In Situ Strategy to Encapsulate Antibiotics in a Bioinspired CaCO3

Subscriber access provided by Northern Illinois University

Article

An in situ Strategy to Encapsulate Antibiotics in a Bioinspired CaCO3 Structure Enabling pH-Sensitive Drug Release Apt for Therapeutic and Imaging Applications Gousia Begum, Thuniki Naveen Reddy, K. Pranay Kumar, Koude Dhevendar, Shashi Singh, Miriyala Amarnath, Sunil Misra, Vijaya K. Rangari, and Rohit Kumar Rana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07177 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

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

ACS Applied Materials & Interfaces

An in situ Strategy to Encapsulate Antibiotics in a Bio-inspired CaCO3 Structure Enabling pHSensitive Drug Release Apt for Therapeutic and Imaging Applications Gousia Begum,a Thuniki Naveen Reddy, a K. Pranay Kumar,c Koude Dhevendar, c Shashi Singh,b Miriyala Amarnath,b Sunil Misra,c Vijaya K. Rangari,d and Rohit Kumar Ranaa,* a

Nanomaterials Laboratory, Inorganic and Physical Chemistry Division, Indian Institute of Chemical Technology, Hyderabad-500 007, India. b

c

Centre for Cellular and Molecular Biology, Hyderabad- 500 007, India.

Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad500 007, India.

d

Department of Materials Science and Engineering, Tuskegee University, Tuskegee, AL 36088, USA

KEYWORDS: bio-inspired, antibacterial, antibiotic, anticancer, fluorescence, calcium carbonate

ACS Paragon Plus Environment

1


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

Page 2 of 25

ABSTRACT: Herein we demonstrate a bio-inspired method involving macromolecular assembly of anionic polypeptide with cationic peptide-oligomer that allows for in-situ encapsulation of antibiotics like tetracycline in CaCO3 microstructure. In a single step one-pot process the encapsulation of the drug occurs under desirable environmentally benign conditions resulting in drug loaded CaCO3 microspheres. Whilst this tetracycline-loaded sample exhibits pH dependent in-vitro drug-release profile and excellent antibacterial activity, the encapsulated drug or the dye-conjugated peptide emits fluorescence suitable for optical imaging and detection, thereby making it a multitasking material. The efficacy of tetracycline loaded calcium carbonate microspheres as pH dependent drug delivery vehicles is further substantiated by performing cell viability experiments using normal and cancer cell lines (in vitro). Interestingly, the pHdependent drug release enables selective cytotoxicity towards cancer cell lines as compared to the normal cells, thus has the potential for further development of therapeutic applications.

INTRODUCTION Substantial use of antibiotics can cause several side effects after their absorption into the blood stream; one of them is bacterial resistance. Therefore, control of drug dosing is imperative for maximizing therapeutic effect while minimizing side effects. In this regard, stimulus-responsive materials are attractive in drug delivery because of their ability to release the entrapped molecules in response to slight changes in environment, such as light,1-3 pH,4 temperature,5-6 etc. In this study, we report a methodology for encapsulation of such therapeutics in a pH dependent soluble CaCO3 capsules as a novel means for controlled transportation. CaCO3 when compared to other inorganic materials is ideal as drug delivery system for many therapeutics because of its pH tunable solubility, biocompatibility and biodegradability. As a biomineral CaCO3 is


2

Page 3 of 25

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


bioresorbable and insoluble at physiological pH but soluble under acidic conditions. Since the pH around many of the tumors and endolysosomes is acidic,7 CaCO3 can be used as a pHdependent vehicle to deliver therapeutics through the blood stream to the target tissue.8-9 Zhao et al. demonstrated fabrication of an amorphous calcium carbonate/doxorubicin@ silica (ACC– DOX@silica) nanoreactor, which releases DOX in the weakly acidic microenvironment of cancer cells resulting in efficient cell death.10 Moreover the CaCO3, which is being a widely used excipient in oral drug formulations, has been found to stabilize amorphous state of active pharmaceutical ingredients like celecoxib.11 As a result of pore-induced amorphization of the drug in the vaterite particles, a five to six fold enhancement in solubility of the drug was observed. Recently, it has been shown that porous calcium carbonate carriers in the form of polycrystalline vaterite are useful in photodynamic therapy (PDT), wherein a fast degradation of the carriers below pH 7 has been proposed to be critical for cancer treatment.12 Hierarchical calcium carbonate scaffolds have also been evaluated for osteoregenerative applications demonstrating their potential for bone regeneration.13 Herein we demonstrate a facile one-pot encapsulation method to produce CaCO3 microspheres with core-shell morphology under very mild conditions in an aqueous medium conducive for insitu encapsulation of the drug tetracycline (Scheme 1). Tetracycline is one of the most potent broad spectrum therapeutic molecules, which is used extensively to treat bacterial infections associated with bone diseases.14 Local administration of tetracycline is recognized to increase bone regeneration in periodontal defects due to its anti-collagenolytic effect. It also promotes the growth of alveolar bone in periodontal therapy.15 Various matrices for encapsulation and controlled release of tetracycline includes poly (DL-lactide-co-glycolide) microspheres, PLGA films and chitosan microspheres.16-18 Gedanken et al., reported a sonochemical method to


3


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

Page 4 of 25

produce tetracycline loaded BSA microspheres.19 While poly(methyl methacrylate) beads are widely used for treatments, its removal after exhaustion of the antibiotic activity has been a major drawback.20 In case of silica based SBA-15 materials it has been shown that surface modification with Fe3+ ions is required for improved adsorption of tetracycline.21 Similarly presence of phosphonium cation functionalities in hydrothermally prepared mesoporous phosphosilicates are found to facilitate tetracycline adsorption.19 Moreover, most of these methods are either complicated or have disadvantages that the obtained materials lack structural integrity and hence limit their practical applications. Therefore, it is highly desirable to develop facile and mild method for encapsulation of therapeutic molecules like tetracycline, which overcomes the major drawbacks associated with traditional post synthesis encapsulation strategies. Our methodology relies on a self-assembly property of polypeptides in presence of suitable counter anions to mineralize CaCO3 structures, 23 while it simultaneously facilitates the entrapment of tetracycline in situ (Scheme 1). Typically, poly(L-asp) was found to form spherical aggregates in presence of penta(L-lys) as the counter cation. Addition of CaCl2 and (NH4)2CO3 to a suspension of these aggregates results in formation of CaCO3 microspheres at room temperature. Our approach is to use the assembled structures formed from interaction of anionic polypeptides with cationic peptide oligomers as counter cations, to effectively act as an entrapping medium for encapsulation of the tetracycline drug. To load the drug, the tetracycline was first added to the suspension of poly(L-asp) - penta(L-lys) aggregates. These peptide coacervates are thought to interact with tetracycline via ionic/hydrophobic interactions to facilitate its penetration and subsequent addition of CaCl2 and (NH4)2CO3 can wrap them up to form the drug entrapped CaCO3 microspheres (see experimental).


4

Page 5 of 25

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


Scheme 1. Schematic illustration of the in situ encapsulation process during the mineralization and assembly of CaCO3. The bottom image is a schematic representation of possible interactions of various components present in the structure. EXPERIMENTAL SECTION Materials. Penta(L-lysine hydrobromide) (penta(L-lys), 1-5 kDa), Poly(L-aspartic acid sodium salt) (poly(L-asp), 15-50 kDa and 5-15 kDa) and Tetracycline were procured from SigmaAldrich and were used as received. Calcium chloride (CaCl2) and ammonium carbonate (NH4)2CO3 were obtained from Qualigen Fine Chemicals, India. All the solutions were prepared using de-ionized water (18.2 MΩ, Millipore water purification system). Synthesis of Tetracycline containing CaCO3 microstructures. The synthesis of CaCO3 microstructures as described above was modified to have in situ entrapment of tetracycline. In a


5


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

Page 6 of 25

typical synthesis to 200 µL of poly(L-asp) (2 mg/mL), 200 µL of penta(L-lys) (2 mg/mL) was added which resulted in a turbid solution. To this salt polymer aggregate 250 µL of tetracycline solution in 0.01M HCl was added followed by addition of 250 µL of CaCl2 and then 250 µL of (NH4)2CO3 in a molar ratio of 0.23 poly(L-asp): 0.15 penta(L-lys): 0.022 - 0.11 Tetracycline: 1 Ca2+: 1 CO32-. This mixture was then aged for 30 min at room temperature. The precipitate then obtained was centrifuged and washed 5 – 6 times with de-ionized water until there was no drug found in the supernatant and dried at room temperature. From UV-vis spectroscopy the tetracycline loading efficiency in the CaCO3 microspheres was determined. It was found that the loading efficiency can be increased from 14.94 to 62.27 wt. % by increasing the initial concentration of tetracycline added during the synthesis. Also the dispersibility of the sample prepared at the molar concentration of 0.23 poly(L-asp): 0.15 penta(L-lys): 0.022 Tetracycline: 1 Ca2+: 1 CO32- (Figure 1a) was better than prepared with 0.23 poly(L-asp): 0.15 penta(L-lys): 0.11 Tetracycline: 1 Ca2+: 1 CO32- (Figure S1, Supporting Information). The bare CaCO3 microspheres were prepared by following the above procedure but without using tetracycline. The in vitro time dependent drug release studies were carried in PBS buffer (pH = 7.4 and 6.0) at 37 0C. At different time intervals a known amount of aliquot was taken out from the medium, which was then replaced by an equal amount of fresh PBS buffer. The percentage release of drug was calculated by finding out the concentration of drug in released medium at particular time intervals by extrapolating its absorbance in the standard curve. Antimicrobial Activity. Antimicrobial activity of tetracycline loaded CaCO3 spheres and bare tetracycline was tested against the Gram-negative bacterial strains of E. coli (MTCC 443), K. pneumoniae (MTCC 618) and P. aeroginosa (MTCC 1688) and also with number of Grampositive bacterial strains of B. subtilis (MTCC 441), S. aureus (MTCC 96), and S. epidermidis


6

Page 7 of 25

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


(MTCC 435) obtained from Institute of Microbial Technology, Chandigarh. Cultures of test organism were maintained on Nutrient agar slants and were sub-cultured in Petri dishes prior to testing. The media used was Nutrient agar and broth procured from Himedia Laboratories, Mumbai. Determination of Minimum Inhibitory Concentration (MIC) for Antimicrobial Activity Studies. The Minimum Inhibitory concentration (MIC) was estimated by broth dilution method (NCCLS Methods, 2000).24For determining the MIC of tetracycline loaded CaCO3 microstructures required for the inhibition of bacterial growth, broth dilution method was used. Nutrient broth was supplemented with different concentration of tetracycline loaded CaCO3 microstructures (5–100 µg/ml) and inoculated with bacterial suspension to obtain 106 colony forming units (CFU)/ml. The MIC was determined after 24 h of incubation at 37 0C by observing the visible turbidity and measuring the optical density of these culture broths at 600 nm. Antimicrobial Activity Studies by Zone of Inhibition Method. in vitro antibacterial activity of the tetracycline loaded CaCO3 microstructures was studied against various bacterial strains by agar well diffusion method.25 Bacterial culture was prepared by growing a single colony overnight in nutrient broth and adjusting the turbidity to 0.5 McFarland standard. Mueller– Hinton agar (MHA) plates were inoculated with this bacterial suspension and various volumes of tetracycline loaded CaCO3 microstructures corresponding to different amounts (25 and 50 µg) were added to a center well with a diameter of 8 mm. Control plates were maintained with sterile saline loaded wells. These plates were incubated at 37 0C for 24 h and the zone of inhibition (ZOI) was measured by subtracting the well diameter from the total inhibition zone diameter. Monitoring Drug Release and Antimicrobial Activity by Confocal Microscopy. Bacterial culture was incubated for various time intervals with tetracycline loaded CaCO3 microstructures


7


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

Page 8 of 25

at the concentration of 25 µg/ml in the nutrient broth. At various time intervals bacterial suspension was placed on a slide with a cover slip and imaged using Leica Confocal System SP5. With an excitation using 405 nm laser line, the emissions were collected with 450 BP. in vitro MTT test. The MTT assay was performed to determine the cell viability in response to CaCO3 coated TC and pure TC treatment following the earlier published standard protocol.26 In this study for comparison of toxicity, one normal cell line CHO and three different cancer cell lines (A549, MDA-MB-231 and Hela) were used. These cell lines were obtained from ATCC (Manassas, VA) and maintained in DMEM medium supplemented with 10% (FBS) Fetal Bovine Serum. Characterizations. Field Emission Scanning Electron Microscopic (FE-SEM) analyses of the prepared materials were performed by using JEOL-7610F instrument. Powder XRD (X-Ray diffraction) patterns were recorded on a PAN analytical (empyrean, UK) X-Ray Diffractometer using CuKα (λ=1.5406 Å) radiation at 45 kV and 30 mA with a standard monochromator, equipped with a Ni filter to avoid CuKβ interference. The confocal imaging was carried out under Leica Confocal System TCS SP5 with 63 X 1.4 NA objective and with a gain value of 850-950 V. Optical sectioning was done at 0.5 mm intervals with 1 AU pinhole and three of the central sections were projected using the LAS AF software. For tetracycline loaded CaCO3 sample the 405 nm laser line was used for excitation and the emissions were collected with 450 BP. Dynamic Light Scattering (DLS) measurements to determine the particle size and Zeta potential to determine the charge on the particles in the colloidal state were done using a Malvern Zetasizer (Nano-ZS) instrument equipped with the detector at 173° angle and with a 668 nm laser source. FT-IR spectra of the solid samples were recorded on a Bruker alpha spectrometer


8

Page 9 of 25

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


equipped with a DTGS KBr detector over range of 4000 cm-1- 400 cm-1. The UV-vis spectroscopic analysis was carried on Varian Cary 5000 spectrophotometer. RESULTS AND DISCUSSION As illustrated in Scheme 1, the supramolecular assembly of the anionic polypeptide with cationic peptide oligomer as counter cations was utilized to encapsulate the tetracycline drug. Then the polypeptide mediated mineralization of CaCO3 from CaCl2 and (NH4)2CO3 resulted in drug entrapped microsphere structures. The morphological characterizations of thus fabricated structures were carried out by electron microscopy. As shown in Figure 1a & b, the FE-SEM analysis illustrated the spherical morphology of the synthesized material and the spheres were of 0.5-1 µM diameters The DLS study also confirmed the size distribution of the particle in the same range (Figure 1c). The Zeta potential (ζ) value of the particles in the water was found to be -11.2 mV (Figure S2, Supporting Information). The Energy Dispersive X-ray spectroscopic (EDX) elemental analysis revealed the presence of nitrogen in addition to calcium, carbon and oxygen indicating the presence of either or both polypeptides and tetracycline (Figure 1d). Further to locate tetracycline in the microsphere, its fluorescence was analyzed under a confocal fluorescence microscope. As seen in Figure 1e, clearly the fluorescence originating from the microspheres confirmed the encapsulation of tetracycline in the structure. Moreover the fluorescence as seen in the inset image of an individual sphere under focus (Figure 1e) and in Figure S1(Supporting Information), indicated that the entrapped tetracycline molecules were in some cases confined to the shell wall of the microspheres forming a ring like structure. For other cases, a distribution of the tetracycline throughout the sphere was observed. As earlier shown by us, the mineralization of CaCO3 results in microspheres having either a distinct shell wall, or a


9


solid sphere.23 Therefore, the particles with a shell containing tetracycline show a ring like structure, whereas the solid particles look total green in the confocal image.

(f) 0.35 0.28

TC Tet CA

1413 1480 1605 1642

0.21 0.14 0.07

866 1076

(g) 66000 Intensity

(e)

Absorbance

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

Page 10 of 25

44000

22000

0.00 500 1000 1500 2000 -1 Wavenumber (cm )

450

540 630 720 Wavelength (nm)

Figure 1. Microscopic and spectroscopic characterization of tetracycline loaded CaCO3: (a) & (b) FE-SEM images at different magnifications; (c) Size distribution of the particles in a colloidal form obtained by DLS measurement; (d) EDX elemental mapping on the selected area indicated in

the inset; (e) Confocal microscopic image (inset: higher magnification image of the marked area in red depicts the presence of tetracycline in the shell wall of a sphere under focus); (c) FT-IR spectra (Tet, TC and CA represent free tetracycline, tetracycline loaded CaCO3 and bare CaCO3, respectively); (d) Fluorescence spectra (at an excitation wavelength of 360 nm).


10

Page 11 of 25

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


The X-ray diffraction pattern of tetracycline loaded CaCO3 microspheres displayed three distinct diffraction peaks (2θ) at 22.9° (110), 28.6° (111) and 31.4° (112), indexed to vaterite phase of CaCO3 (JCPDS Card No. 33-0268) (Figure S3, Supporting Information). The FT-IR spectrum of the tetracycline loaded sample (Figure 1f) exhibited characteristic bands for the vaterite phase of CaCO3 at 866 cm-1. The band at 1076 cm-1 due to the symmetric stretch in non-centrosymmetric structure (ν1) indicates the presence of an amorphous phase of CaCO3 as well. The split band centered at 1450 cm-1 attributed to asymmetric stretching vibration of the carbonate ion (ν3), which results from lack of symmetry around the carbonate ion is another characteristic feature of amorphous phase of CaCO3.27-28 In addition to the characteristic bands for amorphous and vaterite phases of CaCO3, the FT-IR band at 1642 cm-1 assignable to amide linkages is indicative of the presence of both polypeptides and tetracycline in the matrix. These polypeptides, which are used for the mineralization and encapsulation process, get entrapped into the CaCO3 structure. Furthermore, the FT-IR spectrum displays a strong band at 1605 cm-1 assignable to the C-C stretching vibration from aromatic ring of tetracycline. Characterization by fluorescence studies revealed a broad fluorescence band around 500-600 nm when excited at 360 nm, characteristics of tetracycline, (Figure 1g).29 To understand the evolution of the spherical morphology we performed few control experiments. When the CaCO3 mineralization was carried out in absence of poly(L-asp) or penta(L-lys) keeping other reagents and parameters same, in either of the cases only randomly oriented particles were formed without any morphological assembly (Figure S4, Supporting Information). This indicates that the cumulative effect of these reactants in the formation process. Poly(L-asp) has been shown to undergo supramolecular aggregation in presence of penta(L-lys), which further acts as template for the formation of spherical particles.23 Although poly(L-asp) by itself


11


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

Page 12 of 25

is capable of mineralizing CaCO3 spheres by virtue of its supramolecular assembly with Ca2+ ions,30 but the presence of tetracycline in our control reaction plausibly obstructed the assembly process and as a result of which there was no such structural transformation took place. This was further supported by the fact that in contrast to the formation of rhombohedra shapes for CaCO3 mineralized in absence of any additive,23 when we used tetracycline as the only additive it resulted in particulate structures without any typical morphology (Figure S4(c), Supporting Information). Therefore, we believe that the tetracycline, in a similarity to that is reported for certain calcite binding peptides and macromolecules, 31 may cause surface specific interactions that influence the morphological growth of the CaCO3 crystals. Nevertheless, in case of the microspheres, it is the penta(L-lys) by virtue of its ionic interaction with poly(L-asp) provides structural stability to these spheres. This assembly process not only keeps the assembled nanoparticles together, but also allows in-situ encapsulation of tetracycline in the spheres. Thus prepared tetracycline entrapped CaCO3 spheres were examined for antimicrobial activity against both Gram-positive and Gram-negative microbial strains. As shown in Figure S5 (Supporting Information) the minimum inhibitory concentration (MIC) values for various strains were estimated to be in the order S. aureus ~ S. epidermidis ~ E. coli < P. aeroginosa ~ K. pneumoniae < B. subtilis. From the disk diffusion assay the zone of inhibition (in mm) at a concentration of 25 and 50 µg of the tetracycline in tetracycline loaded CaCO3 for various strains was calculated (Figure 2). Both MIC and zone of inhibition studies demonstrate that the antibacterial efficacy of the tetracycline is similar to that of bare tetracycline and hence its activity is not affected by loading into the CaCO3 matrix. It has been reported that the bioavailability of tetracycline can be affected when it complexes with Ca2+ ions. 32-,33 In our case, the interference by Ca2+ ions with tetracycline could be excluded probably for the two reasons.


12

Page 13 of 25

(a) 5

MIC (µg/ mL)

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


TC Tet

4 3 2 1 0 1

2

3

4

Species

5

6

(b)

Figure 2. Antimicrobial studies: (a) Minimum inhibitory concentrations (MIC); and (b) Visual images of zones of inhibition studies using tetracycline loaded CaCO3 microspheres (Tetracycline to Ca2+ molar ratio of 0.11 : 1.0) and control condition for various bacterial strains (1) B. subtilis, (2) S. aureus, (3) E.coli, (4) P. aeroginosa, (5) K. pneumoniae, and (6) S. epidermidis. Tet and TC represent free tetracycline and tetracycline loaded CaCO3 microspheres respectively and within the parenthesis is given the amount of sample in µg.


13


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

Page 14 of 25

Firstly, the interaction of Ca2+ with the polypeptides and the other reason is the pH (7.2-7.4) of the experimental medium, where the tetracycline predominantly exists as zwitterionic species rather than its complex with the metal ion (Ca2+).23 The zwitterionic form readily passes through the cell membrane, while the complex with metal ion reduces its uptake. Therefore, the entrapped tetracycline could be released from CaCO3 to effectively interact with the bacteria. Confocal imaging of the tetracycline loaded CaCO3 microspheres inoculated with bacterial suspension was performed to further monitor the antibacterial activity (Figure 3a & Figure S6, Supporting Information). We observed a time dependent uptake of the drug by the bacterial strands. The uptake of the tetracycline by E.coli strands can be seen from the emanating fluorescence at 30 min of incubation, and the uptake increases with time. After 40 hours of incubation the presence of cell debris indicates the destruction of the bacteria. The drug release from the tetracycline loaded CaCO3 microspheres and its pH dependency was studied. The in vitro drug release in PBS buffer (pH 7.4 and 6.0) at 37 0C shows that there is an initial burst release of the drug followed by a slow release up to 360 h (Figure 3b & Figure S7, Supporting Information). As reported earlier, the burst release even at pH 7.4 could be explained by the water-induced dissolution/phase transition of the amorphous calcium carbonate in aqueous environments.10 (Note: The FT-IR spectrum of the tetracycline loaded CaCO3 sample (Figure 1c) exhibits presence of both vaterite and amorphous phase). When monitored under a confocal fluorescence microscope, the release of the drug could be further ascertained from the reduction in fluorescence from the microspheres with time as shown in Figure 3(c & d). After 360 h almost 94% of the drug is released at pH=6.0, whereas only 61% of the drug is released from the microspheres at pH=7.4. This behaviour in turn can be attributed to pH dependent


14

Page 15 of 25

solubility of CaCO3 wherein the acidic medium results in dissolution of the matrix thereby allowing most of the encapsulated drug to be released. (a)

(b) % Drug release

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


96

72

48

24 0

(c)

pH=7.4 pH=6.0

2

4

20 40 Time (in hours)

80

360

(d)

Figure 3. Controlled release of tetracycline from tetracycline loaded CaCO3 microspheres: (a) Confocal microscopic images of E. coli incubated with tetracycline loaded CaCO3 microspheres (0.022 Tetracycline: 1 Ca2+ ) for (i) 30 min (ii) 5 h and (iii) 40 h. (b) Drug release profile of tetracycline loaded CaCO3 microspheres in PBS buffer at pH=7.4 and 6.0. Confocal microscopic


15


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

Page 16 of 25

images of tetracycline loaded CaCO3 microspheres (c) before and (d) after (360 hrs) release of the tetracycline at pH=7.4. (Insets show magnified images of the marked area in red). When the pH of the medium was further decreased to 4.5, 100% of drug was released within 24 hours (Figure S7 (c & d), Supporting Information). The FE-SEM analysis of the samples after drug release illustrated morphological deformations and the extent of deformation was more for the sample incubated at lower pH due to the increased solubility of CaCO3 in the acidic medium (Figure S8, Supporting Information). Moreover, this type of release profile is highly desirable for treatment of bacterial infections associated with bones where a immediate high dose is required followed by a slow release. The fluorescence either from the entrapped tetracycline 34 or the dye tagged peptides can as well be utilized in molecular imaging and cell biology and thus making the mineralized materials suitable for multitasking applications. To further substantiate pH-dependent drug release, the in vitro cytotoxicity of bare tetracycline and tetracycline loaded calcium carbonate microspheres was measured in a normal (CHO) and three cancer cell lines (A549, MDA-MB-231, HeLa). The extracellular microenvironment around the cancer cells is acidic due to the formation of lactate from glucose known as Warburg effect. 35 The difference in the pH of extracellular microenvironments of normal and cancer cells would therefore gives us an opportunity to investigate efficacy of tetracycline loaded calcium carbonate microspheres as pH dependent drug delivery vehicles. The Chinese hamster ovary (CHO) cells, adenocarcinomic human alveolar basal epithelial cells (A549), human breast adenocarcinoma cells (MDA-MB-231) and cervical cancer cells (HeLa) were used for the cytotoxicity studies. The IC50 (half maximal inhibitory concentration) values for various cell lines indicate relatively greater cell inhibition in the case of all the three cancer cell lines in comparison to normal cells (Figure 4). Tetracyclines are known to inhibit the activity of matrix


16

Page 17 of 25

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


metalloproteinases (collagenases and gelatinases), reduce the mitochondrial biogenesis and also cause depolarization of the mitochondrial membrane and thus inducing apoptotic pathway to programmed cell death. 36-39 A higher percentage of cell inhibition in case of all the cancer cells compared to normal cells tested in our study is therefore due to the more availability of tetracycline around cancer cells, which in turn is the result of acid induced dissolution of calcium carbonate microspheres. Therefore, the CaCO3 spheres not only allow encapsulation of drug molecules, but also enables pH-dependent delivery particularly suitable for cancer therapeutic applications.

Figure 4: The half maximal inhibitory concentrations (IC50) of bare tetracycline (Tet) and tetracycline loaded CaCO3 microspheres (TC) for CHO, A549, MDA-MB-231 and HeLa cells. CONCLUSIONS We demonstrated that a macromolecular assembly of anionic polypeptide with cationic peptide oligomer creates confined structure suitable for encapsulation of guest molecules such as drugs for biomedical applications. Such an easily adaptable method overcomes limitations of traditional encapsulation strategies, as it requires no post-treatments and multi-step processes but


17


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

Page 18 of 25

allows for in situ encapsulation of the drug molecule at ambient conditions without affecting the mineralization process. The tetracycline-loaded sample exhibited antibacterial activity together with pH dependent in-vitro drug-release profile. The potential of tetracycline loaded calcium carbonate microspheres as pH dependent drug delivery vehicles was further verified by measuring the cytotoxicity of drug-loaded particles towards normal and cancer cell lines. Interestingly, the IC50 (half maximal inhibitory concentration) values indicated relatively greater cell inhibition in the case of cancer cell lines in comparison to normal cells. The antibacterial activity and pH-dependent drug release can further lead to designing drug delivery vehicles for therapeutic applications. In addition the fluorescence either from the encapsulated tetracycline or the dye tagged peptides can as well be used in imaging applications, particularly to detect and locate malignant tissue in bone lesions. ASSOCIATED CONTENT Supporting Information. Confocal imaging for monitoring the drug and bacteria interactions, SEM images of CaCO3 particles, minimum inhibitory concentrations (MIC) of tetracycline loaded CaCO3 microspheres and bare tetracycline for various bacterial strains, UV-vis spectra for tetracycline released from CaCO3 microspheres. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Fax: (+91) 4027-160-921, E-mail: [email protected] ACKNOWLEDGMENT


18

Page 19 of 25

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


The financial support from CSIR, India (NanoSHE, BSC-0112) and DST, India (SB/FT/CS111/2014) are greatly acknowledged. The authors are grateful to Dr. S.V. Manorama and Y. Swarnalatha for helping in materials characterization. REFERENCES (1) Murata, K.; Aoki, M.; Suzuki, T. ; Harada, T.; Kawabata, H.; Komori, T.; Ohseto, F.; Ueda, K.; Shinkai, S. Thermal and Light Control of the Sol-Gel Phase Transition in Cholesterol-Based Organic Gels. Novel Helical Aggregation Modes As Detected by Circular Dichroism and Electron Microscopic Observation. J. Am. Chem. Soc. 1994, 116, 6664 –6676. (2) Wang, Z.; Wang, P.; Tang, X. Synthesis of Light-Induced Expandable Photoresponsive Polymeric Nanoparticles for Triggered Release. ChemPlusChem 2013, 78, 1273–1281. (3) Ahmed, S. A. ; Sallenave, X.; Fages, F.; Mieden-Gundert, G.; Müller, W. M.; Müller, U.; Vögtle, F.; Pozzo, J. L. Multiaddressable Self-Assembling Organogelators Based on 2HChromene and N-Acyl-1,ω-amino Acid Units. Langmuir 2002, 18, 7096–7101. (4) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C.; Pitkeathly, M. ; Radford, S. E. Responsive Gels formed by the Spontaneous Self-assembly of Peptides into Polymeric Beta-sheet Tapes. Nature 1997, 386, 259–262. (5) Li, S. K.; D’Emanuele, A. On-off Transport through a Thermoresponsive Hydrogel Composite Membrane. J. Control. Release 2001, 75, 55–67. (6) Kong, G.; Anyarambhatla, G.; Petros, W. P. ; Braun, R. D.; Colvin, O. M. ; Needham, D.; Dewhirst, M. W. Efficacy of Liposomes and Hyperthermia in a Human Tumor Xenograft Model: Importance of Triggered Drug Release. Cancer Res. 2000, 60, 6950–6957.


19


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

Page 20 of 25

(7) Tycko, B.; Maxfield, F. R. Rapid Acidification of Endocytic Vesicles Containing α2Macroglobulin. Cell 1982, 28, 643–651. (8) Lauth, V.; Maas, M.; Rezwan, K. Coacervate-directed synthesis of CaCO3 microcarriers for pH-responsive delivery of biomolecules. J. Mater. Chem. B 2014, 2, 7725-7731. (9) Zhou, C.; Chen, T.; Wu, C.; Zhu, G.; Qiu, L.; Cui, C.; Hou, W.; Tan. W. Aptamer CaCO3 Nanostructures: A Facile, pH-Responsive, Specific Platform for Targeted Anticancer Theranostics. Chem. Asian J. 2015, 10, 166 – 171. (10) Zhao, Y.; Luo, Z.; Li, M.; Qu, Q.; Ma, X.; Yu, S.-H.; Zhao, Y. A Preloaded Amorphous Calcium Carbonate/Doxorubicin@Silica Nanoreactor for pH-Responsive Delivery of an Anticancer Drug. Angew. Chem. Int. Ed. 2015, 54, 919 –922. (11) Forsgren, J.; Andersson, M.; Nilsson, P.; Mihranyan, A. Mesoporous Calcium Carbonate as a Phase Stabilizer of Amorphous Celecoxib – An Approach to Increase the Bioavailability of Poorly Soluble Pharmaceutical Substances. Adv. Healthcare Mater. 2013, 2, 1469–1476. (12) Svenskaya, Y.; Parakhonskiy, B.; Haase, A.; Atkin, V.; Lukyanets, E.; Gorin, D.; Antolini, R. Anticancer Drug Delivery System Based on Calcium Carbonate Particles Loaded with a Photosensitizer. Biophys. Chem. 2013, 182, 11–15. (13) Yu, H. -D.; Zhang, Z. -Y.; Win, K. Y.; Chan, J.; Teoh, S. H.; Han, M.-Y. Bioinspired Fabrication of 3D Hierarchical Porous Nanomicrostructures of Calcium Carbonate for Bone Regeneration. Chem. Commun. 2010, 46, 6578 – 6580.


20

Page 21 of 25

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


(14) Griffin, M. O.; Fricovsky, E.; Ceballos, G.; Villarreal, F. Tetracyclines: A Pleitropic Family of Compounds with Promising Therapeutic Properties. Review of The Literature. Am J Physiol Cell Physiol. 2010, 299, C539– C548. (15) Park, Y. J.; Lee, Y. M.; Park, S. N.; Lee, J. Y.; Ku, Y.; Chung, C. P.; Lee, S. J. Enhanced Guided Bone Regeneration by Controlled Tetracycline Release from Poly(L-lactide) Barrier Membranes. J. Biomed. Mater. Res. 2000, 51, 391–397. (16) Bittner, B.; Mäder, K.; Kroll, C.; Borchert, H. -H.; Kissel, T. Tetracycline-HCl-loaded Poly(dl-lactide-co-glycolide) Microspheres Prepared by a Spray Drying Technique: Influence of γ-irradiation on Radical Formation and Polymer Degradation. J. Control. Release 1999, 59, 23– 32. (17) Webber, W. L.; Lago, F.; Thanos, C.; Mathiowitz, E. Characterization of Soluble, SaltLoaded, Degradable PLGA Films and Their Release of Tetracycline. J. Biomed. Mater. Res. 1998, 41, 18–29. (18) Hejazi, R.; Amiji , M. Stomach-specific Anti-H. Pylori Therapy. I: Preparation and Characterization of Tetracyline-loaded Chitosan Microspheres. Int. J. Pharm. 2002, 235, 87– 94. (19) Avivi, S.; Nitzan, Y.; Dror, R.; Gedanken, A. An Easy Sonochemical Route for the Encapsulation of Tetracycline in Bovine Serum Albumin Microspheres. J. Am. Chem. Soc., 2003, 125, 15712–15713. (20) Kanellakopoulou, K.; Bourboulis, E. J. G. Carrier Systems for the Local Delivery of Antibiotics in Bone Infections. Drugs, 2000, 59, 1223–1232.


21


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

Page 22 of 25

(21) Vu, B. K.; Shin, E. W.; Snisarenko, O.; Jeong, W. S.; Lee, H. S. Removal of the Antibiotic Tetracycline by Fe-impregnated SBA-15. Korean J. Chem. Eng 2010, 27, 116–120. (22) Das, S. K.; Bhunia, M. K.; Chakraborty, D.; Khuda-Bukhsh, A. R.; Bhaumik, A. Hollow Spherical Mesoporous Phosphosilicate Nanoparticles as A Delivery Vehicle for An Antibiotic Drug. Chem. Commun. 2012, 48, 2891–2893. (23) Begum, G.; Rana, R. K. Bio-inspired Motifs via Tandem Assembly of Polypeptides for Mineralization of Stable CaCO3 Structures. Chem. Commun. 2012,48, 8216–8218. (24) Linday, M. E. Practical Introduction to Microbiology. 177 (London: E & F.N Spon Ltd. 1962). (25) NCCLS Methods for Dilution Antimicrobial susceptibility tests for bacteria, that grows aerobically. Eighth ed. NCCLS, Villanova, Pa., 2009, Approved Standard M 07-A 8. (26) Nethi, S. K. ; Mukherjee, S.; Veeriah, V.; Barui, A. K.; Chatterjee, S.; Patra, C. R. Bioconjugated gold nanoparticles accelerate the growth of new blood vessels through redox signaling. Chem. Commun. 2014, 50, 14367-14370. (27) Addadi, L.; Raz, S.; Weiner, S. Taking Advantage of Disorder: Amorphous Calcium Carbonate and Its Roles in Biomineralization. Adv. Mater. 2003, 15, 959–970. (28) Huang, S. –C. ; Naka, K.; Chujo, Y. A Carbonate Controlled-Addition Method for Amorphous Calcium Carbonate Spheres Stabilized by Poly(acrylic acid)s. Langmuir 2007, 23, 12086–12095.


22

Page 23 of 25

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


(29) Schneider, S.; Schmitt, M.O.; Brehm, G.; Reiher, M.; Matousek, P.; Towrie, M. Fluorescence Kinetics of Aqueous Solutions of Tetracycline and Its Complexes with Mg2+ and Ca2+. Photochem. Photobiol. Sci. 2003, 2, 1107–1117. (30) Zhang, Z. -P.; Gao, D. -A.; Zhao, H.; Xie, C. -G.; Guan, G. –J.; Wang, D. -P.; Yu, S. -H. Biomimetic Assembly of Polypeptide-Stabilized CaCO3 Nanoparticles. J. Phys. Chem. B, 2006, 110, 8613– 8618. (31) Pokroy, B.; Quintana, J. P.; Caspi, E. N.; Berner, A.; Zolotoyabko, E. Anisotropic Lattice Distortions in Biogenic Aragonite. Nat. Mater., 2004, 3, 900–902. (32) Dürckheimer, W.; Tetracyclines: Chemistry, Biochemistry, and Structure-Activity Relations. Angew. Chem. Int. Ed. 1975, 14, 721-734. (33) Zhang, Y.; Boyd, S. A. ; Teppen, B. J.; Tiedje, J. M.; Li, H. Role of Tetracycline Speciation in the Bioavailability to Escherichia coli for Uptake and Expression of Antibiotic Resistance. Environ. Sci. Technol. 2014, 48, 4893–4900. (34) McLeay, J. F.; Walske, B. R. Tetracycline Fluorescence in Bone Lesions. J. Bone Joint Surg. Am. 1960, 42, 940– 944. (35) Gatenby, R. A.; Gillies, R. J. Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer 2004, 4, 891-899. (36) Sapadin, A. N.; Fleischmajer, R. Tetracyclines: Nonantibiotic properties and their clinical implications. J. Am. Acad. Dermatol. 2006, 54, 258–265.


23


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

Page 24 of 25

(37) Pershadsingh, H. A. ; Martin, A. P.; Vorbeck, M. L.; Long Jr., J. W.; Stubbs, Jr. E. B. Ca2+dependent depolarization of energized mitochondrial membrane potential by chlorotetracycline (auromycin). J. Biol. Chem. 1982, 257, 481-12 484.. (38) Lamb, R.; Ozsvari1, B.; Lisanti, C. L.; Tanowitz, H. B.; Howell, A.; Martinez-Outschoorn, U. E.; Sotgia, F.; Lisanti, M. P. Antibiotics that Target Mitochondria Effectively Eradicate Cancer Stem Cells, Across Multiple Tumor Types: Treating Cancer like an Infectious Disease. Oncotarget. 2015, 6, 4569-4584. (39) Lokeshwari, B. L.; Selzeri, M. G.; Zhu, B.-Q.; Block, N. L.; Golub, L. M.; Inhibition of Cell Proliferation, Invasion, Tumor Growth and Metastasis by an Oral non-Antimicrobial Tetracyclie Analog (COL-3) in a Metastatic Prostate Cancer Model. Int. J. Cancer 2002, 98, 297–309.


24

Page 25 of 25

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


Table of Contents Graphic and Synopsis

In a bio-inspired method macromolecular assembly of polypeptides allows for in-situ encapsulation of tetracycline during mineralization of CaCO3 microstructures favorable for multitasking applications


25

In Situ Strategy to Encapsulate Antibiotics in a Bioinspired CaCO3

Recommend Documents