Formulation development, stability and anticancer efficacy of core-shell cyclodextrin nanocapsules for oral chemotherapy with camptothecin

Summary Background: The aim of this study was to design and evaluate hybrid cyclodextrin (CD) nanocapsules intended for the oral delivery of the anticancer agent camptothecin (CPT) in order to maintain drug stability in the body and to improve its eventual bioavailability. For this reason, an amphiphilic cyclodextrin (CD) derivative per-modified on the primary face 6OCAPRO was used as core molecule to form nanocapsules with the nanoprecipitation technique. Nanocapsules were further coated with the cationic polymer chitosan to improve the cellular uptake and interaction with biological membranes through positive surface charge. Nanocapsules were evaluated for their in vitro characteristics such as particle size, zeta potential, drug loading and release profiles followed by cell culture studies with the MCF-7 and Caco-2 cell line evaluating their anticancer efficacy and permeability. The CD nanocapsules were imaged by scanning electron microscopy (SEM). The concentration of CPT entrapped in nanocapsules was determined by reversed phase HPLC. The in vitro release study of CPT was performed with a dialysis bag method under sink conditions mimicking the gastric and intestinal pH. The hydrolytic stability of CPT in nanocapsules was investigated in simulated gastric and intestinal fluids (SGF, SIF). Results: The mean particle sizes of both anionic and cationic CPT-loaded nanocapsules were in the range of 180–200 nm with polydispersity indices lower than 0.400 indicating monodisperse size distribution of nanocapsules with favourable potential for intracellular drug delivery to tumour cells. Surface charges of anionic and cationic nanocapsules were demonstrated as −21 mV and +18 mV, respectively. The stability of CPT in simulated release media, SGF and SIF were maintained suggesting the improved protection of the drug molecule from rapid hydrolysis degradation or gastrointestinal pH in nanocapsule oily core. Furthermore CD nanocapsules showed higher anticancer efficacy than CPT solution against the MCF-7 cell line. Permeation of CPT across Caco-2 cells was found to be 3 fold higher when incorporated in hybrid CD nanocapsules compared with a DMSO solution. Conclusion: Oral CD nanocapsules indicating increased oral bioavailability might be a promising strategy to maintain the physiological stability and to improve the oral bioavailability of problematic anticancer drugs such as CPT which may contribute to patient quality of life and drug efficacy in cancer therapy.


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Organic solvent was then evaporated under vacuum at 40 °C for 30 min to obtain nanocapsule dispersion at the final desired volume 10 mL. Nanocapsules were recovered by centrifugation at 3500 rpm at room temperature for 15 min. Preformulation studies were performed to determine the effect of formulation parameters such as oil concentration, ratio of organic to aqueous phase and CD concentration on nanoparticle characteristics such as mean diameter, polydispersity and zeta potential. According to the pre-formulation results, optimum formulation parameters were selected and CPT (95% HPLC powder, MW: 348.35 g/mole, Sigma-Aldrich) loaded nanocapsules were prepared with the same parameters dissolving CPT (10% w/w of the polymer) in organic phase. To obtain positively charged nanocapsules 0.025% w/v of Protasan™ (MW < 200 kDa, Novamatrix) was added to aqueous phase.

Physicochemical characterization of CPT loaded NCs
Particle size and zeta potential Mean particle size and polydispersity index (PDI) of the CPT-loaded CD nanocapsules were determined by dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS series, UK) at a scattering angle of 173°. Zeta potential of nanocapsules was measured in mV with Malvern Zetasizer Nano ZS at an angle of 12° and 25 °C.
Each measurement was carried out in triplicate.

Imaging of nanocapsules
The morphology of nanocapsules was determined by using Scanning Electron Microscope (FEI Nova™ Nano SEM 430, ABD). Samples were mounted on the metal S3 stubs and coated with 100 Å thick layer of a Gold-Palladium alloy. Then the particles were imaged at 5 to 20 kV. 1.2 to mimic the physiological conditions. The system was placed in a shaking water bath at 37 °C with an agitation speed of 100 rpm. At specific time periods (0,5., 1., 1,5., 2., 6., 24., 48. and 72.hours), 500 µL of sample was taken from the medium and replaced by the same volume of fresh medium. The cumulative percentage of drug released for each time point was calculated as a percentage of the total drug incorporated into the nanocapsules.

Stability of NCs in simulated gastrointestinal fluids (SGF &SIF)
It is also necessary to determine the physical stability of drug loaded nanocapsules NaOH was added to adjust the pH of the media. SGF without enzyme was prepared by dissolving 100 mg of NaCI in 50 mL deionized water containing 0.35 mL concentrated HCl. For each formulation; 1 mL of nanoparticle dispersion was added to 9 mL of simulated media and incubated in for 2 hours for SGF and for 6 hours for SIF. Mean particle size, PDI and zeta potential values were measured before and after incubation.

Cytotoxicity of blank CD NCs against L929 cell line
The were taken as control with 100% cell viability. Experiments were performed in triplicate and results are expressed as mean ±SD.

In vitro transport studies across Caco-2 cell line
In vitro transport studies were carried out across the human adenocarcinoma Caco-2 cell line, which is a widely used model for intestinal drug absorption and mimic the gastrointestinal barrier for oral chemotherapy in the recent years.
The culture medium was added to both the apical (500 μL) and basolateral ( where dQ/dt is the permeability rate, Co is the initial concentration on the apical side and A is the surface area of the monolayer.

Statistical analysis
The results were expressed as mean ± SD. The significance of differences was estimated by Student's t-test. P value less than 0.05 (P < 0.05) was considered to indicate a statistically significant difference.