Biophysical approach to the effect of liposomes encapsulated chloramphenicol on lenticular changes induced by bacterial endophthalmitis. In-vivo study.
Sherif S. Mahmoud* and Hesham A. Rahman
Biophysics* and Physiological optics departments, Research Institute of Ophthalmology.
2 El-Ahram Street, Giza, Egypt.
Bacterial endophthalmitis is one of the most serious infections of the eye which may results in complete visual loss if not treated properly. Staphylococcous aurous (staph. a.), is the most causative bacteria for endophthalmitis. The present study aims to investigate the biophysical changes associated with staph. a. induced endophthalmitis and, its treatment with liposomal chloramphenicol against the traditional treatment with conventional form; considering the time as an important factor in the severity of damage. The lipoidal data indicated that lens fibers suffered from changes in their stability and fluidity; this was associated to changes in total protein concentration. It can be concluded that liposomal chloramphenicol did not enhance the biophysical changes induced in rat lenses by endophthalmitis when compared with the conventional form; unless the treatment starts at 2 hours postinfection or before the surgical operation.
Introduction
Mycotic eye infection is an important and serious condition which generally has poor visual prognosis. Faliure to treat it early may lead to deeper penetration and loss of the eye (1). Staphylococci – normal inhabitants of the body surface of man and animals – are among the most important bacteria which cause disease in humans(2). Endophthalmitis is a postoperation complication of phacoemulsification surgery. It is the result of staphylococcus aureus (staph. a.) infection (3). This bacteria – generally – produce two forms of disease; acute inflamation and acute toxaemia (4). Staph.a. produces toxins, these toxins contribute to severity of endophthamitis by accelerating the severity of retinal damage (5). Postoperative endophthalmitis occurs with an incidence of 0.05 to 0.33 % ( 6 ).
Antibiotic chloramphenicol (CAP), is one of the most effective drugs for the treatment of intraocular infections. It has a broad antimicrobial spectrum, being effective against wide varity of gram-positive and gram-negative organisms, and has good penetration characteristics for ocular tissues(7). The present work aimed to investigate the efficiency of liposomal encapsulated CAP against the free drug (conventional form) for the treatment of staph.a. infected eye; monitored by the biophysical changes that take place in lenses.
Materials and Methods
Staphylococcus aureus –isolated from patients- was cultured on agar, then preserved in 0.9 % normal saline solution. The bacteria in this case (salinated staph.a.) is impotent. Activation was achieved by culturing on normal browth (active staph.a. i.e pathogenic).
Albino rats – Rattus norvegicus – weighing 200 – 300 g were used in this study. Experimental endophthalmitis was established by intravitreal injection of rat eyes with pathogenic staph.a. (104 cfu/20 ml saline) then, rats were categorized into five groups (I – V); each group composed of 10 rats. For all groups the right eyes received liposomes encapsulated CAP while, the left ones received free CAP. Group I is the staph.a. infected group without any antibiotic treatments. On the other hand, group II received the antibiotic treatment just before infection with staph.a. ( this group mimic the surgical situation). The other groups III, IV and V received antibiotic treatment 2, 24 and 48 hours postinfection and, this treatment repeated once daily (20 mg CAP whether encapsulated or not) via subconjunctival injection for two weeks. Rat eyes were removed and the previously weighed lenses were homogenized separatley in one normal sodium hydroxide (1 N NaOH). Total protein was estimated in lens homogenates using the procedure of Lowry et al. (1951)(8). The rest of the homogenates were subjected to lipid extraction and purification according to the procedure of Folch et al. (1954)(9). Total cholesterol as well as total phospholipids were estimated in the purified lipids according to Zigman et al. (1984)(10) and Broekhyse (1968) (11) respectively.
Finally, liposomes encapsulated CAP were prepared as previously described by Bangham et al. (1970) (12). To compare the obtained data; normal rats (five rats=10 eyes) were involved. Student’s “t” test was used to evaluate the obtained results.
Results
Preliminary experiments on the absorption characteristics of antibiotic CAP ( in 1 N NaOH) show that CAP has two absorption maxima at 220 nm and 280 nm.
On the other hand, lens proteins (in 1 N NaOH) also has two absorption maxima at 220 nm and 280 nm. Due to these overlapped bands, and in order to understand the interaction of CAP – whether encapsulated or not – with staph.a. and proteins it was necessary to investigate spectrophotometrically all these possible interactions. These experiments are summarized in figure (1-a). Liposomes encapsulated with CAP studied separatly or incubated with impotent staph.a. was characterized by absorption maximum at 220 nm. Free CAP when incubated with impotent staph.a. shows two bands – as the free CAP incubated alone – at 220 nm and 280 nm. Impotent staph.a. shows one absorption maximum at 192 nm.
Further experiments (figure 1-b) indicate that pathogenic staph.a. when incubated with either empty liposomes or encapsulated with CAP was characterized by one absorption maximum at 226-228 nm region, but differ in its magnitude. The interaction between liposomal CAP and pathogenic staph.a. was charaterized by lower magnitude relative to that with empty liposomes. Therefore the absorption band at 226-228 nm region reflects the viability of staph.a. On the other hand, when pathogenic staph.a. incubated with free CAP, the interaction pattern was completely different. There was two absorption bands; one at the wavelength interval 192-214 nm with lmax at 200 nm and the other at ≈315nm. In addition to absorption shoulder at 280 nm due to CAP. This implies that free CAP interact positively with pathogenic staph. a. and resulted in inactive bacteria (band at lmax=200 nm) and another interaction complex at ≈315 nm.
According to the formentioned results figure 2 (a-e) reflects the pathogenesity of staph.a. as determined in different studied groups. It is clear that the infected group (fig.2a) characterized by highly pathogenic staph.a. (O.D.=1.18). In group II which mimic the surgical situation (fig.2b); free CAP found to be markedly effective when compared with encapsulated one. When infected rat eyes treated with CAP whether encapsulated or not after 2 hours, both forms have the same effect (fig.2c). Liposomal CAP was found to be more effective than free CAP in case of starting treatment after 24 hours post-infection (fig.2d). On the other hand, free CAP was significantly effective than encapsulated one when the treatment starts after 48 hours post-infection (fig.2e) and, this is due to the sustain release of CAP from liposomes while free CAP is more effective because of its direct interaction with staph.a.
Figure (3) illustrates the total protein estimated in rat lenses of all groups. It is clear that the left eyes which received free CAP of all groups were characterized by significant decrease in their protein content as compared with the normal. On the other hand, the right eyes (treated with Liposomal CAP) showed significant increased protein content except for that of group II.
The typical absorption spectra of lens lipids prepared from all studied groups
(Fig. 4) indicate the existence of absorption maximum at 230 nm region resulted
from the diene conjugated structures – primary lipid peroxidation products (1ry LPO),
it is found in lenses received both treatments; liposomal CAP and free CAP. There is
another peak at 274 nm corresponds to triene conjugates – secondary molecular lipid
peroxidation products ( 2nd LPO). The peak at 206 nm region corresponds to absorption
of isolated double bonds of hydrocarbon chains. The absorption peak at 230 nm was
virtually absent in lipids extracted from normal lenses and that one at 274 nm had
significantly smaller amplitude relative to lipids from other groups.
The estimated lipid peroxidation products are summarized in figure (5). The right eyes
of groups II and III were characterized by decreased LPO – whether primary or secondary –
relative to the left ones. For group IV the right eyes showed increased ratio of 1 ry as well as
2 nd LPO relative to left ones. In group V the observed changes were increased 1 ry LPO
associated with decreased 2 nd LPO in the liposomal CAP treated eyes than that in free
CAP treated ones.
Total phospholipids concentration (figure 6) was significantly higher (P<0.001) in all groups relative to the normal one. While, the cholesterol concentration (figure 7) was subjected to fluctuated changes among the studied groups; significantly increased in pre-infected group (p<0.001), after 2 hours (p<0.05) and 48 hours groups (p<0.001) and, significantly decreased (p<0.001) in the infected group as well as after 24 hours group.
Discussion
Liposome-entrapped drugs exhibit superior pharmacological properties to these observed with conventional formulations(13). Reduced dosage, allergic and immunmological reactions, increased cellular permeabilities and delayed drug eliminations represent some of the advantages of using liposomes as drug delivery agents(14). A major advantage of applying liposomes in the eye is their ability to evade initially the mononuclear phagocytes that make the intravenous use of liposomes so challenging. The treatment with Liposomal CAP was associated with increased protein content when compared with free CAP. This gives the imptus to speculate that staph.a. utilize the liposomes themselves as nutritional source so, the metabolic rate increased and resulted in increased protein content. Infection with staph.a. leads to decreased stability of lens fibers as indicated by cholesterol concentrations in infected group (figure 7). The lens try to maintain its normal function, therfore, the increased phospholipids and/or cholesterol seems to buffer the changes induced by staph.a.; this is applied for both types of treatments. If the increase in cholesterol and phospholipids concentrations was due to increased metabolic rate of staph.a. their estimated values should show some homogenity, but this is not the situation, which support the idea that liposomal CAP increase the stability of lenses in case of group II (surgical mimic ), and after 2 hours post-infection to enable the lens performing normal function.
The obtained data clearly indicate that both types of antibiotic has its own benefits as well as its drawbacks. So, it can be concluded that the use of liposomal CAP should be before operating the surgery or maximally after 2 hours post-infection otherwise, free CAP (conventional) is more effective.
Acknowledgement
The authors would like to thank Prof. Nadia Soliman head of microbiology department, Research Institute of Ophthalmology, Giza, Egypt for the assistance with the microbiological facility.
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