Characterization, pharmacokinetics, in vitro cytotoxicity of oxytetracycline-loaded CaCo₃ nanoparticle and its antibacterial and antibiofilm effects against Corynebacterium pseudotuberculosis isolated from goats
Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis which is one of the most important bacterial diseases of goats causing significant economic losses with high prevalence worldwide. Currently, the treatment of the disease is via the administration of antibiotics co...
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/90478/1/FPV%202020%2010%20IR.pdf http://psasir.upm.edu.my/id/eprint/90478/ |
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| Summary: | Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis
which is one of the most important bacterial diseases of goats causing significant
economic losses with high prevalence worldwide. Currently, the treatment of the
disease is via the administration of antibiotics combined with surgical excision,
flushing and draining of the abscesses. This treatment protocol fails because the
antibiotic does not get to the causative agent which is walled away in pus and excision
further spreads the organism into the environment. For effective therapy, an improved
method of drug delivery is therefore necessary. The main aim of the present study was
to determine the effect of cockle shell derived calcium carbonate aragonite
nanoparticle encapsulated oxytetracycline against Corynebacterium
pseudotuberculosis isolated from goat caseous lymphadenitis. Calcium carbonate
aragonite nanoparticle (CS-CaCO3NP) was synthesized from cockle shell using top
down method and oxytetracycline (OTC) was loaded into it. Characterization to
ensure that the CS-CaCO3NP and OTC-CS-CaCO3NP produced had the desired
properties was done using Zeta analysis, transmission electron microscopy (TEM),
field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD),
Fourier transform infra-red spectroscopy (FTIR) and Brunauer-emmett-teller (BET)
surface area analysis. Then, the in vitro cytotoxicity evaluation of CS-CaCO3NP,
OTC-CS-CaCO3NP and OTC was explored using MTT and Trypan blue assay in
NIH3T3 cells. The antibacterial and antibiofilm effects of CS-CaCO3NP, OTC-CSCaCO3NP
and OTC against C. pseudotuberculosis were also investigated using
minimum inhibitory assay (MIC) and minimum biofilm eradication concentration
(MBEC) assays. The antibacterial mode of action of OTC-CS-CaCO3NP on
planktonic C. pseudotuberculosis was assessed using high resolution transmission
electron microscopy (HR-TEM) while the antibiofilm effect was investigated using scanning electron (SEM) and fluorescent microscopy. Furthermore, for the
pharmacokinetics study of OTC-CS-CaCO3NP and OTC, a total of 100, 5-6 weeks
old female BALB/c mice divided into two groups of 50 mice each were used. They
were administered 10mg/kg of OTC-CS-CaCO3NP and OTC, respectively. At specific
time intervals of 0, 5, 10, 15, 30 minutes and 1, 2, 6, 24 and 48 hrs, five mice from
each group were sacrificed. Blood, liver and kidneys were collected.
The results revealed that the synthesized CS-CaCO3NP and OTC-CS-CaCO3NP had
a homogeneously spherical appearance on TEM with a mean diameter of 29.90 (nm)
and -19.9 (mV) zeta potential which increased to 62.40 nm and -23.5 (mV),
respectively after loading with OTC. OTC crystallinity and functionality within CSCaCO3NP
were maintained as showed by XRD and FTIR spectral peaks. The
formulation of OTC-CS-CaCO3NP in ratio 1:4 with drug encapsulating efficiency
(71%) was used for in vitro release study. OTC was sustainably released from OTCCS-
CaCO3NP over a period of 96 hours with pH 4 having the highest drug release
percentage (98.2%). Cytotoxicity assay revealed that OTC-CS-CaCO3NP had
significantly higher cell viability (P < 0.05) compared to OTC in NIH3T3 cells.
Loading OTC into CS-CaCO3NP reduced OTC cytotoxicity in NIH3T3 cells. MTT
assay overestimated the cytotoxicity of CS-CaCO3NP, OTC-CS-CaCO3NP and OTC
when compared to trypan blue assay. The minimum inhibitory concentration (MIC)
for OTC-CS-CaCO3NP and OTC was 125 μg/ml and 500 μg/ml while the minimum
biofilm eradication concentration (MBEC) was 250 μg/ml and >2000 μg/ml,
respectively. However, CS-CaCO3NP alone demonstrated no antibacterial activity.
HR-TEM revealed that the antimicrobial mechanism of action of OTC-CS-CaCO3NP
was due to damage to the outer envelope of C. pseudotuberculosis while SEM and
fluorescent microscope showed digestion and death of the bacteria cells within C.
pseudotuberculosis biofilms. Pharmacokinetic studies demonstrated that OTC-CSCaCO3NP
had a slower elimination rate (0.135 1/hr), longer half-life (5.133 hr),
increased area under the curve (AUC) (46.68 μg/ml*h) and increased volume of
distribution (1.587 mg/kg/μg/ml) than OTC. Thus, OTC-CS-CaCO3NP is a safe and
biocompatible alternative antibiotic delivery system whose antibacterial efficacy is
more pronounced compared to OTC. |
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