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In vitro controlled release of Rifampicin through liquid-crystalline folate nanoparticles Rohan Parmar 1 , Rahul Misra ∗,1 , Sanat Mohanty Department of Chemical Engineering, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India
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Article history: Received 13 October 2014 Received in revised form 19 March 2015 Accepted 24 March 2015 Available online xxx Keywords: Rifampicin Control release Liquid-crystalline folate Alveolar macrophage Tuberculosis
a b s t r a c t Rifampicin is one of the frontline drugs for tuberculosis therapy but poor bioavailability of Rifampicin in combination with other anti-tuberculosis drugs is a subject of concern. Nano-based formulations for sustained release of anti-tubercular drugs have been shown to increase antibacterial efficacy and pharmacokinetic behavior. In the present study, liquid-crystalline folate nanoparticles were designed for sustained delivery of Rifampicin and its in vitro release study is reported. Liquid-crystalline nanoparticles of biocompatible folate ions consist of self assembled structures, resulting in high encapsulation, controlled release and low drug losses of about 20-30%, which is significant in itself. This study reports the size-control method of forming Rifampicin encapsulated folate nanoparticles as well as the parameters to control the release profiles of Rifampicin through these nanoparticles. These designs are able to present sustained release for over 25 days. The effect of different parameters such as nanoparticles size, type of cross-linking cation, cross-linking cation concentration and drug-loading on Rifampicin release was studied in vitro. The intracellular uptake and low cytotoxicity of nanoparticles by alveolar macrophages was also demonstrated using fluorescence microscopy and MTT assay respectively. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis is a highly contagious and persistent bacterial infection caused by Mycobacterium tuberculosis. Rifampicin is one of the four drugs along with Isoniazid, Pyrazinamide and Ethambutol used in combination therapy to treat tuberculosis. Rifampicin inhibits the bacterial RNA synthesis by binding to the beta-subunit of bacterial DNA dependent RNA polymerase (DDRP). Inhibition of DDRP leads to blocking of the initiation chain formation in RNA synthesis. It is one of the most effective anti-tuberculosis agents available and is a frontline drug for the treatment of tuberculosis. However, poor bioavailability of Rifampicin in combination with the other three drugs is a subject of concern. This poor bioavailability leads to sub-therapeutic levels of Rifampicin in the body which increases the risk of developing multiple drug resistance tuberculosis (MDR-TB) [1]. There is a significant potential advantage of using nanoparticles as a drug delivery medium in tuberculosis. When administered intravenously, the nanoparticles in the size range of 100–400 nm follow the route of other foreign particles and get endocytosed by resident macrophages and monocytes. On the other hand, in case
∗ Corresponding author. Tel.: +91 9582708534. E-mail address: [email protected] (R. Misra). 1 Both authors have equal contribution.
of infection caused by intracellular persisting microbes (like M. tuberculosis); macrophages become reservoirs for pathogens, thus representing targets for delivery of antimicrobial agents. Hence, nanoparticles improve drug delivery to macrophages, increasing the amount of drug reaching the target site, which allows the reduction of overall therapeutic dose and decrease of side-effects [2–5]. A sizeable number of studies have been done to show the effectiveness of sustained release of anti-tubercular drugs through various nanoparticles both in vivo and in vitro [6–9]. These studies have been summarized in supplementary information. Folic acid is a chromonic molecule which self-assembles to form liquid-crystalline solution. Our previous studies have shown that folic acid self assembles in the form stacks even at low concentrations of 0.1 wt % [10]. It represents a class of materials that exhibit high-order self-assembly behavior which is driven primarily by enthalpic interactions. Chromonics are structurally composed of aromatic rings with hydrophilic groups at the periphery and show liquid crystalline behavior in aqueous solutions. Folic acid derivatives are observed to behave like chromonic molecules and exhibit liquid crystalline behavior. Folic acid is a natural vitamin which is ingested by humans as part of their daily diet. It is found in green leafy vegetables, dried beans, peas and citrus fruits. It is essential for the formation of new cells and DNA in our body. Thus, using folic acid as a drug delivery carrier has a lower risk of toxicity and side effects [11].
http://dx.doi.org/10.1016/j.colsurfb.2015.03.051 0927-7765/© 2015 Elsevier B.V. All rights reserved.
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Folate nanoparticles can be engineered from liquid crystalline folate solution by mixing it with HPMC (Hydroxypropyl Methylcellulose) and their size can be controlled by varying the relative concentrations of HPMC and folic acid [12]. Hydroxypropyl Methylcellulose (HPMC) is a non-toxic, semi-synthetic, inert; viscoelastic polymer used as a food additive, as well as an excipient and controlled-delivery component in oral medicaments [13]. Its structure is shown in Fig. S1c. 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 forming a two-phase system. When folate is mixed with HPMC for a sufficient amount of time nano domains of size 100–400 nm are formed [12]. These domains are cross-linked with multivalent salts like Calcium Chloride or Zinc Chloride to form stable nanoparticles. During the cross-linking process, multivalent ions (Ca2+ or Zn2+ ) exchange with the monovalent cation (Na+ ) in the folate solution. When suspended in release medium containing monovalent salts (saline, phosphate buffered saline etc.) the folate assembly is disrupted leading to release of folic acid and the encapsulated drug. 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. The advantage of using liquid crystalline folate nanoparticles as a drug delivery medium is that folate forms highly ordered structures which lead to comparatively low drug losses as compared to other drug delivery mediums. Rifampicin is encapsulated in this ordered structure through intercalation within the folate stacks which implies that it participates in the folate self-assembly. This ordered structure is present in the nanoparticles developed. The nanoparticles are cross-linked with multivalent cation to keep them stable. The drug molecules can be released in a controlled manner by disrupting this folate assembly. This paper presents folate nanoparticles as a novel material for sustained release of Rifampicin, an anti-tubercular drug, in the treatment of tuberculosis. The present study discusses the method to encapsulate Rifampicin in folate nanoparticles and understand the parameters by which Rifampicin can be released in a controlled manner through these nanoparticles under different conditions. Dynamic Light Scattering (DLS) and Scanning Electron Microscope (SEM) techniques are used to characterize the nanoparticles developed while the concentration of released Rifampicin is determined by a measuring the absorbance value with UV-visible spectrophotometer. This study also addresses the cellular uptake and cytotoxicity of folate nanoparticles by alveolar macrophages through fluorescence microscopy and MTT assay respectively. 2. Materials and methods 2.1. Materials
solution of folic acid 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. It has been reported in 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 [14]. Rifampicin (PubChem: CID 5381226) was purchased TCI Chemicals, India. Fig. S1b shows the chemical structure of Rifampicin. Normal saline (0.8% NaCl solution) was used as a release medium in all the experiments to perform the release studies. 2.2. Encapsulation of Rifampicin in liquid-crystalline folate assembly Rifampicin is added to liquid-crystalline folate solution at loading amount of 10% and 30% of folic acid concentration. This solution is continuously stirred for 1 h at 300 rpm. To study the effect of Rifampicin on folate self-assembly, X-Ray diffraction was performed with the 5% folate solution loaded with 0.5% and 1.5% Rifampicin. 2.3. Preparation of Rifampicin encapsulated folate nanoparticles The desirable size range of the nanoparticles in tuberculosis drug delivery has been reported to be 100–400 nm [15]. Rifampicin encapsulated folate nanoparticles were prepared in this size range by changing the relative concentrations of folate and HPMC. 5% liquid crystalline folate solution was mixed with 1%, 5% and 10% HPMC in the ratios (w/w) of folic acid to HPMC. This mixture of folate solution and HPMC was mixed by continuous stirring at 800–900 rpm for 6 h at room temperature. 2.4. Cross-linking of nanoparticles Multivalent cationic salts (aqueous solutions of 10% CaCl2 and ZnCl2 ) 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 800–900 rpm for 8 h at room temperature. This resultant solution was centrifuged (Eppendorf centrifuge 5810R) at 10,000 rpm for 10 min. The obtained pellet is suspended and washed with de-ionized water and centrifuged again at 10,000 rpm for 10 min. This pellet is re-suspended in deionized 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 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. Encapsulation efficiency of Rifampicin
Folic acid (molecular formula: C19 H19 N7 O6 ; molecular weight: 441.3974 g/mol; PubChem: CID 6037) and HPMC (Hydroxypropyl Methylcellulose) (molecular formula: C12 H20 O10 ; molecular weight: 324.2848 g/mol) were purchased from Central Drug House (CDH) New Delhi. An aqueous stock solution of 5 wt% folic acid and 10 wt% HPMC was prepared using de-ionized water. Folic acid does not dissolve in water by itself; however in the presence of sodium hydroxide it forms the liquid crystalline solutions. The stock
Encapsulation efficency =
In each experiment, mass balance of Rifampicin was carried out to determine the amount of drug encapsulated in the folate nanoparticle. To separate the HPMC and salts that were not bound, a centrifugation step was performed after cross-linking the nanodomains with multivalent salts. During this step, drug loss was calculated by analyzing the drug concentration in the supernatant with the help of a UV–vis spectrophotometry. Accounting for the losses during the method of preparation, encapsulation efficiency was calculated by the given formula:
Initial amount of drug added − Amount of drug lost during processing × 100 Initial amount of added
Encapsulation efficency (%) =
Amount of drug in pellet × 100 Initial amount of drug loaded
(1)
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2.6. In-vitro Rifampicin release
2.10. Characterization of nanoparticles and Rifampicin release
For different experiments, 8 mg of lyophilized nanoparticles were taken in vials and suspended in 2 ml release medium (0.8% Normal Saline).The nanoparticle dispersions in closed vials were kept at room temperature (30–32 ◦ C) at static conditions in the absence of light. At different time points (0–27 days), supernatant samples were collected after centrifugation of the medium at 3000 rpm for 5 min. These supernatants were analyzed by a measuring absorbance of the solution at 473 nm (for Rifampicin) via UV visible spectrophotometer. To detect any particles in supernatant, DLS measurements were performed with supernatant to check the presence of nanoparticles. The absence of particles by DLS suggests that very less particles were present in the supernatant. After sampling, the entire medium was removed and replaced with the same amount of fresh release media (0.8% Normal Saline).
2.10.1. Dynamic light scattering (DLS) 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 deionized water and sonicated during 1 min. The analyses were performed at a scattering angle of 90◦ with refractive index 1.33 and at a temperature of 25 ◦ C.
2.7. Culture of alveolar macrophages (NR8383 cells) NR8383 cells (purchased from National Centre of Cell Science (NCCS), Pune, India) were used as cultured alveolar macrophages. They were cultured in F-12K medium containing 10% fetal bovine serum. The media was supplemented with 100 mg/l penicillin and 100 mg/l streptomycin. Cultures were maintained at 37 ◦ C under 5% CO2 and 95% relative humidity. 2.8. In vitro cytotoxicity studies The cytotoxic effect of Rifampicin and folate nanoparticles on alveolar macrophages was studied using MTT assay. NR8383 cells were seeded into 96 wells of tissue culture plate having 180 l of complete media and were incubated for 18 h. Folate nanoparticles loaded with different concentration of Rifampicin were exposed to cells and incubated at 37 ◦ C in a humidified incubator maintained with 5% CO2 . In order to study the cytotoxic effect of folic acid, the concentration of folate nanoparticles were also varied. Moreover, cytotoxicity of folate nanoparticles without Rifampicin was also tested as a control. The cell viability was estimated by 3-(4,5-dimethylthiazol)-2-diphenyltetrazolium bromide (MTT) assay [16] after 24 h. Two controls were included along with the test compounds to check any error while performing the experiment, growth of cells, and preparing the test compounds. a) 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. b) 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. 2.9. In vitro cellular uptake studies Cellular uptake of folate nanoparticles by alveolar macrophages was studied using fluorescence microscopy. NR8383 cells were seeded into 35 mm cell culture plates and incubated in the medium supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C with 5% CO2 . After 8 h, the cells were washed with incomplete media and were incubated with 12 g/ml RIF loaded folate nanoparticles. After 12 h of incubation, the cells were washed to remove free nanoparticles and fluorescence microscopy was performed on Nikon Eclipse Ti-V Inverted Fluorescence Microscope IX71 equipped with a DS/Fi2 U3 color camera (excitation filter 465–495 nm).
2.10.2. Scanning electron microscope (SEM) The morphology of nanoparticles was observed by scanning electron microscopy (Zeiss EVO 50). A drop of the nanoparticles 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×. 2.10.3. UV–vis spectrophotometry To determine the concentration of the released Rifampicin in the release medium absorbance values at 473 nm for Rifampicin was recorded. Halo DB-20 UV-Visible double beam spectrophotometer was used for this process. A spectrophotometric method of determination was developed to quantify the concentration of released Rifampicin in the medium. 3. Results and discussion 3.1. Method development for detection of Rifampicin in presence of folic acid When folate nanoparticles disintegrate, both folic acid and Rifampicin will be release into the medium. Therefore, a method of analysis was developed in order to determine the concentration of Rifampicin in the presence of folic acid in the release medium using UV-Vis spectrophotometry. Wavelength scan was performed separately for known concentrations of folic acid, Rifampicin and both the components in mixture. Pure folic acid solution showed its characteristic peak at 281 nm while pure Rifampicin showed multiple peaks at 236 nm, 256 nm, 334 nm and 473 nm (refer to supplementary data). However when a wavelength scan of solution of equal concentrations of folic acid and Rifampicin was performed it was found that 281 nm peak of folic acid was predominant and a distinct peak of Rifampicin was observed at 473 nm which increased with increase in concentrations (Fig. 1). Hence, 473 nm was selected as an ideal wavelength to measure absorbance of Rifampicin for determining its concentration in the release medium. A calibration curve was plotted between absorbance and concentration for Rifampicin at this wavelength from samples containing folate and Rifampicin in mixture at different concentration. 3.2. Participation of Rifampicin in folate self-assembly Previous studies have shown that guest molecules can be a part of chromonic liquid-crystalline solutions [17]. X-ray diffraction was performed on samples prepared from 0.5% and 1.5% Rifampicin added to 5% liquid crystalline folate solution so as to check whether Rifampicin is encapsulated in folate assembly or it disrupts and does not take part in the folate assembly. No significant change was observed in the characteristic peaks of folate assembly at any concentrations (0.5 and 1.5%) of Rifampicin. The XRD profiles of folate mixtures with Rifampicin molecules (Fig. 2) show that the Rifampicin molecules do not disrupt the assembly and become part of the assembled stacks. In addition, the nanoparticles formed from these liquid crystalline solutions maintain the order. Hence, we hypothesize that when the nanoparticles disintegrate, any guest
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of size range 340 nm (cross-linked with Ca2+ ) and 380 nm (crosslinked with Zn2+ ). Low folate, high HPMC leads to size range of 140 nm (cross-linked with Ca2+ ) and 110 nm (cross-linked with Zn2+ ). Fig. 5 shows SEM images of the nano-domains formed. SEM image exhibits the nano-domains of size range of 100–150 nm with irregular morphology. 3.4. Encapsulation efficiency and drug loss
Fig. 1. Wavelength scan of mixture of folic acid and Rifampicin (mixed in equimolar concentrations) at different concentrations.
molecules included in the assembly would also be released at the same rate as the folate molecules, thus allowing better control of release. 3.3. Formation of Rifampicin encapsulated folate nanoparticles An extensive research has been carried out by us previously in understanding the formation of folate nanoparticles by HPMC with respect to thermodynamics aspects and stability [12]. We have also reported that size of the nanoparticles can be controlled by controlling the relative concentrations of folate and HPMC in the solution. In the present study, the nanoparticles of Rifampicin encapsulated liquid crystalline folate solutions have been developed in the similar manner and hydrodynamic size of these nanoparticles has been determined by dynamic light scattering (DLS). The DLS data shows that the nanoparticles formed after cross-linking had a particle size distribution of ±20 nm around the mean particle size (refer to supplementary data). DLS data indicates increasing trend in the size distribution of nanoparticles with decrease in the concentration of HPMC. Table 1 shows that for a particular folate concentration, an increase in the HPMC concentration leads to decrease in size of the nanoparticles. High folate and low HPMC concentrations lead to nanoparticles
With the help of mass-balance, the amount of drug encapsulated was determined. The amount of drug loss is accounted for during the preparation of nanoparticles. Using this data (Table 2), the encapsulation efficiency was calculated by Eq. (1). Table 2 indicates the method of forming different size of nanoparticles as well as the drug loss during formation. There is 7–8% change in drug loss and encapsulation efficiency with the change in size of the nanoparticles, keeping the cross-linking cation concentration same. However, the concentration of the cross-linking agent affects the loss of drug significantly. The data (Table 2) indicates that the drug loss is reduced from 80% to 30% on increasing the concentration of cross-linking agent (CaCl2 , in this example) from 1% to 5%. Reducing the amount of drug loading does not seem to have an effect on encapsulation efficiency. It is hypothesized that as crosslinking concentration is increased, the folate assembly along with drug molecules is more strongly bound. Therefore the possibility of losing the drug molecules decreases at higher concentrations of cross-linking. 3.5. Rifampicin 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 cross-linker concentration on Rifampicin release (equivalently, nanoparticle break up) is presented. 140 nm size folate nanoparticles were cross-linked with different concentrations of calcium chloride in solution. It was observed that as the concentration of cross-linking agent was increased from 1% to 10%, percent, the release of Rifampicin decreased from 80% to 39% in 628 h (refer to supplementary data). Hence the general trend suggests that at higher concentrations of cross-linking cation, the folate particles are more “strongly” bound and the folate assemblies break up more slowly. 3.6. Rifampicin release: effect of nanoparticle’s size Past studies have shown that the size of nanoparticle affects the release rate of the encapsulated drug [18]. Experiments were carried out to observe the variation in release rates with nanoparticles of size 343 nm, 241 nm and 140 nm. Fig. 3 shows that upto 49% of Rifampicin is released with 343 nm size folate nanoparticles in the period of 628 h, while 39% release was obtained with 140 nm sized folate nanoparticles. It is hypothesized that higher surface area of smaller particles which favors more effective cross-linking. Hence, release of Rifampicin may be slower with decreasing size of folate nanoparticles. 3.7. Rifampicin release: effect of drug loading
Fig. 2. XRD profile of Rifampicin with folic acid showing the participation of Rifampicin in the ordered structure of folate assembly.
For administering a specific amount of drug to a particular site, it is reported that the quantity of nanocarrier can be reduced, if that nanocarrier has a higher drug loading capacity [19]. Therefore, a release study was carried out with 140 nm folate nanoparticle loaded with Rifampicin concentration at 10% and 30% of folic acid concentration. It was observed (refer to supplementary data) that the percent release of Rifampicin was effectively the same on decreasing the drug loading amount from 30% to 10% during
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Table 1 Concentration of compounds used in formation of folate nanoparticles of desired size. Concentration of folate, drug and HPMC
Hydrodynamic size of the nanoparticles
Folate % (g/100 ml)
HPMC % (g/100 ml)
Rifampicin % (g/100 ml)
Cross-linked withCalcium (nm)
Cross linked with Zinc (nm)
2.50 0.83 0.46
5.0 8.30 9.09
0.75 0.25 0.14
342 ± 20 241 ± 20 140 ± 20
381 ± 20 224 ± 20 110 ± 20
is even lower than what was observed with ZnCl2 as the crosslinking agent. 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 [20]. 3.9. Delivery of Rifampicin to alveolar macrophages (AMs): cellular uptake studies
Fig. 3. Effect of nanoparticle’s size on the percent release of Rifampicin (different size of nanoparticles cross-linked with 10% CaCl2 as cross-linking agent).
the period of 628 h. Thus, lower loading levels can achieve similar release rates with higher quantity of total carrier administered. 3.8. Rifampicin release: effect of cross-linking cation Since different multivalent cations interact differently with the folate anions, it could be argued that the choice of cross-linking agent might also affects the release rates of Rifampicin. Release rates studies were carried out with 140 nm size nanoparticles crosslinked with different divalent and trivalent cross-linking agents, namely ZnCl2 , CaCl2 and FeCl3 . Fig. 4 shows that high release of Rifampicin was achieved in CaCl2 in comparison to ZnCl2 . In over 383 hrs, more than 38% of Rifampicin has been released from 140 nm sized nanoparticles cross-linked with CaCl2 , whereas only 21% release was observed in case of ZnCl2 cross-linked nanoparticles. Rifampicin release from nanoparticles cross-linked with FeCl3
A major step in tuberculosis pathogenesis is the ability of the M. tuberculosis to enter and replicate within the macrophages of its human host. During primary infection with the organism, aerosol-droplet nuclei containing small numbers of M. tuberculosis are deposited in the alveoli of the lung and are phagocytized by alveolar macrophages (AMs). M. tuberculosis enters the alveolar macrophages via receptor mediated phagocytosis. In contrast to most microbes; M. tuberculosis is resistant to the biocidal mechanisms of AMs and proliferates in the AMs using them as incubator. In the standard treatment of tuberculosis, antibiotics are given via oral route. These drugs are distributed to many tissues via circulatory system. Hence the antibiotics accumulated in alveolar macrophages are not sufficient to kill the underlying M. tuberculosis. Thus, there is a need to develop a drug delivery system to transport anti-tuberculosis drugs into alveolar macrophages selectively and more efficiently [21,22]. In order to deliver Rifampicin in alveolar macrophages, folate nanoparticles were exposed to cultured macrophages and their uptake was studied using fluorescence microscopy. Past studies have reported that zinc ions in bound state exhibit fluorescence [23]. 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, fluorescence spectroscopy was used to see the uptake of folate nanoparticles in alveolar macrophages with time. Zinc crosslinked folate nanoparticles (200 ± 20 nm) were exposed to cultured macrophages and fluorescence was detected at different time intervals over the period of 24 h. After exposure at different time intervals, cultured macrophages when observed under fluorescence microscope, bright fluorescence (green) was observed in cells. Fig. 5A–C shows both bright field and dark field images at 10 h, 16 h and 24 h respectively. These images show intracellular uptake of folate nanoparticles phagocytized by alveolar macrophages. Moreover, alveolar macrophages also represent folate receptors on their surface (particularly folate receptor ). Therefore, folate receptor mediated phagocytosis can be the probable mechanism for intracellular uptake. 3.10. In vitro cytotoxicity of folate nanoparticles on alveolar macrophages (AMs)
Fig. 4. Effect of cross-linking cation on Rifampicin release (120 ± 20 nm folate particle cross-linked 10% cross-linking agent).
For an effective nanocarrier, it is required to deliver the drug to AMs efficiently and the drug delivered should prevent the infection of M. tuberculosis without being toxic to macrophages. Therefore, to test the cytotoxic effect of our delivery system, cultured
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Fig. 5. Bright field and dark field fluorescence images of alveolar macrophages with cellular uptake of folate nanoparticles at (A) 10 h, (B) 16 h and (C) 24 h.
macrophages were exposed to folate nanoparticles loaded with different concentrations of Rifampicin. To compare the cytotoxicity, free Rifampicin and folate nanoparticles without Rifampicin was also exposed to cultured macrophages. After 24-h exposure, cytotoxicity was studied by MTT assay. Following treatment of RFP, folate nanoparticles without RFP, and RFP loaded folate nanoparticles, the cell viability of all experiment samples was over 80% (Fig. 6). The nanoparticles show a slight cell viability decrease in a concentration-dependent manner from 10 to 20 g/ml folate concentration (refer to supplementary data). This clearly suggests that folate nanoparticles show low cytotoxicity to alveolar macrophages at different levels of Rifampicin loading.
amount of drug released into the solution, we calculate the disintegration/stability of the particles in solution. The particles are extremely stable (show very little release) in acidic solutions. Storage at different temperature does not affect the release rates significantly. In buffer solutions with monovalent cations, we see
3.11. Stability of Rifampicin loaded folate particles The role of the cross-linking solution using multi-valent ions is significant. In the absence of cross-linking, we lose almost all the folate domains/particles in the HPMC-folate solution, when washed with de-ionized water. Once cross-linked, these particles are stable over 30 days. Cross-linked lyophilized particles do not change their sizes significantly when suspended in deionized water over 24 h and lose less than 9% of their mass over 30 days (refer to supplementary data). Further, these cross-linked particles are stable in acidic solution as well. For release studies, the particles are suspended in different buffer solutions, solutions of different pH, etc. By measuring the
Fig. 6. MTT assay showing cell viability of alveolar macrophages at different RFP loading levels with folate concentration of 10 g/ml. (positive control: 5% DMSO; negative control: growth media).
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115 53 21 15 17 12 8 139 98 75
26.032 9.539 4.913 1.658 4.444 4.180 1.966 24.960 8.889 4.515
76.36 83.97 79.49 80.49 71.91 67.64 31.81 73.22 78.25 73.06
that changing the cross-linking cation (size, valency) affects the rate in an expected manner (higher valency ions show slower release). Increasing the concentration of the cross-linking group also reduces the release rate and increases stability. Increasing the concentration of the monovalent ions, however, increases the release rates. We also see that when release studies are undertaken with constant shaking, there is a small but significant increase in release owing to reduced mass transfer barriers, improved dispersion or both. Based on these trends, we hypothesize that the cross-linking cations coordinate with the anions of the folate assemblies without significant change in self-assembly structure (as seen by XRD of the cross-linked particles). These coordination bonds are strong enough to keep the particles stable and ‘fix’ their size and structure under neutral environments. The particles have zeta potential of −20 to −40 mV which indicates moderate stability in the medium. The zeta potential increases with increase in the cross-linking concentration (refer to supplementary data). These particles, while stable in acidic pH or deionized water, where there are few available monovalent ions that can exchange with the cross-linking groups, begin to disintegrate when monovalent ions are available for exchange. This explains increase in release rates in buffer solutions with monovalent ions, as well as with increase concentration of monovalent ions or reduced concentration of cross-linking. 4. Conclusion The present study reports a method to design and control the size of Rifampicin encapsulated folate nanoparticles in the size range of 100–350 nm. It has been shown that drug molecules can be encapsulated in these nanoparticles with low drug loss of 20-30 %. Rifampicin molecules intercalate between or within the ordered folate stacks of the nanoparticles, which are cross-linked with a multivalent cation. Cross-linking cations play major role in keeping the drug encapsulated folate assembly intact. Due to exchange of ions present in release medium with the cross-linking cation, this assembly is disrupted. The rate of disruption of this assembly (measured by the rate of release of Rifampicin) is dependent on the size of the nanoparticles and its concentration. These nanoparticles can deliver the drug efficiently to macrophages without being toxic to macrophages. 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 of Rifampicin.
150 60 30 10 30 30 30 150 60 30 0.75 0.25 0.14 0.05 0.14 0.14 0.14 0.75 0.25 0.14
34.09 11.36 6.18 2.06 6.18 6.18 6.18 34.09 11.36 6.18
8.059 1.821 1.267 0.402 1.736 2.000 4.214 9.130 2.471 1.665 5.0 8.30 9.09 9.09 9.09 9.09 9.09 5.0 8.30 9.09 2.50 0.83 0.46 0.46 0.46 0.46 0.46 2.50 0.83 0.46 Calcium (10%) Calcium (10%) Calcium (10%) Calcium (10%) Calcium (5%) Calcium (3%) Calcium (1%) Zinc (10%) Zinc (10%) Zinc (10%) 20 20 20 20 20 20 20 20 20 20 ± ± ± ± ± ± ± ± ± ± 342 241 140 120 140 140 140 381 224 110
The supplementary data is available at the end of the manuscript. Table S1 summarizes the different nanoparticles mediated anti-tubercular drug delivery system previously studied. Fig. S1 represents structure of folic acid, Rifampicin and HPMC. Fig. S2 shows the wavelength scan of pure folic acid and Rifampicin as a reference. Fig. S3 shows the DLS data for size distribution of folate particles. Fig. S4 represents the SEM image of folate particles. Fig. S5 shows the effect of cross-linker concentration of Rifampicin release while Fig. S6 represents the effect of drug loading on Rifampicin release. The cytotoxicity of folate particles at 20 g/ml Rifampicin loading has been studied by MTT assay in Fig. S7. FDH = folate + drug + HPMC.
Drug encapsulated (mg) Weight of dry pellet (mg) Drug loss (mg) Amount of drug after cross-linking (mg) Amount of drug after adding HPMC (mg) RIF % Folate %
HPMC %
7
Supplementary information
Cross-linking cation Nano-particle size (nm)
Drug amount during encapsulation Method of development Nanoparticle specifications
Table 2 Mass balance of Rifampicin (RIF) encapsulated, lost and encapsulation efficiency achieved in the method of nanoparticle formation.
Drug amount after encapsulation and loss
Encapsulation percentage (%)
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Acknowledgments The authors would like to thank Department of Science and Technology, Government of India (Grant no. RP2519) for financial support of this project. We also thank to Ms. Mohita Upadhyay and Kusuma School of Biological Sciences, IIT-Delhi for providing facilities to conduct cell line studies.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.colsurfb.2015.03.051. References [1] L.C. du Toit, V. Pillay, M.P. Danckwerts, Tuberculosis chemotherapy: current drug delivery approaches, Respir. Res. 7 (2006) 118–136. [2] S. Gelperina, K. Kisich, M.D. Iseman, L. Heifets, The potential advantages of nanoparticle drug delivery systems in chemotherapy of tuberculosis, Am. J. Respir. Crit. Care Med. 172 (2005) 1487–1490. [3] K. Peters, et al., Preparation of a clofazimine nanosuspension for intravenous use and evaluation of its therapeutic efficacy in murine Mycobacterium avium infection, J. Antimicrob. Chemother. 45 (1) (2000) 77–83. [4] I.A. Bakker-Woudenberg, et al., Liposomes with prolonged blood circulation and selective localization in Klebsiella pneumoniae-infected lung tissue, J. Infect. Dis. 168 (1) (1993) 164–171. [5] D.L. Clemens, et al., Targeted intracellular delivery of antituberculosis drugs to Mycobacterium tuberculosis-infected macrophages via functionalized mesoporous silica nanoparticles, Antimicrob. Agents Chemother. 56 (5) (2012) 2535–2545. [6] A. Zahoor, S. Sharma, G.K. Khuller, Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis, Int. J. Antimicrob. Agents 26 (4) (2005) 298–303. [7] R. Pandey, G.K. Khuller, Solid lipid particle-based inhalable sustained drug delivery system against experimental tuberculosis, Tuberculosis 85 (2005) 227–234. [8] R. Pandey, A. Zahoor, S. Sharma, G.K. Khuller, Nanoparticle encapsulated anti tubercular drugs as a potential oral drug delivery system against murine tuberculosis, Tuberculosis 83 (2003) 373–378. [9] P. Deol, G.K. Khuller, Lung specific stealth liposomes: stability, biodistribution and toxicity of liposomal anti-tubercular drugs, Biochim. Biophys. Acta 41 (1997) 1211–1214. [10] G. Motkar, M. Lonare, O. Patil, S. Mohanty, Self-assembly of folic acid in aqueous media, AIChE J. 59 (2013) 1360–1368. [11] A.M. Shuaibi, G.P. Sevenhuysen, J.D. House, The importance of using folate intake expressed as dietary folate equivalents in predicting folate status, J. Food Compos. Anal. 22 (1) (2009) 38–43.
[12] R. Misra, M. Lonare, R. Gupta, S. Mohanty, Engineering chromonic nanoparticles from liquid-crystalline folate solutions, Sci. Adv. Mater. 6 (2014) 1–9. [13] D.J. de Silva, J.M. Olver, Hydroxypropyl methylcellulose (HPMC) lubricant facilitates insertion of porous spherical orbital implants, Ophthalmic Plast. Reconstr. Surg. 21 (4) (2005) 301–302. [14] F. Ciuchi, G. Di Nicola, H. Franz, G. Gottarelli, P. Mariani, M.G. Ponzi Bossi, G.P. Spada, Self recognition and self-assembly of folic acid salts: columnar liquid crystalline polymorphism and the column growth process, J. Am. Chem. Soc. 116 (1994) 7064–7071. [15] A. Swami, J. Shi, S. Gadde, A.R. Votruba, N. Kolishetti, O.C. Farokhzad, Nanoparticles for Targeted and Temporally Controlled Drug Delivery. Nanostructure Science and Technology: Multifunctional Nanoparticles for Drug Delivery Applications, 2012, pp. 9–29. [16] G. Fotakis, J.A. Timbrell, In vitro cytotoxicity assays: comparison of LDH, neutral red. MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride, Toxicol. Lett. 160 (2006) 171–177. [17] K. Mundy, J.C. Sleep, J.E. Lydon, The intercalation of ethidium bromide in the chromonic lyotropic phases of drugs and nucleic acids, Liq. Cryst. 19 (1995) 107–112. [18] L. Mu, S.S. Feng, A novel controlled release formulation for the anticancer drug paclitaxel (Taxol): PLGA nanoparticles containing vitamin E TPGS, J. Control. Release 86 (1) (2003) 33–48. [19] T. Govender, S. Stolnik, M.C. Garnett, L. Illum, S.S. Davis, PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug, J. Control. Release 57 (2) (1999) 171–185. [20] M. Ahmadi, A. Fattahi, On the binding of Mg2+ , Ca2+ , Zn2+ and Cu2+ metal cations to 20-deoxyguanosine: changes on sugar puckering and strength of the N-glycosidic bond, Sci. Iran. 18 (2011) 1343–1352. [21] J. Chuan, Y. Li, L. Yang, X. Sun, Q. Zhang, T. Gong, Z. Zhang, Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles, J. Nanopart. Res. 15 (2013) 1634. [22] L.E. Des Jardin, T.M. Kaufman, B. Potts, B. Kutzbach, H. Yi, L.S. Schlesinger, Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FccRII and the mannose receptor, Microbiology 148 (2002) 3161–3171. [23] P. Jiang, Z. Gua, Fluorescent detection of zinc in biological systems: recent development on the design of chemosensors and biosensors, Coord. Chem. Rev. 248 (2004) 205–229.
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In vitro controlled release of Rifampicin through liquid-crystalline folate nanoparticles Rohan Parmar 1 , Rahul Misra ∗,1 , Sanat Mohanty Department of Chemical Engineering, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 19 March 2015 Accepted 24 March 2015 Available online xxx Keywords: Rifampicin Control release Liquid-crystalline folate Alveolar macrophage Tuberculosis
a b s t r a c t Rifampicin is one of the frontline drugs for tuberculosis therapy but poor bioavailability of Rifampicin in combination with other anti-tuberculosis drugs is a subject of concern. Nano-based formulations for sustained release of anti-tubercular drugs have been shown to increase antibacterial efficacy and pharmacokinetic behavior. In the present study, liquid-crystalline folate nanoparticles were designed for sustained delivery of Rifampicin and its in vitro release study is reported. Liquid-crystalline nanoparticles of biocompatible folate ions consist of self assembled structures, resulting in high encapsulation, controlled release and low drug losses of about 20-30%, which is significant in itself. This study reports the size-control method of forming Rifampicin encapsulated folate nanoparticles as well as the parameters to control the release profiles of Rifampicin through these nanoparticles. These designs are able to present sustained release for over 25 days. The effect of different parameters such as nanoparticles size, type of cross-linking cation, cross-linking cation concentration and drug-loading on Rifampicin release was studied in vitro. The intracellular uptake and low cytotoxicity of nanoparticles by alveolar macrophages was also demonstrated using fluorescence microscopy and MTT assay respectively. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis is a highly contagious and persistent bacterial infection caused by Mycobacterium tuberculosis. Rifampicin is one of the four drugs along with Isoniazid, Pyrazinamide and Ethambutol used in combination therapy to treat tuberculosis. Rifampicin inhibits the bacterial RNA synthesis by binding to the beta-subunit of bacterial DNA dependent RNA polymerase (DDRP). Inhibition of DDRP leads to blocking of the initiation chain formation in RNA synthesis. It is one of the most effective anti-tuberculosis agents available and is a frontline drug for the treatment of tuberculosis. However, poor bioavailability of Rifampicin in combination with the other three drugs is a subject of concern. This poor bioavailability leads to sub-therapeutic levels of Rifampicin in the body which increases the risk of developing multiple drug resistance tuberculosis (MDR-TB) [1]. There is a significant potential advantage of using nanoparticles as a drug delivery medium in tuberculosis. When administered intravenously, the nanoparticles in the size range of 100–400 nm follow the route of other foreign particles and get endocytosed by resident macrophages and monocytes. On the other hand, in case
∗ Corresponding author. Tel.: +91 9582708534. E-mail address: [email protected] (R. Misra). 1 Both authors have equal contribution.
of infection caused by intracellular persisting microbes (like M. tuberculosis); macrophages become reservoirs for pathogens, thus representing targets for delivery of antimicrobial agents. Hence, nanoparticles improve drug delivery to macrophages, increasing the amount of drug reaching the target site, which allows the reduction of overall therapeutic dose and decrease of side-effects [2–5]. A sizeable number of studies have been done to show the effectiveness of sustained release of anti-tubercular drugs through various nanoparticles both in vivo and in vitro [6–9]. These studies have been summarized in supplementary information. Folic acid is a chromonic molecule which self-assembles to form liquid-crystalline solution. Our previous studies have shown that folic acid self assembles in the form stacks even at low concentrations of 0.1 wt % [10]. It represents a class of materials that exhibit high-order self-assembly behavior which is driven primarily by enthalpic interactions. Chromonics are structurally composed of aromatic rings with hydrophilic groups at the periphery and show liquid crystalline behavior in aqueous solutions. Folic acid derivatives are observed to behave like chromonic molecules and exhibit liquid crystalline behavior. Folic acid is a natural vitamin which is ingested by humans as part of their daily diet. It is found in green leafy vegetables, dried beans, peas and citrus fruits. It is essential for the formation of new cells and DNA in our body. Thus, using folic acid as a drug delivery carrier has a lower risk of toxicity and side effects [11].
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Folate nanoparticles can be engineered from liquid crystalline folate solution by mixing it with HPMC (Hydroxypropyl Methylcellulose) and their size can be controlled by varying the relative concentrations of HPMC and folic acid [12]. Hydroxypropyl Methylcellulose (HPMC) is a non-toxic, semi-synthetic, inert; viscoelastic polymer used as a food additive, as well as an excipient and controlled-delivery component in oral medicaments [13]. Its structure is shown in Fig. S1c. 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 forming a two-phase system. When folate is mixed with HPMC for a sufficient amount of time nano domains of size 100–400 nm are formed [12]. These domains are cross-linked with multivalent salts like Calcium Chloride or Zinc Chloride to form stable nanoparticles. During the cross-linking process, multivalent ions (Ca2+ or Zn2+ ) exchange with the monovalent cation (Na+ ) in the folate solution. When suspended in release medium containing monovalent salts (saline, phosphate buffered saline etc.) the folate assembly is disrupted leading to release of folic acid and the encapsulated drug. 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. The advantage of using liquid crystalline folate nanoparticles as a drug delivery medium is that folate forms highly ordered structures which lead to comparatively low drug losses as compared to other drug delivery mediums. Rifampicin is encapsulated in this ordered structure through intercalation within the folate stacks which implies that it participates in the folate self-assembly. This ordered structure is present in the nanoparticles developed. The nanoparticles are cross-linked with multivalent cation to keep them stable. The drug molecules can be released in a controlled manner by disrupting this folate assembly. This paper presents folate nanoparticles as a novel material for sustained release of Rifampicin, an anti-tubercular drug, in the treatment of tuberculosis. The present study discusses the method to encapsulate Rifampicin in folate nanoparticles and understand the parameters by which Rifampicin can be released in a controlled manner through these nanoparticles under different conditions. Dynamic Light Scattering (DLS) and Scanning Electron Microscope (SEM) techniques are used to characterize the nanoparticles developed while the concentration of released Rifampicin is determined by a measuring the absorbance value with UV-visible spectrophotometer. This study also addresses the cellular uptake and cytotoxicity of folate nanoparticles by alveolar macrophages through fluorescence microscopy and MTT assay respectively. 2. Materials and methods 2.1. Materials
solution of folic acid 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. It has been reported in 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 [14]. Rifampicin (PubChem: CID 5381226) was purchased TCI Chemicals, India. Fig. S1b shows the chemical structure of Rifampicin. Normal saline (0.8% NaCl solution) was used as a release medium in all the experiments to perform the release studies. 2.2. Encapsulation of Rifampicin in liquid-crystalline folate assembly Rifampicin is added to liquid-crystalline folate solution at loading amount of 10% and 30% of folic acid concentration. This solution is continuously stirred for 1 h at 300 rpm. To study the effect of Rifampicin on folate self-assembly, X-Ray diffraction was performed with the 5% folate solution loaded with 0.5% and 1.5% Rifampicin. 2.3. Preparation of Rifampicin encapsulated folate nanoparticles The desirable size range of the nanoparticles in tuberculosis drug delivery has been reported to be 100–400 nm [15]. Rifampicin encapsulated folate nanoparticles were prepared in this size range by changing the relative concentrations of folate and HPMC. 5% liquid crystalline folate solution was mixed with 1%, 5% and 10% HPMC in the ratios (w/w) of folic acid to HPMC. This mixture of folate solution and HPMC was mixed by continuous stirring at 800–900 rpm for 6 h at room temperature. 2.4. Cross-linking of nanoparticles Multivalent cationic salts (aqueous solutions of 10% CaCl2 and ZnCl2 ) 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 800–900 rpm for 8 h at room temperature. This resultant solution was centrifuged (Eppendorf centrifuge 5810R) at 10,000 rpm for 10 min. The obtained pellet is suspended and washed with de-ionized water and centrifuged again at 10,000 rpm for 10 min. This pellet is re-suspended in deionized 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 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. Encapsulation efficiency of Rifampicin
Folic acid (molecular formula: C19 H19 N7 O6 ; molecular weight: 441.3974 g/mol; PubChem: CID 6037) and HPMC (Hydroxypropyl Methylcellulose) (molecular formula: C12 H20 O10 ; molecular weight: 324.2848 g/mol) were purchased from Central Drug House (CDH) New Delhi. An aqueous stock solution of 5 wt% folic acid and 10 wt% HPMC was prepared using de-ionized water. Folic acid does not dissolve in water by itself; however in the presence of sodium hydroxide it forms the liquid crystalline solutions. The stock
Encapsulation efficency =
In each experiment, mass balance of Rifampicin was carried out to determine the amount of drug encapsulated in the folate nanoparticle. To separate the HPMC and salts that were not bound, a centrifugation step was performed after cross-linking the nanodomains with multivalent salts. During this step, drug loss was calculated by analyzing the drug concentration in the supernatant with the help of a UV–vis spectrophotometry. Accounting for the losses during the method of preparation, encapsulation efficiency was calculated by the given formula:
Initial amount of drug added − Amount of drug lost during processing × 100 Initial amount of added
Encapsulation efficency (%) =
Amount of drug in pellet × 100 Initial amount of drug loaded
(1)
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2.6. In-vitro Rifampicin release
2.10. Characterization of nanoparticles and Rifampicin release
For different experiments, 8 mg of lyophilized nanoparticles were taken in vials and suspended in 2 ml release medium (0.8% Normal Saline).The nanoparticle dispersions in closed vials were kept at room temperature (30–32 ◦ C) at static conditions in the absence of light. At different time points (0–27 days), supernatant samples were collected after centrifugation of the medium at 3000 rpm for 5 min. These supernatants were analyzed by a measuring absorbance of the solution at 473 nm (for Rifampicin) via UV visible spectrophotometer. To detect any particles in supernatant, DLS measurements were performed with supernatant to check the presence of nanoparticles. The absence of particles by DLS suggests that very less particles were present in the supernatant. After sampling, the entire medium was removed and replaced with the same amount of fresh release media (0.8% Normal Saline).
2.10.1. Dynamic light scattering (DLS) 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 deionized water and sonicated during 1 min. The analyses were performed at a scattering angle of 90◦ with refractive index 1.33 and at a temperature of 25 ◦ C.
2.7. Culture of alveolar macrophages (NR8383 cells) NR8383 cells (purchased from National Centre of Cell Science (NCCS), Pune, India) were used as cultured alveolar macrophages. They were cultured in F-12K medium containing 10% fetal bovine serum. The media was supplemented with 100 mg/l penicillin and 100 mg/l streptomycin. Cultures were maintained at 37 ◦ C under 5% CO2 and 95% relative humidity. 2.8. In vitro cytotoxicity studies The cytotoxic effect of Rifampicin and folate nanoparticles on alveolar macrophages was studied using MTT assay. NR8383 cells were seeded into 96 wells of tissue culture plate having 180 l of complete media and were incubated for 18 h. Folate nanoparticles loaded with different concentration of Rifampicin were exposed to cells and incubated at 37 ◦ C in a humidified incubator maintained with 5% CO2 . In order to study the cytotoxic effect of folic acid, the concentration of folate nanoparticles were also varied. Moreover, cytotoxicity of folate nanoparticles without Rifampicin was also tested as a control. The cell viability was estimated by 3-(4,5-dimethylthiazol)-2-diphenyltetrazolium bromide (MTT) assay [16] after 24 h. Two controls were included along with the test compounds to check any error while performing the experiment, growth of cells, and preparing the test compounds. a) 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. b) 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. 2.9. In vitro cellular uptake studies Cellular uptake of folate nanoparticles by alveolar macrophages was studied using fluorescence microscopy. NR8383 cells were seeded into 35 mm cell culture plates and incubated in the medium supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C with 5% CO2 . After 8 h, the cells were washed with incomplete media and were incubated with 12 g/ml RIF loaded folate nanoparticles. After 12 h of incubation, the cells were washed to remove free nanoparticles and fluorescence microscopy was performed on Nikon Eclipse Ti-V Inverted Fluorescence Microscope IX71 equipped with a DS/Fi2 U3 color camera (excitation filter 465–495 nm).
2.10.2. Scanning electron microscope (SEM) The morphology of nanoparticles was observed by scanning electron microscopy (Zeiss EVO 50). A drop of the nanoparticles 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×. 2.10.3. UV–vis spectrophotometry To determine the concentration of the released Rifampicin in the release medium absorbance values at 473 nm for Rifampicin was recorded. Halo DB-20 UV-Visible double beam spectrophotometer was used for this process. A spectrophotometric method of determination was developed to quantify the concentration of released Rifampicin in the medium. 3. Results and discussion 3.1. Method development for detection of Rifampicin in presence of folic acid When folate nanoparticles disintegrate, both folic acid and Rifampicin will be release into the medium. Therefore, a method of analysis was developed in order to determine the concentration of Rifampicin in the presence of folic acid in the release medium using UV-Vis spectrophotometry. Wavelength scan was performed separately for known concentrations of folic acid, Rifampicin and both the components in mixture. Pure folic acid solution showed its characteristic peak at 281 nm while pure Rifampicin showed multiple peaks at 236 nm, 256 nm, 334 nm and 473 nm (refer to supplementary data). However when a wavelength scan of solution of equal concentrations of folic acid and Rifampicin was performed it was found that 281 nm peak of folic acid was predominant and a distinct peak of Rifampicin was observed at 473 nm which increased with increase in concentrations (Fig. 1). Hence, 473 nm was selected as an ideal wavelength to measure absorbance of Rifampicin for determining its concentration in the release medium. A calibration curve was plotted between absorbance and concentration for Rifampicin at this wavelength from samples containing folate and Rifampicin in mixture at different concentration. 3.2. Participation of Rifampicin in folate self-assembly Previous studies have shown that guest molecules can be a part of chromonic liquid-crystalline solutions [17]. X-ray diffraction was performed on samples prepared from 0.5% and 1.5% Rifampicin added to 5% liquid crystalline folate solution so as to check whether Rifampicin is encapsulated in folate assembly or it disrupts and does not take part in the folate assembly. No significant change was observed in the characteristic peaks of folate assembly at any concentrations (0.5 and 1.5%) of Rifampicin. The XRD profiles of folate mixtures with Rifampicin molecules (Fig. 2) show that the Rifampicin molecules do not disrupt the assembly and become part of the assembled stacks. In addition, the nanoparticles formed from these liquid crystalline solutions maintain the order. Hence, we hypothesize that when the nanoparticles disintegrate, any guest
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of size range 340 nm (cross-linked with Ca2+ ) and 380 nm (crosslinked with Zn2+ ). Low folate, high HPMC leads to size range of 140 nm (cross-linked with Ca2+ ) and 110 nm (cross-linked with Zn2+ ). Fig. 5 shows SEM images of the nano-domains formed. SEM image exhibits the nano-domains of size range of 100–150 nm with irregular morphology. 3.4. Encapsulation efficiency and drug loss
Fig. 1. Wavelength scan of mixture of folic acid and Rifampicin (mixed in equimolar concentrations) at different concentrations.
molecules included in the assembly would also be released at the same rate as the folate molecules, thus allowing better control of release. 3.3. Formation of Rifampicin encapsulated folate nanoparticles An extensive research has been carried out by us previously in understanding the formation of folate nanoparticles by HPMC with respect to thermodynamics aspects and stability [12]. We have also reported that size of the nanoparticles can be controlled by controlling the relative concentrations of folate and HPMC in the solution. In the present study, the nanoparticles of Rifampicin encapsulated liquid crystalline folate solutions have been developed in the similar manner and hydrodynamic size of these nanoparticles has been determined by dynamic light scattering (DLS). The DLS data shows that the nanoparticles formed after cross-linking had a particle size distribution of ±20 nm around the mean particle size (refer to supplementary data). DLS data indicates increasing trend in the size distribution of nanoparticles with decrease in the concentration of HPMC. Table 1 shows that for a particular folate concentration, an increase in the HPMC concentration leads to decrease in size of the nanoparticles. High folate and low HPMC concentrations lead to nanoparticles
With the help of mass-balance, the amount of drug encapsulated was determined. The amount of drug loss is accounted for during the preparation of nanoparticles. Using this data (Table 2), the encapsulation efficiency was calculated by Eq. (1). Table 2 indicates the method of forming different size of nanoparticles as well as the drug loss during formation. There is 7–8% change in drug loss and encapsulation efficiency with the change in size of the nanoparticles, keeping the cross-linking cation concentration same. However, the concentration of the cross-linking agent affects the loss of drug significantly. The data (Table 2) indicates that the drug loss is reduced from 80% to 30% on increasing the concentration of cross-linking agent (CaCl2 , in this example) from 1% to 5%. Reducing the amount of drug loading does not seem to have an effect on encapsulation efficiency. It is hypothesized that as crosslinking concentration is increased, the folate assembly along with drug molecules is more strongly bound. Therefore the possibility of losing the drug molecules decreases at higher concentrations of cross-linking. 3.5. Rifampicin 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 cross-linker concentration on Rifampicin release (equivalently, nanoparticle break up) is presented. 140 nm size folate nanoparticles were cross-linked with different concentrations of calcium chloride in solution. It was observed that as the concentration of cross-linking agent was increased from 1% to 10%, percent, the release of Rifampicin decreased from 80% to 39% in 628 h (refer to supplementary data). Hence the general trend suggests that at higher concentrations of cross-linking cation, the folate particles are more “strongly” bound and the folate assemblies break up more slowly. 3.6. Rifampicin release: effect of nanoparticle’s size Past studies have shown that the size of nanoparticle affects the release rate of the encapsulated drug [18]. Experiments were carried out to observe the variation in release rates with nanoparticles of size 343 nm, 241 nm and 140 nm. Fig. 3 shows that upto 49% of Rifampicin is released with 343 nm size folate nanoparticles in the period of 628 h, while 39% release was obtained with 140 nm sized folate nanoparticles. It is hypothesized that higher surface area of smaller particles which favors more effective cross-linking. Hence, release of Rifampicin may be slower with decreasing size of folate nanoparticles. 3.7. Rifampicin release: effect of drug loading
Fig. 2. XRD profile of Rifampicin with folic acid showing the participation of Rifampicin in the ordered structure of folate assembly.
For administering a specific amount of drug to a particular site, it is reported that the quantity of nanocarrier can be reduced, if that nanocarrier has a higher drug loading capacity [19]. Therefore, a release study was carried out with 140 nm folate nanoparticle loaded with Rifampicin concentration at 10% and 30% of folic acid concentration. It was observed (refer to supplementary data) that the percent release of Rifampicin was effectively the same on decreasing the drug loading amount from 30% to 10% during
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Table 1 Concentration of compounds used in formation of folate nanoparticles of desired size. Concentration of folate, drug and HPMC
Hydrodynamic size of the nanoparticles
Folate % (g/100 ml)
HPMC % (g/100 ml)
Rifampicin % (g/100 ml)
Cross-linked withCalcium (nm)
Cross linked with Zinc (nm)
2.50 0.83 0.46
5.0 8.30 9.09
0.75 0.25 0.14
342 ± 20 241 ± 20 140 ± 20
381 ± 20 224 ± 20 110 ± 20
is even lower than what was observed with ZnCl2 as the crosslinking agent. 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 [20]. 3.9. Delivery of Rifampicin to alveolar macrophages (AMs): cellular uptake studies
Fig. 3. Effect of nanoparticle’s size on the percent release of Rifampicin (different size of nanoparticles cross-linked with 10% CaCl2 as cross-linking agent).
the period of 628 h. Thus, lower loading levels can achieve similar release rates with higher quantity of total carrier administered. 3.8. Rifampicin release: effect of cross-linking cation Since different multivalent cations interact differently with the folate anions, it could be argued that the choice of cross-linking agent might also affects the release rates of Rifampicin. Release rates studies were carried out with 140 nm size nanoparticles crosslinked with different divalent and trivalent cross-linking agents, namely ZnCl2 , CaCl2 and FeCl3 . Fig. 4 shows that high release of Rifampicin was achieved in CaCl2 in comparison to ZnCl2 . In over 383 hrs, more than 38% of Rifampicin has been released from 140 nm sized nanoparticles cross-linked with CaCl2 , whereas only 21% release was observed in case of ZnCl2 cross-linked nanoparticles. Rifampicin release from nanoparticles cross-linked with FeCl3
A major step in tuberculosis pathogenesis is the ability of the M. tuberculosis to enter and replicate within the macrophages of its human host. During primary infection with the organism, aerosol-droplet nuclei containing small numbers of M. tuberculosis are deposited in the alveoli of the lung and are phagocytized by alveolar macrophages (AMs). M. tuberculosis enters the alveolar macrophages via receptor mediated phagocytosis. In contrast to most microbes; M. tuberculosis is resistant to the biocidal mechanisms of AMs and proliferates in the AMs using them as incubator. In the standard treatment of tuberculosis, antibiotics are given via oral route. These drugs are distributed to many tissues via circulatory system. Hence the antibiotics accumulated in alveolar macrophages are not sufficient to kill the underlying M. tuberculosis. Thus, there is a need to develop a drug delivery system to transport anti-tuberculosis drugs into alveolar macrophages selectively and more efficiently [21,22]. In order to deliver Rifampicin in alveolar macrophages, folate nanoparticles were exposed to cultured macrophages and their uptake was studied using fluorescence microscopy. Past studies have reported that zinc ions in bound state exhibit fluorescence [23]. 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, fluorescence spectroscopy was used to see the uptake of folate nanoparticles in alveolar macrophages with time. Zinc crosslinked folate nanoparticles (200 ± 20 nm) were exposed to cultured macrophages and fluorescence was detected at different time intervals over the period of 24 h. After exposure at different time intervals, cultured macrophages when observed under fluorescence microscope, bright fluorescence (green) was observed in cells. Fig. 5A–C shows both bright field and dark field images at 10 h, 16 h and 24 h respectively. These images show intracellular uptake of folate nanoparticles phagocytized by alveolar macrophages. Moreover, alveolar macrophages also represent folate receptors on their surface (particularly folate receptor ). Therefore, folate receptor mediated phagocytosis can be the probable mechanism for intracellular uptake. 3.10. In vitro cytotoxicity of folate nanoparticles on alveolar macrophages (AMs)
Fig. 4. Effect of cross-linking cation on Rifampicin release (120 ± 20 nm folate particle cross-linked 10% cross-linking agent).
For an effective nanocarrier, it is required to deliver the drug to AMs efficiently and the drug delivered should prevent the infection of M. tuberculosis without being toxic to macrophages. Therefore, to test the cytotoxic effect of our delivery system, cultured
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Fig. 5. Bright field and dark field fluorescence images of alveolar macrophages with cellular uptake of folate nanoparticles at (A) 10 h, (B) 16 h and (C) 24 h.
macrophages were exposed to folate nanoparticles loaded with different concentrations of Rifampicin. To compare the cytotoxicity, free Rifampicin and folate nanoparticles without Rifampicin was also exposed to cultured macrophages. After 24-h exposure, cytotoxicity was studied by MTT assay. Following treatment of RFP, folate nanoparticles without RFP, and RFP loaded folate nanoparticles, the cell viability of all experiment samples was over 80% (Fig. 6). The nanoparticles show a slight cell viability decrease in a concentration-dependent manner from 10 to 20 g/ml folate concentration (refer to supplementary data). This clearly suggests that folate nanoparticles show low cytotoxicity to alveolar macrophages at different levels of Rifampicin loading.
amount of drug released into the solution, we calculate the disintegration/stability of the particles in solution. The particles are extremely stable (show very little release) in acidic solutions. Storage at different temperature does not affect the release rates significantly. In buffer solutions with monovalent cations, we see
3.11. Stability of Rifampicin loaded folate particles The role of the cross-linking solution using multi-valent ions is significant. In the absence of cross-linking, we lose almost all the folate domains/particles in the HPMC-folate solution, when washed with de-ionized water. Once cross-linked, these particles are stable over 30 days. Cross-linked lyophilized particles do not change their sizes significantly when suspended in deionized water over 24 h and lose less than 9% of their mass over 30 days (refer to supplementary data). Further, these cross-linked particles are stable in acidic solution as well. For release studies, the particles are suspended in different buffer solutions, solutions of different pH, etc. By measuring the
Fig. 6. MTT assay showing cell viability of alveolar macrophages at different RFP loading levels with folate concentration of 10 g/ml. (positive control: 5% DMSO; negative control: growth media).
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115 53 21 15 17 12 8 139 98 75
26.032 9.539 4.913 1.658 4.444 4.180 1.966 24.960 8.889 4.515
76.36 83.97 79.49 80.49 71.91 67.64 31.81 73.22 78.25 73.06
that changing the cross-linking cation (size, valency) affects the rate in an expected manner (higher valency ions show slower release). Increasing the concentration of the cross-linking group also reduces the release rate and increases stability. Increasing the concentration of the monovalent ions, however, increases the release rates. We also see that when release studies are undertaken with constant shaking, there is a small but significant increase in release owing to reduced mass transfer barriers, improved dispersion or both. Based on these trends, we hypothesize that the cross-linking cations coordinate with the anions of the folate assemblies without significant change in self-assembly structure (as seen by XRD of the cross-linked particles). These coordination bonds are strong enough to keep the particles stable and ‘fix’ their size and structure under neutral environments. The particles have zeta potential of −20 to −40 mV which indicates moderate stability in the medium. The zeta potential increases with increase in the cross-linking concentration (refer to supplementary data). These particles, while stable in acidic pH or deionized water, where there are few available monovalent ions that can exchange with the cross-linking groups, begin to disintegrate when monovalent ions are available for exchange. This explains increase in release rates in buffer solutions with monovalent ions, as well as with increase concentration of monovalent ions or reduced concentration of cross-linking. 4. Conclusion The present study reports a method to design and control the size of Rifampicin encapsulated folate nanoparticles in the size range of 100–350 nm. It has been shown that drug molecules can be encapsulated in these nanoparticles with low drug loss of 20-30 %. Rifampicin molecules intercalate between or within the ordered folate stacks of the nanoparticles, which are cross-linked with a multivalent cation. Cross-linking cations play major role in keeping the drug encapsulated folate assembly intact. Due to exchange of ions present in release medium with the cross-linking cation, this assembly is disrupted. The rate of disruption of this assembly (measured by the rate of release of Rifampicin) is dependent on the size of the nanoparticles and its concentration. These nanoparticles can deliver the drug efficiently to macrophages without being toxic to macrophages. 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 of Rifampicin.
150 60 30 10 30 30 30 150 60 30 0.75 0.25 0.14 0.05 0.14 0.14 0.14 0.75 0.25 0.14
34.09 11.36 6.18 2.06 6.18 6.18 6.18 34.09 11.36 6.18
8.059 1.821 1.267 0.402 1.736 2.000 4.214 9.130 2.471 1.665 5.0 8.30 9.09 9.09 9.09 9.09 9.09 5.0 8.30 9.09 2.50 0.83 0.46 0.46 0.46 0.46 0.46 2.50 0.83 0.46 Calcium (10%) Calcium (10%) Calcium (10%) Calcium (10%) Calcium (5%) Calcium (3%) Calcium (1%) Zinc (10%) Zinc (10%) Zinc (10%) 20 20 20 20 20 20 20 20 20 20 ± ± ± ± ± ± ± ± ± ± 342 241 140 120 140 140 140 381 224 110
The supplementary data is available at the end of the manuscript. Table S1 summarizes the different nanoparticles mediated anti-tubercular drug delivery system previously studied. Fig. S1 represents structure of folic acid, Rifampicin and HPMC. Fig. S2 shows the wavelength scan of pure folic acid and Rifampicin as a reference. Fig. S3 shows the DLS data for size distribution of folate particles. Fig. S4 represents the SEM image of folate particles. Fig. S5 shows the effect of cross-linker concentration of Rifampicin release while Fig. S6 represents the effect of drug loading on Rifampicin release. The cytotoxicity of folate particles at 20 g/ml Rifampicin loading has been studied by MTT assay in Fig. S7. FDH = folate + drug + HPMC.
Drug encapsulated (mg) Weight of dry pellet (mg) Drug loss (mg) Amount of drug after cross-linking (mg) Amount of drug after adding HPMC (mg) RIF % Folate %
HPMC %
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Supplementary information
Cross-linking cation Nano-particle size (nm)
Drug amount during encapsulation Method of development Nanoparticle specifications
Table 2 Mass balance of Rifampicin (RIF) encapsulated, lost and encapsulation efficiency achieved in the method of nanoparticle formation.
Drug amount after encapsulation and loss
Encapsulation percentage (%)
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Acknowledgments The authors would like to thank Department of Science and Technology, Government of India (Grant no. RP2519) for financial support of this project. We also thank to Ms. Mohita Upadhyay and Kusuma School of Biological Sciences, IIT-Delhi for providing facilities to conduct cell line studies.
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