In vitro control release, cytotoxicity assessment and cellular uptake of methotrexate loaded liquid-crystalline folate nanocarrier

Biomedicine & Pharmacotherapy 69 (2015) 102–110

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Original article

In vitro control release, cytotoxicity assessment and cellular uptake of methotrexate loaded liquid-crystalline folate nanocarrier Rahul Misra a,*, Mohita Upadhyay b, Vivekanandan Perumal b, Sanat Mohanty a a

Advance Materials & Nanoscience Laboratory, Department of Chemical Engineering, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India b Kusuma School of Biological Sciences, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 October 2014 Accepted 9 November 2014

Folate molecules self-assemble in the form of stacks to form liquid-crystalline solutions. Nanocarriers from self-assembled folates are composed of highly ordered structures, which offer high encapsulation of drug (95–98%), controlled drug release rates, active cellular uptake and biocompatibility. Recently, we have shown that the release rates of methotrexate can be controlled by varying the size of nanoparticles, cross-linking cation and cross-linking concentration. The present study reports the in vitro cytotoxic behavior of methotrexate loaded liquid-crystalline folate nanoparticles on cultured HeLa cells. Changing drug release rates can influence cytotoxicity of cancer cells. Therefore, to study the correlation of release rate and cytotoxic behavior, the effect of release controlling parameters on HeLa cells was studied through MTT assay. It is reported that by controlling the methotrexate release, the survival rates of HeLa cells can be controlled. Released methotrexate kills HeLa cells as effectively as free methotrexate solution. The co-culture based in vitro cellular uptake study through fluorescence microscopy on folate receptor positive and negative cancer cells shows that the present nanocarrier has the potential to distinguish cancer cells from normal cells. Overall, the present study reports the in vitro performance of self-assembled liquid-crystalline folate nanoparticles, which will be a platform for further in vivo studies and clinical trials. ß 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Folate nanoparticles Liquid-crystalline Cytotoxicity Self-assembly Chromonics Cellular uptake

1. Introduction In the field of cancer research, in particular, nanoparticles are being studied as therapeutic and diagnostic tools to better understand, detect, and treat cancer. Nanoparticle mediated controlled chemotherapy is also one strategy, which claims to make conventional chemotherapy more effective. In general, conventional chemotherapy is effective for treatment of cancer, but due to direct administration of drug, patient experiences extremely high levels of drug in the body. These levels (higher than therapeutic levels required) are high enough to kill maximum number of cancer cells, but at the same time, these levels are also toxic to the normal healthy tissues of the body. Past studies have reported several serious off-target side effects [1,2]. At low doses, conventional chemotherapy will be ineffective against the tumor, whereas, at excessive doses, the toxicity will be intolerable to the patient. Moreover, there is a significant increment in these side * Corresponding author. Tel.: +919582708534. E-mail address: [email protected] (R. Misra). http://dx.doi.org/10.1016/j.biopha.2014.11.012 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.

effects with repetitive cycles of drug administration (alternative days, weeks etc.) [3,4]. Modified release pattern of chemotherapeutic drugs can enhance the therapeutic effects on malignant cells with reduced toxicity on normal cells by maintaining a constant therapeutic window. This can possibly be achieved through controlled drug delivery strategies. Controlled drug delivery is desirable in order to achieve multiple but constant therapeutic levels in chemotherapy for prolonged time without drug re-administration. This can reduce toxic levels as observed in conventional chemotherapy. Different classes of materials have been explored in the past two decades to develop such controlled drug delivery vehicles, which are biocompatible and effective in nature [5–10]. Liquid-crystalline folate nanocarriers represent such class of novel materials. Folic acid is a natural vitamin B. Being a part of dietary supplements, it is biocompatible in nature. It has been reported that higher amount of folic acid does not cause any harm as it is excreted out through the urine [11]. Thus, there is lower risk of toxicity and side effects. The daily requirement of folate in the body for an average adult is reported to be 400–500 mcg/day to support

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various functions, like cell growth, DNA methylation [12,13]. Cancer cells, in general overexpress folate receptors on their surface [14–16]. This characteristic property has been used by researchers in the past, to direct nanoparticles towards tumors by attaching folic acid as a ligand on the surface of nanoparticles [17–19]. Liquid-crystalline folate nanoparticles, being composed of ordered structure, offers high encapsulation of drugs. As these nanoparticles are formed of folic acid, they will be biocompatible and non-toxic in nature. We also hypothesize that they do not require any extra step of targeting using folate ligands. Moreover, in chemotherapy, drugs are administered along with a supplement of folic acid to help the growth of healthy cells, which are targeted by the toxic effects of the drug [2,3]. As these nanoparticles disintegrate, both folic acid and encapsulated drug will be released into the medium. The release of folic acid along with the methotrexate will automatically help the growth of healthy cells off-targeted by the nanoparticles, if any. Therefore, folic acid provides strong motivation to be explored as a nanocarrier for controlled chemotherapy. Folic acid exhibits liquid-crystalline behavior in aqueous solution by forming a self-assembly in the form of ordered stacks and columns [20–22]. The nanoparticles of liquid-crystalline folates exhibit highly ordered structures with the potential to encapsulate drugs along with the controlled release of those drugs. Our previous studies [23,24] have shown that folate self-assembles even at low concentration of 0.1 wt % and, we have reported a method to engineer folate nanoparticles from liquid-crystalline folates [25]. Moreover, an extensive study on controlled release of methotrexate from liquid-crystalline folate nanoparticles has been reported recently by our group [26]. This study has shown that the release rates of methotrexate can be controlled by controlling the size of nanoparticles, cross-linking cation and cross-linking concentration. The present study reports the in vitro cytotoxic and cellular uptake assessment of methotrexate loaded folate nanoparticles on cultured HeLa cells. The role of release rate controlling parameters on cell survival was studied and cytotoxicity of MTX-folate nanoparticle over free MTX was compared. This study also shows the potential of folate nanoparticles in distinguishing cancer cells from normal cells through co-culture method [27]. The influence of methotrexate release rates on cytotoxicity of HeLa cells was studied. A comparative cellular uptake study was performed on folate receptor positive and negative cancer cells, which show that the mechanism of uptake via folate receptor-mediated endocytosis. Dynamic light scattering (DLS) and scanning electron microscope (SEM) techniques are used to characterize the nanoparticles developed while the concentration of released methotrexate is determined by a method developed by high performance liquid chromatography technique. The cytotoxicity studies were carried using MTT assay while the cellular uptake was studied through fluorescence microscopy. 2. Materials and methods 2.1. Materials Folic acid (molecular formula: C19H19N7O6; molecular weight: 441.3974 g/mol; PubChem:CID 6037) and hydroxy propyl methyl cellulose (HPMC) (molecular formula: C12H20O10; molecular weight: 324.2848 g/mol) were purchased from Central Drug House (CDH), New Delhi. Methotrexate (molecular formula: C20H22N8O5; molecular weight: 454.44 g/mol; PubChem: CID 126941) was purchased from TCI Chemicals India Ltd, New Delhi. Methotrexate is an antimetabolite and anti-folate drug, which is widely used in the majority of cancer types during conventional chemotherapy [28]. Fig. 1a–c shows the chemical structure of folic acid, methotexate and HPMC

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Fig. 1. Chemical structure of (a) folic acid, (b) methotrexate (MTX), (c) hydroxy propyl methyl cellulose (HPMC).

respectively. Normal saline (0.8% NaCl solution) was used as a release medium for in vitro release studies of methotrexate. (All concentrations reported in this study are in weight/weight basis). 2.2. Preparation of liquid-crystalline folate solution Folic acid by itself does not dissolve in water, however, in the presence of NaOH, it forms the liquid-crystalline solutions. It has been reported in the past that folic acid molecules get completely ionized by NaOH and can be dissolved in water easily. Liquidcrystalline behavior is observed between the pH values 6.5–7.5 [23]. The stock solution of folic acid was neutralized by adding 1 N NaOH solution dropwise till the solution turned liquid-crystalline (visually) while ensuring that the pH was less than 7.0. 2.3. Preparation of methotrexate encapsulated folate nanoparticles Folate nanoparticles can be engineered using HPMC as a tool. HPMC is a water-soluble cellulosic biocompatible polymer [29], which is used in the food industry as additives, emulsifiers, thickening and suspending agents as well as in pharmaceutical industry. Due to difference in the nature of interactions of folic acid and HPMC in aqueous solution, folate nanoparticles are formed. In aqueous state, folate forms two-phase system with HPMC as folate ions with aromatic rings prefer to interact with themselves, rather than with HPMC. Liquid-crystalline folates when mixed with HPMC get dispersed into nano-domains; which on cross-linking with multivalent salts forms stable nanoparticles. We have extensively studied the designing of folate nanoparticles from liquid-crystalline folate solutions in terms of phase behavior, size distribution and thermodynamics aspects previously [25]. It has been reported by us that the size of the folate nanoparticles can be controlled by the choice of folate or HPMC concentration, with

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little impact of the cross-linking cation on the size. Moreover, folates in the nanoparticles largely maintain their ordered structure. Subsequently, our recent study has shown the detailed analysis of methotrexate loading in folate nanoparticles along with the encapsulation efficiency (about 95%) and participation of methotrexate in folate assembly with the help of X-ray diffraction and in vitro release [26]. In the present study, the nanoparticles of methotrexate encapsulated liquid-crystalline folate solutions have been developed using same method. Methotrexate loading amount was kept at 10% and 30% of folate concentration. Liquid-crystalline folate solution with methotrexate was mixed with HPMC polymer for 6 h at 500–600 rpm. This solution was cross-linked with multivalent salt. Following cross-linking, the size of the nanoparticles were determined with the help of dynamic light scattering technique (DLS). For all further analysis, the mean particle size is used to describe trends with changing concentrations. After cross-linking, a centrifugation and washing step is performed to remove the excess HPMC and unbound salts. Further, the pellet obtained after centrifugation was lyophilized (Christ Alpha 1-4 LD Plus Freeze Dryer) at –49 8C and 0.002 mbar vacuum pressure. 2.4. Characterization 2.4.1. Dynamic light scattering The size distribution of the nanoparticles developed was determined by dynamic light scattering technique using a particle size analyzer (Malvern Zetasizer nano ZS 90). For the measurements, 1 mL of the nanoparticle suspension was dispersed in 5 mL of de-ionized water and sonicated during 1 min. The analyses were performed at a scattering angle of 908 with refractive index 1.33 and at a temperature of 25 8C. 2.4.2. High performance liquid chromatography (HPLC) To determine the concentration of released methotrexate in the release medium, measurements were made with the help of HPLC technique. In our previous study [26] on methotrexate release, we have developed a method of analysis with the help of reversedphase HPLC (Thermo Scientific UHPLC Dionex Ultimate 3000) to detect the methotrexate and folic acid in aqueous mixtures. A combination of phosphate/citrate buffer and acetonitrile in the ratio 90:10 was used as a mobile phase. The samples were detected with the help of a UV detector at 302 nm. Each sample (5 mL) was filtered by 0.2 mm membrane filters (Axiva Nylon 66) and passed through C18 5 mm column (Acclaim 120 4.6  250 mm) as a stationary phase for 15 min at flow rate of 1 mL/min. In the present study, same method was used to determine the concentration of released methotrexate. 2.4.3. In vitro control release of methotrexate Recently, we have reported that the release of methotrexate from folate nanoparticles can be controlled by controlling the size of nanoparticles, cross-linking cation and cross-linking concentration as designing parameters [26]. However, release rates were also dependent on pH of the release medium and nature of release medium. In the present study, we have shown the change in release rates of methotrexate with changing parameters using 0.8% NaCl as release medium. For different experiments, 0.04 g of lyophilized nanoparticles were taken in vials and suspended in 10 mL release medium. At different time points (0–30 days), supernatant samples were collected after centrifugation of the medium at 3000 rpm for 5 min. These supernatants were analyzed by a method developed with the help of HPLC. Methotrexate concentration in the medium was calculated using a calibration curve developed at various concentrations. To detect any particles in supernatant, DLS measurements were performed with supernatant to check the

presence of nanoparticles. The absence of particles in the supernatant by DLS suggests that few particles were present if any. After sampling, the entire medium was removed and replaced with the same amount of fresh release media. 2.4.4. Cell culture The human cervix carcinoma (HeLa), human osteosarcoma (MG63) and mouse fibroblast (NIH-3T3) cell lines were cultivated for in vitro experiments. Cell lines were obtained from the National Centre for Cell Sciences (NCCS) Pune, India. It was cultured in Dulbecco Modified Eagle’s medium (DMEM) and Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum, 100 units/mL penicillin and 100 mg/mL streptomycin, 4 mM L-glutamine and incubated under 5% CO2 and 95% humidified atmosphere at 37 8C. 2.4.5. Cytotoxicity The effect of release controlling parameters on in vitro cytotoxicity was studied on HeLa cells. HeLa cell line was seeded into 96-wells of tissue culture plate having 180 mL of complete media and was incubated for 18 h. In different experiments, MTX-folate nanoparticles of different size, cross-linking cation, cross-linking concentration and MTX loading were prepared and added to the cells and incubated at 37 8C in a humidified incubator maintained with 5% CO2. The cell viability was estimated by 3-(4,5-dimethylthiazol)-2diphenyltetrazolium bromide (MTT) assay [30] at different time points between 2–24 h. Three controls were included along with the test compounds to check any error while performing the experiment, growth of cells, and preparing the test compounds:  positive control: 5% DMSO (dimethyl sulfoxide) was added as positive control to compare the killing efficiency of drug along with the errors in preparing drug compounds. It is known that DMSO is a cytotoxic agent, hence, remarkable cytotoxicity should be achieved in this case;  negative control: only growth medium was added to check any error related to culturing, seeding and growth of cells. Highest growth is expected in this case if no error has been occurring in maintaining the cells;  solvent control: solvent which is used for preparing aqueous drug compounds and suspending nanoparticles. It is used as a control to check any effect of solvent on the growth of cells. It contains de-ionized water and cross-linking ions.

2.4.6. In vitro cellular uptake studies A comparative uptake study was performed between folate receptor (FR) positive and negative cells as well as between cancer cells and normal cells. Three cell lines, HeLa (human cervical cancer cell, FR overexpressing), NIH-3T3 (mouse fibroblast cell, normal cell) and MG-63 (carcinomic human osteosarcoma, FR negative), were seeded into 35 mm cell culture plates and incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 8C with 5% CO2. Nanoparticle uptake by cells was studied using fluorescence microscopy. After 8 h, the cells were washed with incomplete media and were incubated with 25 mg/mL MTX loaded folate nanoparticles. After 12 h of incubation, the cells were washed to remove free nanoparticles and fluorescence microscopy was performed on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD (excited at 470 nm). 3. Results and discussion 3.1. Size distribution of methotrexate loaded folate nanoparticles For cancer drug delivery, the desirable size range of the nanoparticles has been reported to be 30–300 nm [31]. Methotrexate

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Table 1 Concentration of compounds used in formation of folate nanoparticles of desired size. Concentration of folate, drug and HPMC

Size of the nanoparticles

Folate % (g/100mL)

HPMC % (g/100 mL)

Methotrexate % (g/100 mL)

Cross-linked with zinc

Cross-linked with calcium

2.5 0.85 0.45

6 8 9.5

0.25 0.085 0.045

315  20 nm 220  20 nm 105  20 nm

335  20 nm 210  20 nm 125  20 nm

PDI

Intensity (%)

0.37 0.48 0.51

61.2 78.6 62.9

Fig. 2. Size distribution of MTX-folate nanoparticles with the help of dynamic light scattering technique (DLS). The size of MTX-folate nanoparticles can be controlled by controlling the relative concentrations of folate and HPMC. DLS graphs showing the size of folate nanoparticles at (a) 0.45% folate, 9.5% HPMC; (b) 0.85% folate, 8% HPMC; (c) 2.5% folate, 6% HPMC; cross-linked with 10% ZnCl2.

3.2. In vitro methotrexate release In our recent study [26], it has been reported that methotrexate release rates from folate nanoparticles can be controlled by changing the size of nanoparticles, cross-linking cation and cross-linking concentration. Moreover, the release rates were dependent on nature of release medium and pH. When nanoparticles are suspended in release medium, there is an exchange of ions present in release medium with the cross-linked cation in nanoparticles. This leads to disruption of folate assembly and therefore, methotrexate is released into the medium. With changing size of nanoparticles and cross-linking cation, the disruption of folate assembly can be regulated. For example, due to more ionic affinity of zinc ions than calcium, zinc cross-linked folate assembly disrupts slowly as

compared to calcium cross-linked nanoparticles. Similarly, due to more surface area of smaller nanoparticles, they get more intensely cross-linked than bigger particles. Therefore, the drug release from smaller nanoparticles is slower than larger particles [26]. Methotrexate release rates were determined for the present study, by varying the size and cross-linking of particles. Fig. 3 shows the change in release profiles of methotrexate with changing size of nanoparticles and cross-linking cation. The release rates of methotrexate can be increased by increasing the size of nanoparticles as well as by cross-linking with calcium instead of zinc. 3.3. Determining cytotoxic concentrations of free folic acid and methotrexate To study the cytotoxicity of released drug accurately, it was necessary to determine the concentrations at which folic acid and 100

Percent release of Methotrexate (MTX)

encapsulated folate nanoparticles were prepared in this size range by changing the relative concentrations of folate and HPMC. In the present study, folate nanoparticles containing methotrexate have been prepared by the method reported in our previous study [25,26]. Also, it has been shown (Table 1) that the size of these nanoparticles can be controlled by choosing the appropriate concentration of folate and HPMC, as reported earlier in our studies. Following cross-linking, the size of the nanoparticles were determined with the help of dynamic light scattering technique (DLS). In general, for a particular folate concentration, an increase in the HPMC concentration leads to decrease in size of the nanoparticles. DLS data (Fig. 2) indicates increasing trend in the size distribution of nanoparticles with decrease in the concentration of HPMC. Table 1 shows that high folate and low HPMC concentrations lead to nanoparticles of size range 300–350 nm; while low folate, high HPMC leads to size range of 80–150 nm with little impact of crosslinking cation. The nanoparticles formed after cross-linking had a particle size distribution of  20 nm around the mean particle size, as shown in Fig. 2. For all further analysis, the mean particle size is used to describe trends with changing concentrations.

80

105+20 220+20 315+20 125+20 210+20 335+20

nm nm nm nm nm nm

(Zinc cross-linked) (Zinc cross-linked) (Zinc cross-linked) (Calcium cross-linked) (Calcium cross-linked) (Calcium cross-linked)

60

40

20

0

s rs our hou 4h 12

ay days days days days days days days 1d 2 7 4 11 20 24 30

Time Fig. 3. In vitro release profiles of methotrexate with change in release controlling parameters.

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% survival of HeLa cells

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60

40

20

0 l l l l l l l l l l l l l l l /m /m /m /m /m /m /m /m /m /m /m /m tro tro tro ng pg pg ng ng ng ng pg pg ng ng ng on on on 7 0 0 c c 2 0 0 0 0 7 0 2 0 c 0 0 t 1 0 0 0 0 0 1 0 X 1 3 n 3 1 1 ive ive FA FA X 3 T X 1 TX 3 MT MTX TX FA Solve osit egat FA FA FA M MT M M P N Concentration of samples (FA: Folic acid; MTX: Methotrexate) Fig. 4. Cytotoxicity of folic acid and methotrexate within concentration range of picograms to nanograms per mL.

methotrexate are cytotoxic to HeLa cells. After overnight incubation of cultured HeLa cells, the growth medium was removed and the cells were washed with 1 X PBS (phosphate buffer saline). Different concentrations of folic acid and methotrexate were prepared in the range of micrograms, nanograms and picograms. All of these samples were added to the wells containing adhered HeLa cells in triplicates and cytotoxicity was determined within each concentration range through MTT assay. Both folic acid and methotrexate shows negligible cytotoxicity in the range of picograms and nanograms (Fig. 4), whereas significant cytotoxicity was observed at concentrations in micrograms (Fig. 5). Methotrexate is cytotoxic even at lower concentrations of 1 mg/mL, whereas folic acid is cytotoxic only at higher concentrations of 12 mg/mL and above. These results not only provide the cytotoxic concentrations of both the compounds but also suggest that it is safe to use folic acid as a carrier molecule due to its negligible cytotoxicity in lower concentrations as compared to methotrexate, which is structurally similar to folic acid. Solvent

control and negative control shows no cytotoxicity, while 80% cytotoxicity was observed in positive control. 3.4. Comparitive cytotoxicity of methotrexate loaded folate nanocarrier over free methotrexate For an effective designing of nanocarrier, it is necessary that a drug loaded nanocarrier should have an equivalent cytotoxicity comparable to free drug. To investigate this, comparative cytotoxic behavior of bound methotrexate (methotrexate loaded folate nanoparticles) and free methotrexate was studied over time on HeLa cells. Then, 200  20 nm size folate nanoparticles were loaded with 50% MTX and cross-linked with calcium ions. The total concentration of 12 mg/mL MTX-folate nanoparticles loaded with 6 mg/mL methotrexate were added to the wells containing adhered HeLa cells. Aqueous mixture of folic acid and methotrexate at same concentration was also exposed to HeLa cells simultaneously. Moreover, to study the cytotoxicity of the carrier itself, folate nanoparticles

Fig. 5. Cytotoxicity of folic acid and methotrexate at different concentrations in micrograms per mL. Red circle indicating the lowest concentration at which cytotoxicity is observed (FA: folic acid; MTX: methotrexate).

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% survival of HeLa cells

00 10

80 8

60 6 MT TX loaded folate nanoparticles (20 00+20nm) Aq queous mixture of o free MTX and fo olic acid Fo olate nanoparticle es without MTX (2 200+20nm) So olvent control Po ositive control Ne egative control

40 4

20 2

0 0

2

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8

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22 2

24

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Time ((hours) Fig. 6. Comparative cytotoxic effect of released MTX to free MTX with time at the concentration of 6 mg/mL on HeLa cells. (Positive control: 5% DMSO; negative control: DMEM growth media).

(200  20 nm) without methotrexate was also added in different wells at same concentration as a control. DMSO (5%) was added as a positive control, while DMEM growth media was negative control. Aqueous solvent of nanoparticle suspension was used as a solvent control. All the compounds were added in triplicates. The cytotoxicity was determined over time with the help of MTT assay. With free drug, the cytotoxicity increases from 15% to 45% in 2 h to 24 h, respectively. Whereas, with MTX loaded folate carrier, no significant cytotoxicity was observed during initial 6 h but later, it was observed that the cytotoxicity increases after 6–8 h (Fig. 6) and picks up similar killing rates as with free drug. With free drug mixture, the cells are directly exposed to cytotoxic concentration from initial hours. On the other hand, with MTX-folate nanoparticles, drug is present in bound form, which is released over time and leads to cytotoxicity with increase in concentration in the medium. Folate carrier without drug, which was used as a control did not show any significant cytotoxicity. This confirms that the decrease in viability observed with time is due to released methotrexate and there is negligible participation of folic acid in cytotoxicity observed. No cytotoxicity was observed in solvent control and negative control. 3.5. Cytotoxicity of methotrexate loaded folate nanocarrier: effect of cross-linking cation We have seen that the cross-linking cation is an important parameter to regulate the release rates of methotrexate from nanoparticles. Therefore, it is hypothesized that by regulating the release rates, the cytotoxicity can also be controlled. Therefore, to understand the role of cross-linking cation in cytotoxicity, MTXfolate nanoparticles cross-linked with different cations were exposed to HeLa cells and cytotoxicity was determined at different time intervals. The total concentration of 12 mg/mL MTX-folate nanoparticles (200  20 nm) loaded with 6 mg/mL methotrexate were cross-linked individually with 1% CaCl2 and 1% ZnCl2. The solvent control contains the de-ionized water along with equal amount of cross-linking cations. DMSO (5%) was added as a positive control, while DMEM growth media was negative control. All the compounds were added to the wells in triplicates. The cytotoxicity observed (Fig. 7) with calcium cross-linked nanoparticles was higher in comparison to zinc cross-linked nanoparticles. This is consistent with the results obtained in

release studies that show higher initial methotrexate release from calcium cross-linked particles, leading to lower cell survival. Less than 10% cytotoxicity was observed in solvent control, while no cytotoxicity was observed in negative control. 3.6. Cytotoxicity of methotrexate loaded folate nanocarrier: effect of cross-linking concentration We have shown that cross-linking concentration inversely regulate the release rates of methotrexate. With an increase in cross-linking concentration, the folate assembly gets intact more strongly resulting in slower release of methotrexate. To study the influence of cross-linking concentration on cytotoxic behavior, MTX-folate nanoparticles (200  20 nm) were cross-linked with 0.5% 1%, 5% and 10% CaCl2. The total concentration of 12 mg/mL nanoparticles loaded with 6 mg/mL methotrexate was added to wells containing adhered HeLa cells. The solvent control contains the equal concentrations of cross-linking cations in aqueous solvent for nanoparticle suspension. DMSO (5%) was added as a positive control, while DMEM growth media was negative control. All the compounds were added to the wells in triplicates. It was observed (Fig. 8) that there was a reduction in cytotoxicity with increase in cross-linking concentration from 0.5% to 5%. This is due to decrease in initial release rates of methotrexate with increase in cross-linking concentration. However, an increase in cytotoxicity was recorded with 10% CaCl2. The solvent control respective to 10% CaCl2 also show significant cytotoxicity. This is expected since at higher concentrations of cross-linking agent, the cation itself can be cytotoxic to HeLa cells. Only 10–15% cytotoxicity was observed with other solvent control. 3.7. Folate receptor-mediated cellular uptake It is known that folate receptor can capture folic acid from the extracellular medium and transport it inside the cell via receptormediated endocytosis [32–35]. It is the major mechanism of nanoparticles uptake by cancer cells as most of malignant cells have been found to overexpress folate receptors on their surface. Past studies have used folic acid as a ligand to facilitate the entry of nanoparticles into the cancer cells [17–19]. However, folate nanoparticles offer an advantage where no such targeting is required as the nanoparticles in present case is composed of folic

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% survival of HeLa cells

100

80

60

Calcium cross-linked folate nanoparticle (200+20nm) Zinc cross-linked folate nanoparticle (200+20nm) Solvent control (with Calcium ions) Solvent control (with Zinc ions) Positive control Negative control

40

20

0 0

2

4

6

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Time (hours) Fig. 7. Comparative cytotoxic effect of cross-linking cation on HeLa cells. (Positive control: 5% DMSO; negative control: DMEM growth media).

acid itself. To confirm this, cellular uptake studies were carried out on cancers cells expressing folate receptors and cells which do not express folate receptors. Past studies have reported that zinc ions in bound state exhibit fluorescence [36]. This characteristic of zinc ions has been employed to study the uptake of folate nanoparticles. It has been seen that folate nanoparticles cross-linked with zinc fluoresce by themselves. Therefore, florescence spectroscopy was used to see the uptake of folate nanoparticles in cancer cells with time. Zinc cross-linked folate nanoparticles (200  20 nm) were exposed to HeLa cells and fluorescence was detected at different time intervals over the period of 24 h. Calcium cross-linked folate nanoparticles were also exposed to HeLa cells similarly as a control. Fluorescence microscopic images (Fig. 9) shows increase in fluorescence (red) with time, which indicates uptake of the nanoparticles. Whereas, no fluorescence was detected with calcium cross-linked folate nanoparticles.

3.8. In vitro distinguishing cancer cells and normal cells To test the applicability of folate nanoparticles in distinguishing cancer cells from normal cells, uptake studies were performed on a cancer cell and normal cells using co-culture technique. NIH-3T3 was selected as normal cells while, HeLa cell line was used as cancer cells. Due to difference in morphology, HeLa cells can be differentiated from NIH-3T3 cells. HeLa cells have nearly round morphology whereas, NIH-3T3 show elongated appearance. Moreover, NIH-3T3 is folate receptor negative while, like other cancer cells, HeLa cells overexpress folate receptors. Both NIH-3T3 and HeLa cells were co-cultured together in the same culture plate for 14 h. The cells were incubated with folate nanoparticles (200  20 nm) for 12 h and fluorescence imaging was performed. Fig. 10 shows the bright field and dark field image of cells. Bright field image clearly show the HeLa cells and NIH-3T3 cells.

% survival of HeLa cells

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80

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0 0

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22

24

26

Time (hours) Fig. 8. Comparative cytotoxic effect of cross-linking concentration on HeLa cells. (Positive control: 5% DMSO; negative control: DMEM growth media).

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Fig. 9. Fluorescence over time exhibited by HeLa cells exposed zinc cross-linked folate nanoparticles.

Fig. 10. Folate nanoparticles distinguishing cancer cells from normal cells. a: bright field image of HeLa/NIH-3T3 co-culture. NIH-3T3 cells showing elongated morphology, while HeLa cells showing round morphology; b: bright fluorescence (red) observed in HeLa but not in NIH-3T3.

While, distinct fluorescence was observed between both the cells. HeLa cells produce bright fluorescence, whereas no fluorescence was observed in elongated NIH-3T3 cells. This suggests that the present folate nanoparticle system is suited for the simple discrimination of FR-positive cancerous cells from normal cells. Moreover, this kind of co-culture study will also be useful in evaluating other types of cells. 4. Conclusion The cytotoxic concentration of folic acid and methotrexate indicates that it is safe to use folic acid as a carrier molecule at lower to moderate concentrations. Increase in cytotoxicity was observed with time as the release of drug was increased. As hypothesized, cross-linking significantly affected cytotoxicity pattern. Due to higher release rates, calcium cross-linked nanoparticles exhibited higher cytotoxicity than zinc cross-linked nanopartcles. Moreover, on increasing the cross-linking concentration, the cytotoxicity was reduced due to decrease in release rates. However, this can be true for a limiting concentration of cations (10% in case of CaCl2). Above certain concentration, crosslinking cation may also play role in cytotoxicity. Finally, the study also shows that there is a marked uptake of these nanoparticles in by HeLa cells. Increase in fluorescence indicates active targeting by folate nanocarriers. Acknowledgement The authors would like to thank Department of Science and Technology, Government of India for support for this project.

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