Nanoprecipitation Process Research Articles

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Flash nanoprecipitation of poly(styrene-co-acrylonitrile) colloids in the presence of hydrophobic organoplatinum and their derived Pt-carbon nanocomposites for oxygen reduction reaction

Herein, we present a flash nanoprecipitation process for one-step formation of soft colloidal particles, in which platinum (II) acetylacetonate is entrapped within poly(styrene-co-acrylonitrile) nanospheres. The protection of the polymer matrix in the flash nanoprecipitated composites would ensure that the monodisperse platinum nanoparticles are uniformly dispersed in the carbon matrix after carbonization. Electrochemical characterization shows that the obtained Pt-carbon nanocomposites (denoted as Pt@NC) exhibit a high catalytic performance and low polarization loss toward oxygen reduction reaction (ORR) in an alkaline electrolyte via a favorable four-electron pathway.

Hybrid nanoparticles as a new technological approach to enhance the delivery of cholesterol into the brain

Restoration of the Chol homeostasis in the Central Nervous System (CNS) could be beneficial for the treatment of Huntington’s Disease (HD), a progressive, fatal, adult-onset, neurodegenerative disorder. Unfortunately, Chol is unable to cross the blood–brain barrier (BBB), thus a novel strategy for a targeted delivery of Chol into the brain is highly desired.This article aims to investigate the production of hybrid nanoparticles composed by Chol and PLGA (MIX-NPs) modified with g7 ligand for BBB crossing.We described the impact of ratio between components (Chol and PLGA) and formulation process (nanoprecipitation or single emulsion process) on physico-chemical and structural characteristics, we tested MIX-NPs in vitro using primary hippocampal cell cultures evaluating possible toxicity, uptake, and the ability to influence excitatory synaptic receptors. Our results elucidated that both formulation processes produce MIX-NPs with a Chol content higher that 40%, meaning that Chol is a structural particle component and active compound at the same time. The formulation strategy impacted the architecture and reorganization of components leading to some differences in Chol availability between the two types of g7 MIX-NPs. Our results identified that both kinds of MIX-NPs are efficiently taken up by neurons, able to escape lysosomes and release Chol into the cells resulting in an efficient modification in expression of synaptic receptors that could be beneficial in HD.

Near-infrared fluorescent pyrrolopyrrole cyanine derivatives and colloidal nanoparticles with tunable optical properties for in vivo bioimaging

Near infrared (NIR) fluorescent molecules with light absorption and emission above 700 nm are important for noninvasive and deep-tissue imaging for disease diagnosis and imaging-guided surgery. Nevertheless, currently available NIR fluorophores with high fluorescence quantum yields and robust photostability remain rather limited. Here we report a series of π-extended pyrrolopyrrole cyanine (PPCy) derivatives with tunable light absorption and emission in the NIR spectral region. The extent of the intramolecular charge transfer shows significant effect on the optical bandgap, the Stokes shift and the fluorescence quantum yield. These hydrophobic PPCy derivatives were dispersed as colloidal nanoparticles into water via a nanoprecipitation process. The nanoparticles composed of spirofluorene-flanked PPCy derivatives exhibit the optimal optical properties, effective tumor-targeting, and high fluorescence contrast for in vivo bioimaging of xenografted tumor-model mice.

Encapsulation of Bioactive Compounds (Monocaprin and Monolaurin) into Polymeric Nanoparticles

The dextran-covered poly (lactic acid) (PLA) polymeric nanoparticles were prepared by a nanoprecipitation process for the encapsulation of monocaprin (MC) or monolaurin (ML). The concentration of PLA/MC or ML was varied to evaluate the best condition for preparation. The miscibility of various PLA/MC or ML blends was studied using differential scanning calorimetry. The result showed that at concentrations of 50/50 wt/wt, the PLA/ML blend was partly miscible and the nanoparticle suspension produced large amounts of macroscopic aggregates after nanoprecipitation. Conversely, the miscibility of PLA/MC blends progressively increased with increasing amounts of PLA. Therefore, the best concentration to encapsulate the MC was the PLA/MC 90/10 (wt/wt), according to the miscibility results.

Protein-Based Nanoparticle Preparation via Nanoprecipitation Method.

Nanoparticles are nowadays largely investigated in the field of drug delivery. Among nanoparticles, protein-based particles are of paramount importance since they are natural, biodegradable, biocompatible, and nontoxic. There are several methods to prepare proteins containing nanoparticles, but only a few studies have been dedicated to the preparation of protein- based nanoparticles. Then, the aim of this work was to report on the preparation of bovine serum albumin (BSA)-based nanoparticles using a well-defined nanoprecipitation process. Special attention has been dedicated to a systematic study in order to understand separately the effect of each operating parameter of the method (such as protein concentration, solvent/non-solvent volume ratio, non-solvent injection rate, ionic strength of the buffer solution, pH, and cross-linking) on the colloidal properties of the obtained nanoparticles. In addition, the mixing processes (batch or drop-wise) were also investigated. Using a well-defined formulation, submicron protein-based nanoparticles have been obtained. All prepared particles have been characterized in terms of size, size distribution, morphology, and electrokinetic properties. In addition, the stability of nanoparticles was investigated using Ultraviolet (UV) scan and electrophoresis, and the optimal conditions for preparing BSA nanoparticles by the nanoprecipitation method were concluded.

Single-Component Oligomer Nanoparticle-Based Size-Dependent Dual-Emission Modulation

Multichromophoric oligomers offer a versatile platform for nanoparticle multicolor fluorescent modulation. A donor–acceptor–donor (D–A–D) type oligomer (DDBTD), with blue emitting antenna and red emitting core, is chosen to assemble into fluorescent colloidal nanoparticles (FCNs) using a nanoprecipitation method. By modulating the DDBTD concentrations in good solvent, the DDBTD nanoparticles with average diameters ranging from sub-10 to 300 nm are obtained by the nanoprecipitation process in aqueous solution. Interestingly, multicolor photoluminescence (PL) can be realized from bright blue (∼440 nm) to rose red (∼630 nm) based on FCNs size control. The size-dependent PL originates from the aggregation-enhanced fluorescence resonance energy transfer (FRET) from diphenyl-aminofluorenyl antenna unit (blue emitter) to benzothiadiazole-based core (red emitter). Furthermore, the lifetime measurement of the FCNs in excited state shows a size-dependent behavior, which confirms that the size-dependent multicolor P...

Controlling and Predicting Nanoparticle Formation by Block Copolymer Directed Rapid Precipitations.

Nanoparticles have shown promise in several biomedical applications, including drug delivery and medical imaging; however, quantitative prediction of nanoparticle formation processes that scale from laboratory to commercial production has been lacking. Flash NanoPrecipitation (FNP) is a scalable technique to form highly loaded, block copolymer protected nanoparticles. Here, the FNP process is shown to strictly obey diffusion-limited aggregation assembly kinetics, and the parameters that control the nanoparticle size and the polymer brush density on the nanoparticle surface are shown. The particle size, ranging from 40 to 200 nm, is insensitive to the molecular weight and chemical composition of the hydrophobic encapsulated material, which is shown to be a consequence of the diffusion-limited growth kinetics. In a simple model derived from these kinetics, a single constant describes the 46 unique nanoparticle formulations produced here. The polymer brush densities on the nanoparticle surface are weakly dependent on the process parameters and are among the densest reported in the literature. Though modest differences in brush densities are observed, there is no measurable difference in the amount of protein adsorbed within this range. This work highlights the material-independent and universal nature of the Flash NanoPrecipitation process, allowing for the rapid translation of formulations to different stabilizing polymers and therapeutic loads.

Enhanced transdermal delivery of meloxicam by nanocrystals: Preparation, in vitro and in vivo evaluation

Meloxicam (MLX) is efficient in relieving pain and inflammatory symptoms, which, however, is limited by the poor solubility and gastrointestinal side effects. The objective of this study is to develop a nanocrystal formulation to enhance transdermal delivery of MLX. MLX nanocrystals were successfully prepared by the nanoprecipitation technique based on acid-base neutralization. With poloxamer 407 and Tween 80 (80/20, w/w) as mixed stabilizers, MLX nanocrystals with particle size of 175 nm were obtained. The crystalline structure of MLX nanocrystals was confirmed by both differential scanning calorimetry and X-ray powder diffractometry. However, the nanoprecipitation process reduced the crystallinity of MLX. Nanocrystals increased both in vitro and in vivo transdermal permeation of MLX compared with the solution and suspension counterparts. Due to the enhanced apparent solubility and dissolution as well as the facilitated hair follicular penetration, nanocrystals present a high and prolonged plasma MLX concentration. And 2.58- and 4.4-fold increase in AUC0→24h was achieved by nanocrystals comparing with solution and suspension, respectively. In conclusion, nanocrystal is advantageous for transdermal delivery of MLX.

Size-controlled fabrication of zein nano/microparticles by modified anti-solvent precipitation with/without sodium caseinate.

Zein-based nano/microparticles have been demonstrated to be promising carrier systems for both the food industry and biomedical applications. However, the fabrication of size-controlled zein particles has been a challenging issue. In this study, a modified anti-solvent precipitation method was developed, and the effects of various factors, such as mixing method, solvent/anti-solvent ratio, temperature, zein concentrations and the presence of sodium caseinate (SC) on properties of zein particles were investigated. Evidence is presented that, among the previously mentioned factors, the mixing method, especially mixing rate, could be used as an effective parameter to control the size of zein particles without changing other parameters. Moreover, through fine-tuning the mixing rate together with zein concentration, particles with sizes ranging from nanometers to micrometers and low polydispersity index values could be easily obtained. Based on the size-controlled fabrication method, SC-coated zein nanoparticles could also be obtained in a size-controlled manner by incubation of the coating material with the already-formed zein particles. The resultant nanoparticles showed better performance in both drug loading and controlled release, compared with zein/SC hybrid nanoparticles fabricated by adding aqueous ethanol solution to SC solution. The possible mechanisms of the nanoprecipitation process and self-assembly formation of these nanoparticles are discussed.

Size controlled fabrication of cellulose nanoparticles for drug delivery applications

Spherically shaped cellulose nanoparticles with average particle diameter range between 70 and 365 nm were fabricated from cotton fibers by a simple nanoprecipitation method. The average particle diameter of cellulose nanoparticles could be precisely controlled by modulating the synthesis conditions such as cellulose solution concentration and volume ratio of solvent/non-solvent. Methylene blue (MB) as a model hydrophilic drug was loaded onto cellulose nanoparticles via the in-situ nanoprecipitation process. The loading efficiency and release profile of MB were observed to be affected by the average particle diameter of cellulose nanoparticles.

Design and Solidification of Fast-Releasing Clofazimine Nanoparticles for Treatment of Cryptosporidiosis.

Clofazimine, a lipophilic (log P = 7.66) riminophenazine antibiotic approved by the US Food and Drug Administration (FDA) with a good safety record, was recently identified as a lead hit for cryptosporidiosis through a high-throughput phenotypic screen. Cryptosporidiosis requires fast-acting treatment as it leads to severe symptoms which, if untreated, result in morbidity for infants and small children. Consequently, a fast-releasing oral formulation of clofazimine in a water-dispersible form for pediatric administration is highly desirable. In this work, clofazimine nanoparticles were prepared with three surface stabilizers, hypromellose acetate succinate (HPMCAS), lecithin, and zein, using the flash nanoprecipitation (FNP) process. Drug encapsulation efficiencies of over 92% were achieved. Lyophilization and spray-drying were applied and optimized to produce redispersible nanoparticle powders. The release kinetics of these clofazimine nanoparticle powders in biorelevant media were measured and compared with those of crystalline clofazimine and the currently marketed formulation Lamprene. Remarkably improved dissolution rates and clofazimine supersaturation levels up to 90 times equilibrium solubility were observed with all clofazimine nanoparticles tested. Differential scanning calorimetry indicated a reduction of crystallinity of clofazimine in nanoparticles. These results strongly suggest that the new clofazimine nanoparticles prepared with affordable materials in this low-cost nanoparticle formulation process can be used as viable cryptosporidiosis therapeutics.

Synthesis and Nanoprecipitation of HEMA-CLn Based Polymers for the Production of Biodegradable Nanoparticles.

The control over the size distribution and stability of polymeric nanoparticles (NPs) is crucial in many of their applications, especially in the biomedical field. These characteristics are typically influenced by the production method and the nature of the starting material. To investigate these aspects, the controlled radical polymerization of functionalized methacrylates constituted by 2-hydroxyethyl methacrylate (HEMA) functionalized with a controlled number of ε-caprolactone (CL) units (HEMA-CLn), was carried out via reversible addition–fragmentation chain transfer polymerization (RAFT) in solution. The living reaction allows for good control over the molar mass of the final polymer with a low molar mass dispersity. The obtained polymer solutions were nanoprecipitated in order to produce NPs suitable for drug delivery applications with narrow particle size distribution and a wide size range (from 60 to 250 nm). The NP synthesis has been performed using a mixing device, in order to control the parameters involved in the nanoprecipitation process. As already seen for similar systems, the size of the produced NPs is a function of the polymer concentration during the nanoprecipitation process. Nevertheless, when the polymer concentration is kept constant, the NP size is influenced by the chemical structure of the polymer used, in terms of the presence of PEG (poly(ethylene glycol)), the degree of RAFT polymerization, and the length of the caprolactone side chain. These characteristics were also found to influence the stability and degradation properties of the produced NPs.

Drug Delivery Nanoparticles with Locally Tunable Toxicity Made Entirely from a Light-Activatable Prodrug of Doxorubicin.

A major challenge facing nanoparticle-based delivery of chemotherapy agents is the natural and unavoidable accumulation of these particles in healthy tissue resulting in local toxicity and dose-limiting side effects. To address this issue, we have designed and characterized a new prodrug nanoparticle with controllable toxicity allowing a locally-delivered light trigger to convert the payload of the particle from a low to a high toxicity state. The nanoparticles are created entirely from light-activatable prodrug molecules using a nanoprecipitation process. The prodrug is a conjugate of doxorubicin and photocleavable biotin (DOX-PCB). These DOX-PCB nanoparticles are 30 times less toxic to cells than doxorubicin, but can be activated to release pure therapeutic doxorubicin when exposed to 365nm light. These nanoparticles have an average diameter of around 100nm and achieve the maximum possible prodrug loading capacity since no support structure or coating is required to prevent loss of prodrug from the nanoparticle. These light activatable nanoparticles demonstrate tunable toxicity and can be used to facilitate future therapy development whereby light delivered specifically to the tumor tissue would locally convert the nanoparticles to doxorubicin while leaving nanoparticles accumulated in healthy tissue in the less toxic prodrug form.

Cyclic hexapeptide-conjugated nanoparticles enhance curcumin delivery to glioma tumor cells and tissue.

Glioma has one of the highest mortality rates among primary brain tumors. The clinical treatment for glioma is very difficult due to its infiltration and specific growth locations. To achieve improved drug delivery to a brain tumor, we report the preparation and in vitro and in vivo evaluation of curcumin nanoparticles (Cur-NPs). The cyclic hexapeptide c(RGDf(N-me) VK)-C (cHP) has increased affinity for cells that overexpress integrins and was designed to target Cur-NPs to tumors. Functional polyethyleneglycol-modified poly(d,l-lactide-co-glycolide) (PEG-PLGA) conjugated to cHP was synthesized, and targeted Cur-NPs were prepared using a self-assembly nanoprecipitation process. The physicochemical properties and the in vitro cytotoxicity, accuracy, and penetration capabilities of Cur-NPs targeting cells with high levels of integrin expression were investigated. The in vivo targeting and penetration capabilities of the NPs were also evaluated against glioma in rats using in vivo imaging equipment. The results showed that the in vitro cytotoxicity of the targeted cHP-modified curcumin nanoparticles (cHP/Cur-NPs) was higher than that of either free curcumin or non-targeted Cur-NPs due to the superior ability of the cHP/Cur-NPs to target tumor cells. The targeted cHP/Cur-NPs, c(RGDf(N-me)VK)-C-modified Cur-NPs, exhibited improved binding, uptake, and penetration abilities than non-targeting NPs for glioma cells, cell spheres, and glioma tissue. In conclusion, c(RGDf(N-me)VK)-C can serve as an effective targeting ligand, and cHP/Cur-NPs can be exploited as a potential drug delivery system for targeting gliomas.

A novel multiscale model for the simulation of polymer flash nano-precipitation

Numerous models describe the flash nano-precipitation (FNP) process to form polymer nanoparticles, however most of them are based on equilibrium approaches and are not capable of predicting kinetically stable configurations. Moreover, since FNP occurs through solvent-displacement, the way in which the solvent and anti-solvent are mixed plays an important role, which is often overlooked. Here we propose a multiscale approach, which combines molecular dynamics (MD), a Smoluchowski population balance equation (PBE) and computational fluid dynamics (CFD), to model the FNP process, from the atomistic-scale up to the macro-scale. The particle formation process is not described with the usual nucleation-and-growth approach, but as Brownian aggregation of the polymer molecules into nanoparticles. Being the final nanoparticles amorphous, no energy barrier to the aggregation process is considered, whereas the effects of both turbulent mixing and turbulent aggregation on the evolution of the nanoparticles are accounted for. The main novelty of this work is that the aggregation kernel appearing in the PBE, coupled in turn with CFD, is calculated from MD simulations, following the multiscale modeling paradigm. The model is tested on the FNP of poly-ε-caprolactone nanoparticles in acetone-water mixtures. Predictions for the final mean nanoparticle size are found in good agreement with experiments, especially at high initial polymer concentrations, where the hypothesis of no energy barrier is more realistic.

Assembly of Macrocycle Dye Derivatives into Particles for Fluorescence and Photoacoustic Applications

Optical imaging is a rapidly progressing medical technique that can benefit from the development of new and improved optical imaging agents suitable for use in vivo. However, the molecular rules detailing what optical agents can be processed and encapsulated into in vivo presentable forms are not known. We here present the screening of series of highly hydrophobic porphyrin, phthalocyanine, and naphthalocyanine dye macrocycles through a self-assembling Flash NanoPrecipitation process to form a series of water dispersible dye nanoparticles (NPs). Ten out of 19 tested dyes could be formed into poly(ethylene glycol) coated nanoparticles 60-150 nm in size, and these results shed insight on dye structural criteria that are required to permit dye assembly into NPs. Dye NPs display a diverse range of absorbance profiles with absorbance maxima within the NIR region, and have absorbance that can be tuned by varying dye choice or by doping bulking materials in the NP core. Particle properties such as dye core load and the compositions of co-core dopants were varied, and subsequent effects on photoacoustic and fluorescence signal intensities were measured. These results provide guidelines for designing NPs optimized for photoacoustic imaging and NPs optimized for fluorescence imaging. This work provides important details for dye NP engineering, and expands the optical imaging tools available for use.

Core/Shell Nanocomposites Produced by Superfast Sequential Microfluidic Nanoprecipitation

Although a number of techniques exist for generating structured organic nanocomposites, it is still challenging to fabricate them in a controllable, yet universal and scalable manner. In this work, a microfluidic platform, exploiting superfast (milliseconds) time intervals between sequential nanoprecipitation processes, has been developed for high-throughput production of structured core/shell nanocomposites. The extremely short time interval between the sequential nanoprecipitation processes, facilitated by the multiplexed microfluidic design, allows us to solve the instability issues of nanocomposite cores without using any stabilizers. Beyond high throughput production rate (∼700 g/day on a single device), the generated core/shell nanocomposites harness the inherent ultrahigh drug loading degree and enhanced payload dissolution kinetics of drug nanocrystals and the controlled drug release from polymer-based nanoparticles.

Optimizing Delivery Characteristics of Curcumin as a Model Drug via Tailoring Mean Diameter Ranges of Cellulose Beads

Spherical cellulose beads with tailored mean diameter between micrometer (20–80 μm) and nanometer (40–200 nm) ranges were fabricated from regenerated cellulose of paper wastes via water-in-oil (W/O) microemulsion and nanoprecipitation processes, respectively. The mean diameter ranges of cellulose beads were precisely controlled via modulating fabrication parameters such as stirring speed, surfactant concentration, cellulose concentration, and reaction temperature. By tailoring their mean diameter ranges and using curcumin as a model drug, cellulose beads with enhanced loading capacities and optimized release kinetic profiles of curcumin were fabricated.

Smart Release Nano-formulation of Cytochrome C and Hyaluronic Acid Induces Apoptosis in Cancer Cells.

Herein we tested a nanosized cancer-cell targeted delivery system based on cytochrome c (Cyt c) and hyaluronic acid. Cyt c was chosen since it is a per se non-toxic protein but causes apoptosis when delivered to the cytoplasm of target cells. Hyaluronic acid was employed to create the nanosized delivery system with passive targeting capability in order to exploit the enhanced permeation and retention (EPR) effect and active targeting capability of hyaluronic acid. In addition, our goal was to incorporate a smart release strategy to only promote protein release upon reaching its target.Nanoparticles were formed by a simple yet precise nanoprecipitation process based on desolvation. They were physically characterized to select precipitation conditions leading to adequate size, shape, protein bioactivity, and protein loading to produce a feasible targeted cancer treatment. We synthesized nanoparticles of around 500 nm diameter with a 60% protein loading and more than 80% of protein bioactivity. In vitro, cumulative release of 92% of Cyt c was observed after 8 h under conditions mimicking the reductive intracellular environment, while under non-denaturing conditions only 20% was released. The nanoparticles displayed a selective cytotoxic effect on cancer cells. After 6 h of incubation with the nanoparticles, hyaluronic acid receptor over expressing A549 human lung adenocarcinoma cells showed a viability of ca. 20% at 0.16 mg/ml of Cyt c concentration. Only a negligible effect was observed on viability of COS-7 African green monkey kidney fibroblast, a normal cell line notoverexpressing the hyaluronic acid receptor. Confocal microscopy confirmed that the drug delivery system indeed delivered Cyt c to the cytoplasm of the target cells. We conclude that we were able to create a smart stimuli-responsive targeted drug delivery system with significant potential in cancer therapy.