Elsevier

Carbohydrate Polymers

Volume 85, Issue 4, 1 July 2011, Pages 803-808
Carbohydrate Polymers

Preparation, characterization and in vitro release of chitosan nanoparticles loaded with gentamicin and salicylic acid

https://doi.org/10.1016/j.carbpol.2011.03.051Get rights and content

Abstract

Salicylic acid (SA) and gentamicin (GM) loaded nanoparticles of chitosan (CS) cross-linked with tripolyphosphate (TPP) were prepared and used to inhibit the ototoxicity of GM. The prepared nanoparticles were characterized by FT-IR spectroscopy to confirm the cross-linking reaction between CS and cross-linking agent. X-ray diffraction (XRD) was performed to reveal the crystalline nature of the drug after encapsulation. Up to 24.67 ± 2.06% of SA and 26.64 ± 3.92% of GM were loaded into the nanoparticles and the average size of nanoparticles ranged from 148 ± 8.6 to 345.0 ± 12.9 nm. The nanoparticles formed were spherical in shape with high zeta potentials (higher than +30 mV). In vitro release studies in phosphate buffer saline (pH 7.4) showed an initial burst effect and followed by a slow drug release. The drug release followed Weibull equation and a non-Fickian transport. The amounts of SA and GM released from the nanoparticles met the dosage ratio requirement for inhibiting ototoxicity of GM by SA.

Introduction

Gemtamycin (GM) is an aminoglycoside antibiotic used for treating many types of bacterial infections, particularly those caused by Gram-negative bacteria. However, the side effects of GM, i.e. ototoxicity and nephrotoxicity, restrict its clinical application (Song, Sha, & Schacht, 1998). A number of researches have suggested that the free radicals were involved in the ototoxicity of GM (Hirose et al., 1997, Polat et al., 2006, Ryals et al., 1997, Talaska et al., 2006). Therefore, the free radical scavengers, such as glutathione, salicylic acid (SA), aspirin (acetyl salicylic acid), 2,3-dihydroxybenzoic acid, aminoguanidine and edaravone have been used to inhibit the ototoxicity of GM successfully (Lautermann et al., 1995, Maekawa et al., 2009, McFadden et al., 2003, Severinsen et al., 2006). Aspirin has been used to antagonize GM ototoxicity in clinical trials (Chen et al., 2007).

In practice, GM is injected once daily while SA needs to be injected twice a day (Sha & Schacht, 2000). The multiple injections bring inconvenience for the patients. In addition, once the free radicals generate, they will immediately react with the vicinal macromolecules due to their highly activity. In order to effectively inhibit the ototoxicity of the free radicals of GM, scavengers need to be available to react with the free radicals as soon as they are produced. Separating injections may result in poor coordination of GM and SA in vivo, and consequently reduce the antagonism of SA.

A suitable delivery system such as the site-specific and controlled release delivery can improve the bioavailability of the drug and minimize their side effects (Govender et al., 2000). Research showed that nanoparticles were able to protect the drugs from degradation and controlled the release of the encapsulated or adsorbed drugs (Atyabi, Moghaddam, Dinarvand, ZohuriaanMehr, & Ponchel, 2008). The nanoencapsulation of medicinal drugs (nanomedicines) also has many advantages in the enhancement of absorption into a selected tissue and improvement of intracellular penetration, bioavailability and retention time, and therefore, both the cost of the drug use and the risk of the toxicity for patient could be reduced (Kumari, Yadav, & Yadav, 2010).

Polymeric materials such as chitosan (CS), poly-d, l-lactide-co-glycolide (PLGA) and polylactic acid (PLA) are used for synthesis of nanoparticles. CS is a naturally occurring and abundantly available polysaccharide. It is the N-deacetylation product of chitin (Cooney et al., 2009, Peesan et al., 2005). The relative molecular weight of CS ranges from hundreds of thousands to millions (Fu, Huang, Zhai, Li, & Liu, 2007). CS and its derivatives have been widely used in pharmaceutical and medical areas, due to its favorable biological properties such as good biocompatibility, biodegradability, antibacterial activity (No, Kim, Lee, Park, & Prinyawiwatkul, 2006), wound healing acceleration ability, fungistatic, anticancerogen, anticholesteremic and low toxicity (Fan, Hu, & Shen, 2009). LD50 of CS for laboratory mice is 16 g/kg body weight, which is close to that of sugar or salt (Agnihotri, Mallikarjuna, & Aminabhavi, 2004). CS derivatives are also found to possess good antioxidant activity (Lin & Chou, 2004). Therefore, CS nanoparticles are suitable to be used for the drug and gene delivery (Gan et al., 2005, Wan et al., 2009, Wang et al., 2008). In addition, CS has special properties such as protonization under acidic condition due to its amino groups and can be cross-linked with anions such as tripolyphosphate (TPP) to form nanoparticles. The preparation of CS nanoparticles via the cross-linking method was found to be simple and mild.

In present study, the compound CS nanoparticles were synthesized by an ionic cross-linking method. GM and SA were loaded into the nanoparticles. The compound nanoparticles could release GM and SA simultaneously, and hence enhance the antagonism of SA. The physicochemical properties of CS nanoparticles were investigated using various analytical techniques such as transmission electron microscope (TEM), scanning electron microscope (SEM), X-ray diffraction (XRD) and FT-IR spectroscopy, and the drug release capability in vitro was also investigated.

Section snippets

Materials

Chitosan (CS, Deacetylation degree 95%, molecular weight 80 kDa) was purchased from Golden-shell Biochemical Co. Ltd. (Zhejiang, China). Gentamicin was purchased from North China Pharmaceutical Co. Ltd. (Hebei, China). Salicylic acid was purchased from Hezhong Bio-Chemical Co. Ltd. (Wuhan, China). Tripolyphosphate was purchased from Wenzhou Dongsheng Chemical reagent Co. Ltd. (Zhengjiang, China). All other materials and reagents used in the study were analytical grade.

Preparation of compound CS nanoparticles

CS nanoparticles were

Preparation of CS nanoparticles containing GM and SA

Preparation of the CS nanoparticles containing SA and GM with different mass ratios of CS to TPP from 3:1 to 7:1 was investigated. The concentration of CS was 0.2% (w/v). The feed mass ratio of SA (0.1%, w/v) to GM (0.2%, w/v) was selected as 1.5:1, and the pH of CS solution was 5.0. Table 1 shows the effect of CS/TPP mass ratios on the EE, LC, size and zeta potential values of CS nanoparticles. It was found that the EE and LC of the nanoparticles were decreased with increasing the CS/TPP mass

Conclusion

The GM and SA loaded CS nanoparticles were successfully prepared by cross-linking with TPP. The nanoparticles were stable and spherical in shape with a narrow size distribution. Different percentage entrapment efficiency and loading efficiency of GM and SA were obtained by varying the mass ratio of CS to TPP, the drug to polymer ratio, the pH of CS solution and the feed ratio of SA to GM. The in vitro release studies indicated that the feed ratio of SA to GM influenced the release rate of GM

Acknowledgements

Thus research was supported by Chongqing University Postgraduates’ Science and Innovation Fund (201005A1A0010333) and 211 Project Innovation Personnel Training Plan Items of Chongqing University (S-09103). We would like to thank Lu Ju and Li Fei (Laboratory of Electron Microscopy of Third Military Medical University) for they help in the nanoparticles characterization by SEM and TEM.

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