are innate immune system cells that can efficiently and effectively produce these cytokines and chemokines [14,15,16,17,18,19,20,21,22,23,24,25]. In the lesions of the patients suffering from autoimmune disorders, infectious diseases and cancer, macrophages have been widely detected [14,15,16,17,18,19,20,21,22,23,24,25]. These cells are responsible for the inflammation associated with the disease conditions [14,15,16,17,18,19,20,21,22,23,24,25]. Inflammation is actually a necessary process of immune cell migration to the region along with the production of pro-inflammatory cytokines such as TNFα, IL1β, IL6, GMCSF, IL12p40, IL17A, IL22, IL21, and IFNγ [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33].

These cytokines aim to regulate the function of the immune system cells together with the tissue resident cells to eliminate the source of the danger stimulus [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Afterwards, anti-inflammatory cytokines such as IL10 and TGFβ are produced for the resolution of the inflammation which could damage the tissue if it becomes chronic or persistent [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Then, the tissue healing process starts [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33].

Macrophages are major cell types of the immune system that can function at both ends of the spectrum [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. This quality makes them unique since they can be pushed towards pro-inflammatory or anti-inflammatory mode depending on the case [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Moreover, they can also alter the activities of other immune system cells including neutrophils, T cells, B cells, and natural killer cells either by cytokine production or antigen presentation [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Therefore, targeting these cells to regulate the overall immune system function would be a great starting point [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33].

Regulation of the immune system is immunomodulation and it creates a major advantage for immunotherapy applications. Design and screening of immunomodulatory agents would support the field to create different options of potential medicines for the therapy [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Schiff bases are well known for their anti-tumor, anti-microbial, and anti-inflammatory properties [34,35,36,37,38]. In the light of the previous findings, we generated copper bearing Schiff base derivatives and characterized them in our studies [39,40,41]. They exerted a light responsive nature which makes them good candidates for photodynamic therapy (PDT) applications [39,40,41].

In our current study, we are presenting a group of unique copper bearing Schiff bases’ anti-microbial and anti-inflammatory activities in dark and after PDT application. These molecules had strong anti-microbial activities against gram negative and gram positive bacteria. Moreover, they had anti-inflammatory properties on the mammalian macrophages based on the cytokine secretion levels. Both of these properties got more substantial with light exposure. The results of this study are, for the first time, suggesting anti-inflammatory and anti-microbial PDT potential of these novel copper Schiff bases.

Material and Methods

Cell Culture and Growth

ATCC RAW 264.7 cells were utilized and they were grown in 10% FBS, 1% 100 μg/ml penicillin, and 100 μg/ml streptomycin mixture of antibiotics supplemented into RPMI 1640 media. A 37 °C 5% CO2 incubator was used for the incubations [26,27,28,29,30,31,32,33].

Macrophage Activation and Photo-induction Studies with Schiff Bases and Lipopolysaccharide

Twenty-four well plates were used for the cell stimulations and the macrophages were plated in 1 mL in 106 cells/well concentration. In order to let the cells adhere and rest, the plates were incubated overnight in the 37 °C 5% CO2 incubator. Schiff bases and salicylic acid were used in10 and 100 μg/mL final concentrations and put into appropriate wells with or without 1 μg/mL lipopolysaccharide (LPS) from Enzo Life Sciences. The same volume of DMSO was put into the negative control wells which lacked the stimulant LPS as well as Schiff Bases. The plates were incubated for 24 h and then the media of each well were collected into eppendorf tubes for ELISA. The cells of each well were counted with Trypan Blue Staining protocol [26,27,28,29,30,31,32,33]. Three different experiments were repeated for the same set up to draw statistical relevance. The same experimental set up was used for three different conditions: dark (0 min), 5 min, and 10 min of Xenon light exposure after addition of the stimulant and Schiff bases into appropriate wells. Twenty-four-hour stimulation was done after the light exposure [26,27,28,29,30,31,32,33].

ELISA

TNFα ELISA (BD Biosciences) was conducted by following the guidelines of the BD Biosciences. A detailed ELISA protocol was given in our previous studies: [26,27,28,29,30,31,32,33].

Microtiter Broth Dilution Test for Anti-microbial Activity Measurements

A range of Schiff base concentrations were applied to colonies of E.coli (ATCC 25922) and S.aureus (ATCC 29213) that were grown in Mueller Hinton Broth (MHB) overnight and whose concentrations were adjusted with PBS according to 0.5 McFarland absorbance measurements. Neomycin control and Schiff bases were tested on both bacteria in 1, 10, 50, and 100 μg/mL concentrations to calculate IC50 values on each bacteria. McFarland adjusted 100 uL bacterial samples were plated into sterile 96 well plates with 100 uL fresh MHB in each well. Schiff bases and neomycin were added into the appropriate wells and the plates were incubated on a 37 °C shaker for 18 h. Absorbance values were measured at 600 nm for each plate and each compound’s absorbance was measured by itself to subtract it during the IC50 calculations. Three different experiments were repeated for the same set up. The same experimental set up was used for three different conditions: dark (0 min), 5 min, and 10 min of Xenon light exposure after addition of the Schiff bases into appropriate wells. Eighteen-hour stimulation was done after the light exposure [26,27,28,29,30,31,32,33].

Synthesis of Novel Copper Bearing Schiff Bases

Copper(II) complexes, [Cu2(L1)2(CH3COO)2](L1-Cu),[Cu(L2)(CH3COO)].2H2O(L2-Cu) of the two new Schiff base ligands (HL1and HL2) derived from 2-hydroxy-3-methoxy benzaldehyde and 3-ethoxy-2-hydroxybenzaldeyhde with N,N′-dimethylethane-1,2-diamine have been prepared according to our previous publication: [39]. The Cu(II) complexes were obtained by using HL1 ligand for L1-Cu and HL2 ligand for L2-Cu.The synthesized compounds were characterized through elemental analyses and spectral methods. The spectral data of the compounds are given in Figs. S1-S10 in Supporting information (SI).

(HL1): Yield: 94%, color: yellow liquid. M.W.: 222.29 g/mol. Anal. Calc. For C12H18N2O2: C, 64.84; H, 8.16; N, 12.60%. Found: C, 64.51; H, 8.08; N, 12.34%. IR data (KBr pellet, cm−1): 3424 (O-H) aromatic, 2941-2774 (C-H) aliphatic, 1632 (CH=N), 1466 (C-C) aromatic, 1255 (C-O) phenolic. 1H-NMR (600 MHz, DMSO-d6) δ ppm: 13.84 ppm (s, -C-OH, 1H), 8.51 ppm (s, =CH-N, 1H), 7.00-6.74 ppm (m, Ar-H, 3H), 3.77 ppm (s, -O-CH3, 3H), 3.67 ppm (t, -C=N-CH2-, 2H), 2.53-2.51 ppm (t, -N-CH2-, 2H), 2.18 ppm (s, -N-CH3, 6H).13 C-NMR (150 MHz, DMSO-d6) δ ppm: 166.76 (C=N), 153.38–115.00(Ar-C), 59.76-45.71 (Aliphatic-C), 56.46 (OCH3).

(HL2): Yield: 91%, color: dark yellow liquid, M.W.: 236.32 g/mol. Anal. Calc. For C13H20N2O2: C, 66.07; H, 8.53; N, 11.85%. Found: C, 65.98; H, 8.48; N, 11.64%. IR data (KBr pellet, cm−1): 3441 (O-H) aromatic, 2977-2784 (C-H) aliphatic, 1634 (CH=N), 1465 (C-C) aromatic, 1252 (C-O) phenolic. 1H-NMR (600 MHz, DMSO-d6) δ ppm: 13.82 ppm (s, -C-OH, 1H), 8.49 ppm (s, -N=CH-,1H), 7.00-6.71 ppm (m, Ar-H, 3H), 4.03-3.99 ppm (q, -O-CH2-CH3, 2H), 3.67 ppm (t,-C=N-CH2-, 2H), 2.53–2.51 ppm (t, -N-CH2-, 2H), 2.19 ppm (s, -N-CH3, 6H),1.32 ppm (t, -O-CH2-CH3, 3H). 13C-NMR (150 MHz, DMSO-d6) δ ppm: 166.80 (C=N), 153.44–116.49 (Ar-C), 64.36-39.56 (Aliphatic-C), 59.78(OCH2CH3), 15.27 (OCH2CH3).

L1-Cu: Yield: 89%, color: green. M.P.: 202–204 °C. Anal.Calcd. for C28H40N4O8Cu2: C, 48.90; H, 5.86; N, 8.15; Cu: 18.48. Found: C, 48.92; H, 5.82; N, 7.48; Cu: 17.10%. IR data (cm−1): 3345 (water), 3040-2828 (C-H) aliphatic, 1624 (CH=N), 1604-1470 (C-C) aromatic, 1376-1327 (C-O) phenolic, 576(M-O), 460(M-N).

L2-Cu: Yield: 92%, color: dark green. M.P.: 138 °C. Anal. Calcd. for C16H30N2O6Cu: C, 46.88; H, 7.38; N, 6.83; Cu: 15.50. Found: C, 46.81; H, 6.52; N, 6.69; Cu: 14.83%. IR data (cm−1): 3465-3394 (water), 2983-2873 (C-H) aliphatic, 1635 (CH=N), 1586-1468 (C-C) aromatic, 1442-1384 (C-O) phenolic, 534 (M-O), 463 (M-N).

Statistical Analysis

Student t test was applied to each data set on GraphPad Prism Software version 5 [26,27,28,29,30,31,32,33].

Results

Copper Bearing Schiff Bases Did Not Alter the Cell Viability of Macrophages

Schiff bases HL1, L1-Cu, HL2, and L2-Cu (Fig. 1) were used in 10 μg/mL and 100 μg/mL concentrations in the presence and absence of LPS (Fig. 2). Xenon light was shone on the plates for 5 and 10 min, and the results were compared with their dark toxicities (Fig. 2). Our compounds were safe at the used concentrations since they did not lead to any change in the cell viability of macrophages after 24 h of incubation period (Fig. 2). Moreover, Xenon light exposure for different periods did not cause any change in the cell viabilities as well (Fig. 2).

Fig. 1
figure 1

Chemical structures of all compounds

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Fig. 2
figure 2

Live cell percentages after 24 h incubation with 10 and 100 μg/mL Schiff bases with or without LPS. The same experimental set up was used for three different conditions: dark (0 min), 5 min and 10 min of Xenon light exposure after addition of the Schiff bases into appropriate wells. N = 3

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Copper Bearing Schiff Bases Were Anti-Inflammatory on Activated Macrophages and this Property Got More Substantial upon Xenon Light Treatment

A total of 10 and 100 μg/mL concentrations of the bases were used in the absence of LPS in order to measure their possible immunostimulatory role (Fig. 3). They did not activate the macrophages by themselves at those concentrations (Fig. 3). When these compounds were used together with stimulant LPS, they lead to a significant and substantial decrease in pro-inflammatory TNFα secretion by macrophages compared with only LPS-treated control wells in dark (Fig. 3). At 100 μg/mL concentration, these compounds were as effective as salicylic acid control in terms of their anti-inflammatory activities (Figs. 3 and 4). Five minutes of Xenon light lead to a substantial change in their anti-inflammatory potential which became very stark after 10 min of Xenon light exposure (Fig. 3). In total, 10 μg/mL of Schiff base was more effective than salicylic acid control upon light exposure as an anti-inflammatory agent on the macrophages (Figs. 3 and 4).

Fig. 3
figure 3

TNFα ELISA was done for the media of each well after 24 h incubation of macrophages with 10 and 100 μg/ml of Schiff bases with or without 1 μg/ml LPS. DMSO was added into negative control wells, 1 μg/ml of LPS and DMSO were added into positive control wells. The same experimental set up was used for three different conditions: dark (0 min), 5 min and 10 min of Xenon light exposure after addition of the Schiff bases into appropriate wells. Statistical analysis for each data set was student t test, *p < 0.001, ** p < 0.0005, ***p < 0.0001, N = 3

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Fig. 4
figure 4

TNFα ELISA was done for the media of each well after 24 h incubation of macrophages with 10 and 100 μg/ml of salicylic acid with or without 1 μg/ml LPS. DMSO was added into negative control wells, 1 μg/ml of LPS and DMSO were added into positive control wells. Salicylic acid is not light responsive so this set was done only in the dark. Statistical analysis for each data set was student t test, *p < 0.001, **, p < 0.0005, ***p < 0.0001, N = 3

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Copper Bearing Schiff Bases Could Eliminate Both Gram Negative E.coli and Gram Positive S.aureus Effectively

A range of Schiff base concentrations were applied separately to gram negative E.coli and gram positive S.aureus (Tables 1 and 2). Microtiter broth dilution assay gave the IC50 values for each compound against both bacteria (Tables 1 and 2). Compared with Neomycin, our compounds had a strong anti-bacterial activity which did not require Xenon light exposure (Tables 1, 2, and 3).

Table 1 IC50 values of Schiff base derivatives on gram negative E.coli
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Table 2 IC50 values of Schiff base derivatives on gram positive S.aureus
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Table 3 IC50 values of Neomycin on gram negative E.coli and gram positive S.aureus
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Discussion

Regulation of the immune system cell functions to eliminate a disease condition has gathered more attention recently as immunotherapy [1,2,3,4,5,6,7,8,9,10,11,12,13]. This type of therapy has been tried in versatile disease conditions including different types of cancer to infectious disease and autoimmune disorders [1,2,3,4,5,6,7,8,9,10,11,12,13]. The aim of the immunotherapy is using our own tools, immune system cells, against the danger of interest [1,2,3,4,5,6,7,8,9,10,11,12,13]. Modulation of the immune system cell function is a complicated process and requires more research [1,2,3,4,5,6,7,8,9,10,11,12,13].

Immunomodulatory compounds are great tools that can be used to regulate and control the immune cell activities [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Moreover, having an immunomodulatory chemical whose activity can further be controlled and fine-tuned by photodynamic activation would bring an immense medicinal potential out [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. Schiff bases are good candidates for immunomodulatory PDT applications that are aiming to manipulate the immune cell functions by light exposure without damaging the immune cells [34,35,36,37,38,39,40,41]. Schiff base derivatives’ anti-cancer, anti-proliferative, anti-inflammatory, and anti-microbial activities have been long studied [34,35,36,37,38,39,40,41]. Based on those studies, we generated a group of Schiff bases that bear copper and chemically characterized them [39,40,41]. In this study, we tested these novel compounds’ anti-inflammatory potential on macrophages.

Macrophages are one of the primary cell types of the immune system that are heavily involved in inflammation [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. These cells were the target of our study since they can produce cytokines and present antigens to other immune cells to regulate their activities and change overall immune response against a certain danger element [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. TNFα is a pro-inflammatory cytokine that is produced by macrophages and is involved in immune reactions against bacterial and viral infections as well as tumor cells [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33].

Our compounds had anti-inflammatory activity in dark but upon light treatment this activity got more substantial. These results overlap with the previous findings suggesting the anti-inflammatory role of Schiff base derivatives. In this study, we are showing the anti-inflammatory PDT potential of Schiff base derivatives that bear copper. In Schiff base HL1 in the absence of copper, there was a stark dark anti-inflammatory activity (Fig. 2). This was changed by the addition of copper in L1-Cu (Figs. 1 and 2). L1-Cu did not have as strong as HL1’s dark anti-inflammatory activity but upon light treatment it caught up an anti-inflammatory potential as effective as that of HL1(Fig. 2). Therefore, addition of copper to Schiff base structure created a PDT agent (Fig. 1). This was not the case for HL2 since it did not have a strong dark anti-inflammatory potential as that of HL1 (Figs. 1 and 2). Addition of copper did not alter its PDT-induced anti-inflammatory activity since both HL2 and L2-Cu had similar dark and PDT-induced effective and stark anti-inflammatory activities (Figs. 1 and 2). It is important to note that a small change of structure from HL1 to HL2made a substantial change in their dark anti-inflammatory potential (Figs. 1 and 2). Addition of a Methyl group (HL2) decreased the dark anti-inflammatory potential of HL1 (Figs. 1 and 2). Overall, our compounds were stronger than a common anti-inflammatory active ingredient, salicylic acid in their anti-inflammatory activities which became more substantial and obvious upon PDT application (Figs. 2 and 3).

Furthermore, we also tested these compounds’ anti-microbial activities on gram negative E.coli and gram positive S.aureus (Tables 1, 2, and 3). These compounds had strong dark anti-microbial activities which increased by light exposure but did not change substantially (Table 1, 2, and 3). These compounds were stronger than Neomycin in terms of their anti-microbial activities without the need of PDT (Table 1, 2, and 3).

In conclusion, these Schiff base derivatives have immunomodulatory PDT potential that can be utilized in the field of medicine. Addition of copper to the Schiff base structure led to a change in its activity and made it PDT-responsive. Having PDT-responsive immunomodulatory compounds will enable efficient and more controlled therapy against inflammatory disorders, infections, as well as cancer [33,34,35,36,37,38,39,40,41,42,43,44]. We are currently investigating their mechanism of action at cellular level and designing more Schiff base derivatives that would have higher PDT responsiveness with better efficiencies.