Structurally Tuned Antimicrobial Mesoporous Hydroxyapatite

Dec 14, 2016 - *E-mail: [email protected]. ... with an aspect ratio of ∼13.2 and their mesoporous nanorods (small and large pore) by fine regu...
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Structurally Tuned Antimicrobial Mesoporous Hydroxyapatite Nanorods by Cyclic Oligosaccharides Regulation to Release of Drug for Osteomyelitis Murugesan Selvakumar, P. Senthil Kumar, Bodhisatwa Das, Santanu Dhara, and Santanu Chattopadhyay Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01190 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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Structurally Tuned Antimicrobial Mesoporous Hydroxyapatite Nanorods by Cyclic Oligosaccharides Regulation to Release of Drug for Osteomyelitis

M. Selvakumar,1,2 P. Senthil Kumar,3 Bodhisatwa Das,4 Santanu Dhara,4 and Santanu Chattopadhyay1*

1

Indian Institute of Technology, Rubber Technology Centre, Kharagpur-721302, India. 2

Department of Mechanical Engineering, Center for Rapid Prototyping based 3D Tissue/Organ printing, Pohang University of Science and Technology (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, Korea 3

VHNSN College, Department of Chemistry, Virudhunagar-626001, Tamil Nadu, India. 4

Indian Institute of Technology, School of Medical Science and Technology, Kharagpur-721302, India. *

Authors to whom correspondence should be addressed E-Mail: [email protected] (*S. Chattopadhyay)

ABSTRACT This work unveils a straightforward and controlled biomimetic synthesis (combined modified coprecipitation and sonochemistry method) of rod-like 2D hydroxyapatite (HA) nanoparticles with having the aspect ratio of ~13.2 and its mesoporous nanorods (small and large pore) by fine regulation of morphology using β-Cyclodextrin (β-CD) oligomer. From the various methodical characterization results like WXRD, HRTEM, BET and FTIR, it comprehensively established that β-CD acts as an effective nucleating agent by virtue of strong adsorption of α-Dglucopyranoside moieties of β-CD on the surface of calcium phosphate as well as owing to electrostatic interactions due to toroid and cyclic structure of β-CD. Besides, the improved the

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crystallinity of HA nanorod, as well as induced crystal growth of nanorod along the [110] crystallographic direction was observed from the HRTEM micrographs. Consequently, a plausible mechanism also been proposed for the nucleation and growth of the nanorods followed by their crystallization. Subsequently, the prepared tuned mesoporous nanorods were employed as a drug carrier and delivery systems using ciprofloxacin (CFX) drug for the treatment of osteomyelitis. Eventually, CFX encapsulated βCD-MPHA nanocrystals showcases a greater drug loading capability (~79%). It also shows sustained release characteristics (in vitro). Furthermore, antimicrobial activity showed a significant zone of inhibition ~33mm of the CFX-loaded mesoporous frame, especially for the βCD-MPHA nanocrystals. Hemolysis assay with human erythrocytes displays good blood compatibility (less than ~1 % of hemolysis) of the prepared various HA nanorods. Besides, the robust cell proliferation at day 7 as well as zero toxicity level of the prepared different HA nanocrystals was confirmed by MTT assays and monitoring cell adhesion as well as cell morphology (cytoskeleton) by fluorescence microscopy using osteoblast cells. Thus, these structurally tuned nanocrystals along with combinatorial properties envisage a broad range of potential biomedical applications particularly since as a biomaterial for osteomyelitis therapy. Likewise, it can also be used as nanofillers in fabricating bionanocomposite with a suitable matrix for bone tissue engineering, where the infection is major problems of osteoblast proliferation.

KEYWORDS: nanohydroxyapatite; mesoporous; nanorod; β-Cyclodextrin; osteomyelitis; hemolysis.

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1. INTRODUCTION At present, the most challenging task in the era of rapid growth of nanotechnology as well as in biomedical engineering is the synthesis of novel nano-biomaterials with precise architectural manipulation of the structures and hence tailoring their technical properties.1 Among various nanomaterials, Hydroxyapatite (HA) is key and gifted bioceramics used in extensive biomedical applications in different allied areas especially in orthopedic, drug delivery, dental implant, maxillofacial therapies and hard tissue applications.2,

3

This is because of its

peculiar physico-chemical properties, non-toxicity, non-inflammatory properties, excellent biocompatibility, and kinetic stability in biological fluids, non-immunogenicity, and hemocompatibility.4 The chemical composition and microstructure (morphology) of the HA crystals are mimics the filler of human bone where collagen is the matrix. It has combinations of minerals like Ca, O, P and an empirical formula of HA ceramics are Ca10(PO4)6(OH)2 and it is cited as calcium phosphate.5 Due to its versatile morphologies and physical properties of HA crystals, it has also attracted to other applications like used as the strengthener for biomaterials along with biocompatible matrix like collagen, bone paste for regenerations, sensors, bio photocatalysts and targeted drug delivery.6,

7

For every individual biomedical application, the

preferred or desired nanoparticle shape with size and fine-tuning the morphology of HA nanocrystals is the important top step. For instance, tuned mesoporous HA crystals are solely dedicated for targeted drug delivery applications. Likewise, for the reinforcement function with a judicious matrix to achieve the desired physico-mechanical properties, HA should have a high surface area or high aspect ratio. Recently, substantial progress and the significant amount of interest has been devoted by the researchers on the tailor-made morphology of fabrication of HA ceramics such as spherical (1D), nanorod (2D), platelets, nanorings, flower (3D) and nanowire, etc. A plenty of techniques primarily chemical co-precipitation, hydrothermal method,8,

9

sol-gel technique, sonochemical

synthesis,10 electrospinning,11 ball milling, mechanochemical synthesis have been adapted to prepare HA nanocrystals for various bulk morphology. Among all, 2D rod-like nano-

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hydroxyapatite (nHA) is highly endorsed by most of the biomedical applications as it is bioinspired, and it replicates the morphology (microstructure) of most of the bones of the human body.12 In natural bone, the nHA rods are embedded in the collagen matrix with an average rod length of 50 nm.12,

13

On the other hand, mesoporous nHA nanorods have also attracted the

attention of many researchers due of its hierarchical morphology combined with tuned textural characteristics. These includes percentage of crystallinity, surface functionalities, higher surface areas, tailor-made pores (large and small size), and manipulated surface roughness, which are the key factor to be considered in advanced and targeted drug delivery applications.14 These mesoporous architecture nanocrystals have the great prospect to encapsulate the wide range of pharmaceutical drugs as physically and chemically also for various therapeutic as well as for regenerative medicines in bone tissue regeneration. In recent years, hydroxyapatite nanorod with high aspect ratio has been successfully fabricated by the template-assisted hydrothermal method,15 emulsion methods, etc. In contrast, ionic surfactants, non-ionic surfactant, oligomeric surfactant like polypropylene glycol (PPG) have been widely used as templates or as a surfaceregulating polymer for the synthesis of nHA nanocrystals with tailored morphology. Qianjun He et al15 successfully prepared mesoporous HA nanocrystals by the nano silica template-supported method for the controlled release of large pharmaceutics. In another study, Ine´s S. Neira et al16 prepared HA with various effective morphology such as hexagonal prism shape (3D), fine-plate type (3D), plate needle (3D) and needle structure (2D) by urea based surfactant-assisted hydrothermal

technique.

Subsequently,

Cuimiao

Zhang

et

al17

reported

hexadecyltrimethylammonium bromide (CTAB) and trisodium citrate assisted hydrothermal process for the fabrication of hydroxyapatite crystals with versatile morphologies from micro to nanostructures. They also have fabricated various unique shapes like 2D morphologies of nanorods and nanowires, 1D morphologies of microspheres, microsheets, and 3D morphologies of microflowers. Likewise, Jevtic M et al10 established method of the preparation for plate-like hydroxyapatite as well as nanorod morphology and that grow along the (001) crystal face by sonochemical homogeneous precipitation method. Pramanik et al14 also successfully synthesized the dendrimer functionalized mesoporous HA by using CTAB as a porogen.

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From the detailed literature study, it is realized that surfactants containing carbonyl or alcohol functionalities can enhance the rate of nHA formation by promoting the nucleation sites of HA crystal through electrostatic attraction with positively charged ions of Ca2+.18, 19 From this essence, we have attempted to use β-CD for the present investigation for the biomimetic synthesis of nHA as non-ionic surfactant which has both the moieties like bearing hydrophilic functional moieties (–O–, –OH– and C=O) and bearing hydrophobic moieties as well (–CH2– and –CH–,). In addition to this, β-CD compounds made up of oligosaccharides (sugar molecules), and it can form host-guest complexes with hydrophobic molecules. Hence, it anticipates that β-CD can enhance the biological properties of the HA crystals by reacting on the surfaces. Besides, these multiple functionalities of the oligosaccharides (β-CD) will helps in increasing the intermolecular interactions among the HA nanocrystals. These intermolecular interactions may strengthen the matrix by the formation of the strong interface as well as it helps in limiting the crystallization of nHA crystal. Nevertheless, no open literature connected to tuned mesoporous nHA nanorod with high aspect ratio is available so far and their applications in drug delivery system. Focusing on mild reaction temperature at normal atmosphere solution conditions (aqueous medium) for the synthesis of tuned mesoporous nHA nanorods with high aspect ratio is the subject matter of this present investigation. A combined modified coprecipitation and sonochemical method with surface-regulating oligomer β-CD without changing the pH is adapted to prepare tuned mesoporous nanorods. The formation mechanism of rod-like tuned mesoporous nHA and the effect of β-CD on the crystal nucleation and growth have also been discussed. The ability to use these structurally tuned mesoporous nanorods for drug delivery applications are subsequently examined. The mesoporous nanorods provide an exceptionally high CFX drug encapsulation with sustained-release characteristics. Lastly, the cell proliferation (biocompatibility), cytotoxicity, blood compatibility as well as percentage hemolysis also investigated to proof their potential for biomedical applications.

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2. EXPERIMENTAL DETAILS Chemicals For the purpose of synthesizing HA nanocrystals, Calcium nitrate tetrahydrate (Ca(NO3)2.4H2O) and diammonium hydrogen phosphate (DAHP) ((NH4)2HPO4) were taken as the starting materials for Ca and P, respectively (Merck, Mumbai-India). Non-ionic surfactant, βCyclodextrin (β-CD) oligomer, which has a

Mn ~1134,98 g/mol) and Ciprofloxacin

hydrochloride monohydrate (C17H18FN3O.HCl.H2O) drug were supplied by Sigma-Aldrich, USA. Deionized water (DW) with double distillation was used throughout HA synthesis. Synthesis of various HA nanorods The temperature of the reaction was optimized as 60 °C after many trails as well as from our earlier works to acquire a rod-like morphology of 2D HA crystals.1, 4, 5 In a typical reaction, each salt of Ca and P precursors were dissolved separately in 500 ml of Deionized water (DW) with 0.2 (M) to match the Ca:P molar ratio at about ~1.6. By incorporation of the necessary amount of ammonia solution, we have kept the pH of the both (Ca and P) the solution was at about ~11 until the reaction completion. For the synthesis of β-CD regulated HA nanorods and it’s mesoporous, we have followed the in-situ method and five weight percentage of the β-CD oligomer was taken on Ca, P precursor salts. It was dissolved in the solution of Ca precursor (calcium nitrate) as a non-ionic surfactant. Afterward the solution of DHAP salt was mixed gradually (drop-wise) to the calcium nitrate solution at a temperature of 60 °C and meantime with the help of NH4OH, the pH was maintained at about ~12 as mentioned in the scheme 2. Subsequently, the white colour solution started to form upon addition of DHAP solution into calcium nitrate solution. The reaction of a white milky solution was stirred continuously with the mechanical stirrer (2000 rpm) for 6hrs. After aging of 6hrs, the solution was washed a number of times using DI water until pH shows at around ~7 (neutral) and then the precipitate was filtered with centrifuge method (3500 rpm). The filtered powder was dried out in vacuum oven at 70 °C for 18 hrs followed by calcinations of those at 300 °C for 6 hrs. After that, the calcined samples

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were taken for the characterization. Thus, the prepared rods-like HA without and with β-CD regulation are designated as NRHA and βCD-NRHA, respectively. On the other hand, we have used ultrasound-assisted modified co-precipitation technique along with the reaction conditions as mentioned earlier with and without β-CD regulation in order to synthesis the mesoporous HA nanocrystals. In the meantime, throughout the process of reaction, a titanium gun type ultrasonic probe having a diameter of 8 mm along with highintensity (25 kHz and 900 W) was directly inserted into the reaction solution beaker as mentioned in scheme 1&2. These parameters are optimized from our earlier reports.4 The probe was functioning in a cycle of 3 seconds ON and 2 seconds OFF mode (intestinally) until the reaction completion.

Scheme 1. Synthesis setups and mechanism for the formation of MPHA and βCD-MPHA nanocrystals: (a) Strong cavitations effect, (b) Bubble formation along with HA crystal, (c) Gas escaping by rapid cooling and (d) formed β-CD wrapped mesoporous nanorods (βCD-MPHA). After completion of reaction about 6 hrs, purposely the reaction beaker was cooled down instantly using an ice bath. Remaining other procedures such as centrifugal filtration process, calcination temperature, and time of the total gelatinous precipitates was followed the same condition as mentioned earlier for the HA nanorod preparation. Hereafter, the prepared rod-like mesoporous HA without and with β-CD regulations are designated as MPHA and βCD-MPHA, respectively. All HA nanocrystals are formed according to the following reaction:

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10Ca(NO3)2 4H2O + 6NH4H2PO4 + 2NH4OH → Ca10(PO4)2(OH)2 + 8NH4NO3+12HNO3

Scheme 2. Flowchart for the mesoporous hydroxyapatite nanorods

3. CHARACTERIZATION of VARIOUS nHA NANOCRYSTALS Fourier transform infrared (FTIR) spectroscopy was applied for the various HA nanocrystals to reveal the functional groups present in it. We have used KBr pellet technique for this analysis using Bruker Equinox 55 spectrophotometer in the range of 4000-500 cm−1. It has a resolution of 0.5 cm−1 and each spectrum was averages of 64 scans. A Wide-angle X-ray diffraction (WXRD) was done to look at the structure of the crystal and their corresponding phases of the synthesized HA nanocrystals using Philips PW-1710 X-ray diffractometer (Eindhoven, The Netherlands). We have scanned in the 2θ range of 10-80° using voltage and beam current of 40 kV and 20 mA, respectively. Differential scanning calorimetry (DSC) was also employed for the various HA nanocrystals by a differential scanning calorimeter (TA instruments, Model DSC Q200, USA) to reveal the crystallinity changes. About ~6 mg of different HA powders in a sealed Al pans and it were purposefully experienced a series of

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heating to take out the internal stress in it. During first heating, it was heated from room temperature to 150 ºC with 10 °C min−1 rate of heating. Subsequently, the same rate of cooling (10 °C min-1) was applied to bring the system to room temperature. Then the last heating cycle, they system was again heated from 25 °C to 400 ºC with 10 °C min−1 rate of heating. This second heating data was used for the interpretation of DSC results. Bulk morphology of various HA nanocrystals was visualized by high-resolution transmission electron microscopy (HRTEM). WE have used JEM 2100 JEOL transmission electron microscope with an operating voltage of at 200 kV and lanthanum hexa-boride target was used with a beam current of 116 µA. Moreover, energy dispersive X-ray spectroscopy (EDAX), which is connected to the HRTEM was used to find the Ca/P ratio of the various HA nanocrystals. X-ray photoelectron spectroscopy (XPS) [PHI 5000 Versa Probe II (ULVAC-PHI Inc., Japan)] was employed to have an idea about the chemical composition as well as the wrapping up of β-CD on HA nanocrystals since XPS is a very surface sensitive technique. A very micro-focused beam of around 100 µm monochromatic Al Kα radiation of energy of 1486.6 eV was used along with beam current of 25W and 15 kV for this analysis. The textural characteristics of representative mesoporous HA nanorods (MPHA and βCD-MPHA) were examined with the help of Micromeritics Tristar 3000 porosimeter, Australia. HA powders were dried completely (degas) at 125 °C for 3 hrs and then the nitrogen adsorption trial at -190 °C using a surface area analyzer was employed. Brunauer-Emmett-Teller (BET) technique was used to have an idea about the specific surface area of the mesoporous HA nanorods. On the other hand, a Barrett-Joyner-Halenda (BJH) method was employed to reveal the pore parameters such as average pore diameter and its volume. Ciprofloxacin hydrochloride (CFX) drug was used to study the drug encapsulation (physically) and its release characteristics using MPHA and βCD-MPHA nanocrystals as the drug carrier. For this, representative HA nanocrystals (MPHA and βCD-MPHA) were taken about 0.5g and it was dispersed in H2O followed by ultrasonication for 30 mts to ensure the better dispersion of nanocrystals. Separately, CFX drug solution was also prepared by dissolving CFX in double distilled water (20-wt% concentration). Later, both the solution was mixed and given gentle shaking about 6 hrs and just kept ideal for a day in order to bind the CFX drug into

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the mesoporous nanocrystals. Afterward, the precipitate was filtered with centrifuge method (3500 rpm), washed and dried well. This physical adsorption of CFX into the mesoporous HA nanocrystals was studied by FTIR technique through their structural characteristic band changes. The % of drug encapsulation into HA nanocrystals by the empirical equation (Supporting Information, S1). Subsequently, Drug release profile was investigated in a PBS solution with pH 7.4 at room temperature. About ~200 mg of the drug encapsulated MPHA, and βCD-MPHA nanocrystals was mixed well in 100 ml of PBS solution at the sterile condition. The solution were underwent UV analysis (wavelength of 274 nm and Perkin Elmer lambda-25 UV-Vis) at various intervals to know the changes in the concentration of the solutions. The results are averages of 3 results. Malvern Nano Zetasizer ZS instrument (25 °C with 4 mW He-Ne laser (λ =632.8 nm) was used to determine the water stability of the HA nanocrystals after CFX drug encapsulation into mesoporous HA nanocrystals. Standard disk diffusion technique was employed to study the in vitro antibacterial activity of the CFX embedded HA nanocrystals. To prepare a disk with 8 mm in diameter and 0.5 mm in thickness, powder samples were compressed at ∼40 MPa for 10 minutes. Representative human pathogens such as Pseudomonas aeruginosa (P. aeroginosa) and Staphylococcus aureus (S. aureus) was chosen since they are highly responsible for the osteomyelitis and it was cultured on agar plates. The prepared disks were kept on this plate and incubated for 12 h after the complete culture of those pathogens. Finally, the microbial zone of inhibition was captured using camera measured after the incubation period (12 h) and documented. MTT was performed by using MG-63 cells from NCCS, Pune-India for the cellular responses (toxicity and cell proliferation) of the various HA nanocrystals. In Dulbecco’s Modified Eagle Medium (DMEM) cells were cultured. 10% fetal bovine serum (FBS) were added to the cultured cells as a supplement, and it was trypsinized and re-suspended in fresh media. Biocompatible polymer of Poly-l-lysine surface coated coverslips were used for the cell seeding/cultured and 96 well plates with a cell seeding density of 1×104 well-1. In order not to disturb the cell monolayer, culture mediums was changed the regular time period. Beside this, cell proliferation was observed by performing MTT assay. MTT solution of about 5 mg mL-1

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was prepared by dissolving MTT dye in PBS buffer solution. Multiple washing using PBS solution was done after removal of the cell culture media. Subsequently, 50µl of prepared MTT solution was added to each well and then an addition of 500 µl of DMSO after 4hr for removing formed purple formazan crystals. Results are interpreted using Student’s t-test and P-values of < 0.05. Fluorescence microscope (Carl Zeiss inverted optical microscope) was used to visualize the morphology (cytoplasm) of the proliferated cells (MG-63). Further, the cell-seeded cover slips were counterstained for nuclei by DAPI-Hoecht-33342 (Life Technologies). Finally, microscopic images were captured taken using Fluorescence microscope (Carl Zeiss inverted optical microscope). Blood compatibility test was carried out for the various prepared HA nanocrystals by following our previously reported work. Absorbance at 570 nm was observed for all the samples after centrifugation for measuring hemolytic activity using Biorad microplate reader model 550, Japan. 0% lysis and 100% lysis in 1% Triton X-100 surfactant were used to get the result for the control samples. By using this following empirical equation, % of Hemolysis was determined: % Hemolysis = [(sample absorbance - negative control) / (positive control - negative control)] × 100

4. RESULTS and DISCUSSION 4.1 Characterization of synthesized various HA nanocrystals The FTIR spectra of the various HA nanocrystals (NRHA, βCD-NRHA, MPHA, and βCD-MPHA) are displayed in Figure 1(a-b) to get an idea about the functional group presence in it. Degenerated vibration (triply) mode of P–O bond was at around 1041 cm−1 followed by nondegenerate stretching mode of P–O bond (symmetric) of HA nanocrystals was received at around 947 cm−1. The prominent sharp peaks observed at around 1031 and 1082 cm−1 are ascribed as from the PO43− groups. Likewise, the two different modes such as stretching and liberation are observed for the structural hydroxyl groups of HA nanocrystals at around 3569 and 629 cm−1, respectively. On the other hand, various vibration modes of moderate peaks received at around 1042, 944, 601 and 568 cm−1. These are attributing for the PO43− groups.

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Figure 1. FTIR spectra of the various HA nanocrystals (a) without β-CD regulated synthesis and (b) with β-CD regulated synthesis. Another type of degenerated bending mode (triply) of O–P–O bond is received at around 602 and 568 cm−1 with sharply. Additionally, the intense peaks of structural and vibration mode for hydroxyl group (-OH) of HA nanocrystals is received at around 632 and 3571 cm−1.20 The IR peaks (moderate) for the adsorbed water also received at around 1633 cm−1 and water from the crystal lattice is received at around 3427 cm−1 (broadband). On the contrary, the peak assignments for confirming the β-CD regulated preparation of HA as well as wrap up of β-CD on the surface of the HA nanocrystals are manifest from the identification of many peaks like an intense peak at around 2964 cm-1 for CH stretching (asymmetric). Similarly, for the CH3 deformation (symmetric) was received at around 1258 cm−1 (figure 1b). The functional group of HA nanocrystals is confirmed from these peak combinatorial assignments as well as from the literature.20-22

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Figure 2. XPS plot of the representative HA nanocrystals. XPS is one of the major surface characterization technique, and it was employed to get insight into the quantitative idea about the surface modification of the prepared HA nanocrystals by βCD. Figure 2 displays an XPS plot of the prepared representative nanorods such as hydroxyapatite (NRHA) and β-CD wrapped HA nanocrystals (βCD-NRHA). It reveals major elements of HA nanocrystals like Ca, P, and O by noticing their distinctive peaks. Various binding energies peaks (sharp) at 975, 434 and 347 eV are confirming the different state of Ca like Calcium Ca (LMM), Ca2s and Ca2p, respectively. For the oxygen elements, the intense band of binding energy received at 532 eV for O1s. Finally, the confirmation for the P element also revealed. The binding energies at various positions like 192 and 132 eV are assigned to P2s and P2p bands of HA nanocrystals. On the other hand, for the surface modification of the prepared HA nanocrystals by β-CD are confirmed by observing the binding energy at 288 eV from the C1s of the carbon chains (inset of figure 2). This finding evidently argues the interaction between β-CD polymer chains and the surface of HA nanocrystals. The typical WXRD patterns of the various HA nanocrystals crystals are shown in Figure 3(a-b). It shows the various crystalline type of diffraction peaks at various 2θ regions was observed for all HA nanocrystals and those are close matches with the standard data of JCPDS Card No. 09–0432 as well as with the literature. In contrast, the various peaks at around 26, 32,

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33 and 40° are attributed to (002), (211), (300) and (310) crystal planes, respectively.20,

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22, 23

Intense and strong signals as revealed in the figure (3a) for β-CD regulated HA indicate the formation of strong crystalline HA. However, other than these, the 2θ region of the various crystal plane peaks of pristine HA such as (211), (300), (202), are closely coming with the β-CD regulated synthesized or surface coated HA nanocrystals. Therefore, bulk properties of the β-CD assisted synthesized HA would remain the same. In addition to that, the in-situ β-CD assisted method has not induced any additional changes in HA nanorods. To gain insight into the crystallite changes of the prepared nanocrystals, we also have attempted to reveal the consequence of the crystallite size and its strain in the lattice owing to dislocations using empirical equations related diffracted peak broadening. Scherrer formula and Williamson−Hall isotropic strain mode were employed to reveal the lattice strain and crystallite sizes24,

25

(Supporting Information. S2) and it summarized in Table 1. Likewise, the effect β-CD on crystal growth of various HA nanocrystals, the degree of crystallinity (Xc) was determined with the help of the following equation26 (1), which is directly related to Full Width Half Maximum (FWHM) and the values are tabulated in table1:

 0.24   X c =   β 002 

3

-------- (1)

Where, β002 is the FWHM of (002) miller`s crystal plane. A significant improvement in the crystallinity is observed for the β-CD regulated various HA nanocrystals. Crystallite size of the β-CD regulated HA nanocrystals increases significantly as compared to other HA crystals (without β-CD). This increase is more prominent for the rod-as if HA crystals compared to mesoporous rods. Lattice strain also tends to decrease relatively for the various βCD-HA crystals (Table 1).

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Figure 3. WXRD profile of the various HA nanocrystals (a) without β-CD regulated synthesis and (b) with β-CD regulated synthesis The degree of crystallinity also decreases significantly for the β-CD regulated mesoporous HA nanocrystals synthesis (βCD-NRHA and βCD-MPHA).This observation is reliable with the result of Teng et al27, Klinkaewnarong et al,28 and A. Martins et al.29 According to them, surface energy reduces once the smaller nanocrystals are fused during synthesis as well as during calcinations. These are the feasible reason behind for the changes in the crystallite size of the HA nanorod of β-CD regulated synthesis. Moreover, ‘a & b’ axes are identical and the ‘c’ axis lies along the nanorod axis in a hexagonal close packed (HCP) unit cell of hydroxyapatite crystal structure which leads to an increase of crystallinity. Furthermore, the increase in crystallinity of the HA nanorod of β-CD regulated synthesis was studied my Differential scanning calorimetry (DSC) for the better understanding. Figure S1 (supporting information) shows the sharp melting peaks (endo) with the varied heat of fusion (∆Hf) for the various HA nanocrystals. It found that the area under the peak, as well as the melting enthalpy or heat of fusion (∆Hf) of the crystallization peak at around 140 °C is changes tremendously especially for the HA nanorods (βCD-NRHA and βCD-MPHA) of β-CD regulated synthesis. Thus, it confirms that β-CD plays a vital role in crystallization of HA nanocrystals as well as in crystal growth. A plausible mechanism for the HA crystal nucleation and its growth is discussed in the subsequent section (HRTEM micrographs). HRTEM photomicrographs of the prepared various HA nanocrystals are shown in Figures 4 and 5. It demonstrates that HA nanocrystals show well-

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defined rod-like 2D structure (figure 4a-b) for the preparation method at a temperature of 60°C. Nevertheless, the aspect ratio of the HA nanorods is vary from the both the methods (with and without β-CD regulation).

Table 1. Crystallite size, degree of crystallinity and % lattice strain determination by different methods for various HA nanocrystals Sample

(hkl)



Xs (nm)

D (nm) 10.4

% lattice strain (W-H method) 7.8×10-4

Degree of crystallinity (Xc) 3.72

NRHA

002

26.09

9.8

βCD-NRHA

002

25.90

28.7

35.1

4.1×10-4

4.25

MPHA

002

26.06

10.1

10.8

8.1×10-4

2.71

βCD-MPHA

002

26.02

32.1

33.4

7.1×10-4

2.98

In contrast, the HA crystals synthesized by the β-CD regulated method broadcast a very high aspect ratio of ~13.1 (βCD-NRHA, figure 4b) and for the HA nanorods prepared by simple precipitation technique shows ~3.8 (NRHA). Secondly, the HA nanorods prepared by combined methods (sonochemistry and precipitation method) broadcast a 2D rod shape mesoporous frame (figure 5). Here also the β-CD regulated synthesis broadcast high aspect ratio ~14.7 (βCDMPHA) while the non-regulated β-CD synthesis (MPHA) shows less aspect ratio ~6.1 (figure 5a-e). Besides, the stoichiometry of Ca/P = ~1.5 of prepared HA nanorod has been observed in all the cases from EDX studies (figure 4e & 5e). More detailed information about the textural properties of the prepared mesoporous frames (MPHA and βCD-MPHA) were looked through BET surface area analysis and BJH model, respectively. The specific surface area of these nanocrystals MPHA and βCD-MPHA are found to be 21.6 m²/g and 42.1 m²/g, respectively (Figure S2 and S3, Supporting Information). Both the mesoporous nanorods broadcast a large range of mesopore sizes (5-40 nm) with broad distribution (pore) which has been obtained from BJH model, more closely, average pore diameter shows 7.4 nm for the MPHA nanocrystals and 13.6 nm for the β-CD regulated synthesis (the βCD-MPHA). Furthermore, the pore volume for the MPHA nanocrystals has 0.04 cm³/g (Figure 7b). While β-CD regulated synthesis (the βCDMPHA) exhibits 0.14 cm³/g.

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Figure 4. HRTEM photomicrographs (a) NRHA (b) βCD-NRHA nanocrystals (c) SAED patterns of NRHA (d) SAED patterns of βCD-NRHA (e) EDX spectrum acquired from the NRHA nanorods and (f) Consequent lattice parameter of βCD-NRHA nanocrystals at higher magnification.

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Figure 5. HRTEM photomicrographs (a) MPHA nanocrystals (b) Magnified MPHA nanocrystals (c) SAED patterns of MPHA (d) βCD-MPHA nanocrystals (e) Magnified βCDMPHA nanocrystals (f) SAED patterns of βCD-MPHA (g) Corresponding lattice parameter at higher magnification of βCD-MPHA nanocrystals and (h) EDX spectrum acquired from the MPHA mesoporous frame. 4.2. Formation Mechanisms of βCD-NRHA and βCD-MPHA nanocrystals 4.2.1 HA nanorod with high aspect ratio The mechanisms for the constitution HA nanorods with greater aspect ratio and its tuned mesoporous frame (small and large) were examined especially for the β-CD regulated method. It has observed from the figure 4(b) that β-CD greatly influences the crystal growth because the aspect ratio of the rod-like 2D HA nanocrystals has changed enormously at a temperature of 60

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°C, due to various causes. As discussed earlier, β-CD is a non-ionic surfactant that has both hydrophobic entities as well as hydrophilic bearing functionalities. Besides, it can form hostguest complexes with hydrophobic molecules. Furthermore, it has the ability to form the long aliphatic chains along with hydrophilic functionalities disclosed in aqueous dispersion while hydrophobic entities anchored in the same aqueous solution. Oxygen atoms of β-CD multiple functionalities facilitate the binding with Ca2+ ions in a combination of β-CD oligomer and Ca precursor in aqueous solution.30 Due to the multiple functionalities and electrostatic interactions of β-CD, plenty of Ca2+ ions can able to accumulate on the inner and outer surface of β-CD toroid structure. Followed by this, the nucleation of hydroxyapatite crystal formation would start on the β-CD molecular surface once complete dissolution of hydroxyapatite is increased. Then, the O atoms of β-CD long chains will act as the nucleation site for HA.31 After nucleation of the HA crystals, it likely to acquire inside the cone structure of β-CD along the c-axis resulting in longer HA rod nanocrystals. Subsequently, these polymer chains wrap onto the surface of nHA crystals32 as shown in Scheme 3 according to ions-nucleation-growth-aggregation model. For our curiosity, we also have performed this synthesis at around 85 °C by maintaining the same pH condition. It ended up in a dissociated rod-like morphology as can be realized from the HRTEM photomicrographs as shown in figure S4 (Supporting information). It may due to a fact that the template is completely collapsing the morphology by degradation of the template (β-CD).

4.2.2 Mesoporous HA nanorod with high aspect ratio The mechanism for the establishment of the various mesoporous frames nanorod morphologies (MPHA and βCD-MPHA) by sonochemistry assisted modified co-precipitation method has also been established. With and without β-CD regulated synthesis gives mesoporous structure as seen from the Figure 5a-e. However, the pore size is varying from each other. A plausible mechanism for the formation of tuned (large and small) mesoporous nanorods rationalized as assimilates: First, during precipitation reaction numerous foam/bubbles would start to form as it has the combined effect of an intense cavitations effect from the ultrasonic probe and instant generation of heat. Thereafter, the initial nucleation of the HA crystal also will start very quickly since the reaction is experiencing combinatorial effects.33 Once crystal

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nucleation starts, the subsequent growth of crystal will initiate in the region of the bubbles since the system has already filled up by the huge amount of foams. Soon after, the bubbles will get blast since fluctuation of heating and cooling in the reaction as the ultrasonic probe is functioning like three seconds on and two seconds off. As a result, the pores with mesoporous structure will be formed in crystal where the bubbles blasted or getting away of gases (quick escape) in the reaction.33, 34

35

. The major reason for variance in the average pore sizes of the

mesoporous frames are pressure, viscosity, the rate of bubble blasting and temperature of the reaction.36 More to the point, β-CD assisted synthesis broadcast bigger in average pore size of the mesoporous HA nanorods due the effect of cyclic (cup) and toroid structure of β-CD. 33, 34, 3739

Scheme 3. Schematic illustration of the mechanism of formation of the various HA nanocrystals. Probable scheme for the various aspects of formation mechanism of the HA nanocrystals is demonstrated in scheme 1 and 3. SAED patterns (figure 4c&d and 5c&f) obtained from the HRTEM reveals that the polycrystalline nature (random orientation of planes) of the all the samples even for the β-CD regulated synthesis (βCD-NRHA and βCD-MPHA) as it shows acquit

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ring patterns. In addition to this, crystal growth of the hexagonal closed packed HA ceramics was grown in [110] direction and it is lateral to the long c-axis axis. As a whole, the fabrication of such a structurally tuned morphologies of HA nanorods with β-CD based polymeric surfactant made this work more fascinating.

4.3 Drug Loading and Release Profiles of MPHA and βCD-MPHA nanocrystals Drug loading efficiency of mesoporous hydroxyapatite nanocrystals was examined by the aforementioned procedures. The percentage drug loading of the βCD-MPHA and MPHA nanocrystals were calculated as 79 % and 66%, respectively. This directly implicit to significantly greater CFX drug encapsulation capability of the βCD-MPHA nanorods as compared MPHA nanocrystals. It is because of their physical-chemical characteristics or properties such as porous internal structures, large pore volume with broad distribution and high surface area. On top this, wrapping of β-CD chains on the mesoporous nanorods leads to electrostatic interaction and hydrogen bonding interactions between CFX drug and HA nanorods are also considered as main physical-chemical characteristic contributors to the more CFX adsorption/loading of the βCD-MPHA nanocrystals.15,

40

The physically CFX loaded

hydroxyapatite nanocrystals were also examined using spectroscopy results (FTIR-Figure 6). Important IR peaks of two types HA nanorod and CFX are observed for both the CFX embedded HA nanocrystals. The mesoporous HA nanorods encapsulated with CFX drugs displays a combination IR bands of HA ceramics, which we have already discussed in the earlier section as well as the typical bands for the CFX are C=C double bond at 1602 followed by -CH2- bending at around wavenumber of 1461, and -CH2- stretching at 2995 cm-1 and CN stretching at 864 cm1

. Moreover, the peak shifting towards lower wavenumber side in the drug-loaded both the

nanocrystals was also observed. It implied that some sort of physical interaction had taken place between drugs and nanocrystals. A zeta potential result further reinforces the dispersivity of drug-encapsulated nanocrystals in H2O by noticing a decrease in potential surface charges.

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Figure 6. FTIR spectra of CFX loaded mesoporous nanocrystals. The negative surface potential of −20.8 mV was obtained for the MPHA nanorods and it turns out to be even more negative of −31.4 mV for the β-CD regulated synthesized nanocrystals (βCD–MPHA.15 It is expected that higher loading capacities of βCD-MPHA would help in more sustained drug release than the MPHA. Figure 7a also reveals the same trend. However, the rate of releasing CFX drug is lower for the βCD-MPHA loaded one compared to MPHA nanorods. Nevertheless, βCD-MPHA nanocrystals display the higher amount of CFX releases due to various factors such as larger average pore diameter cum distribution (broad) and of course the efficiency of drug encapsulation.15 The drug is released gradually over a period for the βCDMPHA leading to controlled releasing of the drug. Moreover, the amount of drug released in one day is the minimum inhibition concentration sufficient to inhibit the bone infections (osteomyelitis).26 On the other hand, the CFX release from both the HA nanocrystals displays a typical two-stage mechanism. There is a rapid burst of the drug in the initial days (1-10) and then it becomes normalized (controlled way).41 CFX release at some stage in the early stage is like the type of burst release as can be seen from the figure 7a. This is due to the fact that CFX drug moieties that are placed (adsorbed) on the HA crystals surfaces did not strongly bind with the HA nanocrystals.42

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Figure 7 (a) CFX drug release profiles and (b) Average Pore size analysis of the mesoporous frames In addition, the rate of CFX release slow due to slackly accumulated drugs in the nanocrystals and it is also speculated that due to the presence of an immobilized -COOH moieties that reacts with calcium ions in the HA nanorods lead to complex formation with CFX drugs.43,

44

Overall, constant and extended release of ciprofloxacin was experienced for the

prepared mesoporous nanocrystals. Over the period, nearly 90% of the encapsulated CFX drug was prolifically released from the βCD-MPHA nanocrystals whereas only ~30% of the loaded ciprofloxacin was released in MPHA as because of the factors of physical-chemical properties. Significant quantities of ciprofloxacin drug remain in MPHA nanocrystals, and it will helpful in extensive antibiotic medicine for the bone infections.

4.4 Antibacterial Activity (in vitro) In general, S. aureus and P. aeruginosa are the most familiar micro-organism, which causes bone infections (osteomyelitis). Antimicrobial activities of the CFX embedded HA nanocrystals along with control HA against S. aureus and P. aeruginosa are displayed in Figure 8. It broadcast no zone of inhibition was observed for the pristine HA pellet. As expected, the CFX loaded βCD-MPHA and MPHA nanocrystals showed a high level of the zone of inhibition ~33mm as can be seen clearly from the figure 8. This is due to a fact of the considerable quantity of CFX release causes. Thus, CFX incorporated βCD-MPHA nanocrystals indicates higher the zone of inhibition or relatively better antimicrobial activity than CFX loaded MPHA nanocrystals. Furthermore, the kinetics of antibacterial activity of both the crystals (MPHA and

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βCD-MPHA) also have performed by noticing the zone of inhibition for every 4 hr for the S. aureus microorganism only for the better understanding. The results are presented in figure S5. It depicts that the rate of the zone of inhibition at 4hr and 8hr are better and increasing for the CFX embedded βCD-MPHA nanocrystals compared to MPHA since it has more amount of drug in it. Nevertheless, the slightly higher zone of inhibition was also observed for the βCD-MPHA nanocrystals after a certain period (12hr) compared to MPHA due to the higher amount of CFX drug release characteristics. Besides, the quantity of CFX release in 24 hrs (1 day) from the synthesized mesoporous nanorods is beyond the minimal inhibitory concentration (MIC) of CFX (.25-2 µg/ml). Mechanism for this ciprofloxacin causes is exactly like fluoroquinolone type of antibiotic drugs.45-47

Figure 8. Zone of inhibition around the CFX-loaded HA mesoporous nanocrystals against S. aureus and P. aeruginosa cultures along with pristine HA nanocrystals. 4.5 Intracellular Uptake and Cytotoxicity with Osteoblast Cells

Figure 9. % Cell viability of osteoblast cells on various HA nanocrystals (n = 3, P < 0.05).

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The MTT assay was performed for the various HA nanocrystals by osteoblast-like MG63 cells to get more idea about the biological functioning and that may determine the fate of the implanted bone graft. Figure 9 shows the cell viability (%) of various HA nanocrystals as a function of various times (1, 3 and 7 day). It is inferred that the cells are proliferated furiously for the various morphology of HA nanocrystals after 7 days. Robust cell proliferation especially for the βCD-MPHA nanocrystals and it is dependent on the crystal surface properties like surface area, and pore size. According to their surface texture properties, % cell viability has increased more particularly for the mesoporous HA one compared to HA nanorod. Nevertheless, all morphologies of HA nanocrystals show better cell growth and spreads well lead to no toxicity. Among all, mesoporous frame show enormous cell growth as because of many factors especially physical-chemical properties: high surface areas, surface roughness, and surface coated oligosaccharides of βCD as well as porosity of the mesoporous nanocrystals.

Figure 10. DAPI (nucleolus) and rhodamine (cytoplasm) counter stained fluorescent photomicrographs of osteoblast cells cultured on the various HA nanocrystals as a function of days (1, 3 and 7 days).

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The photo-microscopic images (fluorescence) of cells on various HA nanocrystals for 1, 3 and 7 days are shown in Figure 10. It is found that cells are greater spreading and adherence on the HA nanocrystals surfaces. Specifically, cells on the mesoporous surfaces (MPHA and βCD– MPHA) were more voluminous cum dense and adulterated cytoskeleton are clearly visible with well organized after 7th days of cell culture. On the other hand, traditional HA crystals surface like rod-like morphologies, the cells exhibited less proliferation of the cells. Major factors physical-chemical property like surface area and energy, osteoconductivity and porosities are the facets to enhance the intracellular uptake without cytotoxicity leading to the improved bioactivity of the biomaterials. Overall, in vitro cellular activity suggests that this fabricated novel structurally tuned HA nanocrystals does not show or induce any toxicity. Besides an extended delivery of drug scheme is essential for the osteomyelitis diseases and it is accomplished in the current findings of mesoporous frames.

4.6 In vitro Hemocompatibility Assay

Figure 11. In vitro hemocompatibility assay of various HA nanocrystals along with the positive control. Many works of literature reported that HA nanocrystals could also be used for biosensing activity.48 For hematological biosensing applicability, the prepared HA nanocrystals were investigated for hemolysis and shown in figure 11. It demonstrates that apart from mesoporous frame there was no significant change of hemolysis of various HA nanocrystals. Mesoporous HA nanocrystals show less than 0.5% Hemolysis, which shows that it is antithrombotic or hemolytic in nature.49, 50 Nevertheless, all HA nanorods including mesoporous morphologies are displaying

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less than 2% hemolysis, which is recommended by the International Organization for Standardization/Technical Report 7406 of bio-related products. 51, 52 These percentage hemolysis results give an idea about other potential biomedical applications like biosensing, drug delivery, etc of the fabricated structurally tuned HA nanocrystals.52-54

5. CONCLUSIONS In summing up, a novel bioinspired synthesis was formulated for the preparation of structurally tuned various hydroxyapatite nanocrystals including mesoporous frame. The prepared HA nanocrystals were characterized extensively using various techniques. Various results have proven that β-CD plays a key role in HA nanorod crystal growth. Plausible mechanisms for the nanorod constitution with high aspect ratio (~13-14) and mesoporous frame have been established. Mesoporous structures with tuned physical-chemical characteristics were efficiently used for the intracellular delivery and sustained release of CFX drug for bone infections (osteomyelitis). Remarkably, it has high payload of CFX drug (~79%) due to the presence of tuned mesoporous nanorods (large and small pores). A high level of the zone of inhibition ~33mm around the CFX-loaded HA mesoporous nanocrystals were also observed. Besides, the prepared HA crystals have no cytotoxicity at all with human osteoblast cells and it proved their excellent biocompatibility. Consequently, all HA crystals were found to be causing less than 1% hemolysis that shows their antithrombotic or hemolytic in nature. Thus, this novel synthesis strategy opens up a new way to control the shape, structure, size and pores of the HA nanocrystals for various biomedical applications. Especially as a bone replacement biomaterial along with blended characteristics of HA ceramics and ciprofloxacin drug for the intervention of osteomyelitis and nanofiller in bionanocomposite with a suitable matrix for bone tissue engineering where an infection is the major problems.

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6. ASSOCIATED CONTENT Supporting Information Percentage of drug loading (calculation), crystallite size determination for various HA nanocrystals, DSC second heating plot, BET results, HRTEM photomicrograph of HA nanorod synthesized at 85 °C and kinetics of antibacterial activity of CFX loaded various HA

nanrods. 7. ACKNOWLEDGEMENT Authors are thankful to G. Sathishkumar, Bharathidasan University, Department of Biotechnology and Genetic Engineering as well as to Dineshkumar Ramalingam, Department of Biotechnology,

Indian

Institute

of

Technology

Kharagpur

for

their

assistance

in

hemocompatibility assay and antibacterial activity.

8. REFERENCES (1) Selvakumar, M.; Srivastava, P.; Pawar, H. S.; Francis, N. K.; Das, B.; Sathishkumar, G.; Subramanian, B.; Jaganathan, S. K.; George, G.; Anandhan, S.; Dhara, S.; Nando, G. B.; Chattopadhyay, S., On-Demand Guided Bone Regeneration with Microbial Protection of Ornamented SPU Scaffold with Bismuth-Doped Single Crystalline Hydroxyapatite: Augmentation and Cartilage Formation. Acs Appl Mater Inter 2016, 8, (6), 4086-4100. (2) Wang, Y. M.; Ren, X. X.; Ma, X. M.; Su, W.; Zhang, Y. P.; Sun, X. S.; Li, X. D., AlginateIntervened Hydrothermal Synthesis of Hydroxyapatite Nanocrystals with Nanopores. Crystal Growth & Design 2015, 15, (4), 1949-1956. (3) Yang, P.; Quan, Z.; Li, C.; Kang, X.; Lian, H.; Lin, J., Bioactive, luminescent and mesoporous europium-doped hydroxyapatite as a drug carrier. Biomaterials 2008, 29, (32), 4341-4347. (4) Selvakumar, M.; Pawar, H. S.; Francis, N. K.; Das, B.; Dhara, S.; Chattopadhyay, S., Excavating the Role of Aloe Vera Wrapped Mesoporous Hydroxyapatite Frame Ornamentation in Newly Architectured Polyurethane Scaffolds for Osteogenesis and Guided Bone Regeneration with Microbial Protection. ACS Appl Mater Interfaces 2016, 8, (9), 5941-60. (5) Selvakumar, M.; Jaganathan, S. K.; Nando, G. B.; Chattopadhyay, S., Synthesis and Characterization of Novel Polycarbonate Based Polyurethane/Polymer Wrapped Hydroxyapatite Nanocomposites: Mechanical Properties, Osteoconductivity and Biocompatibility. J Biomed Nanotechnol 2015, 11, (2), 291-305. (6) Ma, J.; Qin, J. L., Graphene-like Zinc Substituted Hydroxyapatite. Crystal Growth & Design 2015, 15, (3), 1273-1279. (7) Hou, Z.; Yang, P.; Lian, H.; Wang, L.; Zhang, C.; Li, C.; Chai, R.; Cheng, Z.; Lin, J., Multifunctional hydroxyapatite nanofibers and microbelts as drug carriers. Chemistry–A European Journal 2009, 15, (28), 6973-6982. (8) Wu, F.; Lin, D. D.; Chang, J. H.; Fischbach, C.; Estroff, L. A.; Gourdon, D., Effect of the materials properties of hydroxyapatite nanoparticles on fibronectin deposition and conformation. Crystal Growth & Design 2015.

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(9) Yang, Y. S.; Wu, Q. Z.; Wang, M.; Long, J.; Mao, Z.; Chen, X. H., Hydrothermal Synthesis of Hydroxyapatite with Different Morphologies: Influence of Supersaturation of the Reaction System. Crystal Growth & Design 2014, 14, (9), 4864-4871. (10) Jevtic, M.; Mitric, M.; Skapin, S.; Jancar, B.; Ignjatovic, N.; Uskokovic, D., Crystal structure of hydroxyapatite nanorods synthesized by sonochemical homogeneous precipitation. Crystal Growth & Design 2008, 8, (7), 2217-2222. (11) Cui, W. G.; Li, X. H.; Chen, J. G.; Zhou, S. B.; Weng, J., In Situ Growth Kinetics of Hydroxyapatite on Electrospun Poly(DL-lactide) Fibers with Gelatin Grafted. Crystal Growth & Design 2008, 8, (12), 4576-4582. (12) Zhou, H.; Lee, J., Nanoscale hydroxyapatite particles for bone tissue engineering. Acta Biomater 2011, 7, (7), 2769-2781. (13) Zhou, Y. Y.; Yao, H. C.; Wang, J. S.; Wang, D. L.; Liu, Q.; Li, Z. J., Greener synthesis of electrospun collagen/hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering. Int J Nanomed 2015, 10, 3203-3215. (14) Pramanik, N.; Imae, T., Fabrication and Characterization of Dendrimer-Functionalized Mesoporous Hydroxyapatite. Langmuir 2012, 28, (39), 14018-14027. (15) He, Q. J.; Pan, L. M.; Wang, Y. W.; Meldrum, F. C., Bioinspired Synthesis of Large-Pore, Mesoporous Hydroxyapatite Nanocrystals for the Controlled Release of Large Pharmaceutics. Crystal Growth & Design 2015, 15, (2), 723-731. (16) Neira, I. S.; Kolen'ko, Y. V.; Lebedev, O. I.; Van Tendeloo, G.; Gupta, H. S.; Guitian, F.; Yoshimura, M., An Effective Morphology Control of Hydroxyapatite Crystals via Hydrothermal Synthesis. Crystal Growth & Design 2009, 9, (1), 466-474. (17) Zhang, C. M.; Yang, J.; Quan, Z. W.; Yang, P. P.; Li, C. X.; Hou, Z. Y.; Lin, J., Hydroxyapatite Nano- and Microcrystals with Multiform Morphologies: Controllable Synthesis and Luminescence Properties. Crystal Growth & Design 2009, 9, (6), 2725-2733. (18) Cui, W. G.; Li, X. H.; Xie, C. Y.; Zhuang, H. H.; Zhou, S. B.; Weng, J., Hydroxyapatite nucleation and growth mechanism on electrospun fibers functionalized with different chemical groups and their combinations. Biomaterials 2010, 31, (17), 4620-4629. (19) Toworfe, G. K.; Composto, R. J.; Shapiro, I. M.; Ducheyne, P., Nucleation and growth of calcium phosphate on amine-, carboxyl- and hydroxyl-silane self-assembled monolayers. Biomaterials 2006, 27, (4), 631-642. (20) Khan, A. S.; Ahmed, Z.; Edirisinghe, M. J.; Wong, F. S. L.; Rehman, I. U., Preparation and characterization of a novel bioactive restorative composite based on covalently coupled polyurethanenanohydroxyapatite fibres. Acta Biomater 2008, 4, (5), 1275-1287. (21) Ramay, H. R. R.; Zhang, M., Biphasic calcium phosphate nanocomposite porous scaffolds for load-bearing bone tissue engineering. Biomaterials 2004, 25, (21), 5171-5180. (22) Choi, H. W.; Lee, H. J.; Kim, K. J.; Kim, H. M.; Lee, S. C., Surface modification of hydroxyapatite nanocrystals by grafting polymers containing phosphonic acid groups. J Colloid Interf Sci 2006, 304, (1), 277-281. (23) Liu, Y. K.; Hou, D. D.; Wang, G. H., A simple wet chemical synthesis and characterization of hydroxyapatite nanorods. Mater Chem Phys 2004, 86, (1), 69-73. (24) Jamwal, D.; Kaur, G.; Raizada, P.; Singh, P.; Pathak, D.; Thakur, P., Twin-Tail Surfactant Peculiarity in Superficial Fabrication of Semiconductor Quantum Dots: Toward Structural, Optical, and Electrical Features. J Phys Chem C 2015, 119, (9), 5062-5073. (25) Selvakumar, M.; Mahendran, A.; Bhagabati, P.; Anandhan, S., Thermodynamic Miscibility and Thermal and Mechanical Properties of Poly(ethylene-co-vinyl acetate-co-carbon monoxide)/Poly(vinyl chloride) Blends. Adv Polym Tech 2015, 34, (1). (26) Kumar, G. S.; Govindan, R.; Girija, E. K., In situ synthesis, characterization and in vitro studies of ciprofloxacin loaded hydroxyapatite nanoparticles for the treatment of osteomyelitis. J Mater Chem B 2014, 2, (31), 5052-5060.

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(27) Teng, S. H.; Shi, J. J.; Chen, L. J., A novel method to synthesize large-sized hydroxyapatite rods. J Cryst Growth 2006, 290, (2), 683-688. (28) Klinkamnarong, J.; Swatsitang, E.; Maensiri, S., Nanocrystalline hydroxyapatite powders by a chitosan-polymer complex solution route: Synthesis and characterization. Solid State Sci 2009, 11, (5), 1023-1027. (29) Martins, M. A.; Santos, C.; Almeida, M. M.; Costa, M. E. V., Hydroxyapatite micro- and nanoparticles: Nucleation and growth mechanisms in the presence of citrate species. J Colloid Interf Sci 2008, 318, (2), 210-216. (30) Roy, N.; Bhowmick, A. K., Tailor-Made Fibrous Nanohydroxyapatite/Polydimethylsiloxane Composites: Excavating the Role of Nanofiller Aspect Ratio, Amorphicity, and Noncovalent Surface Interaction. J Phys Chem C 2012, 116, (15), 8763-8772. (31) Roy, N.; Bhowmick, A. K., Tailor-Made Fibrous Hydroxyapatite/Polydimethylsiloxane Composites: Insight into the Kinetics of Polymerization in the Presence of Filler and Structure-Property Relationship. J Phys Chem C 2012, 116, (50), 26551-26560. (32) Huang, F. Z.; Shen, Y. H.; Xie, A. J.; Zhu, J. M.; Zhang, C. Y.; Li, S. K.; Zhu, J., Study on synthesis and properties of hydroxyapatite nanorods and its complex containing biopolymer. J Mater Sci 2007, 42, (20), 8599-8605. (33) Zhu, J. J.; Xu, S.; Wang, H.; Zhu, J. M.; Chen, H. Y., Sonochemical synthesis of CdSe hollow spherical assemblies via an in-situ template route. Adv Mater 2003, 15, (2), 156-+. (34) Wang, S. F.; Gu, F.; Lu, M. K., Sonochemical synthesis of hollow PbS nanospheres. Langmuir 2006, 22, (1), 398-401. (35) Liang, T.; Qian, J. C.; Yuan, Y.; Liu, C. S., Synthesis of mesoporous hydroxyapatite nanoparticles using a template-free sonochemistry-assisted microwave method. J Mater Sci 2013, 48, (15), 5334-5341. (36) Lin, K. L.; Wu, C. T.; Chang, J., Advances in synthesis of calcium phosphate crystals with controlled size and shape. Acta Biomater 2014, 10, (10), 4071-4102. (37) Liu, J.; Liu, F.; Gao, K.; Wu, J. S.; Xue, D. F., Recent developments in the chemical synthesis of inorganic porous capsules. J Mater Chem 2009, 19, (34), 6073-6084. (38) Prouzet, E.; Cot, F.; Boissiere, C.; Kooyman, P. J.; Larbot, A., Nanometric hollow spheres made of MSU-X-type mesoporous silica. J Mater Chem 2002, 12, (5), 1553-1556. (39) Peng, Q.; Dong, Y. J.; Li, Y. D., ZnSe semiconductor hollow microspheres. Angew Chem Int Edit 2003, 42, (26), 3027-3030. (40) Devanand Venkatasubbu, G.; Ramasamy, S.; Ramakrishnan, V.; Kumar, J., Nanocrystalline hydroxyapatite and zinc-doped hydroxyapatite as carrier material for controlled delivery of ciprofloxacin. 3 Biotech 2011, 1, (3), 173-186. (41) Matsunaga, K.; Murata, H.; Mizoguchi, T.; Nakahira, A., Mechanism of incorporation of zinc into hydroxyapatite. Acta Biomater 2010, 6, (6), 2289-2293. (42) Nandi, S. K.; Mukherjee, P.; Roy, S.; Kundu, B.; De, D. K.; Basu, D., Local antibiotic delivery systems for the treatment of osteomyelitis - A review. Mat Sci Eng C-Mater 2009, 29, (8), 2478-2485. (43) Yolles, S., Controlled Release of Biologically-Active Agents. Acta Pharm Suec 1976, 13, 32-32. (44) Venkatasubbu, G. D.; Ramasamy, S.; Ramakrishnan, V.; Kumar, J., Nanocrystalline hydroxyapatite and zinc-doped hydroxyapatite as carrier material for controlled delivery of ciprofloxacin. 3 Biotech 2011, 1, (3), 173-186. (45) Dalhoff, A., Antiviral, antifungal, and antiparasitic activities of fluoroquinolones optimized for treatment of bacterial infections: a puzzling paradox or a logical consequence of their mode of action? Eur J Clin Microbiol 2015, 34, (4), 661-668. (46) Hooper, D. C., Mode of action of fluoroquinolones. Drugs 1999, 58, 6-10. (47) Schaechter, M., A paean to Microbiology and molecular biology reviews. Microbiol Mol Biol R 1999, 63, (2), 265-+.

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(48) Li, J. H.; Kuang, D. Z.; Feng, Y. L.; Zhang, F. X.; Liu, M. Q., Glucose biosensor based on glucose oxidase immobilized on a nanofilm composed of mesoporous hydroxyapatite, titanium dioxide, and modified with multi-walled carbon nanotubes. Microchim Acta 2012, 176, (1-2), 73-80. (49) Ostomel, T. A.; Shi, Q. H.; Tsung, C. K.; Liang, H. J.; Stucky, G. D., Spherical bioactive glass with enhanced rates of hydroxyapatite deposition and hemostatic activity. Small 2006, 2, (11), 1261-1265. (50) Ostomel, T. A.; Shi, Q. H.; Stucky, G. D., Oxide hemostatic activity. J Am Chem Soc 2006, 128, (26), 8384-8385. (51) Dobrovoiskaia, M. A.; Clogston, J. D.; Neun, B. W.; Hall, J. B.; Patri, A. K.; McNeil, S. E., Method for analysis of nanoparticle hemolytic properties in vitro. Nano Lett 2008, 8, (8), 2180-2187. (52) Yu, T.; Malugin, A.; Ghandehari, H., Impact of Silica Nanoparticle Design on Cellular Toxicity and Hemolytic Activity. Acs Nano 2011, 5, (7), 5717-5728. (53) Asharani, P. V.; Sethu, S.; Vadukumpully, S.; Zhong, S. P.; Lim, C. T.; Hande, M. P.; Valiyaveettil, S., Investigations on the Structural Damage in Human Erythrocytes Exposed to Silver, Gold, and Platinum Nanoparticles. Adv Funct Mater 2010, 20, (8), 1233-1242. (54) Rejinold, N. S.; Muthunarayanan, M.; Divyarani, V. V.; Sreerekha, P. R.; Chennazhi, K. P.; Nair, S. V.; Tamura, H.; Jayakumar, R., Curcumin-loaded biocompatible thermoresponsive polymeric nanoparticles for cancer drug delivery. J Colloid Interf Sci 2011, 360, (1), 39-51.

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Crystal Growth & Design

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Structurally Tuned Antimicrobial Mesoporous Hydroxyapatite Nanorods by Cyclic Oligosaccharides Regulation to Release of Drug for Osteomyelitis M. Selvakumar, P. Senthil Kumar, Bodhisatwa Das, Santanu Dhara, and Santanu Chattopadhyay

Synopsis At low temperature, structurally tuned hydroxyapatite nanorod with high aspect ratio (~13.1) and its mesoporous frame with tailored pores were fabricated by cyclic oligosaccharides regulated using combined modified co-precipitation method and sonochemistry. This unique nanorods exhibit remarkably high payload of drug and it can be potential candidate for osteomyelitis disease cure in bone tissue engineering applications.

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Crystal Growth & Design

Structurally Tuned Antimicrobial Mesoporous Hydroxyapatite Frameworks by β–Cyclodextrin Regulation for Drug Release 265x202mm (96 x 96 DPI)

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