# The Kinetics of Ampicillin Release from Hydroxyapatite  for  Bones Regeneration

Authors: Giovanilton Ferreira da Silva; José Sílvio Veras Albuquerque; Caroliny Gomes de Oliveira; Ricardo Emilio Ferreira Quevedo Nogueira; Andrea Lopes de Oliveira Ferreira
Journal: International Journal of Chemical Engineering
 (2009)

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## Abstract

Semisynthetic beta-lactam antibiotics are among the most used pharmaceuticals. Their use in veterinary and human medicine is in continuous expansion. There is a growing need for developing bioactive implants. Advantages of implantable drug delivery tools can include high release efficiency, precise dose control, low toxicity, and allow to overcome disadvantages connected with conventional methods. In this respect, hydroxyapatite (HA) is an elective material. It enables to produce architectures similar to those of real bones. Here we studied a kinetic model to describe ampicillin release from HA. In the course of adsorption experiment, ampicillin was dissolved, maintained at30∘C and shaken at 60 strokes/minute. Samples were withdrawn periodically for analysis and then returned to the mixture. Adsorbed amounts were measured by the difference of the concentration of the antibiotics before and after adsorption using UV adsorption at 225 nm. The aim of this work was to evaluate its application as ampicillin delivery carrier.


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## Body

## 1. Introdution

Semisynthetic beta-lactam antibiotics are the most important
class of antibacterial agents. Their use in veterinary and human medicine is in
continuous expansion. Some examples of semisynthetic penicillins and
cephalosporins are amoxicillin, ampicillin, cephalexin, cefadroxil, and cefazolin,
among many others. They have in common the presence of the beta-lactam ring,
responsible for their antimicrobial activity. They irreversibly inhibit the
last step of the bacterial cell wall biosynthesis. The beta-lactam antibiotics
can be described in terms of a beta-lactam nucleus with a side-chain (Figure1).Figure 1
General structure of beta-lactam antibiotics.Many different nuclei and side-chains are found in the
antibiotics that are in use today. Different combinations of side-chains and
nuclei form antibiotics with distinctive properties; for example, replacing the
phenylacetic acid side-chain of penicillin G with D-phenylglycine (PG) results
in the beta-lactam antibiotic ampicillin (Figure2), which in contrast to
penicillin G, is orally stable [1–4]. Ampicillin (6-[2-amino-2-phenylacetamide]
penicillanic acid) is in the penicillin group of which penicillin proper was
the first antibiotic to be used in therapy [5, 6]. It is one of most widely used
semisynthetic beta-lactam antibiotics [7]. It has an estimated market of
20000 ton/year [5].Figure 2
Structure of ampicillin, in highlighted: beta-lactam ring.There is a growing need for developing bioactive implants, due
biomaterials are biocompatible, resorbable, and present osteoconductive
properties. It is known that the use of bone substances has many inherent
disadvantages in practical applications, and it is linked to many surgical
problems [8].Advantages of implantable drug delivery tools can include high
release efficiency, precise dose control, low toxicity, and allow to overcome
disadvantages connected with conventional methods [9]. In this respect,
hydroxyapatite (HA) is an elective material. It enables to produce
architectures similar to those of real bones. Here we studied a kinetic model
to describe ampicillin release from HA. HA analogous to the mineral component
of bones, its properties make it  desirable as implant materials and delivery
agents of drugs. This paper describes the ampicillin adsorption and release
profiles of HA material. The aim of this work was to evaluate its application
as ampicillin delivery carrier.


## 2. Materials and Methods

## 2.1. Materials

Ampicillin was from Aldrich Chem. Co., USA. All other
chemicals were of laboratory grade from different commercial suppliers.


## 2.2. Antibiotic Loaded HA Samples

Ampicillin was used as drug molecules. HA were impregnated
with 25 mM of antibiotic buffer solution at30°C for 48 hours. Ampicillin adsorbed in the HA has
been quantified by spectophotometric analysis. In the course of the release
experiment, ampicillin was dissolved in phosphate buffer to make a stock
solution, maintained at 30°C and shaken at 60 strokes/minute. Kinetic
experiments to determine the amount of ampicillin adsorbed as a function of
contact time were conducted by stirring. Samples were withdrawn periodically
for analysis and then returned to the mixture. Adsorbed amounts were measured
by the difference of the concentration of the antibiotics before and after adsorption
using UV adsorption at 215 nm.


## 2.3. HA Synthesis

HA was synthesized by the aqueous precipitation method from
CaO andH3PO4 as the reagents, and used as a carrier for
charging ampicillin. In this study, apatite nanoparticles were produced by
aqueous precipitation. The starting solution was 0.3 M H3PO4,
0.5 M Ca(OH)2, and 1 M CH3CHCO2HOH. The pH value of the
solution was adjusted to pH 8 by NH4OH additon. The suspensions were
left overnight for ageing. The suspension was then vacuum filtered and washed
in deionised water to remove NH4OH. The powders were dried in an
oven at 100°C overnight. HA powders and carnauba wax were synthesized in stirred
50°C. HA
powders were then synthesize by means of uniaxial pressing (40 MPa) and
convenient thermal treatments. The pellets were heated to 550°C to remove all wax.
Pellets of cilinder shape were produced with size of 19 mm of diameter and 19 mm of length at 1350°C The powders were analyzed
by X-ray diffraction (XRD) and then sintered. Sintered powders were again analyzed
by XRD and scanning electronic  microscopy (SEM) to assess the final phase
composition [10]. Figures 3 and 4 show XRD and SEM of HA, porous of 100 ~ 300 μm were
obtained.Figure 3
X-ray diffraction of HA pellet synthesized at1350°C.Figure 4
Scanning electron micrographs of a sample HA
particle, after synthesis assay.


## 2.4. Solubility Experiments

Solubility of ampicillin was determined following Gude et al.
[11]. The samples were prepared gravimetrically. Glass-flasks with screw caps
filled with the samples were immersed into a thermostated water bath and
stirred. All samples were stirred for at least 4  hours. Subsequently, the mixture
was allowed to settle. The samples were taken with syringes with an attached
0.2 μm filter to avoid entrainment of solids. The
compositions of the liquid phases were analyzed by HPLC.


## 2.5. Analysis

Concentrations of ampicillin were determined using HPLC to
analyze if antibiotic degraded during the assays: C18 column (Waters Nova-Pack,
C18, 60 Å, 4μm, 3.9 × 150 mm); eluent: 35% acetonitrile,  2‰   SDS
(lauryl sodium sulphate), 10 mM H3PO4, 5 mM K2H2PO4, with a flow of 1 mL/min at 25°C and λ=225 nm.


## 2.6. Adsorption Performance

Ampicillin adsorption performance(AP) was defined as follows: (1)%AP=(1−C*Cinitial)×100,  where C* is the
concentration of ampicillin (mM), and Cinitial is the concentration of ampicillin at the beginning of the assays (mM).


## 3. Results and Discussion

## 3.1. Solubility Studies

The solubility of ampicillin was measured for pHs in the range
7.8–8.0, at30°C. The selected range of pHs for the solubility studies was
bracketed by stability of antibiotic. The obtained results, which are shown in
Figure 5, are similar to the ones obtained by other authors [12–14].Figure 5
Solubility of ampicillin as function of pH at30°C.Ampicillin solubility increases with the pH. This behavior can
be explained by its determined values of the acid group pK (2.66) and amine
group pK (7.24) and calculated of its isoelectric point (4.95). Hence, above
the pH correspondent to its isoelectric point the number of ampicillin molecules
with a neutral charge (which is the most insoluble form) decreases, leading to
higher solubility values. This effect becomes more important for pH above 7.0.Our aim is to perform the ampicillin adsorption with its separation
from HA at37°C, what implies to working at sorption conditions where the
solubility of ampicillin (AMP) is the highest possible. Therefore, the obtained
results indicate that for higher pH, the performance of the AMP release might be
better. However, the best value for pH is a tradeoff between AMP solubility
and temperature.Temperature effects on the solubility of ampicillin were also
evaluated by determining the compounds solubility at pH 7.0 and 8.0, at37°C.
The antibiotic solubility slightly increases at 37°C. Most of the solubility
values at 37°C were around 11% higher than those at 30°C. However, operation of
the system at 37°C implies too much lower adsorption rates than at 30°C. It is
believed that an increase of 11% in the ampicillin solubility is not big enough
to justify the operation at such a high temperature. Anyway, these experiments
aimed at only indicating the range of adsorption conditions for studying the kinetic
of the process.


## 3.2. Equilibrium Time

In order to properly evaluate the adsorption process of
ampicillin, the equilibrium curves were determined experimentally. Equilibrium
time depends on adsorption rate, that is, affinity between antibiotic and HA, and
temperature of assay. Figure6 shows the results to ampicillin adsorption at 30
and 37°C, respectively. It can be observed that equilibrium state was obtained
at 8 hours. At 37°C, adsorption rate higher than at 30°C can be explained due
to increasing of mass transfer, because adsorption is not favor at higher
temperatures.Figure 6
Equilibrium assays atCinitial=25 mM, phosphate butter 10 mM , pH 7.5.


## 3.3. pH Effect at Adsorption Isotherms

Figure7 shows the effect of pH at adsorption performance of
ampicillin. It can be observed that AP decreases when pH increases. The amino group of ampicillin is not protoned when
pH increases, so less antibiotic can be adsorbed on HA. Therefore, ampicillin
adsorption slightly improves when pH decreases.Figure 7
pH effect on adsorption ampicillin at30°C.Adsorption isotherms of
ampicillin on HA at30°C and pH 7.0 were shown in Figure 8. The adsorption
isotherms were further correlated to Linear, Freudlich, and Langmuir equations:
(2)q=KlinC,q=KFrCn,q=qmCKLan+C,      where q is the equilibrium adsorption capacity (mg/gHA), C is the initial concentration of
ampicillin (mg/mL), Klin, KFr, and  KLan are Linear,
Freundlich, and Langmuir parameters,  respectively. n is the Freundlich parameter related to the magnitude of
adsorption driving force. qm is the maximum adsorption capacity on HA.
Some assumptions were made to adsorption model: the process operated under
isothermal conditions, HA porosity was constant and homogeneous along as the
particle.Figure 8
Adsorption isotherms of ampicillin on HA at30°C, pH 7.0.The data plotted were fitted
according to the Linear isotherms:Klin=18.46±1.85 and R2=0.958. The Freundlich isotherms were  KFr=52.51±3.48,  n=0.5688±0.036, and R2=0.980, and Langmuir
isotherms were qm=274.05±8.92,  KLan=4.99±0.34, and R2=0.998. The n value of Freundlich
isotherm shows that adsorption process is favorable (n<1) at 30°C. All parameters for the adsorption of ampicillin
were obtained using the nonlinear least squares algorithm of Marquardt [15],
with 95% confidence interval for the parameter estimates.In the process of analysis of
the models, as Freundlich isotherm and as Langmuir isotherm can represent the
isotherm data of HA appropriately in the given concentration range. The Linear
isotherm just presented good fitting at lower concentration. Note that a
constant exist instead of zero in Figure6, which is caused due to higher
concentration of ampicillin. The best fit was obtained when Langmuir model (R2=0.998) was used,
although Freundlich model was also reached good fit.


## 4. Conclusions

Ampicillin solubility was studied at different pH. Higher pH
improved ampicillin solubility. The equilibrium state was obtained at  8 hour of
assay. At37°C, adsorption rate was higher than at 30°C. The best results of
adsorption performance were obtained when pH decreased (7.0). The relation between the adsorption
capacity and the equilibrium solute concentration was analyzed. Linear,
Freundlich, and Langmuir isotherms were used and provided good fit for data.
The best model was achieved with Langmuir isotherm.



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*Source: 273808-2009-04-15.xml*