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A Versatile pH-responsive Chitosan-g-Polycaprolactone/ Maleic Anhydride-Isoniazid Polymeric Micelle to Improve the Bioavailability of Tuberculosis Multi-drugs Rajendran Amarnath Praphakar, Rajadas Sam Ebenezer, Sounderrajan Vignesh, Harshavardhan Shakila, and Mariappan Rajan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00003 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A Versatile pH-responsive Chitosan-g-Polycaprolactone/Maleic AnhydrideIsoniazid Polymeric Micelle to Improve the Bioavailability of Tuberculosis Multi-drugs Rajendran Amarnath Praphakar,a Rajadas Sam Ebenezer,b Sounderrajan Vignesh,b Harshavardhan Shakila,b and Mariappan Rajan,a* aBiomaterials

in Medicinal Chemistry Laboratory, Department of Natural Products

Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai-625021, India bDepartment

of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University,

Madurai-625021, India

*Corresponding Author E-mail: [email protected]; Tel: +91 9488014084, Fax: 0452–2459845


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Abstract The potential of polymeric micelles constructed by coalescing natural and synthetic polymers for tuberculosis (TB) treatment was evaluated in this work. We designed a polymeric micelle to improve the delivery of anti-TB drugs (rifampicin [RF] and isoniazid [INH]). The polymeric core was synthesized in the following order; initially chitosan (CS) was grafted with polycaprolactone (PCL) to form CS-g-PCL followed by amide bond formation with maleic anhydride-isoniazid (MA-INH); finally, CS-g-PCL was conjugated with the MA-INH moiety to form the CS-g-PCL/MA-INH polymeric core. Another anti-TB drug, RF, was loaded onto CS-g-PCL/MA-INH through dialysis. The changes in the nature of functional groups and crystallinity were investigated by Fourier-transform infrared spectroscopy and X-ray diffraction analysis respectively. The shape and size of CS-gPCL/MA-INH and RF-CS-g-PCL/MA-INH were analyzed by dynamic light scattering, Scanning Electron Microscopy, and Transmission Electron Microscopy. The cumulative drug release profiles were measured by UV-Visible spectrophotometry and HPLC analysis. The antimicrobial activity of the loaded micelles was evaluated by finding the

minimum

inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and bacterial cell rupture analyses. The non-toxic nature of the micelles was assessed by ex-vivo studies on U937, L929 cell lines and erythrocytes by performiong MTT assay, apoptosis assay and hemolysis assay. Ex-vivo cellular uptake and in-vivo internalization of the INH and RF containing micelles were tested on U937 cells and zebrafish using fluorescence microscopy analysis. All the observations indicate that the multi-TB drug-loaded polymeric micelle is an safe and effective system for delivery of anti-TB drugs without affecting the mycobactericidal activity. Keywords: chitosan, isoniazid, micelle, rifampicin, tuberculosis, zebrafish



Introduction Tuberculosis (TB) is a chronic lethal communicable disease caused by Mycobacterium tuberculosis.1 Chemotherapy with TB drugs, such as rifampicin (RF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (ETM), effectively clears TB.2,3 The role of RF is to inhibit DNA-dependent RNA polymerases4, whereas INH inhibits mycolicacid biosynthesis.5 However, these drugs are often associated with hepatotoxicity6, require long-term treatment, and distribution of these drugs to non-target organs leads to undesired effects.7,8 There exists a lacuna in efficient drug delivery to reduce the high mortality rate of TB. Currently, a new respirable way of delivery is being used by encapsulating TB drugs within a carrier to overcome the problems caused by conventional methods. Tremendous exploration on nanoparticulate drug delivery systems, have demonstrated advantages of controlled release, prolonged blood circulation, and high drug loads.9 The loading of multi-TB drugs in a single carrier may provide a more beneficial choice by enhancing the therapeutic action with controlled and localized delivery without causing adverse side effects. Some researchers have shown more interest on the delivery of antituberculosis drugs RF and INH using different delivery systems such as composite scaffold, liposomes and microspheres.10,11 Inspite of its slight therapeutic efficiency, these delivery system have failed in releasing the drugs in a controlled and sustained fashion. As an alternative polymeric drug delivery systems has gained recent attention in which, the drugs may be encapsulated or covalently bonded with polymeric scaffolds; thus, the drug loading can be precisely controlled. There are several synthetic and natural polymers used to deliver TB drugs12, such as carrier formation, chitosan13, κ-carrageenan14, sodium alginate15, poly(lactic-co-glycolic acid)16, polycaprolactone (PCL), and polylactic acid.17 Chitosan (CS) containing a repeating structural unit of β-(1,4)-2-amino-2-deoxy-β-D-glucose, has been broadly explored for TB drug delivery applications, since it efficiently escapes from the


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mucociliary clearance from the airways.18,19 PCL, a hydrophobic biodegradable and biocompatible polyester approved by the FDA, has some limitations in biomedical applications due to its slow degradation rate and lack of natural cell recognition sites.20 To overcome these shortcomings, various modifications have been carried out with other polymers, especially with CS, to generate itself into a new amphiphilic polymer with a desirable combination of properties.21,22 Ester linkages between CS and PCL results in a new hybrid polymer (hydrophilic and hydrophobic) that exhibits a synergistic pH response and an enhanced performance. Furthermore these polymeric micelles can be spontaneously formed and used to encapsulate hydrophobic drugs in their inner cores in a stable manner.23–25 Though, the non-covalent encapsulation of multi-TB drugs in a single micelle nanocarrier is a multi-step process, the synergism of physical encapsulation and chemical conjugation could drastically increase loading capacity in a polymeric nanocarrier.26-27 Due to its high drug loading efficiency and delivery without loss, the need for excess dose administration also fades away. These properties of micelles enables it to be considered as potential cargo carriers for effective drug delivery by the pharmaceutical industry. In the present work, amphiphilic CS-g-PCL was fabricated initially and then blended with MA-INH moieties to fabricate an INH-conjugated amphiphilic polymer, CS-gPCL/MA-INH. The CS-g-PCL/MA-INH polymer was used to encapsulate hydrophobic RF in the inner hydrophobic PCL core. This multi-TB drug-based polymeric drug delivery system was characterized by a series of physio-chemical characterizations to analyse various properties such as chemical modifications, crystallinity, morphological change and in vitro drug release profile. Antimicrobial activity was performed on Mycobacterium tuberculosis, Klebsiella pneumoniae and Streptococcus aureus. Cytotoxicity was tested on L929 and U937 cells using MTT assay, while cell uptake studies were conducted in U937 cells. Hemolytic



activity was analyzed against human erythrocytes. Finally, in-vivo localization studies were conducted using zebrafish animal model. 2. Materials and methods 2.1. Materials Chitosan (Mw = 60 kDa, degree of deacetylation (DD) = 99%), ε-caprolactone (ε-CL), maleic anhydride, sodium hydroxide (NaOH), potassium dihydrogen phosphate (KH2PO4), and dimethylaminopyridine (DMAP) were purchased from SRL, India. Rifampicin (RF) and isoniazid (INH) were procured from Himedia laboratories, India. All the chemicals and reagents were used as received from the supplier without further purification. 2.2. Chitosan-grafted-polycaprolactone preparation Chitosan-grafted polycaprolactone was constructed according to a published method.28 In brief; CS (1 gm, 6 mmol) was dissolved in 15 mL of MeSO3H in a glass container at 45°C in a N2 atmosphere. Later, ε-CL (52 mmol) was added to the container and allowed to react under reduced pressure (5 mmHg). Subsequently, the reactor was irradiated for a predetermined time period using microwaves. A dark brown crude mixture was obtained as the product and it was cooled. The crude mixture was then added into the solution consisting of KH2PO4 (17 mL, 0.2 M), NaOH (8 mL, 10 M), and crushed ice (25 g). The filtrate was then purified through dialysis (12000 kDa molecular weight cut-off [MWCO], Himedia, India) against double distilled water for 48 hours. Finally, the product was obtained by lyophilization. 2.3. Maleic anhydride-isoniazid preparation Maleic anhydride-isoniazid (MA-INH) was synthesized using a previously reported procedure.29 1 mm MA solution (0.1 g in 5 mL of acetonitrile) was stirred with DMAP (0.012


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g, 0.1 mm) and INH (0.14 g, 1 mm) on a magnetic stirrer at ambient temperature for 12 hours. Thereafter, 20 mL brine solution was mixed with the above mixture. Ethyl acetate (organic solvent) was introduced further to collect the product. After the completion, the organic layer was completely removed from the aqueous layer and dried in excess of MgSO4. At the end, filtration was done to remove MgSO4 and evaporation was done to remove ethyl acetate. The final product was obtained as a viscous colourless liquid and dried. 2.4. Chitosan-grafted-polycaprolactone/maleic anhydride-isoniazid preparation Chitosan-grafted-polycaprolactone/maleic

anhydride-isoniazid

was

obtained

according to a published method by Lu et al.,.30 In brief, maleic anhydride-isoniazid (0.5 g) was dissolved in water and added dropwise into an aqueous solution of chitosan-gpolycaprolactone (1 g) under magnetic stirring for 24 hours at 60°C. The resulting product was purified through dialysis (12000 kDa molecular weight cut-off [MWCO], Himedia, India) against double distilled water and then dried. 2.5. RF loaded CS-g-PCL/MA-INH preparation The synthesis of RF-CS-g-PCL/MA-INH micelles was carried out according to a previously reported method.31 Briefly, 10 mg of CS-g-PCL/MA-INH and 2 mg of RF was dissolved in 2 mL of dimethyl sulfoxide (DMSO). Subsequently, 1 mL of double distilled water was introduced into this solution and stirred for 30 min. The solution was then dropped into a dialysis bag (12000 kDa molecular weight cut-off [MWCO], Himedia, India) and dialyzed against double distilled water. The dialyzed double distilled water was replaced with fresh double distilled water 3 or 4 times. The RF-CS-g-PCL/MA-INH micelles were then removed from the dialysis bag and centrifuged (13,000 g) for 20 min. Finally, the RF-CS-gPCL/MA-INH micelles were lyophilized (Sub Zero Lyophilizer, Chennai, India) at –40°C.



2.6. FT-IR analysis The sample (10 mg) was placed on a holder and transferred to a FT-IR spectrometer (PerkinElmer Spectrum 100 series). The wavenumber range used for FT-IR measurements was 4000 to 500 cm–1, while the resolution ~ 4 cm–1 and the number of scans ~ 32. 2.7. Zeta potential and particle size analysis The zeta potential was measured using dispersed solutions of the samples with a Zetasizer Nano Series (Malvern, UK) instrument at 37°C; 10 readings were recorded per sample. The particle size was measured using ImageJ software. 2.8. SEM and TEM analysis For scanning electron microscope (SEM) analysis diluted dispersed solutions of CS-gPCL/MA-INH and RF-CS-g-PCL/MA-INH micelles were drop casted onto glass plates and then dried. Later, the dried CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH micelles were sputter coated with Au (thickness ~ 2 nm) and imaged on a SEM (Vega 3SB, TESCAN, Czech Republic) at 5 kV. For Transmission Elctron Microscope (TEM) (Model: JEM-2010, JEOL, Japan) analysis, diluted and dispersed solutions of CS-g-PCL/MA-INH and RF-CS-gPCL/MA-INH micelles were dropped onto copper grid (~200 mesh), dried and observed at at 200 kV. 2.9. Swelling analysis The swelling response of CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH micelles at different pH values (2, 4, 6, 8, and 10) was investigated. For swelling analysis, 100 mg of CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH micelles were placed in beakers containing solutions of different pH values. After 24 h, the weights of the dried CS-g-PCL/MA-INH and


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RF-CS-g-PCL/MA-INH micelles were calculated and the swelling ratio was measured by the following formula:32 Swelling ratio (%) =

swollen micelles weight ― dry micelles weight dry micelles weight

× 100 ……1

2.10. Lysozyme degradation analysis The CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH micelles were dispersed in PBS (pH~7.4) and mixed with a lysozyme enzyme (12 U/mL). Later, the dispersed medium was agitated gently (100 rpm) for 12 days. At the definite time period, the suspension was separated by centrifugation and the dried micelles were weighed. 2.11. In vitro drug release profile The in vitro drug release profile of RF-CS-g-PCL/MA-INH micelles were evaluated by immersing a 50 mg sample into 50 mL of three different PBS solutions (pH 5.5, 6.8, and 7.4). Later, the solution was agitated (50 rpm) at 37°C. Every 24 hours, 3 mL of the medium was removed and replaced with fresh PBS. The amounts of RF and INH in the release medium were analyzed by UV-Vis spectrophotometry (UV-1800, Shimadzu, Japan) and HPLC method as descriced by Franco et al.,.33 The cumulative drug release profiles of RF and INH were calculated as functions of time using the following formula:34 Cumulative drug release (%) =

Amount of drug released Total amount of drug

× 100 ……2

2.12. Determination of the MIC and MBC The MIC and MBC values against Klebsiella pneumoniae and Streptococcus aureus were investigated at 2 μg/mL concentration range. The resultant MIC and MBC of RF, INH, CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH micelles were compared.

2.13. Observation of bacterial cell membrane damage by SEM



The changes in bacterial shape caused by RF-CS-g-PCL/MA-INH micelles were visualized by SEM (VEGA3SB, TESCAN, Czech Republic). Klebsiella pneumoniae and Streptococcus aureus bacterial suspensions were mixed separately with RF-CS-g-PCL/MAINH micelles and incubated for 1 hour at 37°C. The RF-CS-g-PCL/MA-INH micelle-treated bacterial cells were then centrifuged at 13000 rpm for 10 min at 4°C. The samples were then fixed with Sorensen’s phosphate buffer and glutaraldehyde (8%, 1:1 v/v). Later, the samples were treated with osmium tetroxide (4%) mixed with H2O (1:1 v/v). After 12 hours, the samples were washed and dehydrated using water and ethanol respectively. The samples were then analyzed by SEM to envisage the changes in morphology of bacterial cells before and after RF-CS-g-PCL/MA-INH micellar treatment. 2.14. Imaging of bacterial cell surface by TEM The interaction of RF-CS-g-PCL/MA-INH micelles with the cell surface of Klebsiella pneumoniae was observed by TEM. Klebsiella pneumoniae bacterial suspensions were mixed separately with RF-CS-g-PCL/MA-INH micelles and incubated for 1 hour at 37°C. After exposure, the cells were fixed with paraformaldehyde (2%) and glutaraldehyde (2%) at 48°C for 2 hours. The samples were then washed and fixed by using 1% OsO4 at 48°C for 2 hours; the samples were then washed twice with 0.1 M PBS and observed on a TEM (JEM-2010, JEOL, Japan) at 80 kV. 2.15. Anti Mycobacterial Testing by LRP assay: Luciferase Reporter Phage (LRP) assay is a quick tool for drug susceptibility screening.35 The LRP assay was conducted using 2 μg/mL of RF, INH, CS, CS-g-PCL, CS-gPCL/MA-INH, and RF-CS-g-PCL/MA-INH micelles (control ~ DMSO). Further, the LR phage (AETRC21) was treated and the samples were incubated for 4 hours at 37°C. Equal volumes of the cell phage mixture and D-Luciferin (0.3 mM) in sodium citrate buffer (0.05


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M, pH~4.5) were mixed together and the Relative Light Units (RLU) was immediately measured using a luminometer with the integration period of 10 seconds. 50% or more reduction in the RLU with respect to the control was considered as significant. 2.16. Cytotoxicity assay 2.16.1. Cell culture and maintenance U937 and L929 cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. The U937 cells were grown in a RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 100 U of penicillin, and 50 µg/mL of streptomycin. The cells were supplied with fresh complete medium after four days of incubation in a 5% CO2, 95% air, and 37°C environment. L929 cell lines were grown in Dulbecco's Modified Eagle's Medium supplemented with sodium pyruvate and other components as mentioned earlier and incubated under similar growth conditions. All the reagents and growth media were procured from Himedia, India. 2.16.2. MTT assay The effect of RF, INH, CS, CS-g-PCL, CS-g-PCL/MA-INH, and RF-CS-g-PCL/MAINH micelles on the cell viability and growth was tested on cell lines U937 and L929 using a EZcountTM MTT cell assay kit (Himedia, India), which uses 3-(4,5-dimethyl thiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) as its primary component.36 The cells were seeded at a density of 1x103 cells per well in 96-well plates and left overnight under similar growth conditions as mentioned previously. It was then treated at concentrations of 50, 100, 200, and 400 µg/mL of the tested compounds. After 24 hours of incubation, the cells were treated with 10% MTT solution and incubated at 37°C for 3 to 4 hours. Once blue crystals are formed, the reaction was stopped by adding the solubilization buffer. The spectrophotometric readings



were taken at wavelengths of 570nm and 650nm using a Multiskan EX instrument (Thermo Scientific, USA). The percentage of viable cells was calculated using the following formula after subtracting the absorbance value at 650nm from that at 570nm. These measurements were carried out in triplicate. (Absorbance of test ― Absorbance of blank)

Cell viability (%) = (Absorbance of control ― Absorbance of blank) × 100 ……3 2.17. Cell uptake assay To visualize the cellular uptake of RF-CS-g-PCL/MA-INH micelles, the grown U937 cells were seeded onto the sterile cover slips placed in 24-well tissue culture plates. It was then treated with Coumarin 6-labeled RF-CS-g-PCL/MA-INH micelles (1μM) and incubated for 2 hours under the conditions previously mentioned. The cellular uptake was assessed by observing the cells under a fluorescence microscope (OLYMPUS IX71 Inverted Fluorescence Microscope). 2.18. Cell apoptosis assay In cell apoptosis, the condensed state of chromatin was evaluating with Hoechst 33342 and propidium iodide (PI).37 RF-CS-g-PCL/MA-INH micelles treated U937 cells were stored for 48 hours; afterward, the cells were removed by collagenase (type I) and washed with cold PBS. The cell suspension was observed under fluorescence microscope for the appearance of apoptotic cellular morphology. 2.19. Hemolytic activity analysis The effect of RF, INH, CS, CS-g-PCL, CS-g-PCL/MA-INH, and RF-CS-g-PCL/MAINH on human blood was tested on RBCs collected from healthy human volunteers following a protocol described elsewhere.38 In brief; RBCs were collected from whole blood by centrifugation and washed twice using PBS to remove all the serum complement proteins. It


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was then diluted at a ratio of 1:10 using PBS. RBCs (200 µL) were mixed with 1000 µL of the test solutions so that the final concentrations were 50, 100, 200, and 400 µg/mL. Similar volumes of distilled water and PBS were used as the positive and negative controls, respectively. The cell suspensions were gently mixed and incubated at 37°C for 2 hours and centrifuged at 4,000 g for 10 min at 4°C. The supernatant was collected and its optical absorbance at 541 nm was quantified using a Multiskan EX instrument (Thermo Scientific, USA). The measurements were carried out thrice; the percentage of hemolysis was calculated using the following equation: (Absorbance of test ― Absorbance of negative control)

Hemolytic activity (%) = (Absorbance of positive control ― Absorbance of negative control) x 100 …4 2.20. Assessment of in vivo localization in zebrafish An in-vivo localization study was performed by using embryonic zebrafish (Danio rerio). The embryos were taken in a 96-well plate. Coumarin 6-labeled RF-CS-g-PCL/MAINH micelles were dispersed in water and added to the embryos. Finally, the embryos were observed under an inverted microscope (Olympus IX70, Olympus America, Melville, NY) to analyse the localization of RF-CS-g-PCL/MA-INH micelles. 2.21. Statistical Analysis All the numerical data were expressed as mean±standard deviation from individual values. Statistical analysis was performed using GraphPad Prism software version for Windows (GraphPad Software Inc., La Jolla, CA, USA). Statistical analysis with a value of *p < 0.05 were considered significant.

3. Results and discussion



3.1. Synthesis of RF-loaded CS-g-PCL/MA-INH We plan to deliver multi-TB therapeutic agents to TB-affected macrophages in a controlled manner with reduced side effects. Hence, we designed a natural and synthetic polymer-based drug delivery vehicle to deliver first-line anti-TB drugs RF and INH. CS, a well-known natural polymer was grafted with PCL, a synthetic polymer, and a TB drug INH, resulting in a CS-g-PCL/MA-INH core, as shown in Fig.1. The above described polymeric core was used to encapsulate another TB drug RF via dialysis. Owing to hydrophobic interactions, RF can easily interact with the PCL inner core. A graphical representation of the polymeric micelle preparation is presented in Fig.2.


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Figure 1. Preparation of (a) CS-g-PCL, (b) MA-INH, and (c) CS-g-PCL/MA-INH



Figure 2. Schematic of the preparation, interaction and action pathway of RF-CS-gPCL/MA-INH micelles. 3.2. FT-IR analysis RF exhibits characteristic peaks at 3380 (-OH), 2972 (-CH3), 1726 (C=O), 1250 (CO-C), 907, and 642 cm–1, as shown in Fig. S1.39 The FT-IR spectrum of INH in Fig.S1 includes characteristic peaks at 1664 and 1551 cm–1, which could be ascribed to amide I and amide II, respectively. In addition, peaks exist in the range of 1410–669 cm–1.40 The distinctive peaks at 3293 (-OH), 2871 (-CH3), 1661 (amide I), 1586 (amide II), 1424, 1370 and 1024 cm–1 (C-O-C) are ascribed to the characteristic functional groups of CS (Fig.3(a)).41 CS-g-PCL shows peaks at 2939 (-CH3), 1722 (C=O), and 1562 cm–1 (amide II) (Fig.3(b)).42 In the case of CS-g-PCL/MA-INH, absorption peaks appeared at 3209 (-OH), 2935 (-CH3), 1702 (C=O), 1640 (amide I), 1550 (amide II), 1407, and 1249 cm–1 (C-O-C) (Fig.3(c)). In the FT-IR spectrum of RF-CS-g-PCL/MA-INH (Fig. 3(d)), some new peaks appeared in addition to those of CS-g-PCL/MA-INH, demonstrating the existence of RF in the inner core of CS-gPCL/MA-INH.


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Figure 3. FT-IR spectra of (a) CS, (b) CS-g-PCL, (c) CS-g-PCL/MA-INH, and (d) RF-CS-gPCL/MA-INH micelles



3.3. Particle size and zeta potential The particle size of CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH micelles was measured to be 183.4 nm and 211.56 nm respectively (Fig.S4). To predict the physical storage stability of micelle systems, zeta potential is a critically important parameter.43 The higher zeta potential value may offer a repelling force between the micelles, demonstrating the enhanced stability of this colloidal micelle system.44 The zeta potential of CS-g-PCL/MAINH and RF-CS-g-PCL/MA-INH micelles was obtained as 11.2 mV and 26.3 mV respectively (Fig.S4). The change of surface charge from CS-g-PCL/MA-INH to RF-CS-gPCL/MA-INH micelles may influence the interaction between drug and micelle.45 3.4. SEM and TEM analysis The morphological appearance of micelles is an essential factor affecting their applications in the biomedical field. If the surface morphology of micelles is smooth, they can easily penetrate the cell membrane and induce the therapeutic application of encapsulated drugs. Using SEM, an aggregated spheroidal morphology was observed for CS-g-PCL/MAINH, as shown in Fig. 4 (a) and (b). In the case of RF-CS-g-PCL/MA-INH micelles, a distinct non-aggregated spherical form was observed (Fig. 4(c) and (d)). These results corroborate well with the zeta potential values. In the case of CS-g-PCL/MA-INH, the low zeta potential value of the polymer results in an aggregated form while the high zeta potential of RF-CS-g-PCL/MA-INH micelles implies the existence of highly stable spherical micelles with a non-aggregating character. Furthermore, the surface morphologies of CS-g-PCL/MA-INH and RF-CS-gPCL/MA-INH micelles were explored by TEM. Fig.4 (e) and (f) show representative low and high-magnification TEM micrographs of CS-g-PCL/MA-INH. An aggregated spherical morphology without dispersion can be visualized clearly in CS-g-PCL/MA-INH. The results


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recommended superior dispersion without aggregation and a spherical shape for RF-CS-gPCL/MA-INH micelles (Fig.4 (g) and (h)). Unlike CS-g-PCL/MA-INH, RF-CS-g-PCL/MAINH micelles show good dispersion without aggregation, which indicates that the micelles were highly stable; further, their surface morphological characteristics were improved when RF was captured in the core.

Figure 4. SEM micrographs of CS-g-PCL/MA-INH at a resolution of (a) 5 μm and (b) 2 μm and RF-CS-g-PCL/MA-INH micelles at (c) 5 μm and (d) 2 μm. TEM micrographs of CS-gPCL/MA-INH at a resolution of (e) 500 nm and (f) 200 nm and RF-CS-g-PCL/MA-INH micelles at (g) 500 nm and (h) 200 nm. 3.5. Swelling analysis To investigate the pH response of CS-g-PCL/MA-INH carriers with and without RF, swelling analysis was conducted in various pH buffer solutions. The swelling in pH 6 solution was greater than that in other pH media, as illustrated in Fig.S5. In this slightly acidic medium, the anomalous swelling character of CS-g-PCL/MA-INH polymer was observed. At pH 6, the osmotic pressure should increase inside the CS-g-PCL/MA-INH polymer due to the concentration of free H+ and encourage water uptake. In addition,



electrostatic repulsion between carboxyl groups should cause macromolecular chain relaxation and increase swelling. This high swelling ratio can be attributed to the electrostatic repulsive force originating from the negative charge of the ionized carboxyl groups, suggesting that these groups are mainly involved in the pH-sensitive swelling property. It can be determined that the ionizable and/or ionized groups are the key features that administrate the pH-sensitive mechanism.46 In the case of RF-CS-g-PCL/MA-INH, the swelling profile suffered a little due to the well-built structure of the micelles and the presence of hydrophobic RF within the carrier. The swelling profiles of CS-g-PCL/MA-INH and RF-CS-g-PCL/MAINH in different pH solutions suggest that the degradation of the carrier is highly pH dependent and avoids the unusual response to body fluids. This might be because a mild acidic pH can induce CS-g-PCL/MA-INH degradation and improve drug release. 3.6. Lysozyme degradation analysis The degradation of polymeric carriers in the alveolar macrophage environment is based on the cleavage of amide and ester bonds in the polymeric backbone. Incubation of CSg-PCL/MA-INH and RF-CS-g-PCL/MA-INH in lysozyme enzyme would result in the degradation of the CS-g-PCL/MA-INH carrier and a rapid loss of weight. To analyze the degradation of CS-g-PCL/MA-INH by the lysozyme enzyme, biodegradation analysis was conducted on CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH for 12 days. As shown in Fig.S6, the degradation of CS-g-PCL/MA-INH and RF-CS-g-PCL/MA-INH in lysozyme enzyme increased dramatically, indicating a time-dependent degradation behavior for CS-gPCL/MA-INH. The degradation of RF-CS-g-PCL/MA-INH micelles led to little alteration when compared to CS-g-PCL/MA-INH, which might infer that RF-CS-g-PCL/MA-INH will be easily degraded in the macrophage environment.


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3.7. In vitro drug release profile Since, drug release behavior is significantly important for drug delivery systems47, in vitro drug release studies were performed in three different pH ranges. A schematic representation of the micellar behavior in solutions of different pH values is shown in Fig.5a. Owing to the presence of ester bonds, the RF-CS-g-PCL/MA-INH micelles easily degraded in the acidic medium than in basic or neutral media. As depicted in the schematic, the micelles show a better drug release profile for both RF and INH in an acidic environment. Small amounts of INH and RF were released from the RF-CS-g-PCL/MA-INH micelles at pH 6.8 and 7.4, indicating the stability of micelles in a human physiological environment. The releasing profiles evaluation of INH and RF from the RF-CS-g-PCL/MA-INH micelles were determined by UV-Visible spectrophotometry and HPLC technique (Fig. 5, Fig. S14 & Table S1-S3). At day 12, HPLC analysis of the cumulative drug releases of INH and RF at pH 5.5 were observed as 85.19% and 75.78%, respectively (Fig. S-14). As in the case of UVVis spectrum analysis, the release profile of INH and RF at pH 5.5 were obtained 89.21% and 79.37%, respectively. At day 24, the cumulative drug release values of INH and RF at pH 5.5 are 86.73% and 79.32%, respectively (Fig.5b & c). This prompt release performance of RFCS-g-PCL/MA-INH micelles ensures a pH-dependent behavior and this can be utilized very much for macrophage environment.



Figure 5. (a) A schematic representation of the behavior of micelles at different pH values. The cumulative release profiles of (b) INH and (c) RF from RF-CS-g-PCL/MA-INH micelles 3.8. MIC and MBC The calculated MIC and MBC values of RF, INH, CS-g-PCL/MA-INH and RF-CS-gPCL/MA-INH micelles were given in table 1. From the MIC and MBC observation, the RFCS-g-PCL/MA-INH micelles exhibit strong bactericidal activity effect against K.pneumoniae and S.aureus. Due to the existence of multi TB drugs within the polymeric micelle, it shows better performance when compared with pure RF and INH. In addition to the existence of multi-TB drugs, CS also shows anti-bacterial activity and this can be done by the protonation of –NH2 on the C-2 position of CS. This positively charged polymer can bind with negatively charged bacterial cell surface and disrupt the functions of bacterial cells.48,49


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Table 1. MIC (μg/mL) and MBC (μg/mL) values Sample

K.pneumoniae

S.aureus

MIC

MBC

MIC

MBC

RF

>100

195±5

>100

195±5

INH

25.89±0.9

48±3.6

26.66±2.88

48±2

13±1

24±2

17.83±1.04

24±2.17

4.87±0.49

8±0.35

3.19±0.37

4±1

CS-g-PCL/MA-INH RF-CS-g-PCL/MA-INH micelles 3.9. Assessment of bacterial cell damage

The damage incurred on the Klebsiella pneumoniae and Streptococcus aureus bacterial cells due to the action of RF-CS-g-PCL/MA-INH micelles was evaluated by SEM. Control Streptococcus aureus and Klebsiella pneumoniae display a spherical and rod morphology, respectively (Fig.6(a) and (c)). After treatment with RF-CS-g-PCL/MA-INH micelles, the morphologies of Klebsiella pneumoniae and Streptococcus aureus changed in an inhomogeneous manner resulting in the rupture of bacterial cells within one hour of treatment (Fig.6(b) and (d)). This bacterial rupture was further evaluated by TEM analysis. Untreated Klebsiella pneumoniae is shown in Fig.6(e). After treatment, the RF-CS-gPCL/MA-INH micelles adhere on the surface of Klebsiella pneumoniae, as shown in Fig.6(f). The blue arrows indicate the adherence of RF-CS-g-PCL/MA-INH micelles on the bacterial surface.



Figure 6. SEM images of (a) Streptococcus aureus, (b) RF-CS-g-PCL/MA-INH micellestreated Streptococcus aureus, (c) Klebsiella pneumoniae, and (d) RF-CS-g-PCL/MA-INH micelle-treated Klebsiella pneumoniae.TEM images of (e) Klebsiella pneumoniae and (f) RFCS-g-PCL/MA-INH micelle-treated Klebsiella pneumoniae. The blue arrows indicate the interaction of RF-CS-g-PCL/MA-INH micelles with the surfaces of Klebsiella pneumoniae.


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3.10. Anti-TB activity To evaluate the antimycobacterial activity of CS, CS-g-PCL, CS-g-PCL/MA-INH, RF-CS-g-PCL/MA-INH, INH, and RF, an LRP assay was performed. The inhibition of M. tuberculosis H37Rv growth evidenced by the drop in RLU, proved that the RF-CS-gPCL/MA-INH micelles are effective for TB treatment. Consequently, CS-g-PCL/MA-INH also shows therapeutic action because of the ability of the micelles to release INH from their backbones by amide cleavage. The inhibition effect of RF-CS-g-PCL/MA-INH micelles (Fig.7) suggests that a combination of multi-TB drugs can inhibit M. tuberculosis. It should be noted that RF-CS-g-PCL/MA-INH micelles release RF and INH when RF-CS-gPCL/MA-INH micelles were internalized in M. tuberculosis.

Figure 7. Inhibition effect of CS, CS-g-PCL, CS-g-PCL/MA-INH, RF-CS-g-PCL/MA-INH, INH, and RF against M. tuberculosis H37Rv. The RF-CS-g-PCL/MA-INH has highly significant anti-tuberculosis activity similar to the free anti-TB drugs, INH and RF.



3.11. Cell viability assay The toxicity of RF-CS-g-PCL/MA-INH in comparison to that of RF, INH, CS, CS-gPCL, and CS-g-PCL/MA-INH was studied against L929 and U937 cells by MTT assay. In the case of L929 cells, none of the samples were cytotoxic; particularly, CS did not affect cell survival but it promoted cell growth even when the concentration increased to 400 µg/mL. The RF-CS-g-PCL/MA-INH micelles showed a positive impact on L929 cells, which indicated their capability of controlling the side effects of RF-CS-g-PCL/MA-INH micelles (Fig.8(a)). The RF-CS-g-PCL/MA-INH micelles exert a good inhibitory effect on U937 cells, as shown in Fig.8(b). The good cytotoxic profile of RF-CS-g-PCL/MA-INH micelles is due to the presence of combination of multiple TB drugs within the micelle which is released efficiently and inhibits the growth of U937 cells which is of histiocytic lymphoma origin. Another fact is that the released RF and INH from RF-CS-g-PCL/MA-INH micelles can easily penetrate the cells and inhibit cell activity by passive diffusion. Such effective survival of L929 and non-survival of U937 cells after treatment with RF-CS-g-PCL/MA-INH micelles proves the toxic nature of the micelles in the disease environment. 3.12. Time-dependent cell viability The time-dependent effect of RF-CS-g-PCL/MA-INH micelles on the viability of U937 was analyzed at three different time intervals (24, 48, and 72 h) (Fig.S15). Initially, the cell viability improved, after which a dramatic decrease was observed. After 72 h, the cell viability was 88%. This crucial decrease in cell viability indicates the strong cytotoxicity of RF-CS-g-PCL/MA-INH micelles against U937 cells.


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Figure 8. Cell viability of RF, INH, CS, CS-g-PCL, CS-g-PCL/MA-INH, RF-CS-gPCL/MA-INH, and control. (a) L929 and (b) U937 cells. The * denotes the statistically significant difference with P

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