Targeted co-delivery of methotrexate and chloroquine via a pH/enzyme-responsive biocompatible polymeric nanohydrogel for colorectal cancer treatment

Abstract Application of conventional chemotherapy regardless of its unique effectiveness have been gradually being edged aside due to limited targeting capability, lack of selectivity and chemotherapy-associated side effects. To this end, colon-targeted nanoparticles via combination therapy have shown great therapeutic potential against cancer. Herein, pH/enzyme-responsive biocompatible polymeric nanohydrogels based on poly(methacrylic acid) (PMAA) containing methotrexate (MTX) and chloroquine (CQ) were fabricated. PMAA-MTX-CQ exhibited high drug loading capacity of which MTX was 4.99% and was CQ 25.01% and displayed pH/enzyme-triggered drug release behavior. Higher CQ release rate (76%) under simulated acidic microenvironment of tumor tissue whereas 39% of CQ was released under normal physiological conditions. Intestinally, MTX release was facilitated in the presence of proteinase K enzyme. TEM image demonstrated spherical morphology with particle size of less than 50 nm. In vitro and in vivo toxicity assessments indicated that developed nanoplatforms possessed great biocompatibility. These nanohydrogels did not cause any adverse effects against Artemia Salina and HFF2 cells (around 100% cell viability) which highlight the safety of prepared nanohydrogels. There was no death in mice received different concentrations of nanohydrogel through oral administration and less than 5% hemolysis was found in red blood cells incubated with PMAA nanohydrogels. In vitro anti-cancer results showed that combination therapy based on PMAA-MTX-CQ can effectively suppress the growth of SW480 colon cancer cells (29% cell viability) compared to monotherapy. Altogether, these findings suggest that pH/enzyme-responsive PMAA-MTX-CQ could effectively inhibit cancer cell growth and progression via site-specific delivery of its cargo in a safe and controlled manner.


Introduction
Colorectal cancer (CRC) is considered the second most deadly cancer and the thirdmost common malignant tumor which is accounted for 0.9 million deaths and 1.9 million incidence cases in 2020 [1]. Unfortunately, CRC-related mortality and morbidity have dramatically increased worldwide despite all advances in cancer therapy and diagnosis. The high prevalence and incidence rate of CRC are mostly attributed to nutrition, sedentary lifestyle and population aging [2]. Surgery, immunotherapy, radiotherapy, and chemotherapy are considered the most common cancer treatment options. Among them, cancer chemotherapy has displayed an important role in almost all types of cancer, more specifically in CRC therapy aiming at improving the quality and increasing the duration of the individuals [3]. The main challenges concerning cancer chemotherapy are the broad and non-selective distribution of chemotherapeutics. In order to effectively treat the CRC, frequent and series of cycles of anticancer drug administration are required to be accumulated enough in the tumor tissues which may result in massive contamination of the other tissues/organs. In other words, this lack of specificity of anti-cancer drugs and frequent drug administration leads to several toxicological issues that cause severe side effects as well as drug resistance, which ultimately accounted for the failure of chemotherapy [4,5]. To diminish the aforementioned drawbacks, one promising strategy is targeting colon cancer cells using nanotechnology-based drug carriers owing to its positive outcomes [6]. Moreover, according to the previously published studies, substantial advances have been obtained in CRC therapy through the advent of nanotechnology [7]. The surface of drug carriers can be decorated with targeting moieties, such as polysaccharides, transferrin, integrins, antibodies, peptides and folic acid (FA) to improve cellular uptake and intracellular trafficking of anti-cancer drugs within tumor tissues. It has been well documented that folate receptors (FRs) overexpressed over the surface of colorectal cancer cells. Therefore, attachment of FA as an optimal targeting ligand onto the surface of drug nanocarrier could deliver and direct the therapeutic agents to cancer cells intelligently [8]. Methotrexate (MTX), an analog of folic acid, is a chemotherapeutic agent commonly used in the treatment of cancers and other diseases such as artritis reumatoide. To put it briefly, MTX displayed dual functions in CRC therapy (i) targeting ligand and (ii) chemotherapeutic agent [9]. It was demonstrated that the MTX-conjugated nanoparticles exhibited enhanced cellular uptake and therapeutic potential to fight against cancer [9,10]. Similarly, another study conducted by Faghfoori et al. showed MTX-conjugated albumin coated bismuth sulfide nanoparticles could effectively suppress the colon cancer cells by the colon-specific delivery of anticancer drugs while diminishing drug leakage within bloodstream [11]. Currently, there are a limited number of approved nanocarriers in clinics, but it is expected that advances in nanotechnologies near future will likely witness the emergence of a growing number of nanovehicles with therapeutic potential for enhanced cancer treatment. A broad variety of nanosized drug delivery carriers has been used as drug delivery systems in CRC therapy aiming at minimizing chemotherapymediated side effects while augmenting therapeutic efficacy. Among them, polymeric nanoparticles in particular stimuli-responsive polymeric nanohydrogels have gained remarkable attention in site-specific delivery of anticancer drugs to cancer tissues [12]. A growing body of literature has indicated that the intracellular pH of tumor tissues is maintained below the extracellular pH which provides a suitable site for triggering drug release through pH-sensitive nanoparticles. As matter of fact, pH-sensitive polymeric nanohydrogels could efficiently deliver anti-cancer drugs into the tumor tissues in comparison with conventional nanotherapeutics and enable oncologists to treat cancers effectively with the least possible side effects in a safe and controlled manner [13]. Poly(methacrylic acid) (PMAA) is a pH-responsive polymer owing to the abundant carboxylic acid groups (ionizable groups) present in the backbone of polymer structure. These groups allowed the PMAA to contract and swell in response to the changes in the pH value of the exposed microenvironment. Hence, these materials have great potential as drug carriers and for targeted drug delivery. In addition to the pH sensitivity of PMAA nanohydrogels, some other unique features such as excellent biocompatibility, non-toxicity, tunable size, ease of functionalization, and modification make this nanocarrier an ideal candidate for targeted drug delivery [14,15]. Beside these, enzyme-responsive polymer nanohydrogels can also be used as an effective drug delivery which triggering its cargo in response to the lysosomal condition. It has been reported that amide bonds (peptide bonds) dissociate in the lysosome compartment and the amide linkage of MTXconjugated nanomaterials could be cleaved in the presence of enzyme [16]. Additionally, combination therapy (CT) has emerged as a promising treatment modality in cancer eradication. In CT two or more therapeutic agents are involved, and recently it was regarded as a cornerstone of CRC therapy [17]. Combined therapy could enhance the efficacy of CRC treatment due to the additive/or synergistic effects compared to the mono-therapy approach [18]. Chloroquine (CQ), an anti-malarial drug with anti-cancer properties is now broadly used for the treatment of cancers under both in vitro and in vivo conditions. Additionally, CQ has been used to relieve Knot disease systemic lupus erythematosus rheumatoid arthritis [19][20][21]. It has been reported that CQ also can induce apoptosis, inhibit autophagy and cell cycle, gallbladder cancer liver cancer, and lung cancer [22][23][24]. Of note, studies found that CQ in combination with other chemotherapeutic agents could enhance the efficacy of cancer therapy [25]. In this study, a pHresponsive biocompatible and pH/enzyme-responsive MTX-conjugated PMAA containing CQ was fabricated for targeted drug delivery and combination therapy. In vitro anticancer activity of developed nanoplatforms against SW480 colon cancer cells was investigated in detail. Furthermore, in vitro and in vivo toxicities assessments were performed via Artemia Salina lethality assay, hemolysis test, MTT, and oral acute toxicity to unveil whether these nanoparticles are biocompatible or not.

PMAA nanohydrogel preparation
A typical procedure of distillation-precipitation polymerization (DPP) was performed to prepare PMAA nanohydrogels, briefly, MAA (5.808 mmol), bisacrylamide (0.214 mmol), and APS (0.102 mmol) were dissolved in acetonitrile in a dried 50-mL single-necked flask with the aid of ultrasound for 10 min to obtain a homogenous suspension [26]. After that, the reaction mixture was heated from ambient temperature to the boiling state (>90 C) within 30 min and the reaction was ended after about the half of acetonitrile was distilled from the reaction mixture within 1 h. The obtained nanohydrogels were separated and purified three times by repeating ultracentrifugation (12,000 rpm for 10 min)/redispersion cycle in distilled water.

Preparation of pH/enzyme-responsive nanohydrogel
To conjugate the anticancer drugs of MTX onto the pH-sensitive PMAA nanocarrier, first, the carboxylic acid groups of the nanohydrogel should be activated. For this purpose, 190 mg EDC.HCl and 120 mg of NHS were added to the aqueous solution containing 60 mg of PMAA nanohydrogel. Then the reaction mixture was stirred at room temperature for 6 h to activate the carboxylic acid groups. After the activation steps were proceeded, MTX (5 mg) was added to the reaction mixture with gentle shaken (100 rpm) and then kept in a dark for 24 h at 37 C to prepare PMAA-MTX nanohydrogels. The resulting PMAA-MTX products were collected by ultra-centrifuging at 18,000 rpm for 20 min. To obtain impurity-free PMAA-MTX products, the colloidal suspension was re-dispersed and purified using distilled water this process was repeated three times. Ultimately, the crude product was dried under a vacuum at 50 C overnight.

Characterization of prepared nanohydrogel
The chemical structure of fabricated PMAA-MTX nanohydrogels was investigated by Fourier transform infrared spectroscopy (FT-IR) (Bruker, Tensor 27, USA), and 1 H-nuclear magnetic resonance ( 1 H NMR) spectra (BRUKER DRX-250 AVANCE spectrometer) CDCL 3 and tetramethylsilane (TMS) were used as the solvents and internal standard, respectively. Dynamic light scattering (DLS) was applied to measure the zeta potential, mean hydrodynamic diameter, and polydispersity index (PDI) using zetasizer (Malvern Instruments, Worcestershire, UK, model Nano ZS). Furthermore, the morphology of PMAA-MTX-CQ was investigated by CM120 TEM (Philips) transmission electron microscope (TEM) at an accelerating voltage of 200 kV.
Determination of conjugated MTX MTX was chemically conjugated onto the PMAA, and amounts of drug which bonds chemically through an amides linkage, a dialysis membrane (12 kDa) were used to determine MTX conjugated. In this context, PMAA-MTX (3 mg) and a PBS solution containing proteinase K enzyme (1 mg/mL) was immersed in an external solution of 30 mL PBS and then gently shaken (100 rpm) at 37 C. Then, the content of MTX released from PMAA was determined at a wavelength of 304 nm using a UV-Vis spectrophotometer (Thermo Fisher Scientific, Madison, model GENESYSTM 10S).
CQ and loading study CQ drug was encapsulated within PMAA-MTX nanohydrogel and drug loading capacity (DL) and encapsulation efficiency (EE) were determined. Typically, a known amount of PMAA-MTX nanohydrogels (10 mg) and CQ (5 mg) were dispersed in 30 mL of distilled water with constant shaking (100 rpm) at room temperature for 24 h. In order to collect the CQ-loaded PMAA-BSA nanogel, the sample was centrifuged and washed at least three times to remove the unloaded CQ and surface adsorbed CQ. DL was calculated by subtracting the amount of CQ in the supernatant from the amount of the initial drug using a spectrophotometer (Thermo Fisher Scientific, Madison, model GENESYSTM 10S) at 335 nm. DL and EE were determined using the following formula: In vitro drug release MTX was covalently attached to the PMMA surface through amide linkages. To assess the release profile of MTX from PMAA nanohydrogels proteinase K enzyme (mimic the lysosomal condition), was used to dissociate the amide bonds between MTX and PMAA according to a previously published study [11]. Moreover, the MTX release study was carried out at the pH values of acidic and normal physiological 5.5 and 7.4 irrespectively with and without the proteinase K enzyme. Briefly, PMAA-MTX (2 mg) along with a PBS solution containing proteinase K enzyme (1 mg/mL) was added to a dialysis bag (Mw 12 kDa) and immersed in 35 mL of PBS solution with different pH of 5.5 and 7.4 and shaken (100 rpm) at 37 C. Finally, the amount of released MTX at different time intervals was determined at a wavelength of 304 nm using a spectrophotometer. In order to determine the CQ content similar procedure was performed except using the proteinase K enzyme.

In vitro anti-cancer effects of PMAA-MTX-CQ nanohydrogels
The anticancer therapeutic effects of PMAA-MTX-CQ nanohydrogels were investigated using the MTT test toward SW480 colon cancer cells based on previously published research [27]. In brief, in 96-well plates, after the confluency of the cancer cells reached the desired density (7000 per well) the cells were treated with different groups of MTX (2.5, 5 and, 15 lg/mL), CQ (12.5, 25 and 75 lg/mL), PMAA (25, 70 and 210 lg/mL) and PMAA-MTX-CQ with a various concentration equivalent to MTX and CQ drugs. After 24 h of incubation, the culture medium was discarded and then 20 lL of MTT reagent with a concentration of 5 mg/mL was added to each well. After 4 h of incubation, 100 lL DMSO was added to each well and the absorbance of each well was determined at a wavelength of 570 nm using an ELISA reader. Finally, the cell survival rate was calculated by comparing the optical density in the treatment group and the control group.

Biocompatibility study of PMAA nanohydrogels
Artemia salina lethality test One of the commonly used tests for evaluating of the biocompatibility of developed nanocarrier is the Artemia salina lethality assay (ASL). Herein, to determine the possible nanohydrogels-mediated toxicity, the ASL test was performed according to our previous research [28]. This assay possesses several benefits over other toxicological tests such as being inexpensive, rapid, simple, and requires minimal resources that have been commonly applied as a preliminary indication of toxicity [29].

Blood compatibility
Blood compatibility is considered an important feature for any developed nanocarrier and is highly required for materials with direct contact with blood/or blood components in particular red blood cells. Hemolysis assay also called blood compatibility test refers to the induced potential toxicity of substances against Red blood cells [28].
In this assay, after erythrocytes were collected, they were diluted with PBS to prepare an erythrocyte suspension (5% hematocrit). Then erythrocytes were incubated with a broad range of PMAA nanohydrogel concentrations (31.25-1000 lg/mL). After 4 h of incubation at 37 C, the amount of released intracellular hemoglobin (Hgb) as results of erythrocyte membrane destruction was determined. In this context, suspension was then centrifuged at 3000 rpm for 5 min for removing the non-lysed erythrocytes. The absorbance of released Hgb in the supernatant as a result of hemolysis was measured at 540 nm by the spectrophotometric method. Blood compatibility was ascertained by comparing of the hemolysis induced by Sodium dodecyl sulfate (positive control: 100% hemolysis) and PBS (: negative control: no hemolysis). All the experiments were performed in triplicate. The hemolytic effects were calculated as follows: Cell cytotoxicity To confirm the biocompatibility of PMAA nanohydrogels toward healthy cells. MTT assay was performed against HFF2 cell line based on previously published research [27]. In brief, in 96-well plates, after the confluency of the cancer cells reached the desired density (7000 per well), the cells were treated with different concentration of PMAA (ranging from 31.25 to 1000 lg/mL. After 24 h of incubation, the culture medium was discarded and then 20 lL of MTT reagent with a concentrations of 5 mg/mL was added to each well. After 4 h of incubation, 100 lL DMSO was added to each well and the absorbance of each well was determined at a wavelength of 570 nm using an ELISA reader. Finally, the cell survival rate was calculated by comparing the optical density (OD) in the treatment group and the control group. Results were expressed as mean ± SD.
In vivo oral acute toxicity assay Oral acute toxicity mediated by developed PMAA nanohydrogels was exploited using mouse lethality assay (MLS). In this assay, LD 50 is the dose of the tested substance required to kill half the mice. MLS test shows how much of a substance must be orally taken before inducing a hazardous issue/or becomes deadly. 10 Swiss albino adult mice weighing between 20 and 25 g were purchased from the Pasteur Institute of Iran. The mice were placed in the standard cage and kept under standard laboratory conditions, as well as, animals had free access to standard rodent-chow diets and water as per OCED [30]. All experimental protocols were approved by the institutional Ethics Committee of Zanjan University of Medical Sciences. The mice were given by suspension of PMAA nanohydrogels in PBS at concentrations of 17.5, 175, 1750, and 5000 mg/kg using gavage to determine the LD 50 according to the standard procedure of OCED [30]. The mice were monitored for any changes in behavior and physical activity as well as recording the number of death.

Results and discussion
Characterization Synthesis and characterization of MTX-conjugated PMAA nanohydrogels PMAA nanohydrogel as a pH/enzyme-responsive biocompatible polymeric nanoplatforms were prepared for targeted co-delivery of CQ and MTX to fight against colorectal cancer cells. In order to direct the chemotherapeutics into the intended site of action (tumor tissues), MTX as a targeting ligand and anti-cancer drug was conjugated onto the developed pH-responsive nanohydrogels. Synthesizing route of the PMAA, MTX conjugation along with CQ loading were illustrated in Figure 1A and B. In the first step, nanohydrogels were prepared through distillation-precipitation polymerization (DPP) a facile and rapid route as depicted in Figure 1A. In the following step, to diminish off-target toxicities and augment the target-specific colon delivery, MTX, a potent chemotherapeutic, was bonded onto the nanohydrogel surface through amide linkages using the carbodiimide coupling method Figure 1B. The chemical structure and composition of PMMA, MTX and MTX-conjugated PMAA nanohydrogels were characterized using FTIR and 1 H NMR techniques. FTIR is a common characterization technique employed to confirm the nanohydrogesl formation and confirm the MTX-conjugation. In this regard, FTIR spectra of MTX, PMAA, and MTX-conjugated PMAA nanohydrogels are shown in Figure 2A. The FTIR of PMAA-CQ is also shown in Figure 2B. Carboxylic acid groups are predominant and major groups in the structure of PMAA and its corresponding peak is mainly appeared within 2500-3500 cm À1 and related to the O-H stretching, and the presence of the broad peak within about 2700 cm À1 to 3550 cm À1 is attributed to these groups ( Figure 2). Moreover, an intense and sharp peak at around 1710 cm À1 was attributed to the characteristic peak of the carbonyl group (C ¼ O). These findings indicated that PMAA nanohydrogels were successfully prepared and are well supported by previous studies [31]. MTX due to its unique features was used to direct the nanocarrier to cancer cells. In this case, MTX with targeting capability was chemically conjugate on the surface of PMAA using the carbodiimide coupling method. After activation of PMAA carboxylic acid groups, MTX was grafted through the amide bond and MTX leakage through bloodstream is thus minimized. The FTIR spectrum of free MTX and MTXconjugated PMAA is shown in Figure 2. In the FTIR spectrum of free MTX, the characteristic peak of primary amines can be seen at 3436 cm À1 , whereas C-H vibration was assigned at 2962 cm À1 . The peak at 3417 cm À1 is a characteristic peak of primary amine. The peak at 1655 cm À1 is also known as the amide band. Primary amine scissoring peak is observed at 1546 cm À1 whereas wagging peaks related to the À NH2 and -NH is appeared at about 700-740 cm À1 [27,32]. MTX-conjugated PMAA FTIR spectrum indicated the presence of a new carbonyl group at 1690 cm À1 related to the amide bond formation. Besides this, another peak at around 1715 cm À1 is observed which is related to the characteristic peak of unreacted carboxylic acid groups. The stretching vibration of methyl and methylene groups also appeared at 2925 and 2852 cm À1 respectively. Moreover, the bonds at around 1450-1546 cm À1 are assigned to the stretching vibrations of aromatic rings of MTX molecules. All other distinct peaks of free MTX and PMAA were also observed in the PMAA-MTX FTIR spectrum [32]. These results underline the fact that conjugation of MTX onto PMAA was successfully proceed.
Other characterization techniques such as 1 HNMR were also applied to further confirm the conjugation of MTX onto the PMAA. Figure 3 displayed the HNMR spectra of PMAA and MTX-conjugated PMAA. Protons related to the PMAA methyl (-CH 3 ) and methylene (-CH 2 -) groups were observed at around 1.2 and 1.5 ppm and there were no peaks at 5-6 ppm (vinyl group (H 2 -C ¼ C-) which confirms the polymerization process has successfully occurred, as shown in Figure 3. As stated previously MTX played a dual role in anti-cancer drugs and targeting ligands and peaks between 2 ppm to 5 ppm along with 7-8 ppm are the main peaks of MTX in which the latter is attributed to the vinyl bonds in the benzene ring and are shown in PMAA-MTX spectrum. Based on these findings it could be inferred that MTX chemically bonds to the PMAA. [32]. These results are in line with previous results, for example, Fattahi et al. conjugated the MTX to form the lipophilic MTX prodrugs containing ester [32].
Morphology, size and zeta potential Transmission electron microscope (TEM) is one of the important techniques in the characterization of nano-scale entities and is therefore used to analyze the morphology, particle size, and homogeneity of the synthesized pH/enzyme-responsive PMAA-MTX-CQ nanohydrogels. As depicted in TEM images, there were uniform distribution particles along with spherically shaped nanohydrogels. Also, it is obviously demonstrated that the particle size of synthesized PMAA-MTX-CQ nanohydrogels is not exceeded 50 nm, and there was no sign of PMAA-MTX-CQ aggregation ( Figure 4A). Mean hydrodynamic diameter along with zeta potential were also determined by dynamic light scattering. As illustrated by Figure 4B, hydrodynamic diameter and polydispersity index (PSD) were around 174.80 and 0.06, respectively. Interestingly the amount of PSD (less than 0.3) of the PMAA-MTX-CQ nanohydrogels is well consistent with that of showed by TEM images which highlight the homogeneity of developed nanoplatforms. On the contrary, a significant in particle size was observed by comparing the size obtained by DLS and TEM (size obtained by DLS > TEM). These substantial difference is spelled out by the fact that the presence of aqueous in DLS measurements leads to nanohydrogel swelling, while in the TEM technique nanohydrogel is collapsed to the dry state. Furthermore, the zeta potential or surface charge density of as prepared PMAA-MTX-CQ was also determined. This surface characteristic can predict the physical stability of the colloidal system in which the high the zeta potential the more the physical stability of colloidal nanosuspension. The amount of zeta potential was found to be around À14 mV, which indicates great colloidal stability of PMAA-MTX-CQ nanohydrogel and is consistent with previous findings in the literature [27].

In vitro release study and drug loading
To evaluate the quality, efficacy, and safety of PMAA-MTX-CQ systems, in vitro release study of both MTX and CQ drugs was performed. Moreover, to augment intracellular drug trafficking and specific-site delivery while diminishing the unpleasant side effects of the CQ and MTX pH-responsive PMAA nanohydrogels were applied to encapsulate the drug molecules. In this case, several attempts have been conducted aiming at improving remarkable drug accumulation inside target cells. One promising strategy is conjugation drug(s) over or inside the nanocarriers. It was demonstrated that the MTX-conjugated nanoparticles exhibited enhanced cellular uptake and therapeutic potential to fight against cancer. For an instance, Kohler et al. conducted research in which they fabricate MTX conjugated magnetic nanoparticles (MNPs) for enhanced their intracellular uptake. The results indicated that chemical bonds between MTX and MNPs were readily hydrolysable under intracellular conditions and MTX intercellular accumulation is thus increased [33]. Receptor-mediated endocytosis is responsible for MTX conjugated MNPs uptake by cancer cells, due to the MTX (analog of FA). Then lysosomal proteases dissociate the linkage between MNPs and MTX, resulting in MTX trigger release and diminishing off-targeted toxicities. It is believed that presence of proteases in the lysosomal compartment is responsible for cleaving the amide bond between MTX and nanocarriers according to a published study [9]. Herein, to simulated conditions found in the lysosome, fabricated PMAA-MTX nanohydrogels were incubated with proteinase K solution at acidic pH conditions. Figure 5 showed the drug release study of MTX and MTX conjugated PMAA nanohydrogels under different condition. Drug release profile showed that the release rate of MTX drug from MTX conjugated nanohydrogels in the presence of proteinase K enzyme and at pH 5 is higher than that of MTX released from nanohydrogels in the absence of enzyme but similar pH value (5). To put it briefly, approximately 64% of MTX was released from MTX conjugated nanohydrogels in presence of enzyme and an acidic microenvironment during 72 h whereas in the absence of enzyme, only 25% of MTX was released from MTX conjugated nanohydrogels at a similar condition ( Figure 5). Furthermore, the results of MTX release at pH 7.4 from MTX conjugated nanohydrogels confirm the above results, of which the release rate of MTX in The presence of proteinase K enzyme is much higher than the amount of MTX released form MTX conjugated nanohydrogels in the absence of the enzyme. In other words, at acidic pH within 72 h, approximately 70% and of 20% MTX were released from MTX conjugated nanohydrogels in the presence and absence of enzyme, respectively.
Due to the fact that the optimal activity of the proteinase K enzyme at physiological pH 7.4, the amount of MTX released from MTX-conjugated PMAA at this pH it is more than acidic pH 5. Finally, it can be concluded that the release of MTX drug from the prepared nanohydrogels is dependent on enzyme activity and underlined the enzymatic stimuli-responsive properties of developed methotrexate-conjugated nanohydrogels for colon targeted delivery ( Figure 5). These fabulous data share a number of similarities with Nosrati et al.'s findings which according to their obtained results in presence of enzymes the release of MTX drug from developed MTX conjugated MNPs was much faster than that of released under the physiological condition/or absence of enzyme [9]. The amount of MTX conjugated onto the PMAA was determined using UV-vis spectroscopy. It was found that the drug loading capacity (DLC) of MTX was around 4.99%. Moreover, PMAA bears carboxylic acid moieties in its polymeric backbone and due to the negative charge of these groups, CQ could be captured/or absorbed inside the nanohydrogels by electrostatic interaction. Besides this physical entrapment of CQ was facilitated through the diffusion process. Nanohydrogels were swollen in aqueous media and this may lead to more CQ entrapment within nanohydrogels. Hydrogen bonding between the -COOH units and the amines of CQ augment the drug loading as well [26]. Consequently, a high amount of CQ was readily incorporated within the nanohydrogels due to the above-mentioned reasons. UV-vis spectroscopy revealed that CQ loading within developed nanohydrogels was found to be 25.01% which specifies the strong electrostatic absorption between positively charged of CQ and negative charges of (-COOH) units (Table 1).
An efficient propitious nanovehicle should guarantee the controlled accumulation of its cargo as well as be capable of intelligently releasing its payload in response to external stimuli such as enzyme, pH, temperature, and etc. Since tumor tissues possess unique microenvironments of acidic pH, the delivery of old-fashioned drugs based on pH-sensitive polymeric nanohydrogels could efficiently deliver anti-cancer drugs into the tumor tissues in comparison with conventional nanotherapeutics. Additionally, such pH-responsive nanosystems enable the oncologist to treat colon cancers effectively with the least possible side effects in a safe and controlled manner. Accordingly, in vitro CQ release was exploited under both normal and acidic conditions. As shown in Figure 6, the amount of CQ released from developed nanohydrogels in an acidic (pH 5) microenvironment is much more than that of released in physiological conditions. As a matter of fact, 76% and 39% of CQ is released under acidic and normal pH, respectively. As can be seen, a burst CQ release profile was observed and the release of free CQ drug in two environments with different pHs of 7.4 and 5 indicated that the dialysis membrane had no barrier effects for CQ to diffuse across the dialysis bag and pH had no significant effects in diffusion process as well. Due to the burst and more release of the drug in an acidic microenvironment compared to the physiological environment, it can be concluded that the release of CQ from designed nanohydrogels is pH dependent. These findings are in complete agreement with Knipe et al.'s findings in which they developed poly(methacrylic acid-co-N-vinyl-2-pyrrolidone) (P[MAA-co-NVP]) nanogels for pH/Enzyme responsive delivery of siRNA [34]. Based on obtained results, the designed nanosystem could be served as a promising pharmaceutical vehicle for pH/enzyme-responsive delivery of anticancer agents.

In vitro anti-cancer study
It has been widely accepted that the acidic extracellular pH value of malignant tumors is less than that of adjacent healthy tissues due to the hydrolysis of According to their findings, superior anti-cancer activity was detected for the combination of CUR and MTX in nanoparticles formulation (nanoformulation of MTX þ CUR) compared to the combination of both drugs alone (MTX þ CUR) [27].

Biocompatibility study
Cytotoxicity toward normal cells An ideal carrier should not impose any adverse toxicities on normal cells. Therefore, the biocompatibility of developed PMAA nanohydrogels was assessed against HFF2 (normal cells) using MTT the test. As shown in Figure 8A, PMAA over a wide range of concentrations had no unpleasant toxicity even at a high concentration of (1 mg/mL). Indeed, the cell viability rate did not decline significantly by an increment in PMAA concentration. In other words, the survival rate of HFF2 was not affected by PMAA over a broad range of concentrations and this result confirm the MTT results of PMAA nanocarrier against SW480 cells of which PMAA exhibited approximately 100% cell survival rate. This result offers vital evidence for cytocompatibility and corroborates with Sahu et al.'s findings regarding the biocompatibility of developed pH-responsive nanogel for bleomycin delivery [35].

Blood compatibility
Hematotoxicity of prepared nanohydrogels was exploited against blood components in particular red blood cells. As depicted in Figure 8B, PMAA nanohydrogels over wide ranges of tested doses demonstrated insignificant amount of hemotoxic effects. PMAA at 1 mg/mL only induces minor hemolysis (4.98%) which is not exceeded the 5% destruction of the red blood cells membrane. This result highlight the utility of PMAA nanohydrogels as a safe and blood-compatible nanocarrier for purposes of use the delivery of drugs within blood flows. These results fit well with previous results [28,35]. According to the ISO/TR 7406 ethical guidelines, any developed safe biomaterials should not induce more than 5% hemolysis which has further strengthened our confidence in the blood compatibility of developed nanohydrogels [35]. In vivo oral acute toxicity The objective of this assessment is to obtain an insight into the PMAA nanocarrier in vivo oral acute toxicity. To elaborate the in vivo safety of PMAA nanocarrier, a single oral dose (varying doses) was administered to each mice by gavage ( Figure 8C). Interestingly, based on the obtained results oral administration of PMAA had no deleterious effects on mice, of which all mice survived with normal physical activity within one week. According to OCED and Hodge and Sterner scale, the in vivo biosafety of fabricated nanohydrogels is approved for preclinical investigation [30]. Furthermore, these findings are well supported by others findings in the literature [26,32].
Artemia salina lethality assay (ALS) Further biocompatibility analysis was performed to confirm the biosafety of prepared nanohydrogels. ALS assay is rapid, simple and requires no advanced facilities and is selected to be conducted for biocompatibility assessment. The nauplii were exposed to PMAA nanohydrogels at varied concentrations (from 31.25 to 1000 lg/mL) for 24 h and obtained data are depicted by Figure 8D. This assay also confirms the biocompatibility of these nanohydrogels and is in accordance with our initial findings regarding the safety of prepared nanocarrier [26]. Figure 8D indicated that the survival rate of treated nauplii was close to 100% and at a concentration of 1 mg/mL around 90% survival was observed, this negligible toxicity makes the prepared nanohydrogels an ideal pharmaceutical carrier. These results are also completely in line with Rashidzadeh et al. findings in which the prepared nanohydrogels did not cause the significant toxicity against Artemia Salina over wide range of concentrations [26].

Conclusion
Traditional cancer therapy methods such as chemotherapy can cause severe side effects to healthy tissues/organs due to the lack of specificity, series of cycles of drug administration and, broad biodistribution. To suppress the aforementioned drawbacks, the use of combined therapy of chemotherapeutic agents loaded in targeted pH/enzyme-responsive nanovehicles has shown a promising therapeutic potential. Non-toxic and pH/enzyme-responsive MTX-conjugated PMAA containing CQ nanohydrogels were prepared and its biocompatibility along with its anticancer activity were determined as well. Artemia Salina lethality assay, MTT, hemolysis and, oral acute toxicity test showed that the prepared nano-scale entities were practically safe and had excellent biocompatibility. The release of CQ was accelerated under acidic conditions while retarded in normal physiological pH underling the pH-responsiveness properties of developed nanohydrogels while the MTX was released in the presence of enzyme indicating the enzyme-responsiveness features of developed nanohydrogels. Interestingly, PMAA containing MTX and CQ exhibited superior anti-cancer effects against SW480 colon cancer cells compared to the MTX, CQ or, even MTX þ CQ at equivalent dosages and the utility of combined therapy using targeted pH-responsive nanovehicles in cancer therapy is thus confirmed. In summary, such a targeted biocompatible pH/enzyme-responsive PMAA-MTX-CQ could effectively suppress cancer cell growth and proliferation and open the new horizon in colon cancer therapy.