Particle size effect of curcumin nanosuspensions on cytotoxicity, cellular internalization, in vivo pharmacokinetics and biodistribution.

This study aimed to investigate the particle size effect on bioactivity, cellular internalization and bioavailability of curcumin (CUR) nanosuspension (CUR-NS). CUR-NSs of different particle sizes were prepared by two different anti-solvent precipitation methods. CUR-NS with the smallest size showed similar in vitro anticancer activity and bioavailability to the CUR solution, whereas nanosuspensions of larger particle sizes displayed higher in vitro cellular internalization and cytotoxicity, as well as higher in vivo AUC and slower clearance rate after i.v. administration in rats. CUR solution and different sized CUR-NSs reached the highest concentrations in the lung, followed by liver and spleen while the lowest concentration was observed in the brain after i.v. administration in mice. Specifically, CUR-NS of 70nm accumulated more in the brain, whereas CUR-NS of 200nm accrued more in liver and spleen. CUR-NS of 20nm displayed no significant biodistribution difference compared with CUR solution in all tissues.

Physical stability and in vivo brain delivery of polymeric ibuprofen nanoparticles fabricated by flash nanoprecipitation.

Ibuprofen (IBP), a common non-steroidal anti-inflammatory drug (NSAID) with a log P of 3.51, has been shown to possess potential benefit in the treatment of Alzheimer's disease. However, the bioavailability of IBP to the brain is poor, which can be linked to its extensive binding to plasma proteins in the blood. This study aimed to evaluate the nanoparticle production of IBP by flash nanoprecipitation (FNP) technology, and to determine whether the nanoparticles prepared by FNP could enhance the delivery of IBP into the brain. Polymeric IBP nanoparticles were prepared with poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) diblock copolymer as stabilizer under optimized conditions using a four-stream multi-inlet vortex mixer (MIVM). The optimized nanoparticles displayed a mean particle size of around 50 nm, polydispersity index of around 0.2, drug loading of up to 30% and physical stability of up to 34 days. In-depth surface characterization using zeta potential measurement, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) showed that the surfaces of these nanoparticles were covered with the hydrophilic PEG groups from the diblock copolymer. In vivo brain uptake study of the IBP nanoparticles indicated that the particles, when coated with polysorbate 80, displayed an enhanced brain uptake. However, the extent of brain uptake enhancement appeared limited, possibly due to a rapid release of IBP from the nanoparticles into the blood stream following intravenous administration.

Converting nanosuspension into inhalable and redispersible nanoparticles by combined in-situ thermal gelation and spray drying.

While nanoparticulate drugs for deep lung delivery hold promise for particular disease treatments, their size-related physical instability and tendency of being exhaled during breathing remain major challenges to their inhaled formulation development. Here we report a viable method for converting drug nanosuspensions into inhalable, stable and redispersible nano-agglomerates through combined in-situ thermal gelation and spray drying. Itraconazole (ITZ) nanosuspensions were prepared by flash nanoprecipitation, and co-spray dried with two different grades of the gel-forming polymer, methylcellulose (MC M20 and MC M450) as protectants. MC M20 was found superior in protecting ITZ nanoparticles against thermal stress (through nanoparticle entrapment within its gel network structure) during spray drying. In terms of redispersibility, an Sf/Si ratio (i.e., ratio of nanoparticle sizes after and before spray drying) of unity (1.02 ± 0.03), reflecting full particle size preservation, was achieved by optimizing the suspending medium content and spray drying parameters. Formulation components, nanosuspension concentration and spray drying parameters all showed a significant impact on the aerosol performance of the resulting agglomerates, but an absence of defined trends or correlations. Overall, the MC-protected nano-agglomerates displayed excellent in-vitro aerosol performance with fine particle fractions higher than 50% and mass median aerodynamic diameters within the 2-3 µm range, which are ideal for deep lung delivery.

Impact of molecular rearrangement of amphiphilic stabilizers on physical stability of itraconazole nanoparticles prepared by flash nanoprecipitation.

Flash nanoprecipitation (FNP) is a controlled antisolvent precipitation process that has proven effective for consistent production of drug nanoparticles with a defined mean particle size and narrow particle size distribution. However, physical instability of the generated nanoparticles remains a major challenge in the application of this technology in pharmaceutical formulation. Aimed at resolving this problem, the present study has investigated the FNP process and associated stabilization mechanism of itraconazole (ITZ) nanoparticles through in-depth nanoparticle characterization. Results showed that ITZ nanoparticles could be reproducibly produced with a mean particle size <100 nm and a polydispersity index <0.2 in the presence of amphiphilic stabilizers (ASs). Surface analysis of freshly formed nanoparticles by X-ray photoelectron spectroscopy (XPS) revealed initially a disordered packing structure and subsequently a time-dependent molecular rearrangement of incorporated AS towards a micelle-like structure. The faster the molecular rearrangement of AS, the more stable the nanoparticles, as monitored by the change in particle size with time. These findings may have important implications for the selection of effective ASs for formulating stable drug nanoparticles. The present study is the first of its kind to demonstrate the utility of XPS to track the molecular transport of stabilizers in rapidly generated nanoparticles.

Fabrication of doxorubicin nanoparticles by controlled antisolvent precipitation for enhanced intracellular delivery.

Over-expression of ATP-binding cassette transporters is one of the most important mechanisms responsible for multidrug resistance. Here, we aimed to develop a stable polymeric nanoparticle system by flash nanoprecipitation (FNP) for enhanced anticancer drug delivery into drug resistant cancer cells. As an antisolvent precipitation process, FNP works best for highly lipophilic solutes (logP>6). Thus we also aimed to evaluate the applicability of FNP to drugs with relatively low lipophilicity (logP=1-2). To this end, doxorubicin (DOX), an anthracycline anticancer agent and a P-gp substrate with a logP of 1.3, was selected as a model drug for the assessment. DOX was successfully incorporated into the amphiphilic diblock copolymer, polyethylene glycol-b-polylactic acid (PEG-b-PLA), by FNP using a four-stream multi-inlet vortex mixer. Optimization of key processing parameters and co-formulation with the co-stabilizer, polyvinylpyrrolidone, yielded highly stable, roughly spherical DOX-loaded PEG-b-PLA nanoparticles (DOX.NP) with mean particle size below 100nm, drug loading up to 14%, and drug encapsulation efficiency up to 49%. DOX.NP exhibited a pH-dependent drug release profile with higher cumulative release rate at acidic pHs. Surface analysis of DOX.NP by XPS revealed an absence of DOX on the particle surface, indicative of complete drug encapsulation. While there were no significant differences in cytotoxic effect on P-gp over-expressing LCC6/MDR cell line between DOX.NP and free DOX in buffered aqueous media, DOX.NP exhibited a considerably higher cellular uptake and intracellular retention after efflux. The apparent lack of cytotoxicity enhancement with DOX.NP may be attributable to its slow DOX release inside the cells.

Development of highly stabilized curcumin nanoparticles by flash nanoprecipitation and lyophilization.

The influence of critical operating parameters on the Flash Nanoprecipitation (FNP) and resulting material properties of curcumin (CUR) nanoparticles has been evaluated using a confined impinging jets-with-dilution mixer (CIJ-D-M). It has been shown that the mixing rate, molecular weight of polymeric stabilizer (i.e., polyethylene glycol-b-poly(dl-lactide) di-block copolymer; PEG-PLA) and drug-to-copolymer mass ratio all exert a significant impact on the particle size and stability of the generated nanosuspensions. The attainable mean particle size and span of the nanoparticles through optimization of these process parameters were approximately 70nm and 0.85 respectively. However, the optimized nanosuspension was only stable for about two hours after preparation. Co-formulation with polyvinylpyrrolidone (PVP) substantially extended the product lifespan to 5days at ambient conditions and two weeks at 4°C. Results from zeta potential measurement and X-ray photoelectron spectroscopy (XPS) suggested that the enhanced stability is probably due to the formation of an additional protective barrier by PVP around the particle surface, thereby suppressing the dissociation of PEG-PLA from the particles and preventing CUR leakage from inside. Long-term storage stability (>1year) could be achieved by lyophilization of the optimized nanosuspension with Kleptose (hydroxypropyl-ß-cyclodextrin), which was shown to be the only effective lyoprotectant among all the ones tested for the CUR nanoparticles. At an optimal concentration of Kleptose (1.25% w/v), the redispersibility (Sf/Si; ratio of the final and initial particle sizes) and encapsulation efficiency of lyophilized CUR nanoparticles were about 1.22% and 94%, respectively.

Curcumin-conjugated magnetic nanoparticles for detecting amyloid plaques in Alzheimer's disease mice using magnetic resonance imaging (MRI).

Diagnosis of Alzheimer's disease (AD) can be performed with the assistance of amyloid imaging. The current method relies on positron emission tomography (PET), which is expensive and exposes people to radiation, undesirable features for a population screening method. Magnetic resonance imaging (MRI) is cheaper and is not radioactive. Our approach uses magnetic nanoparticles (MNPs) made of superparamagnetic iron oxide (SPIO) conjugated with curcumin, a natural compound that specifically binds to amyloid plaques. Coating of curcumin-conjugated MNPs with polyethylene glycol-polylactic acid block copolymer and polyvinylpyrrolidone by antisolvent precipitation in a multi-inlet vortex mixer produces stable and biocompatible curcumin magnetic nanoparticles (Cur-MNPs) with mean diameter <100 nm. These nanoparticles were visualized by transmission electron microscopy and atomic force microscopy, and their structure and chemistry were further characterized by X-ray diffraction, thermogravimetric analysis, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and Fourier transform infrared spectroscopy. Cur-MNPs exhibited no cytotoxicity in either Madin-Darby canine kidney (MDCK) or differentiated human neuroblastoma cells (SH-SY5Y). The Papp of Cur-MNPs was 1.03 × 10(-6) cm/s in an in vitro blood-brain barrier (BBB) model. Amyloid plaques could be visualized in ex vivo T2*-weighted magnetic resonance imaging (MRI) of Tg2576 mouse brains after injection of Cur-MNPs, and no plaques could be found in non-transgenic mice. Immunohistochemical examination of the mouse brains revealed that Cur-MNPs were co-localized with amyloid plaques. Thus, Cur-MNPs have the potential for non-invasive diagnosis of AD using MRI.

Assessment of the relative performance of a confined impinging jets mixer and a multi-inlet vortex mixer for curcumin nanoparticle production.

The relative performance of two specially designed mixers for nanoparticle production, namely, two-stream confined impinging jets with dilution mixer (CIJ-D-M) and four-stream multi-inlet vortex mixer (MIVM), was evaluated using the model compound, curcumin (CUR), under defined conditions of varying mixing rate and organic solvent. In the absence of turbulent fluctuations, higher mixing rate tended to generate finer particles. Among the three water-miscible organic solvents tested, acetone afforded the smallest particle size and the narrowest particle size distribution. Both mixers were capable of reproducibly fabricating CUR nanoparticles with particle size below 100 nm and high encapsulation efficiency (>99.9%). Specifically, CIJ-D-M yielded nanoparticles with smaller size and polydispersity index while the particles obtained by the MIVM displayed better short-term stability. In addition, CIJ-D-M tended to produce a mixture of irregular nanoaggregates and primary nanoparticles while roughly spherical nanoparticles were generated with the MIVM. The observed particle size and morphological differences could be attributed to the differences in the configuration of the mixing chamber and the related mixing order.