Effect of particle size of calcium phosphate based bioceramic drug ...

Front. Mater. Sci. 2013, 7(3): 261–268 DOI 10.1007/s11706-013-0216-6

RESEARCH ARTICLE

Effect of particle size of calcium phosphate based bioceramic drug delivery carrier on the release kinetics of ciprofloxacin hydrochloride: an in vitro study Swamiappan SASIKUMAR (✉) Materials Chemistry Division, Centre for Excellence in Nanomaterials, School of Advanced Sciences, VIT University, Vellore-632 014, Tamilnadu, India

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2013

ABSTRACT: Hydroxyapatite (HAP) is the constituent of calcium phosphate based bone cement and it is extensively used as a bone substitute and drug delivery vehicle in various biomedical applications. In the present study we investigated the release kinetics of ciprofloxacin loaded HAP and analyzed its ability to function as a targeted and sustained release drug carrier. Synthesis of HAP was carried out by combustion method using tartaric acid as a fuel and nitric acid as an oxidizer. Powder XRD and FTIR techniques were employed to characterize the phase purity of the drug carrier and to verify the chemical interaction between the drug and carrier. The synthesized powders were sieve separated to make two different drug carriers with different particle sizes and the surface topography of the pellets of the drug carrier was imaged by AFM. Surface area and porosity of the drug carrier was carried out using surface area analyzer. The in-vitro drug release kinetics was performed in simulated body fluid, at 37.3°C. The amount of ciprofloxacin released is measured using UV-visible spectroscopy following the characteristic λmax of 278 nm. The release saturates around 450 h which indicates that it can be used as a targeted and sustained release carrier for bone infections. KEYWORDS: ceramic drug delivery system; antibiotic; hydroxyapatite (HAP); porosity; targeted release

1

Introduction

Hydroxyapatite (HAP) is major constituent used in the orthopedic and dental applications because of its structural and chemical similarity to that of human bone. HAP predominates among the class of calcium phosphate bioceramics due to its biocompatibility and osteoconductive characteristics. Hence, HAP is employed as a drug Received June 19, 2013; accepted July 26, 2013 E-mail: [email protected]

delivery carrier both in pure form and as a composite with polymers such as polycaprolactone (PCL) for various drug molecules [1]. Drug delivery systems using bioceramics enhance the bone in growth and regeneration of bone tissues during treatment of bone defects and bone infection [2]. Conventional routes of administration of drugs are not effective due to the poor blood circulation in skeletal tissues and limited penetration of the drug into the bone [3]. Hence, the concentration of many drugs falls below the therapeutic levels in skeletal tissue if the amount of drug is not

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sufficient. As a result, high dosage of the drug is required which may be harmful to the biological system due to the possible toxic side effects as a result of high dosage. Hence local antibiotic delivery systems, which allow the therapeutic agents to be targeted to the disease site with minimum systemic effects, are preferred over conventional routes of drug administration [4]. Non biodegradable drug delivery system like polymethylmethacrylate (PMMA) has been explored as a drug delivery carrier in the form of beads, but it has a drawback of removal of beads after the complete or optimal release of drug which needs two-stage operative procedure which is cumbersome [5]. Hence, as an alternative to non biodegradable polymeric drug delivery carriers a composite of collagen and polylactic acid (PLA) was developed as biodegradable delivery system which was proved to have some clinical success [6]. However, they do not have the ability to replace bone grafting. Most of the biomaterials implanted as an artificial bone in the hard tissues do not have the ability to bond with living bone. Biomimetic bioceramics are suited as drug carriers as they possess good biocompatibility and osteointegration. HAP being a well known bioceramic can be considered as a convenient drug delivery carrier wherever structural considerations need to be satisfied. It scores over other ceramics in terms of cost, its facile synthesis with controlled morphology and porosity, its biocompatibility, ostheophilicity and osteoconductivity. The distinct advantage of using bioceramic drug carriers compared to polymeric drug delivery carrier is its wide variety of synthesis methods with ease [7]. Depending on the processing parameters for a given method especially in combustion method one may be able to control the microstructure, morphology and porosity which are crucial for drug release profile. A well-defined bioceramic drug carrier such as HAP in terms of composition, structure with proper pore size distribution and their interconnectivity can be employed as a sustained drug carrier for patients with chronic arthritis, chronic articular rheumatics and other diseases. Drug loading is also easily done on bioceramic carrier and such in vitro studies have been correlated to in vivo study also [8]. The objective of the present work is to synthesize porous drug delivery carrier suitable for the sustained release of antibiotic and to study the influence of particle size on drug release profile by studying the release kinetics of the antibiotic by in-vitro. In the present study, we report for the first time the effect of particle size on the drug release

kinetics profile of an antibiotic loaded to the bioceramic, HAP synthesized by combustion method which is expected to result in porous products with smaller particle size in comparison to other methods [9–10].

2

Experimental

2.1

Synthesis of drug delivery carriers

An equal volume of one molar calcium nitrate solution is added to one molar tartaric acid with thorough mixing using magnetic stirrer. The pH of the solution is adjusted to 9.5 by adding 1∶1 NH4OH and stoichiometric ratio of (NH4)2HPO4 solution is added which results in the formation of precipitate of amorphous calcium phosphate. The precipitate is dissolved by adding con. HNO3 and the pH is adjusted to 1. Resultant solution is heated to 70°C and stirred until the formation of transparent gel. Gel formed is kept in a preheated muffle furnace at 250°C until it undergoes combustion and forms a black coloured precursor. The precursor is calcined at 900°C for 2 h, which results in pure HAP. 2.2

Drug loading

The synthesized HAP powder is sieve separated using 63 μm sieves in a mechanical sieve shaker. Powder collected after the sieving consists of HAP powder with particle size less than 63 μm and (labeled as Sample 1) and the remaining portion of HAP possesses particle size of greater than 63 μm (labeled as Sample 2). 1 mL of 0.1% (w/v) ciprofloxacin hydrochloride solution and 1 mL of 1% (w/v) polyvinyl alcohol (PVA) solution is added to the 100 mg of Sample 1 and allowed to dry at 40°C. The dried sample is pelletized in a die of 10 mm diameter at a pressure of 6 MPa, using a hydraulic press. Similarly the Sample 2 is also loaded with the same quantity of drug and PVA and pelletized with similar dimension but at the pressure of 12 MPa. The pellet with smaller particle in Sample 1 is given lesser compression load, whereas the bigger particle in Sample 2 is given more compression load. At higher load the pores formed by irregular packing of bigger particles will collapse and leads to closer packing, whereas smaller particles will have a better packing than the bigger particles hence lesser load is applied. By increasing the compression load of bigger particles it is assumed to have lesser porosity with closer packing of particles. Samples were designated by codes reflecting the particle

Swamiappan SASIKUMAR. Effect of particle size of calcium phosphate based bioceramic drug delivery carrier ...

dimension and the compression load. The formulation with particle size less than 63 μm and compression load of 6 MPa is denoted as HAP6S63 and the formulation with particle size more than 63 μm and compression load of 12 MPa is denoted HAP12G63. Whereas, the formulation with particle size less than 63 μm and compression load of 12 MPa is denoted as HAP12S63 and the formulation with particle size more than 63 μm and compression load of 6 MPa is denoted HAP6G63. The above protocol conforms to the standard protocol followed for the release of the chemotherapeutic agent rhodium(II) carboxylate loaded onto HAP [11] and the release of gentamicin sulfate by the HAP composite [12]. 2.3

In vitro release studies

For the long-term in vitro release studies, method described elsewhere [13] is slightly modified and used. In a 50-mL Erlenmeyer flask with a ground-glass stopper, containing 50 mL of buffer (simulated body fluid (SBF)) and the pellet was hung and it was surrounded by a water bath with thermostat maintained at (371)°C. At regular time intervals, 5 mL of the buffer medium was collected and the ciprofloxacin concentration was measured by ultraviolet (UV) spectroscopy by following the characteristic lmax peak (278 nm). The content of flask was replenished with 5 mL of fresh buffer after each withdrawl.

of drug released was estimated using Hitachi, U-2800 spectrophotometer, Japan.

4

Characterization

Phase purity of the synthesized HAP sample was checked by Philips D-500 X-ray diffractometer using Ni filtered Cu Kα radiation. The chemical nature and molecular bond structure of the synthesized samples were determined using Fourier transform infrared spectroscopy (FTIR; Thermo Nicolet, Avatar 330 FTIR spectrometer, USA) studies. The surface topography of the pellets was imaged by atomic force microscopy (AFM; easyScan 2, Nanosurf AG, Switzerland). The Brunauer–Emmett–Teller (BET) technique was employed to analyze the specific average surface area, average pore size of the bioceramic product using fully automated BET surface area analyzer (Micromeritics ASAP2020). The surface morphology was imaged using scanning electron microscopy (SEM; Jeol JSM-5600 LV). The particle size was measured using transmission electron microscopy (TEM; Jeol 200 CX). The release study was carried out in Erlenmeyer flask immersed in water bath with thermostat (RAAGA water bath, India). The amount

Results and discussion

Synthesis of HAP was carried out by combustion reaction by using tartaric acid as a fuel and nitric acid as an oxidizer. Tartaric acid reacts with calcium nitrate and forms a complex which prevents the precipitation of calcium ions as a calcium phosphate in the initial stages of synthesis. After the addition of phosphate source and nitric acid by heating the mixture to 70°C tartaric acid forms a gel network with the entrapped calcium ions and nitrate ions in the network. During the decomposition of dried gel in preheated muffle furnace the gel catches fire due to vigorous redox reaction between the fuel and oxidizer and the local temperature attained is very high which aids the formation of well crystalline HAP. Due to the vigorous exothermic redox reaction between the gel and the oxidizer there will be a copious evolution of gaseous products which makes the material to be highly porous. The success of the process is due to an intimate blending among the constituents and its ability to form the product with lesser particle sizes and highly porous bioceramic which is essential for an effective ceramic drug delivery system. 4.1

3

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Powder X-ray diffraction (XRD) and FTIR analysis

The powder XRD pattern (Curve a in Fig. 1) shows the product formed is single phasic crystals in hexagonal structure with the lattice parameter a = 9.41 Å and c = 6.86 Å. The XRD pattern (Curve b in Fig. 1) of the sieve separated HAP with the particle size less than 63 μm did not show any peak broadening effect which indicates either the absence of nanoparticles or may be due to the agglomeration of particles. The FTIR spectrum (Curve a in Fig. 2) of the final powder shows the characteristic peaks correspondingto OH bending vibrations at 633 cm–1 and its stretching vibrations at 3571 cm–1. Bending vibrations of phosphate group is observed at 473, 571 and 601 cm–1 whereas the stretching vibration of the phosphate group is observed at 962, 1044 and 1089 cm–1. FTIR spectra show that the HAP formed is biomimetic as there is a carbonate substitution in the phosphate site of HAP indicated by the weak bands of the carbonate group at 875 and 1456 cm–1. FTIR spectra (Curve b in Fig. 2) of drug loaded bioceramic carrier shows the characteristic absorption bands at 1260 and 1610 cm–1 of ciprofloxacin hydrochloride attributed to

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the stretching vibration of C–F bond and the vibration of phenyl framework conjugated to –COOH, respectively. The values reported for v(C = O)c (c is carboxylic) and the v(C = O)p (p is pyridone) vibrations of ciprofloxacin hydrochloride are 1707 and 1625 cm–1, respectively. By incorporating the ciprofloxacin in HAP matrix the peak got broadened and observed at 1690 and 1620 cm–1.

Fig. 2 FTIR spectra of HAP before loading ciprofloxacin (a) and after loading ciprofloxacin (b).

Fig. 1 XRD patterns of as synthesized single phasic HAP (a) and sieve separated HAP with particle dimension less than 63 μm (b).

4.2

Microscopic analysis

The products were analyzed by microscopic techniques in order to find out the morphology and particle size. The particle size of the drug carrier decides the drug release profile as the bigger particles will have a less packing efficiency hence releases the drug rapidly. Whereas the morphology plays a crucial role as it is already reported that HAP particles with sharp edges have inflammatory response whereas the particle with spherical morphology resolves the inflammation in faster rate [14]. In addition, morphology of the particles decides the effective packing of the crystals in the pellet which affects the diffusion of drugs through the particles hence the particle size and morphology are the significant parameters which control the release kinetics of the drug. The SEM image (Fig. 3) shows the agglomeration of particles and most of them are in submicron range. Particle size is not uniform and due to agglomeration the porosity of the particle is very much reduced which is an advantage in terms of drug delivery kinetics as it will not favour the rapid diffusion of drug which results in the sustained release of drug from the carrier. But different particle size may not favour the

Fig. 3 SEM image of microcrystalline HAP.

uniform and controlled release as the surface with larger particle size will release the drug in a rapid manner whereas the other part of the surface with lesser particle size will release it in sustained manner which may result in an inconsistent release. The TEM image (Fig. 4) indicates the particles are in the range of submicron and highly agglomerated with various pore sizes and pore volumes. Based on the SEM and TEM images the particles were sieve separated into two halves and loaded with the drug and pelletized using hydraulic press and the pellet was subjected for atomic force microscopic analysis to find out the roughness of the pellet surface. AFM images (Fig. 5) confirm the results of SEM and TEM analysis as it indicates the particles are in sub-micron size range and pores are in nano regime. The surface roughness is found to be comparatively less as the surface does not have any


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and macroporous range. The measurement shows an average pore diameter of 50 nm which shows the product is more of mesoporous in nature. As the porosity controls drug delivery kinetics the macroporous region favors the rapid release of drug whereas the mesoporous and microporous regions were expected to release the drug in sustained mode. 4.4

Fig. 4 TEM image of microcrystalline HAP.

coarser particles which are expected to show a better bioactivity [15]. 4.3

Porosity measurement

The pore size distribution curve (Fig. 6) obtained from BET method shows that the pores are in both mesoporous

In vitro study

It was already reported that the composite of βtricalciumphosphate (TCP)–HAP can be used as a drug carrier [16]. In vitro study of the implants composed of calcium phosphates carried out by Castro et al. shows the release rate decreases almost to half the initial concentration with the increase in compression load. Their result is justified as more compression reduces the voids during packing and also makes the elution of drug slower. They observed the implants compressed at higher compression load will disintegrate to a lesser extent because of their denser, more tightly packed matrix. In their study some of the pellets were disintegrated due to the swelling of

Fig. 5 AFM images of microcrystalline HAP.

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Fig. 8 Cumulative release patterns of ciprofloxacin from HAP12G63 pellets and HAP6S63 pellets for the first 5 h.

Fig. 6 Pore size distribution of the microcrystalline HAP.

ciprofloxacin while hydration. In the present study this problem was rectified by taking the ciprofloxacin hydrochloride salt. The percentage of PVA added as a binder plays a vital role in the prevention of disintegration of the pellets. It is observed that when the quantity of PVA added exceeds 10% in the total composition the pellet shows swelling and disintegration whereas very less quantity of PVA also destabilizes the pellet and gets collapsed in the release medium. The results obtained in the present work is found to be different than that of the results obtained by Castro et al. as the release kinetics graph (Figs. 7 and 8) shows rapid elution of the drug for the sample compressed at high pressure. The contradiction may be explained based on the

Fig. 7 Cumulative release patterns of ciprofloxacin from HAP12G63 pellets and HAP6S63 pellets.

three aspects: (i) instead of β-TCP–HAP composite HAP used as a drug carrier; (ii) the effect of compression load; (iii) the effect of particle size. Considering the first possibility, in physiological environment the dissolution of β-TCP is more when compared to HAP hence the composite with more β-TCP is expected to release the drug rapidly than HAP hence the fast release can not be due to the different carrier material as the present system contains only HAP. Considering the second possibility, the effect of compression load can not be reason for rapid release as the bigger particles was compressed at higher load hence the packing of particles will be almost same of the smaller particles compressed at lesser load. The drug release profile (Fig. 9) proves this argument as HAP6G63 shows a fast release with respect to the sustained release of HAP12S63. Further it is confirmed by the collapse of HAP6G63 pellet after the release of

Fig. 9 Cumulative release patterns of ciprofloxacin from HAP6G63 pellets and HAP12S63 pellets for the first 5 h.


tinny air bubbles which shows the packing is not dense. Considering the third possibility, all parameters and experimental conditions of HAP6S63 and HAP12G63 are similar other than the particle size and hence the difference in the release kinetics may be due to the difference in particle size of HAP6S63 and HAP12G63. The microcrystalline HAP6S63 will be closely packed due to lesser particle size and makes the elution of drug slower whereas well crystalline HAP12G63 is not closely packed which makes the elution of drug faster even though high compression load is applied. The porosity is a critical value in the diffusion of a biological active agent from a matrix [17]. Release kinetics of the drug is mainly controlled by the pore dimensions [18–19]. AFM images (Fig. 5) and pore size distribution curve (Fig. 6) confirms that the drug delivery carrier is mesoporous which may have sustained release when compared to macroporous materials. It is confirmed by the release kinetics graph (Fig. 7) which shows the drug release for the period of one month in sustained mode. Figure 7 shows the quantity of drug released in the first few hours is very high and gradually decreases as the time progresses. This may be due to the elution of drug from entire surface in the first few hours and then the drug present in the core of the pellet starts getting released which takes longer time for the diffusion.

BET FTIR HAP PCL PLA PMMA PVA SBF SEM TCP TEM UV XRD

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Brunauer–Emmett–Teller Fourier transform infrared spectroscopy hydroxyapatite polycaprolactone polylactic acid polymethylmethacrylate polyvinyl alcohol simulated body fluid scanning electron microscopy tricalciumphosphate transmission electron microscopy ultraviolet X-ray diffraction

Acknowledgements The author thanks the management of VIT University and DRDO, Grant in aid scheme, Government of India, for financial assistance and Technology Business Incubator, VIT for FTIR measurements. The author expresses his sincere gratitude to Prof. R. Vijayaraghavan, Assistant Director, Centre for Excellence in Nanomaterials, VIT University for his continuous support and motivation. The author is thankful to Materials Research Centre, Indian Institute of Science for TEM measurements.

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5

Conclusions

gentamicin loaded HAP/TCP bone substitute for prophylactic action: in vitro release validation. Journal of Materials Science:

HAP with mesopores and macropores was successfully synthesized and loaded with ciprofloxacin hydrochloride. In vitro release of ciprofloxacin from the pellet was initially very high and later the implant has a lower release rate that sustained for several days and maximum quantity of the drug got released in the period of one month. From the results it is proved that the particle size plays an important role in the drug release kinetics and carrier with bigger particle releases the drug rapidly whereas the carrier with smaller particle size releases the drug in a sustained manner. The result indicates that the microcrystalline HAP with suitable porosity in the form of bone cement or body implant can be used for sustained release of antibiotics as a local drug delivery carrier.

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Abbreviations

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AFM

atomic force microscopy

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