Low molecular weight chitosan-coated polymeric nanoparticles for sustained and pH-sensitive delivery of paclitaxel

<div><p></p><p>Low molecular weight chitosan (LMWC) is a promising polymer for surface modification of nanoparticles (NPs), which can impart both stealth effect and electrostatic interaction with cells at mildly acidic pH of tumors. We previously produced LMWC-coated NPs via covalent conjugation to poly(lactic-co-glycolic) acid (PLGA-LMWC NPs). However, this method had several weaknesses including inefficiency and complexity of the production as well as increased hydrophilicity of the polymer matrix, which led to poor drug release control. Here, we used the dopamine polymerization method to produce LMWC-coated NPs (PLGA-pD-LMWC NPs), where the core NPs were prepared with PLGA that served best to load and retain drugs and then functionalized with LMWC via polydopamine layer. The PLGA-pD-LMWC NPs overcame the limitations of PLGA-LMWC NPs while maintaining their advantages. First of all, PLGA-pD-LMWC NPs attenuated the release of paclitaxel to a greater extent than PLGA-LMWC NPs. Moreover, PLGA-pD-LMWC NPs had a pH-dependent surface charge profile and cellular interactions similar to PLGA-LMWC NPs, enabling acid-specific NP–cell interaction and enhanced drug delivery to cells in weakly acidic environment. Although the LMWC layer did not completely prevent protein binding in serum solution, PLGA-pD-LMWC NPs showed less phagocytic uptake than bare PLGA NPs.</p></div

pH-dependent zeta-potential profiles of unmodified low molecular weight chitosan (LMCS) (data from the previous study [<b>22</b>]) and zwitterionic chitosan (ZWC) derivatives prepared with different anhydride to amine (An/Am) ratios, chitosan glutamate (C-Gt), and glycol chitosan (Gly-C).

<p>Data are expressed as averages with standard deviations of 3 repeated measurements.</p

Chitosan precipitates (arrows) in the peritoneal cavity.

<p>Mice injected with chitosan glutamate intraperitoneally were examined 7 days after injection. (A) Chitosan precipitates found between the liver and the stomach. (B) Chitosan precipitates stuck on the spleen (top) and the liver (bottom). (C) Lobes of the liver were connected via chitosan residue.</p

Effect of timed application of ZWC or LMCS (all in 2 mg/mL) on MIP-2 production in the LPS-challenged macrophages.

<p>Mouse peritoneal macrophages were incubated with LPS for 0, 2, 4, 8, or 24 hours, and the culture medium was sampled for determination of the MIP-2 level (white bars). In another set, macrophages were incubated with LPS for 0, 2, 4, or 8 hours with LPS and then treated with ZWC or LMCS, and the media were sampled after 24 hours (grey or black bars). Data are expressed as averages with standard deviations of three repeated measurements.</p

Effect of chitosan treatment (all in 2 mg/mL) on the levels of proinflammatory cytokines released from (A) naïve mouse peritoneal macrophages and (B) LPS-challenged macrophages.

<p>Cytokine levels are determined by Milliplex Multi-Analyte Profiling cytokine/chemokine panel. Media of the LPS-challenged macrophages were 10 times diluted prior to analysis. Graphs on the right are displayed in narrow y-scales. ZWC (An/Am = 0.7); C-Gt: chitosan glutamate; Gly-C: glycol chitosan. Data are expressed as averages with standard deviations of three repeated measurements. *: p<0.05; **: p<0.01; ***: p<0.001 vs PBS.</p

Cytology of the peritoneal fluid from different treatment groups using hematoxylin and eosin staining.

<p>(A) PBS; (B) Glutamate buffer; (C) ZWC. (A-C) Peritoneal fluid composed of small macrophages (box) and lymphocytes (arrows). No chitosan precipitates were identified. (D) Glycol chitosan: peritoneal fluid is composed of large macrophages (box) containing chitosan. (E) Chitosan glutamate: peritoneal fluid composed of large macrophages with intracellular eosinophilic chitosan. Extracellular chitosan (Ch) is surrounded by numerous macrophages. All images are of 400× magnification.</p

Incidence of lesions in tissues after intraperitoneal injection of chitosans and buffers.

a<p>Incidence of occurrence: Number of mice with lesion/total number of mice examined.</p

Effect of chitosan treatment (all in 2 mg/mL) on the MIP-2 production from (A) naïve mouse peritoneal macrophages and (B) LPS-challenged macrophages.

<p>MIP-2 level is determined by ELISA. Media of the LPS-challenged macrophages were 10 times diluted prior to analysis. Data are expressed as averages with standard deviations of three repeated measurements. **: p<0.01; ***: p<0.001.</p

Hematoxylin and eosin staining of liver sections of different treatment groups.

<p>(A) PBS (100x); (B) Glutamate buffer (100x); (C) ZWC (100x); (D) Glycol chitosan (100x). (A-D) Normal capsular surface (box). (E) Chitosan glutamate (100x): capsular surface of liver markedly thickened with precipitates of chitosan, which are surrounded by chronic inflammation and mild fibrosis (box). (F) Chitosan glutamate (400x): precipitates of chitosan on the liver surface surrounded by macrophages, fibroblasts, and neutrophils.</p

Endotoxin levels in chitosans.

<p>Endotoxin levels in chitosans.</p