Materials Science and Engineering C 44 (2014) 352–361
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Controlled release of folic acid through liquid-crystalline folate nanoparticles Rahul Misra a, Henna Katyal b, Sanat Mohanty a,⁎ a b
Advance Materials & Nanoscience Laboratory, Department of Chemical Engineering, Indian Institute of Technology-Delhi, HauzKhas, New Delhi 110016, India Department of Nanotechnology, Amity University, Noida, India
a r t i c l e
i n f o
Article history: Received 12 February 2014 Received in revised form 7 April 2014 Accepted 5 August 2014 Available online 12 August 2014 Keywords: Chromonics Folates Cross-linking Control release Nanoparticles
a b s t r a c t The present study explores folate nanoparticles as nano-carriers for controlled drug delivery. Cross-linked nanoparticles of liquid crystalline folates are composed of ordered stacks. This paper shows that the folate nanoparticles can be made with less than 5% loss in folate ions. In addition, this study shows that folate nanoparticles can disintegrate in a controlled fashion resulting in controlled release of the folate ions. Release can be controlled by the size of nanoparticles, the extent of cross-linking and the choice of cross-linking cation. The effect of different factors like agitation, pH, and temperature on folate release was also studied. Studies were also carried out to show the effect of release medium and role of ions in the release medium on disruption of folate assembly. © 2014 Published by Elsevier B.V.
1. Introduction For patient compliance, drug delivery methods enhance the release profile, control absorption, and influence the bio-distribution of a drug administered to the body. Controlled drug delivery aims to release the drug at a specific site for enhanced and prolonged therapeutic effect as well as to maintain the level of therapeutic window to avoid re-administration. In conventional drug delivery methods, it is observed that the drug concentration in the blood varies with time between the maximum concentration (which may represent a toxic level) and a minimum value (below which the drug is ineffective). For example, in chemotherapy, both cancerous and non-cancerous cells are affected by chemotherapeutic agents due to their toxicity levels in the blood [1]. Conventional methods also have been seen to show lower retention and water solubility [2]. Specific distribution, reduced or no toxic effects and lower dosing are other advantages of controlled drug delivery. Thus, achieving a controlled release of drug is significant. Controlled release of drugs is desirable in chemotherapy, rheumatoid arthritis, asthmatic disorders, HIV, diabetes and several other auto-immune diseases. Past studies have shown that it is feasible to target and release drugs at a predetermined site in a controlled manner [3,4]. Nano-carriers present several advantages in controlled drug delivery [5–7]. While numerous lab studies have shown the feasibility of nano-carrier based
⁎ Corresponding author. E-mail address: [email protected] (S. Mohanty).
http://dx.doi.org/10.1016/j.msec.2014.08.025 0928-4931/© 2014 Published by Elsevier B.V.
strategies, there are few clinically proven nanoparticle formulations that show controlled drug release [8–11]. Some successful strategies in the release of anti-cancer are listed in Table 1. There remain some hurdles in controlled drug release with polymer and micellar based nanoparticles that have been extensively used in the past. The drug loss reported with these nano-carriers is in the range of 15–40% [12–15] — thus making this strategy wasteful and expensive. In addition, these nano-carriers are designed to carry drugs in “pockets” within the nanoparticle, thus resulting in “bursts”. Folate is a biocompatible, water soluble vitamin B which is found in green leafy vegetables, dried beans and peas (legumes) as well as citrus fruits and juices. It is essential for the formation of new cells and DNA inside the body. It is ingested by humans as a part of dietary supplements too. Higher amount of folic acid does not cause any harm as it is excreted through urine. Thus, there is lower risk of toxicity and side effects [20–22]. These advantages of folic acid provided strong motivation for using them as a nano-carrier for drug delivery. Fig. 1a represents the molecular structure of folic acid. The present study explores the use of folate nanoparticles as nanocarriers with improved encapsulation and better controlled release. Folate molecules are seen to self-assemble (even at low concentrations) in ordered structures. Moreover, drugs and other guest molecules intercalate into the self-assembled structure resulting in ordered nanoparticles with the promise of better protection and better control of release. Unlike amphiphiles, their self-assemblies do not show any critical micelle concentration or optimum aggregation size and there is no Krafft temperature effect (seen in micelles) to regulate such assemblies [23–27].
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Table 1 Strategies for controlled release of anti-cancer drugs through different nano-carriers. Main drug/formulation
Type of nanoparticle
Highly porous nanocellulose aerogels
Cellulosic nano-carrier Sodium dodecyl sulfate (SDS)
Porous hollow silica nanoparticles Silica nano-carrier PLGA–mPEG nanoparticles of Cisplatin Polymer nanoparticle Camptothecin–iron oxide nanoparticles Magnetic nanoparticle
Release medium
Strategies/phenomenon of release
Interactions between the nanoparticles and the cellulose modulation of the matrix Simulated body fluid (SBF) Entrapment of cefradine inside porous silica Phosphate buffer saline (PBS) In vitro nanoparticle degradation Dulbecco's modified Eagle's medium (DMEM) Intracellular release of the Camptothecin molecules by an external magnetic stimulus
Past studies have shown that other guest molecules could be appropriately chosen to be part of the self-assembled folate liquid-crystalline solutions. These guests molecules generally interact with aromatic ring complexes to participate ordered self assemblies that form [28–38]. In addition, the nanoparticles formed from these liquid-crystalline solutions maintain the order. Hence, when the nanoparticles disintegrate, any guest molecules included in the assembly would also be released at the same rate as the folate molecules. This could result in better control of release compared to nanoparticles with shell–core like structures. While the inclusion of other molecules would somewhat change the release profile on the guest, understanding the baseline behavior of folic acid is necessary to design the release of drug from such nanoparticles. This article reports that folate based nanoparticles are model drug carriers that can be designed for long term sustained release with a high degree of control. These release rates can be controlled by the size and cross-linking of the particles, and by the nature of the release medium. While past studies have explore the release of folates from other nano-carriers [40] or even the use of folate as a surface-modifying functional group [39], this is the first effort to develop folate nanoparticles as nano-carriers.
References [16] [17] [18] [19]
ions with aromatic rings prefer to interact with themselves, rather than with HPMC. Folates when mixed with HPMC get dispersed into nano-domains. This dispersion is exposed to aqueous solutions of multivalent salts to form stable nanoparticles. This process of dispersion also separates the HPMC from the folate nanoparticles. The multivalent cations exchange with the monovalent cations in the liquid crystalline solutions (NaOH in present case). The folate nano-domains cross-linked by multivalent cations are stable even in the absence of HPMC or when suspended in water. The cross-linking keeps the folate nanostructure stable intact. When suspended in release medium consisting of sodium ions at about 0.8% by weight, the folate assembly is disrupted by exchange of an excess of sodium ions with the cross-linking multivalent ions in the
1.1. Folate nanoparticle design and release An extensive study in the design of folate nanoparticles, thermodynamics of particle formation and characterization has been described elsewhere [41]. In brief, folate nanoparticles can be developed from liquid-crystalline folate solution by mixing it with HPMC (Hydroxy Propyl Methyl Cellulose) polymer and their size can be controlled by varying the relative concentrations of both components in the solution. HPMC is a water soluble cellulosic biocompatible polymer [42,43] used in the food industries as additives, emulsifier, and thickening and suspending agents. Fig. 1b represents the chemical structure of HPMC. In aqueous state, folate forms a two phase system with HPMC as folate
Fig. 1. Chemical structure of (a) folic acid and (b) HPMC polymer.
Fig. 2. Effect of relative concentration of folate and HPMC concentration on size distribution of folate nanoparticles. (a) Schematic representation. (b) Contour plot representation. By choosing the appropriate concentrations of both the compounds, folate nanoparticles of desirable size range can be developed.
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HPMC concentration g/100 ml
0.5 0.5 0.5 0.5 0.5 0.5
1.5 3.0 4.5 5.5 7.0 9.0
nanoparticles. This leads to the break-up of the particles and release of folic acid (and any encapsulated host molecule) in a controlled manner. The rate of disruption is influenced by mass transfer of the monovalent cation to the particle surface as well as the exchange kinetics of the monovalent ion with the multivalent ion. This study attempts to quantify the parameters that control the release of folate ions from the nanoparticles.
2. Materials and methods 2.1. Materials Folic acid (molecular formula: C19H19N7O6; molecular weight: 441.3974 g/mol) (PubChem CID 6037) and HPMC (Hydroxy Propyl Methyl Cellulose) (molecular formula: C12H20O10; molecular weight: 324.2848 g/mol) were purchased from Central Drug House (CDH) New Delhi. An aqueous stock solution of 5 wt.% folic acid was prepared using de-ionized water, which was neutralized by adding 1 M NaOH solution drop-wise till the solution turned liquid crystalline (visually) while ensuring that the pH was less than 7.0. 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. Liquid-crystalline behavior is observed between the pH values 6.5 and 7.5 [34]. An aqueous stock solution of 0.5% folate and 10% HPMC was prepared in de-ionized water. Different concentrations of HPMC solution (1.5%, 3%, 4.5%, 5.5%, 7%, and 9%) were prepared from this stock solution as needed to develop nanoparticles. The release mediums used in the study were 0.8% NaCl, Phosphate buffer saline (PBS), and simulated body fluid (SBF). Phosphate buffer saline and simulated body fluid were prepared by the method described by Sambrook et.al [44] and Kokubo et.al [45]. Different experiments were carried out to study the folic acid release in all release mediums. (All concentrations in this study reported are in weight/weight basis.) 2.2. Characterization of nanoparticles and folic acid release 2.2.1. Dynamic light scattering (DLS) The nanoparticle size distribution was determined in de-ionized water at 25 °C by the 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 deionized water and sonicated for 1 min. The analyses were performed at a scattering angle of 90° with a refractive index of 1.33 and at a temperature of 25 °C. 2.2.2. Scanning electron microscope (SEM) The morphology of nanoparticles was observed by scanning electron microscopy (SEM) (Zeiss EVO 50). A drop of the nanoparticle suspension was placed on a metallic surface. After drying under vacuum, the sample was coated with a gold layer. Observations were performed at 10 and 20 kV at magnification of 10k× and 20k×. 2.2.3. Transmission electron microscope (TEM) TEM study was carried out on a FEI-Technai, G2-Model (T-30STWIN) transmission electron microscope with an acceleration voltage of 250 kV. The samples for transmission electron microscope analysis
Fig. 3. Change in size distribution of folate nanoparticles formulated at 0.5% folate concentration and HPMC concentration of (a) 1.5%, (b) 3%, (c) 4.5%, (d) 5.5%, (e) 7% and (f) 9%.
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Fig. 4. (a) SEM and (b) TEM images of nano-domains of folate nanoparticle. SEM image shows the different morphology of nanoparticles in the size range of 200 ± 20 nm, while TEM image shows the textured surface of nanoparticles.
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Fig. 5. (a) Folic acid release with 200 nm sized folate nanoparticle cross-linked at different concentrations. (b) Loss of folic acid while preparing folate nanoparticles at different amounts of cross-linking. (200 nm sized particles).
and HPMC was continuously mixed with the help of a magnetic stirrer at 500–600 rpm for 6 h at room temperature. 2.4. Cross-linking of folate nanoparticles
were prepared by drop-coating folate nanoparticle solution onto a copper grid and subsequent drying at room temperature. 2.2.4. UV–Visible spectroscopy (UV–Vis) To determine the absorbance of folic acid release, UV spectroscopy was performed on a UV–Vis double beam spectrophotometer (Dynamica Halo DB-20) at 281.5 nm with supernatant collected at each time zone by further 30x dilution of the supernatant with deionized water.
Multivalent cationic salts (in separate experiments, either aqueous solutions of 10% ZnCl2 or 10% CaCl2) in the weight ratio 1:10 (Folate– HPMC mixture:Salt solution) were used to form cross-linked folate nanoparticles. Mixing was performed with continuous stirring at 700– 800 rpm for 8 h at room temperature. This resultant solution was centrifuged (Eppendorf centrifuge 5810R) at 10,000 rpm (2236 g) for 10 min. The obtained pellet is suspended and washed with de-ionized
2.3. Preparation of folate nanoparticles Misra et al. [41] reported that the size of the folate nanoparticles can be controlled by the choice of folate or HPMC concentration, with little impact of the cross-linking cation on the size. Fig. 2a and b shows that one can independently choose either the folate concentration or the HPMC concentration to target a certain size. In general, for a particular folate concentration, an increase in the HPMC concentration leads to a decrease in the size of the nanoparticles. High folate and high HPMC concentrations lead to the nanoparticles of the size range 400– 600 nm. High folate and low HPMC concentrations lead to a size range of 800–1000 nm. In the present study, nanoparticles have been designed following the same procedure as reported previously. Folate concentration was kept fixed while HPMC concentration was changed as listed in Table 2. An aqueous solution of 0.5% folate concentration was added to different concentrations of HPMC solution individually. This mixture of folate
Fig. 6. Folic acid release with 200 nm sized folate nanoparticle cross-linked with different cross-linking cations.
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Fig. 7. Folic acid release with different sizes of folate nanoparticles in static mode.
water and centrifuged again at 10,000 rpm for 10 min. This pellet is resuspended in de-ionized water and 1 ml of this suspension was sonicated (Metrex ultrasonic bath sonicator) with 5 ml of de-ionized water for 1 min at room temperature (35 °C). This sonicated mixture is used for size distribution studies by the DLS (dynamic light scattering) technique. Further, the pellet obtained after centrifugation was lyophilized (Christ Alpha 1-4 LD Plus Freeze Dryer) at − 49 °C and 0.002 mbar vacuum pressure to remove any HPMC present. 2.5. Size distribution of developed folate nanoparticles Following cross-linking, the hydrodynamic size of the nanoparticles was characterized with the help of the dynamic light scattering (DLS) technique. In this study, DLS shows that the nanoparticles formed after cross-linking had a particle size distribution of ± 50 nm around the mean particle size, as shown in Fig. 3. For all further analysis, the mean particle size is used to describe trends with changing concentrations. Fig. 3 indicates a decreasing trend in the size distribution of nanoparticles with an increase in the concentration of HPMC. At 0.5% folate concentration, an increase in the HPMC concentration from 1.5% to 9%, leads to a decrease in size from 834 ± 100 nm to 190 ± 30 nm. This is consistent with the trends reported in previous study [41]. Fig. 4 shows SEM and TEM images of the nano-domains formed. The TEM image shows that the surface is not uniform but textured — consistent with a particle made up of ordered material.
Fig. 8. Effect of sodium ion concentration on the release of folic acid (200 nm sized folate nanoparticle cross-linked with 10% ZnCl2).
2.6. In vitro folic acid release The nanoparticle dispersions in closed vials were stored at room temperature in the absence of light. At different time points (0–30 days), the supernatant was sampled and analyzed by UV spectroscopy. Two different modes of release — shaking (release mediums containing nanoparticles were kept under continuous shaking conditions) and static (release medium containing nanoparticles kept undisturbed) were used to analyze the effect of shaking on the release profile. In static mode studies, 0.04 g of lyophilized nanoparticles was taken in a vial and suspended in 10 ml release medium. In shaking mode studies, vials containing suspended nanoparticles were kept in an orbital shaker for continuous mild agitation at 200 rpm. Supernatants were collected after centrifugation of the medium at 3000 rpm for 5 min. The absorbance of released folic acid in the supernatant was determined by UV spectroscopy at 281.5 nm. The folic acid concentration in the medium was calculated using a calibration curve of the folic acid in the corresponding release medium 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.
Fig. 9. Folic acid release from 200 nm sized folate nanoparticle with different release mediums.
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Fig. 10. Comparison of folic acid release in static and shaking modes. Increase in percent release of folic acid at shaking conditions was observed due to higher mass transfer under shaking conditions.
The HeLa cells were obtained from the National Centre for Cell Science (Pune, India) and maintained in DMEM growth medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin– streptomycin at 37 °C under a humidified, 5% CO2 atmosphere. HeLa cells (2 × 104 HeLa cells/well) were cultured in 96-well plates. For determining the cytotoxic concentration of folic acid, cells were exposed to different concentrations of folic acid in the range of picograms to micrograms per ml. At this concentration, the cytotoxicity of folate nanoparticles were studied by exposing nanoparticles to HeLa cells and cell viability was recorded between 2 and 24 h by MTT assay. Similarly, free folic acid at the same concentration was also added to another well as a control. All the experiments were conducted in triplicate. For cellular treatment, nanoparticle suspension (50 μg/ml) was sonicated to avoid precipitation, and then freshly diluted with culture medium to the appropriate concentration. Growth medium was used as negative control while 5% DMSO served as positive control in all experiments.
cross-linker concentration on folate release (equivalently, nanoparticle breakup) is presented. 200 nm sized folate nanoparticles were crosslinked with different concentrations of cross-linking agent (ZnCl2, in present study). It was observed (Fig. 5a) that as the concentration of cross-linking agent was increased from 1% to 10%, the release of folic acid decreased from 55% to 15% in 7 days. This suggests that at higher concentrations of cross-linking cation, the folate particles are more “strongly” bound and the folate assemblies break up more slowly. Moreover, Fig. 5b shows that the concentration of the cross-linking agent also affects the loss of folic acid during processing for the formation of folate nanoparticles. The graph in Fig. 5b indicates that folic acid loss is reduced from 12% to 4% upon increasing the concentration of cross-linking agent (ZnCl2, in this example) from 1% to 10% respectively. As the cross-linking concentration is increased, the amount of liquid crystalline host molecules that are not part of the nanoparticles formed decreases. That is, the yield of the process in the formation of nanoparticles increases. This is significant since in most processes of formulation of nanoparticles, losses are in the range of 15–40% [12–14].
3. Results & discussion
3.2. Folate release: choice of cross-linking cation
3.1. Folate release: impact of cross-linker concentration It has been shown that cross-linking allows the isolation of folate nano-domains and keeps them intact. In this study, the impact of
Since different multivalent cations interact differently with the folate anions, it could be argued that the choice of cross-linking agent might also affect the release rates of folic acid. Release rate studies were carried out with 200 nm sized nanoparticles cross-linked with
Fig. 11. Effect of release medium's pH on folic acid release. Decrease in folic acid release at low pH indicates applicability of system for oral chemotherapy.
Fig. 12. Folic acid release from 200 nm sized folate nanoparticle stored at different temperature conditions.
2.7. Culturing HeLa cells and cytotoxicity assay
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different cross-linking agents, namely ZnCl2, CaCl2, FeCl3, and AlCl3. Fig. 6 shows that high release of folic acid was achieved from ZnCl2 and CaCl2 in comparison to AlCl3 and FeCl3. Over 15 days, more than 50% of folic acid has been released from 200 nm sized nanoparticles cross-linked with CaCl2, whereas only 10% release was observed in the case of FeCl3 cross-linked nanoparticles. The trivalent bound nanoparticles release folates slower than divalent bound nanoparticles. Between the divalent particles, the larger Zn2 + cation bound particles release slower. This is consistent with cation–ligand interactions with other organic acids and studies of such systems have also shown that Zn2 + cations have the strongest ionic affinity among divalent ions [46]. Between the trivalent bound particles, Al3+ has a smaller radius and hence a stronger bond resulting in slower release.
3.3. Effect of nanoparticle's size on folic acid release Release rates are dependent upon the size of the nanoparticles. Several researchers [25–28] have shown that nanoparticle size affects the release rate of the encapsulated drug. Therefore, experiments were carried out to observe the variation in release rates with nanoparticles of size 250 nm, 350 nm, 450 nm and 550 nm. Fig. 7 shows that up to 75% of folic acid is released with 550 nm sized folate nanoparticles in the period of 60 days, while 40% release was obtained with 250 nm sized folate nanoparticles. It is hypothesized that a higher surface area of smaller particles favors more effective cross-linking. Hence, release of folic acid may be slower with decreasing size of folate nanoparticles.
3.4. Effect of ion concentration in release medium Folic acid is released from folate nanoparticles due to the ions present in the release medium. There is an exchange of cross-linked multivalent cations present in folate assembly with the monovalent ions in the release medium leading to release. Therefore, ions play a vital role in the release of folic acid. We investigated the folic acid release due to the effect of ion concentration in release media with 200 nm sized particles cross-linked with 10% ZnCl2. Sodium chloride concentration was varied from 0.3% to 1% in normal saline release medium. It was observed that percent release of folic acid was increased significantly upon increasing the concentration of ions in the release medium. Fig. 8 shows that folic acid release is dependent on Na+ ion concentration in the medium. It is clearly evident that as NaCl concentration was increased from 0.3% to 1%, the percent of folic acid release was also increased from 20% to 30% respectively.
3.5. Effect of release medium on folic acid release The ions in the release medium play a role in the release of the folic acid by disrupting the folate assembly. The ions present in the release medium interact with the ions which have cross-linked the folate stacks. Different release mediums have different combinations and presence of ions, therefore, the effect of the release medium was studied to observe the release rates. The release medium used in the study was 0.8% NaCl, Phosphate buffer saline (PBS), simulated body fluid (SBF) and de-ionized water. Fig. 9 shows that simulated body fluid (SBF) results in the fastest release among these fluids. 50% release is observed with SBF, which is twice the release observed with 0.8% NaCl in 10 days with 200 nm sized nanoparticles. Due to the high monovalent salt content of PBS and SBF, more ions are present in the release medium, and this leads to more disruption of folate assembly. Therefore, high percentage of release was observed in the case of PBS and SBF compared to other release mediums. 3.6. Effect of agitation on folic acid release To understand the circulatory environment of the body, the effect of agitation was studied. Nanoparticles suspended in release medium were kept under mild agitation at 150–200 rpm with the help of an orbital shaker. To compare the release rates with a static system, a similar experiment was carried out in a non-agitated system and it was observed that, the agitation leads to higher release rates. Fig. 10 shows that 200 nm sized nanoparticles, which were kept in shaking mode release 45% folic acid while in static mode release 33% folic acid in 60 days. Accounting for the error bars, there is a distinct difference in release. A similar trend was observed with nanoparticles of different sizes. This may be due to higher mass transfer rates achieved during agitation which leads to more interaction of folate assembly with ions in the release medium, which in turn releases more folic acid. While the shaken environment would not simulate the flow environment accurately, this study is instructive in that it shows the impact of agitation and hence mass transfer effect on the release rate. 3.7. Effect of pH on folic acid release The ions present in the release medium play a role in the release of a drug from a nanoparticle. The pH of the release medium is governed by the ions present in. Therefore it was necessary to investigate the effect of pH on the release of folic acid. 0.8% NaCl solution was used as a release
Intensity
0.5% Folate 0.5% Folate+0.05% Ara-C
5
10
15
20
25
30
35
40
Diffraction angle (2θ)
Fig. 13. Folic acid release from 200 nm sized folate nanoparticle at different temperatures.
Fig. 14. X-ray diffraction pattern of 0.5% folate and folate along with cytarabine (Ara-C). No change in the significant peak suggests the participation of drug in folate assembly.
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Table 3 Mass balance of cytarabine (Ara-C) encapsulated, lost and encapsulation efficiency achieved in the method of nanoparticle formation. Nanoparticle specifications
Method of development
Nanoparticle size
Cross-linking
Folate %
HPMC %
Ara-C %
Initial amount of drug added (g)
Amount of drug after cross-linking (μg)
Drug loss (μg)
Dry weight of cross-linked nanoparticles (g)
Amount of drug in 0.04 g of nanoparticles (μg)
Encapsulation efficiency (%)
500 350 200 500 350 200
Zinc (10%) Zinc (10%) Zinc (10%) Calcium (10%) Calcium (10%) Calcium (10%)
0.5 0.5 0.5 0.5 0.5 0.5
5.5 7.5 9 5.5 7.5 9
0.05 0.05 0.05 0.05 0.05 0.05
0.005 0.005 0.005 0.005 0.005 0.005
4695 4902 4922 4695 4773 4840
157 98 78 305 227 160
0.145 0.216 0.265 0.184 0.271 0.292
1373 908 743 1021 705 663
96.86 98.04 98.44 94.14 95.46 96.81
± ± ± ± ± ±
20 20 20 20 20 20
nm nm nm nm nm nm
Drug amount during encapsulation
Encapsulation efficiency
The data in bold indicates the total drug loss and the drug encapsulated after carrying out mass-balance of drug in formulation process.
body conditions and its influence on the release, the effect of different temperatures around normal body temperature was studied. Similar release studies were carried out in 4 sets at different temperatures of 20°, 30°, 40° and 50 °C with 200 nm sized folate nanoparticles. No significant increase or decrease in percent release was recorded. Fig. 13 shows that the percent release was shifted by only 2–3% on increasing the temperature.
medium and required pH was adjusted by 0.1 N NaOH and 0.1 N HCl. Similar experiments were carried out at different pH values of 0.8% NaCl solution. It was observed that as the release medium becomes acidic the release of folic acid was decreased. Higher release rates were recorded at pH 6–10. This is significant. The nanoparticles release at a much lower rate in an acidic environment suggesting that the encapsulated molecules will perhaps stay protected in such environment. Thus, this delivery system may hold promise for oral delivery where the guest is to be protected in the stomach (Fig. 11).
3.10. Controlled release of an anticancer drug through folate carrier: cytarabine (Ara-C) release
3.8. Effect of storage temperature
The release pattern of folic acid from the folate carrier can be a model to predict the release behavior of encapsulated rugs. To validate this hypothesis, release studies were carried out with cytarabine, an anticancer drug. Cytarabine, also referred as Ara-C (Cytosine Arabinoside) belongs to the class — antimetabolite. It is used for the treatment of leukemic cancer. In the present study, amount of drug loaded is 10% of the carrier concentration. 0.05% Ara-C was added to 0.5% folate solution and continuously stirred for 30 min. It was observed that this solution was in a well mixed state and the XRD profile of the folate and cytarabine mixture (Fig. 14) shows no change in the significant peaks of folate selfassembly. This clearly indicates that cytarabine molecules participate in folate assembly. Folate nanoparticles are formed by adding HPMC to this solution and cross-linking them by the method described in Subsection 2.3. Accounting for the losses during the method of nanoparticle formulation, a
Storing the nanoparticles at different temperatures may affect the activity of the nanoparticles. To study the changes in the release activity, 3 sets of 200 nm sized folate nanoparticles were taken. One set was stored at 4 °C, the second set was exposed to 60 °C while third set was kept at room temperature (30 °C) for 48 h. Release studies were carried out with all the 3 sets of folate nanoparticles. Fig. 12 shows that no significant change in the release was observed except a shift of 2–3% release. No major changes in the release activity of folate nanoparticles were recorded. 3.9. Effect of temperature on folic acid release Temperature is considered as an important parameter to influence the release of drug inside the human body. To simulate the human
Percent release of Cytarabine (Ara-C)
90 80 70
200 ± 20nm (Zinc cross-linked) 350 ± 20nm (Zinc cross-linked) 500 ± 20nm (Zinc cross-linked) 200 ± 20nm (Calcium cross-linked) 350 ± 20nm (Calcium cross-linked) 500 ± 20nm (Calcium cross-linked)
60 50 40 30 20 10 0 5 hours
1 day
2 days
6 days
10 days
17 days
24 days
30 days
Time Fig. 15. Control release of cytarabine in static mode by changing the size of folate nano-carrier and cross-linking cation.
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folates) depends on the choice and concentration of cross-linking cation, and the size of the nanoparticle. These are three parameters that can be independently controlled to control the release rate from the nanoparticle. This study shows that, in addition to these three parameters, the release is also affected by the state of agitation (or flow of release medium around the particle), the nature of the release medium as well as the pH of the release medium. Thus, the nanoparticles need to be designed while accounting for the state of the nano-carrier pathway and release environment. The release from such system will not be affected significantly by a small variation in temperatures (20° to 40 °C) during release conditions or by storing the nanoparticles in different conditions. Thus, the folates present a potential nano-carrier that is biocompatible, can be controlled in the size scale of interest and presents multiple design variables that can be used to control the release profile. At least in in-vitro studies, folate nanoparticles present a strong case as a versatile nano-carrier. Fig. 16. Comparative cytotoxicity of released folic acid from folate carrier and free folic acid with time on HeLa cells (positive control: 5% DMSO; negative control: DMEM growth media).
mass-balance study was performed to determine the encapsulation efficiency. The encapsulation efficiency was calculated as: Encapsulation efficiencyð%Þ ¼
Amount of drug in nanoparticles 100: Initial amount of drug added
The data (Table 3) suggests that only 4–5% of cytarabine has been lost during the method of forming nanoparticles. Therefore, 94–95% of drug has been encapsulated within the folate nano-carrier which is significant in itself. To observe the effect of size and cross-linking cation, cytarabine encapsulated nanoparticles of different sizes were prepared and cross-linked with zinc and calcium cations. In-vitro release studies were performed with all these nanoparticles by suspending them into 0.8% NaCl release medium. The results (Fig. 15) show the control release of cytarabine through a folate nano-carrier and the methods of control are similarly effective. This is in concordance with our hypothesis that the release study of folate from nanoparticles closely follows the release of drugs. 3.11. Cytotoxicity of released folic acid on HeLa cells Initial experiments on cytotoxicity suggested that folic acid is cytotoxic to HeLa cells above 12 μg/ml. To study the cytotoxic behavior of release folic acid from nanoparticles, folate nanoparticles (200 nm) were exposed to the wells containing HeLa cells, at a pre-determined cytotoxic concentration (12 μg/ml). The free folic acid at the same concentration was also added as a control. The cell viability was studied at 2, 6, 10, 20, and 24 h. Fig. 16 shows the difference in impact on exposing folate nanoparticles (cross-linked with Ca2 +) and free folic acid to HeLa cells at this concentration. Bound folic acid shown lower cytotoxicity in comparison to free folic acid during the first 6–7 h. Subsequently, with increased folate release from the nanoparticles, HeLa cells exposed to folate nanoparticles showed similar cell survival as compared to HeLa cells exposed to folate solutions. These results suggest that the released folic acid from nanoparticles is efficient in killing HeLa cells in a controlled manner consistent with its release rate. 4. Conclusion Folate nanoparticles can be designed in the size range of 100– 500 nm even at low concentrations of liquid-crystalline folates. A drug molecule intercalates between or within the ordered folate stacks which are cross-linked with a multivalent cation. Cross-linking cations play a major role in keeping the folate assembly intact. The rate of disintegration of the nanoparticles (measured by the rate of release of
Acknowledgment The authors would like to thank the Department of Science and Technology (SR/S3/CE/027/2011(G)), Government of India as well as the Indian Institute of Technology, Delhi for the support for this project.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Controlled release of folic acid through liquid-crystalline folate nanoparticles Rahul Misra a, Henna Katyal b, Sanat Mohanty a,⁎ a b
Advance Materials & Nanoscience Laboratory, Department of Chemical Engineering, Indian Institute of Technology-Delhi, HauzKhas, New Delhi 110016, India Department of Nanotechnology, Amity University, Noida, India
a r t i c l e
i n f o
Article history: Received 12 February 2014 Received in revised form 7 April 2014 Accepted 5 August 2014 Available online 12 August 2014 Keywords: Chromonics Folates Cross-linking Control release Nanoparticles
a b s t r a c t The present study explores folate nanoparticles as nano-carriers for controlled drug delivery. Cross-linked nanoparticles of liquid crystalline folates are composed of ordered stacks. This paper shows that the folate nanoparticles can be made with less than 5% loss in folate ions. In addition, this study shows that folate nanoparticles can disintegrate in a controlled fashion resulting in controlled release of the folate ions. Release can be controlled by the size of nanoparticles, the extent of cross-linking and the choice of cross-linking cation. The effect of different factors like agitation, pH, and temperature on folate release was also studied. Studies were also carried out to show the effect of release medium and role of ions in the release medium on disruption of folate assembly. © 2014 Published by Elsevier B.V.
1. Introduction For patient compliance, drug delivery methods enhance the release profile, control absorption, and influence the bio-distribution of a drug administered to the body. Controlled drug delivery aims to release the drug at a specific site for enhanced and prolonged therapeutic effect as well as to maintain the level of therapeutic window to avoid re-administration. In conventional drug delivery methods, it is observed that the drug concentration in the blood varies with time between the maximum concentration (which may represent a toxic level) and a minimum value (below which the drug is ineffective). For example, in chemotherapy, both cancerous and non-cancerous cells are affected by chemotherapeutic agents due to their toxicity levels in the blood [1]. Conventional methods also have been seen to show lower retention and water solubility [2]. Specific distribution, reduced or no toxic effects and lower dosing are other advantages of controlled drug delivery. Thus, achieving a controlled release of drug is significant. Controlled release of drugs is desirable in chemotherapy, rheumatoid arthritis, asthmatic disorders, HIV, diabetes and several other auto-immune diseases. Past studies have shown that it is feasible to target and release drugs at a predetermined site in a controlled manner [3,4]. Nano-carriers present several advantages in controlled drug delivery [5–7]. While numerous lab studies have shown the feasibility of nano-carrier based
⁎ Corresponding author. E-mail address: [email protected] (S. Mohanty).
http://dx.doi.org/10.1016/j.msec.2014.08.025 0928-4931/© 2014 Published by Elsevier B.V.
strategies, there are few clinically proven nanoparticle formulations that show controlled drug release [8–11]. Some successful strategies in the release of anti-cancer are listed in Table 1. There remain some hurdles in controlled drug release with polymer and micellar based nanoparticles that have been extensively used in the past. The drug loss reported with these nano-carriers is in the range of 15–40% [12–15] — thus making this strategy wasteful and expensive. In addition, these nano-carriers are designed to carry drugs in “pockets” within the nanoparticle, thus resulting in “bursts”. Folate is a biocompatible, water soluble vitamin B which is found in green leafy vegetables, dried beans and peas (legumes) as well as citrus fruits and juices. It is essential for the formation of new cells and DNA inside the body. It is ingested by humans as a part of dietary supplements too. Higher amount of folic acid does not cause any harm as it is excreted through urine. Thus, there is lower risk of toxicity and side effects [20–22]. These advantages of folic acid provided strong motivation for using them as a nano-carrier for drug delivery. Fig. 1a represents the molecular structure of folic acid. The present study explores the use of folate nanoparticles as nanocarriers with improved encapsulation and better controlled release. Folate molecules are seen to self-assemble (even at low concentrations) in ordered structures. Moreover, drugs and other guest molecules intercalate into the self-assembled structure resulting in ordered nanoparticles with the promise of better protection and better control of release. Unlike amphiphiles, their self-assemblies do not show any critical micelle concentration or optimum aggregation size and there is no Krafft temperature effect (seen in micelles) to regulate such assemblies [23–27].
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Table 1 Strategies for controlled release of anti-cancer drugs through different nano-carriers. Main drug/formulation
Type of nanoparticle
Highly porous nanocellulose aerogels
Cellulosic nano-carrier Sodium dodecyl sulfate (SDS)
Porous hollow silica nanoparticles Silica nano-carrier PLGA–mPEG nanoparticles of Cisplatin Polymer nanoparticle Camptothecin–iron oxide nanoparticles Magnetic nanoparticle
Release medium
Strategies/phenomenon of release
Interactions between the nanoparticles and the cellulose modulation of the matrix Simulated body fluid (SBF) Entrapment of cefradine inside porous silica Phosphate buffer saline (PBS) In vitro nanoparticle degradation Dulbecco's modified Eagle's medium (DMEM) Intracellular release of the Camptothecin molecules by an external magnetic stimulus
Past studies have shown that other guest molecules could be appropriately chosen to be part of the self-assembled folate liquid-crystalline solutions. These guests molecules generally interact with aromatic ring complexes to participate ordered self assemblies that form [28–38]. In addition, the nanoparticles formed from these liquid-crystalline solutions maintain the order. Hence, when the nanoparticles disintegrate, any guest molecules included in the assembly would also be released at the same rate as the folate molecules. This could result in better control of release compared to nanoparticles with shell–core like structures. While the inclusion of other molecules would somewhat change the release profile on the guest, understanding the baseline behavior of folic acid is necessary to design the release of drug from such nanoparticles. This article reports that folate based nanoparticles are model drug carriers that can be designed for long term sustained release with a high degree of control. These release rates can be controlled by the size and cross-linking of the particles, and by the nature of the release medium. While past studies have explore the release of folates from other nano-carriers [40] or even the use of folate as a surface-modifying functional group [39], this is the first effort to develop folate nanoparticles as nano-carriers.
References [16] [17] [18] [19]
ions with aromatic rings prefer to interact with themselves, rather than with HPMC. Folates when mixed with HPMC get dispersed into nano-domains. This dispersion is exposed to aqueous solutions of multivalent salts to form stable nanoparticles. This process of dispersion also separates the HPMC from the folate nanoparticles. The multivalent cations exchange with the monovalent cations in the liquid crystalline solutions (NaOH in present case). The folate nano-domains cross-linked by multivalent cations are stable even in the absence of HPMC or when suspended in water. The cross-linking keeps the folate nanostructure stable intact. When suspended in release medium consisting of sodium ions at about 0.8% by weight, the folate assembly is disrupted by exchange of an excess of sodium ions with the cross-linking multivalent ions in the
1.1. Folate nanoparticle design and release An extensive study in the design of folate nanoparticles, thermodynamics of particle formation and characterization has been described elsewhere [41]. In brief, folate nanoparticles can be developed from liquid-crystalline folate solution by mixing it with HPMC (Hydroxy Propyl Methyl Cellulose) polymer and their size can be controlled by varying the relative concentrations of both components in the solution. HPMC is a water soluble cellulosic biocompatible polymer [42,43] used in the food industries as additives, emulsifier, and thickening and suspending agents. Fig. 1b represents the chemical structure of HPMC. In aqueous state, folate forms a two phase system with HPMC as folate
Fig. 1. Chemical structure of (a) folic acid and (b) HPMC polymer.
Fig. 2. Effect of relative concentration of folate and HPMC concentration on size distribution of folate nanoparticles. (a) Schematic representation. (b) Contour plot representation. By choosing the appropriate concentrations of both the compounds, folate nanoparticles of desirable size range can be developed.
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R. Misra et al. / Materials Science and Engineering C 44 (2014) 352–361 Table 2 Concentration of folate and HPMC in formation of folate nanoparticles of different sizes. Folate concentration g/100 ml
HPMC concentration g/100 ml
0.5 0.5 0.5 0.5 0.5 0.5
1.5 3.0 4.5 5.5 7.0 9.0
nanoparticles. This leads to the break-up of the particles and release of folic acid (and any encapsulated host molecule) in a controlled manner. The rate of disruption is influenced by mass transfer of the monovalent cation to the particle surface as well as the exchange kinetics of the monovalent ion with the multivalent ion. This study attempts to quantify the parameters that control the release of folate ions from the nanoparticles.
2. Materials and methods 2.1. Materials Folic acid (molecular formula: C19H19N7O6; molecular weight: 441.3974 g/mol) (PubChem CID 6037) and HPMC (Hydroxy Propyl Methyl Cellulose) (molecular formula: C12H20O10; molecular weight: 324.2848 g/mol) were purchased from Central Drug House (CDH) New Delhi. An aqueous stock solution of 5 wt.% folic acid was prepared using de-ionized water, which was neutralized by adding 1 M NaOH solution drop-wise till the solution turned liquid crystalline (visually) while ensuring that the pH was less than 7.0. 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. Liquid-crystalline behavior is observed between the pH values 6.5 and 7.5 [34]. An aqueous stock solution of 0.5% folate and 10% HPMC was prepared in de-ionized water. Different concentrations of HPMC solution (1.5%, 3%, 4.5%, 5.5%, 7%, and 9%) were prepared from this stock solution as needed to develop nanoparticles. The release mediums used in the study were 0.8% NaCl, Phosphate buffer saline (PBS), and simulated body fluid (SBF). Phosphate buffer saline and simulated body fluid were prepared by the method described by Sambrook et.al [44] and Kokubo et.al [45]. Different experiments were carried out to study the folic acid release in all release mediums. (All concentrations in this study reported are in weight/weight basis.) 2.2. Characterization of nanoparticles and folic acid release 2.2.1. Dynamic light scattering (DLS) The nanoparticle size distribution was determined in de-ionized water at 25 °C by the 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 deionized water and sonicated for 1 min. The analyses were performed at a scattering angle of 90° with a refractive index of 1.33 and at a temperature of 25 °C. 2.2.2. Scanning electron microscope (SEM) The morphology of nanoparticles was observed by scanning electron microscopy (SEM) (Zeiss EVO 50). A drop of the nanoparticle suspension was placed on a metallic surface. After drying under vacuum, the sample was coated with a gold layer. Observations were performed at 10 and 20 kV at magnification of 10k× and 20k×. 2.2.3. Transmission electron microscope (TEM) TEM study was carried out on a FEI-Technai, G2-Model (T-30STWIN) transmission electron microscope with an acceleration voltage of 250 kV. The samples for transmission electron microscope analysis
Fig. 3. Change in size distribution of folate nanoparticles formulated at 0.5% folate concentration and HPMC concentration of (a) 1.5%, (b) 3%, (c) 4.5%, (d) 5.5%, (e) 7% and (f) 9%.
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Fig. 4. (a) SEM and (b) TEM images of nano-domains of folate nanoparticle. SEM image shows the different morphology of nanoparticles in the size range of 200 ± 20 nm, while TEM image shows the textured surface of nanoparticles.
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Fig. 5. (a) Folic acid release with 200 nm sized folate nanoparticle cross-linked at different concentrations. (b) Loss of folic acid while preparing folate nanoparticles at different amounts of cross-linking. (200 nm sized particles).
and HPMC was continuously mixed with the help of a magnetic stirrer at 500–600 rpm for 6 h at room temperature. 2.4. Cross-linking of folate nanoparticles
were prepared by drop-coating folate nanoparticle solution onto a copper grid and subsequent drying at room temperature. 2.2.4. UV–Visible spectroscopy (UV–Vis) To determine the absorbance of folic acid release, UV spectroscopy was performed on a UV–Vis double beam spectrophotometer (Dynamica Halo DB-20) at 281.5 nm with supernatant collected at each time zone by further 30x dilution of the supernatant with deionized water.
Multivalent cationic salts (in separate experiments, either aqueous solutions of 10% ZnCl2 or 10% CaCl2) in the weight ratio 1:10 (Folate– HPMC mixture:Salt solution) were used to form cross-linked folate nanoparticles. Mixing was performed with continuous stirring at 700– 800 rpm for 8 h at room temperature. This resultant solution was centrifuged (Eppendorf centrifuge 5810R) at 10,000 rpm (2236 g) for 10 min. The obtained pellet is suspended and washed with de-ionized
2.3. Preparation of folate nanoparticles Misra et al. [41] reported that the size of the folate nanoparticles can be controlled by the choice of folate or HPMC concentration, with little impact of the cross-linking cation on the size. Fig. 2a and b shows that one can independently choose either the folate concentration or the HPMC concentration to target a certain size. In general, for a particular folate concentration, an increase in the HPMC concentration leads to a decrease in the size of the nanoparticles. High folate and high HPMC concentrations lead to the nanoparticles of the size range 400– 600 nm. High folate and low HPMC concentrations lead to a size range of 800–1000 nm. In the present study, nanoparticles have been designed following the same procedure as reported previously. Folate concentration was kept fixed while HPMC concentration was changed as listed in Table 2. An aqueous solution of 0.5% folate concentration was added to different concentrations of HPMC solution individually. This mixture of folate
Fig. 6. Folic acid release with 200 nm sized folate nanoparticle cross-linked with different cross-linking cations.
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Fig. 7. Folic acid release with different sizes of folate nanoparticles in static mode.
water and centrifuged again at 10,000 rpm for 10 min. This pellet is resuspended in de-ionized water and 1 ml of this suspension was sonicated (Metrex ultrasonic bath sonicator) with 5 ml of de-ionized water for 1 min at room temperature (35 °C). This sonicated mixture is used for size distribution studies by the DLS (dynamic light scattering) technique. Further, the pellet obtained after centrifugation was lyophilized (Christ Alpha 1-4 LD Plus Freeze Dryer) at − 49 °C and 0.002 mbar vacuum pressure to remove any HPMC present. 2.5. Size distribution of developed folate nanoparticles Following cross-linking, the hydrodynamic size of the nanoparticles was characterized with the help of the dynamic light scattering (DLS) technique. In this study, DLS shows that the nanoparticles formed after cross-linking had a particle size distribution of ± 50 nm around the mean particle size, as shown in Fig. 3. For all further analysis, the mean particle size is used to describe trends with changing concentrations. Fig. 3 indicates a decreasing trend in the size distribution of nanoparticles with an increase in the concentration of HPMC. At 0.5% folate concentration, an increase in the HPMC concentration from 1.5% to 9%, leads to a decrease in size from 834 ± 100 nm to 190 ± 30 nm. This is consistent with the trends reported in previous study [41]. Fig. 4 shows SEM and TEM images of the nano-domains formed. The TEM image shows that the surface is not uniform but textured — consistent with a particle made up of ordered material.
Fig. 8. Effect of sodium ion concentration on the release of folic acid (200 nm sized folate nanoparticle cross-linked with 10% ZnCl2).
2.6. In vitro folic acid release The nanoparticle dispersions in closed vials were stored at room temperature in the absence of light. At different time points (0–30 days), the supernatant was sampled and analyzed by UV spectroscopy. Two different modes of release — shaking (release mediums containing nanoparticles were kept under continuous shaking conditions) and static (release medium containing nanoparticles kept undisturbed) were used to analyze the effect of shaking on the release profile. In static mode studies, 0.04 g of lyophilized nanoparticles was taken in a vial and suspended in 10 ml release medium. In shaking mode studies, vials containing suspended nanoparticles were kept in an orbital shaker for continuous mild agitation at 200 rpm. Supernatants were collected after centrifugation of the medium at 3000 rpm for 5 min. The absorbance of released folic acid in the supernatant was determined by UV spectroscopy at 281.5 nm. The folic acid concentration in the medium was calculated using a calibration curve of the folic acid in the corresponding release medium 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.
Fig. 9. Folic acid release from 200 nm sized folate nanoparticle with different release mediums.
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Fig. 10. Comparison of folic acid release in static and shaking modes. Increase in percent release of folic acid at shaking conditions was observed due to higher mass transfer under shaking conditions.
The HeLa cells were obtained from the National Centre for Cell Science (Pune, India) and maintained in DMEM growth medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin– streptomycin at 37 °C under a humidified, 5% CO2 atmosphere. HeLa cells (2 × 104 HeLa cells/well) were cultured in 96-well plates. For determining the cytotoxic concentration of folic acid, cells were exposed to different concentrations of folic acid in the range of picograms to micrograms per ml. At this concentration, the cytotoxicity of folate nanoparticles were studied by exposing nanoparticles to HeLa cells and cell viability was recorded between 2 and 24 h by MTT assay. Similarly, free folic acid at the same concentration was also added to another well as a control. All the experiments were conducted in triplicate. For cellular treatment, nanoparticle suspension (50 μg/ml) was sonicated to avoid precipitation, and then freshly diluted with culture medium to the appropriate concentration. Growth medium was used as negative control while 5% DMSO served as positive control in all experiments.
cross-linker concentration on folate release (equivalently, nanoparticle breakup) is presented. 200 nm sized folate nanoparticles were crosslinked with different concentrations of cross-linking agent (ZnCl2, in present study). It was observed (Fig. 5a) that as the concentration of cross-linking agent was increased from 1% to 10%, the release of folic acid decreased from 55% to 15% in 7 days. This suggests that at higher concentrations of cross-linking cation, the folate particles are more “strongly” bound and the folate assemblies break up more slowly. Moreover, Fig. 5b shows that the concentration of the cross-linking agent also affects the loss of folic acid during processing for the formation of folate nanoparticles. The graph in Fig. 5b indicates that folic acid loss is reduced from 12% to 4% upon increasing the concentration of cross-linking agent (ZnCl2, in this example) from 1% to 10% respectively. As the cross-linking concentration is increased, the amount of liquid crystalline host molecules that are not part of the nanoparticles formed decreases. That is, the yield of the process in the formation of nanoparticles increases. This is significant since in most processes of formulation of nanoparticles, losses are in the range of 15–40% [12–14].
3. Results & discussion
3.2. Folate release: choice of cross-linking cation
3.1. Folate release: impact of cross-linker concentration It has been shown that cross-linking allows the isolation of folate nano-domains and keeps them intact. In this study, the impact of
Since different multivalent cations interact differently with the folate anions, it could be argued that the choice of cross-linking agent might also affect the release rates of folic acid. Release rate studies were carried out with 200 nm sized nanoparticles cross-linked with
Fig. 11. Effect of release medium's pH on folic acid release. Decrease in folic acid release at low pH indicates applicability of system for oral chemotherapy.
Fig. 12. Folic acid release from 200 nm sized folate nanoparticle stored at different temperature conditions.
2.7. Culturing HeLa cells and cytotoxicity assay
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different cross-linking agents, namely ZnCl2, CaCl2, FeCl3, and AlCl3. Fig. 6 shows that high release of folic acid was achieved from ZnCl2 and CaCl2 in comparison to AlCl3 and FeCl3. Over 15 days, more than 50% of folic acid has been released from 200 nm sized nanoparticles cross-linked with CaCl2, whereas only 10% release was observed in the case of FeCl3 cross-linked nanoparticles. The trivalent bound nanoparticles release folates slower than divalent bound nanoparticles. Between the divalent particles, the larger Zn2 + cation bound particles release slower. This is consistent with cation–ligand interactions with other organic acids and studies of such systems have also shown that Zn2 + cations have the strongest ionic affinity among divalent ions [46]. Between the trivalent bound particles, Al3+ has a smaller radius and hence a stronger bond resulting in slower release.
3.3. Effect of nanoparticle's size on folic acid release Release rates are dependent upon the size of the nanoparticles. Several researchers [25–28] have shown that nanoparticle size affects the release rate of the encapsulated drug. Therefore, experiments were carried out to observe the variation in release rates with nanoparticles of size 250 nm, 350 nm, 450 nm and 550 nm. Fig. 7 shows that up to 75% of folic acid is released with 550 nm sized folate nanoparticles in the period of 60 days, while 40% release was obtained with 250 nm sized folate nanoparticles. It is hypothesized that a higher surface area of smaller particles favors more effective cross-linking. Hence, release of folic acid may be slower with decreasing size of folate nanoparticles.
3.4. Effect of ion concentration in release medium Folic acid is released from folate nanoparticles due to the ions present in the release medium. There is an exchange of cross-linked multivalent cations present in folate assembly with the monovalent ions in the release medium leading to release. Therefore, ions play a vital role in the release of folic acid. We investigated the folic acid release due to the effect of ion concentration in release media with 200 nm sized particles cross-linked with 10% ZnCl2. Sodium chloride concentration was varied from 0.3% to 1% in normal saline release medium. It was observed that percent release of folic acid was increased significantly upon increasing the concentration of ions in the release medium. Fig. 8 shows that folic acid release is dependent on Na+ ion concentration in the medium. It is clearly evident that as NaCl concentration was increased from 0.3% to 1%, the percent of folic acid release was also increased from 20% to 30% respectively.
3.5. Effect of release medium on folic acid release The ions in the release medium play a role in the release of the folic acid by disrupting the folate assembly. The ions present in the release medium interact with the ions which have cross-linked the folate stacks. Different release mediums have different combinations and presence of ions, therefore, the effect of the release medium was studied to observe the release rates. The release medium used in the study was 0.8% NaCl, Phosphate buffer saline (PBS), simulated body fluid (SBF) and de-ionized water. Fig. 9 shows that simulated body fluid (SBF) results in the fastest release among these fluids. 50% release is observed with SBF, which is twice the release observed with 0.8% NaCl in 10 days with 200 nm sized nanoparticles. Due to the high monovalent salt content of PBS and SBF, more ions are present in the release medium, and this leads to more disruption of folate assembly. Therefore, high percentage of release was observed in the case of PBS and SBF compared to other release mediums. 3.6. Effect of agitation on folic acid release To understand the circulatory environment of the body, the effect of agitation was studied. Nanoparticles suspended in release medium were kept under mild agitation at 150–200 rpm with the help of an orbital shaker. To compare the release rates with a static system, a similar experiment was carried out in a non-agitated system and it was observed that, the agitation leads to higher release rates. Fig. 10 shows that 200 nm sized nanoparticles, which were kept in shaking mode release 45% folic acid while in static mode release 33% folic acid in 60 days. Accounting for the error bars, there is a distinct difference in release. A similar trend was observed with nanoparticles of different sizes. This may be due to higher mass transfer rates achieved during agitation which leads to more interaction of folate assembly with ions in the release medium, which in turn releases more folic acid. While the shaken environment would not simulate the flow environment accurately, this study is instructive in that it shows the impact of agitation and hence mass transfer effect on the release rate. 3.7. Effect of pH on folic acid release The ions present in the release medium play a role in the release of a drug from a nanoparticle. The pH of the release medium is governed by the ions present in. Therefore it was necessary to investigate the effect of pH on the release of folic acid. 0.8% NaCl solution was used as a release
Intensity
0.5% Folate 0.5% Folate+0.05% Ara-C
5
10
15
20
25
30
35
40
Diffraction angle (2θ)
Fig. 13. Folic acid release from 200 nm sized folate nanoparticle at different temperatures.
Fig. 14. X-ray diffraction pattern of 0.5% folate and folate along with cytarabine (Ara-C). No change in the significant peak suggests the participation of drug in folate assembly.
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Table 3 Mass balance of cytarabine (Ara-C) encapsulated, lost and encapsulation efficiency achieved in the method of nanoparticle formation. Nanoparticle specifications
Method of development
Nanoparticle size
Cross-linking
Folate %
HPMC %
Ara-C %
Initial amount of drug added (g)
Amount of drug after cross-linking (μg)
Drug loss (μg)
Dry weight of cross-linked nanoparticles (g)
Amount of drug in 0.04 g of nanoparticles (μg)
Encapsulation efficiency (%)
500 350 200 500 350 200
Zinc (10%) Zinc (10%) Zinc (10%) Calcium (10%) Calcium (10%) Calcium (10%)
0.5 0.5 0.5 0.5 0.5 0.5
5.5 7.5 9 5.5 7.5 9
0.05 0.05 0.05 0.05 0.05 0.05
0.005 0.005 0.005 0.005 0.005 0.005
4695 4902 4922 4695 4773 4840
157 98 78 305 227 160
0.145 0.216 0.265 0.184 0.271 0.292
1373 908 743 1021 705 663
96.86 98.04 98.44 94.14 95.46 96.81
± ± ± ± ± ±
20 20 20 20 20 20
nm nm nm nm nm nm
Drug amount during encapsulation
Encapsulation efficiency
The data in bold indicates the total drug loss and the drug encapsulated after carrying out mass-balance of drug in formulation process.
body conditions and its influence on the release, the effect of different temperatures around normal body temperature was studied. Similar release studies were carried out in 4 sets at different temperatures of 20°, 30°, 40° and 50 °C with 200 nm sized folate nanoparticles. No significant increase or decrease in percent release was recorded. Fig. 13 shows that the percent release was shifted by only 2–3% on increasing the temperature.
medium and required pH was adjusted by 0.1 N NaOH and 0.1 N HCl. Similar experiments were carried out at different pH values of 0.8% NaCl solution. It was observed that as the release medium becomes acidic the release of folic acid was decreased. Higher release rates were recorded at pH 6–10. This is significant. The nanoparticles release at a much lower rate in an acidic environment suggesting that the encapsulated molecules will perhaps stay protected in such environment. Thus, this delivery system may hold promise for oral delivery where the guest is to be protected in the stomach (Fig. 11).
3.10. Controlled release of an anticancer drug through folate carrier: cytarabine (Ara-C) release
3.8. Effect of storage temperature
The release pattern of folic acid from the folate carrier can be a model to predict the release behavior of encapsulated rugs. To validate this hypothesis, release studies were carried out with cytarabine, an anticancer drug. Cytarabine, also referred as Ara-C (Cytosine Arabinoside) belongs to the class — antimetabolite. It is used for the treatment of leukemic cancer. In the present study, amount of drug loaded is 10% of the carrier concentration. 0.05% Ara-C was added to 0.5% folate solution and continuously stirred for 30 min. It was observed that this solution was in a well mixed state and the XRD profile of the folate and cytarabine mixture (Fig. 14) shows no change in the significant peaks of folate selfassembly. This clearly indicates that cytarabine molecules participate in folate assembly. Folate nanoparticles are formed by adding HPMC to this solution and cross-linking them by the method described in Subsection 2.3. Accounting for the losses during the method of nanoparticle formulation, a
Storing the nanoparticles at different temperatures may affect the activity of the nanoparticles. To study the changes in the release activity, 3 sets of 200 nm sized folate nanoparticles were taken. One set was stored at 4 °C, the second set was exposed to 60 °C while third set was kept at room temperature (30 °C) for 48 h. Release studies were carried out with all the 3 sets of folate nanoparticles. Fig. 12 shows that no significant change in the release was observed except a shift of 2–3% release. No major changes in the release activity of folate nanoparticles were recorded. 3.9. Effect of temperature on folic acid release Temperature is considered as an important parameter to influence the release of drug inside the human body. To simulate the human
Percent release of Cytarabine (Ara-C)
90 80 70
200 ± 20nm (Zinc cross-linked) 350 ± 20nm (Zinc cross-linked) 500 ± 20nm (Zinc cross-linked) 200 ± 20nm (Calcium cross-linked) 350 ± 20nm (Calcium cross-linked) 500 ± 20nm (Calcium cross-linked)
60 50 40 30 20 10 0 5 hours
1 day
2 days
6 days
10 days
17 days
24 days
30 days
Time Fig. 15. Control release of cytarabine in static mode by changing the size of folate nano-carrier and cross-linking cation.
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folates) depends on the choice and concentration of cross-linking cation, and the size of the nanoparticle. These are three parameters that can be independently controlled to control the release rate from the nanoparticle. This study shows that, in addition to these three parameters, the release is also affected by the state of agitation (or flow of release medium around the particle), the nature of the release medium as well as the pH of the release medium. Thus, the nanoparticles need to be designed while accounting for the state of the nano-carrier pathway and release environment. The release from such system will not be affected significantly by a small variation in temperatures (20° to 40 °C) during release conditions or by storing the nanoparticles in different conditions. Thus, the folates present a potential nano-carrier that is biocompatible, can be controlled in the size scale of interest and presents multiple design variables that can be used to control the release profile. At least in in-vitro studies, folate nanoparticles present a strong case as a versatile nano-carrier. Fig. 16. Comparative cytotoxicity of released folic acid from folate carrier and free folic acid with time on HeLa cells (positive control: 5% DMSO; negative control: DMEM growth media).
mass-balance study was performed to determine the encapsulation efficiency. The encapsulation efficiency was calculated as: Encapsulation efficiencyð%Þ ¼
Amount of drug in nanoparticles 100: Initial amount of drug added
The data (Table 3) suggests that only 4–5% of cytarabine has been lost during the method of forming nanoparticles. Therefore, 94–95% of drug has been encapsulated within the folate nano-carrier which is significant in itself. To observe the effect of size and cross-linking cation, cytarabine encapsulated nanoparticles of different sizes were prepared and cross-linked with zinc and calcium cations. In-vitro release studies were performed with all these nanoparticles by suspending them into 0.8% NaCl release medium. The results (Fig. 15) show the control release of cytarabine through a folate nano-carrier and the methods of control are similarly effective. This is in concordance with our hypothesis that the release study of folate from nanoparticles closely follows the release of drugs. 3.11. Cytotoxicity of released folic acid on HeLa cells Initial experiments on cytotoxicity suggested that folic acid is cytotoxic to HeLa cells above 12 μg/ml. To study the cytotoxic behavior of release folic acid from nanoparticles, folate nanoparticles (200 nm) were exposed to the wells containing HeLa cells, at a pre-determined cytotoxic concentration (12 μg/ml). The free folic acid at the same concentration was also added as a control. The cell viability was studied at 2, 6, 10, 20, and 24 h. Fig. 16 shows the difference in impact on exposing folate nanoparticles (cross-linked with Ca2 +) and free folic acid to HeLa cells at this concentration. Bound folic acid shown lower cytotoxicity in comparison to free folic acid during the first 6–7 h. Subsequently, with increased folate release from the nanoparticles, HeLa cells exposed to folate nanoparticles showed similar cell survival as compared to HeLa cells exposed to folate solutions. These results suggest that the released folic acid from nanoparticles is efficient in killing HeLa cells in a controlled manner consistent with its release rate. 4. Conclusion Folate nanoparticles can be designed in the size range of 100– 500 nm even at low concentrations of liquid-crystalline folates. A drug molecule intercalates between or within the ordered folate stacks which are cross-linked with a multivalent cation. Cross-linking cations play a major role in keeping the folate assembly intact. The rate of disintegration of the nanoparticles (measured by the rate of release of
Acknowledgment The authors would like to thank the Department of Science and Technology (SR/S3/CE/027/2011(G)), Government of India as well as the Indian Institute of Technology, Delhi for the support for this project.
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