Pharmacokinetic control of orally dosed cyclosporine A with mucosal drug delivery system.

This study aimed to control the oral absorption of cyclosporine A (CsA) with the use of a mucosal drug delivery system (mDDS). Mucopenetrating nanocarriers (MP/NCs) and mucoadhesive nanocarriers (MA/NCs) were prepared by flash nanoprecipitation employing polystyrene-block-poly(ethylene glycol) and polystyrene-block-poly(N,N-dimethyl aminoethyl methacrylate), respectively. Their particle distribution in the rat gastrointestinal tract were visualized by fluorescent imaging. Plasma concentrations were monitored after oral administration of CsA-loaded MP/NCs (MP/CsA) and MA/NCs (MA/CsA) to rats. MP/NCs and MA/NCs had a particle size below 200 nm and ζ-potentials of 4 and 40 mV, respectively. The results from in vitro experiments demonstrated mucopenetration of MP/NCs and mucoadhesion of MA/NCs. Confocal laser scanning microscopic images showed diffusion of MP/NCs in the gastrointestinal mucus towards epithelial cells and localization of MA/NCs on the surface of the gastrointestinal mucus layer. In a pH 6.8 solution, rapid and sustained release of CsA were observed for MP/CsA and MA/CsA, respectively. After oral dosing (10 mg-CsA/kg) to rats, amorphous CsA powder exhibited a time to maximum plasma concentration (Tmax) of 3.4 h, maximum plasma concentration (Cmax) of 0.12 µg/mL, and bioavailability of 0.7%. Compared with amorphous CsA powder, MP/CsA shortened Tmax by 1.1 to 2.3 h and increased the bioavailability by 43-fold to 30.1%, while MA/CsA prolonged Tmax by 3.4 to 6.8 h with Cmax and bioavailability of 0.65 µg/mL and 11.7%, respectively. These pharmacokinetic behaviors would be explained by their diffusion and release properties modulated by polymeric surface modification. The mDDS approach is a promising strategy for the pharmacokinetic control of orally administered CsA.

Critical Review: Uptake and Translocation of Organic Nanodelivery Vehicles in Plants.

Nanodelivery vehicles (NDVs) are engineered nanomaterials (ENMs) that, within the agricultural sector, have been investigated for their ability to improve uptake and translocation of agrochemicals, control release, or target specific tissues or subcellular compartments. Both inorganic and organic NDVs have been studied for agrochemical delivery in the literature, but research on the latter has been slower to develop than the literature on the former. Since the two classes of nanomaterials exhibit significant differences in surface chemistry, physical deformability, and even colloidal stability, trends that apply to inorganic NDVs may not hold for organic NDVs, and vice versa. We here review the current literature on the uptake, translocation, biotransformation, and cellular and subcellular internalization of organic NDVs in plants following foliar or root administration. A background on nanomaterials and plant physiology is provided as a leveling ground for researchers in the field. Trends in uptake and translocation are examined as a function of NDV properties and compared to those reported for inorganic nanomaterials. Methods for assessing fate and transport of organic NDVs in plants (a major bottleneck in the field) are discussed. We end by identifying knowledge gaps in the literature that must be understood in order to rationally design organic NDVs for precision agrochemical nanodelivery.

Targeted Delivery of Sucrose-Coated Nanocarriers with Chemical Cargoes to the Plant Vasculature Enhances Long-Distance Translocation.

Current practices for delivering agrochemicals are inefficient, with only a fraction reaching the intended targets in plants. The surfaces of nanocarriers are functionalized with sucrose, enabling rapid and efficient foliar delivery into the plant phloem, a vascular tissue that transports sugars, signaling molecules, and agrochemicals through the whole plant. The chemical affinity of sucrose molecules to sugar membrane transporters on the phloem cells enhances the uptake of sucrose-coated quantum dots (sucQD) and biocompatible carbon dots with ß-cyclodextrin molecular baskets (suc-ß-CD) that can carry a wide range of agrochemicals. The QD and CD fluorescence emission properties allowed detection and monitoring of rapid translocation (<40 min) in the vasculature of wheat leaves by confocal and epifluorescence microscopy. The suc-ß-CDs more than doubled the delivery of chemical cargoes into the leaf vascular tissue. Inductively coupled plasma mass spectrometry (ICP-MS) analysis showed that the fraction of sucQDs loaded into the phloem and transported to roots is over 6.8 times higher than unmodified QDs. The sucrose coating of nanoparticles approach enables unprecedented targeted delivery to roots with ≈70% of phloem-loaded nanoparticles delivered to roots. The use of plant biorecognition molecules mediated delivery provides an efficient approach for guiding nanocarriers containing agrochemicals to the plant vasculature and whole plants.

Flash NanoPrecipitation as an Agrochemical Nanocarrier Formulation Platform: Phloem Uptake and Translocation after Foliar Administration.

The increasing severity of pathogenic and environmental stressors that negatively affect plant health has led to interest in developing next-generation agrochemical delivery systems capable of precisely transporting active agents to specific sites within plants. In this work, we adapt Flash NanoPrecipitation (FNP), a scalable nanocarrier (NC) formulation technology used in the pharmaceutical industry, to prepare organic core-shell NCs and study their efficacy as foliar or root delivery vehicles. NCs ranging in diameter from 55 to 200 nm, with surface zeta potentials from -40 to +40 mV, and with seven different shell material properties were prepared and studied. Shell materials included synthetic polymers poly(acrylic acid), poly(ethylene glycol), and poly(2-(dimethylamino)ethyl methacrylate), naturally occurring compounds fish gelatin and soybean lecithin, and semisynthetic hydroxypropyl methylcellulose acetate succinate (HPMCAS). NC cores contained a gadolinium tracer for tracking by mass spectrometry, a fluorescent dye for tracking by confocal microscopy, and model hydrophobic compounds (alpha tocopherol acetate and polystyrene) that could be replaced by agrochemical payloads in subsequent applications. After foliar application onto tomato plants with Silwet L-77 surfactant, internalization efficiencies of up to 85% and NC translocation efficiencies of up to 32% were observed. Significant NC trafficking to the stem and roots suggests a high degree of phloem loading for some of these formulations. Results were corroborated by confocal microscopy and synchrotron X-ray fluorescence mapping. NCs stabilized by cellulosic HPMCAS exhibited the highest degree of translocation, followed by formulations with a significant surface charge. The results from this work indicate that biocompatible materials like HPMCAS are promising agrochemical delivery vehicles in an industrially viable pharmaceutical nanoformulation process (FNP) and shed light on the optimal properties of organic NCs for efficient foliar uptake, translocation, and delivery.

Formulation and Scale-Up of Fast-Dissolving Lumefantrine Nanoparticles for Oral Malaria Therapy.

Lumefantrine (LMN) is one of the first-line drugs in the treatment of malaria due to its long circulation half-life, which results in enhanced effectiveness against drug-resistant strains of malaria. However, LMN's therapeutic efficacy is diminished due to its low bioavailability when dosed as a crystalline solid. The goal of this work was to produce low-cost, highly bioavailable, stable LMN powders for oral delivery that would be suitable for global health applications. We report the development of a LMN nanoparticle formulation and the translation of that formulation from laboratory to industrial scale. We applied Flash NanoPrecipitation (FNP) to develop nanoparticles with 90% LMN loading and sizes of 200-260 nm. The integrated process involves nanoparticle formation, concentration by tangential flow ultrafiltration, and then spray drying to obtain a dry powder. The final powders are readily redispersible and stable over accelerated aging conditions (50°C, 75% RH, open vial) for at least 4 weeks and give equivalent and fast drug release kinetics in both simulated fed and fasted state intestinal fluids, making them suitable for pediatric administration. The nanoparticle-based formulations increase the bioavailability of LMN 4.8-fold in vivo when compared to the control crystalline LMN. We describe the translation of the laboratory-scale process at Princeton University to the clinical manufacturing scale at WuXi AppTec.

Sequential Flash NanoPrecipitation for the scalable formulation of stable core-shell nanoparticles with core loadings up to 90.

Flash NanoPrecipitation (FNP) is a scalable, single-step process that uses rapid mixing to prepare nanoparticles with a hydrophobic core and amphiphilic stabilizing shell. Because the two steps of particle self-assembly - (1) core nucleation and growth and (2) adsorption of a stabilizing polymer onto the growing core surface - occur simultaneously during FNP, nanoparticles formulated at core loadings above approximately 70% typically exhibit poor stability or do not form at all. Additionally, a fundamental limit on the concentration of total solids that can be introduced into the FNP process has been reported previously. These limits are believed to share a common mechanism: entrainment of the stabilizing polymer into the growing particle core, leading to destabilization and aggregation. Here, we demonstrate a variation of FNP which separates the nucleation and stabilization steps of particle formation into separate sequential mixers. This scheme allows the hydrophobic core to nucleate and grow in the first mixing chamber unimpeded by adsorption of the stabilizing polymer, which is later introduced to the growing nuclei in the second mixer. Using this Sequential Flash NanoPrecipitation (SNaP) technique, we formulate stable nanoparticles with up to 90% core loading by mass and at 6-fold higher total input solids concentrations than typically reported.

Temperature-Responsive Bottlebrush Polymers Deliver a Stress-Regulating Agent In Vivo for Prolonged Plant Heat Stress Mitigation.

Anticipated increases in the frequency and intensity of extreme temperatures will damage crops. Methods that efficiently deliver stress-regulating agents to crops can mitigate these effects. Here, we describe high aspect ratio polymer bottlebrushes for temperature-controlled agent delivery in plants. The foliar-applied bottlebrush polymers had near complete uptake into the leaf and resided in both the apoplastic regions of the leaf mesophyll and in cells surrounding the vasculature. Elevated temperature enhanced the in vivo release of spermidine (a stress-regulating agent) from the bottlebrushes, promoting tomato plant (Solanum lycopersicum) photosynthesis under heat and light stress. The bottlebrushes continued to provide protection against heat stress for at least 15 days after foliar application, whereas free spermidine did not. About 30% of the ∼80 nm short and ∼300 nm long bottlebrushes entered the phloem and moved to other plant organs, enabling heat-activated release of plant protection agents in phloem. These results indicate the ability of the polymer bottlebrushes to release encapsulated stress relief agents when triggered by heat to provide long-term protection to plants and the potential to manage plant phloem pathogens. Overall, this temperature-responsive delivery platform provides a new tool for protecting plants against climate-induced damage and yield loss.

Small-volume in vitro lipid digestion measurements for assessing drug dissolution in lipid-based formulations using SAXS.

Lipid-based formulations improve the absorption capacity of poorly-water-soluble drugs and digestion of the formulation is a critical step in that absorption process. A recent approach to understanding the propensity for drug to dissolve in digesting lipid-based formulations couples an in vitro pH-stat lipolysis model to small-angle X-ray scattering (SAXS) by means of a flow-through capillary. However, the conventional pH-stat apparatus used to measure the extent of lipid digestion during such experiments requires digest volumes of 15-30 mL and drug doses of 50-200 mg, which is problematic for scarce compounds and can require excessive amounts of formulation reagents. This manuscript describes an approach to reduce the amount of material required for in vitro lipolysis experiments coupled to SAXS, for use in instances where the amount of drug or formulation medium is limited. Importantly, this was achieved while maintaining the pH stat conditions, which is critical for maintaining biorelevance and driving digestion to completion. The digestibility of infant formula with the poorly-water-soluble drugs halofantrine and clofazimine dispersed into it was measured as an exemplar paediatric-friendly lipid formulation. Halofantrine was incorporated in its powdered free base form and clofazimine was incorporated both as unformulated drug powder and as drug in nanoparticulate form prepared using Flash NanoPrecipitation. The fraction of triglyceride digested was found to be independent of vessel size and the incorporation of drug. The dissolution of the two forms of clofazimine during the digestion of infant formula were then measured using synchrotron SAXS, which revealed complete and partial solubilisation over 30 min of digestion for the powdered drug and nanoparticle formulations, respectively. The main challenge in reducing the volume of the measurements was in ensuring that thorough mixing was occurring in the smaller digestion vessel to provide uniform sampling of the dispersion medium.

Clofazimine-Loaded Mucoadhesive Nanoparticles Prepared by Flash Nanoprecipitation for Strategic Intestinal Delivery.

PURPOSE: This study was undertaken to develop novel mucoadhesive formulations of clofazimine (CFZ), a drug candidate for the treatment of cryptosporidiosis, with the aim of strategic delivery to the small intestine, the main site of the disease parasites. METHODS: CFZ-loaded nanoparticles (nCFZ) coated with non-biodegradable anionic polymer (nCFZ/A) and biodegradable anionic protein complex (nCFZ/dA) were prepared by Flash NanoPrecipitation (FNP) and evaluated for their physicochemical and biopharmaceutical properties. RESULTS: The mean diameters of nCFZ/A and nCFZ/dA were ca. 90 and 240 nm, respectively, and they showed narrow size distributions and negative ζ-potentials. Both formulations showed higher solubility of CFZ in aqueous solution than crystalline CFZ. Despite their improved dispersion behaviors, both formulations exhibited significantly lower diffusiveness than crystalline CFZ in a diffusion test using artificial mucus (AM). Quartz crystal microbalance analysis showed that both formulations clearly interacted with mucin, which appeared to be responsible for their reduced diffusiveness in AM. These results suggest the potent mucoadhesion of nCFZ/A and nCFZ/dA. After the oral administration of CFZ samples (10 mg-CFZ/kg) to rats, nCFZ/dA and nCFZ/A exhibited a prolongation in Tmax by 2 and >9 h, respectively, compared with crystalline CFZ. At 24 h after oral doses of nCFZ/A and nCFZ/dA with mucoadhesion, there were marked increases in the intestinal CFZ concentration (4-7 fold) compared with Lamprene®, a commercial CFZ product, indicating enhanced CFZ exposure in the small intestine. CONCLUSION: The use of FNP may produce mucoadhesive CFZ formulations with improved intestinal exposure, possibly offering enhanced anti-cryptosporidium therapy.

Sustained release of peptides and proteins from polymeric nanocarriers produced by inverse Flash NanoPrecipitation.

Peptide and protein therapeutics generally exhibit high potency and specificity and are increasingly important segments of the pharmaceutical market. However, their clinical applications are limited by rapid clearance and poor membrane permeability. Encapsulation of the peptide or protein into a nano-scale carrier can modify its pharmacokinetics and biodistribution. This might be employed to promote uptake in desired cell types or tissues, to limit systemic exposure, or to reduce the need for frequent injections. We have recently described inverse Flash NanoPrecipitation (iFNP), a scalable technique to encapsulate water-soluble therapeutics into polymeric nanocarriers, and have demonstrated improvements in therapeutic loading of an order of magnitude over comparable approaches. Here, we describe the formulation parameters that control release rates of encapsulated model therapeutics polymyxin B, lysozyme, and bovine serum albumin from nanocarriers produced using iFNP. Using a neutropenic lung infection mouse model with a multi-drug resistant Acinetobacter baumannii clinical isolate, we demonstrate enhanced therapeutic effect and safety profile afforded by nanocarrier-encapsulated polymyxin B following pulmonary administration. The encapsulated formulation reduced toxicity observed at elevated doses and resulted in up to 2.7-log10 reduction in bacterial burden below that of unencapsulated polymyxin B. These results establish the promise of iFNP as a platform for nanocarrier delivery of water-soluble therapeutics.

Chemistry and Geometry of Counterions Used in Hydrophobic Ion Pairing Control Internal Liquid Crystal Phase Behavior and Thereby Drug Release.

The combination of Flash NanoPrecipitation and hydrophobic ion pairing (HIP) is a valuable approach for generating nanocarrier formulations of ionic water-soluble drugs with controllable release properties dictated by liquid crystalline structuring of the ion pairs. However, there are few examples of this in practice in the literature. This work aims to decipher the influence of the nature of the hydrophobic counterion used in HIP and its consequent impact on liquid crystalline structuring and drug release. The hypothesis of this study was that hydrophobic counterions with different head and tail groups used for FNP with HIP would give rise to different liquid crystalline structures, which in turn would result in different drug release behavior. A cationic, water-soluble antibiotic, polymixin B, was complexed with eight different hydrophobic counterions with varying head and tail groups and encapsulated into nanocarriers 100-400 nm in size prepared using FNP. Sixteen formulations were assessed for internal structure by synchrotron small-angle X-ray scattering, and drug release was measured in vitro in physiological conditions. The liquid crystalline phases formed depended on the counterion head group and tail geometry, drug:counterion charge ratio, and the ionic strength and pH of the release medium. Drug release occurred more rapidly when no liquid crystalline phases were present and more slowly when higher-ordered phases existed. Specific findings include that phosphonic acid counterions lead to the formation of lamellar structures that persisted at pH 2.0 but were not present at pH 7.3. In contrast, sulfonic acids lead to lamellar or hexagonal phases that persisted at both pH 7.3 and 2.0, while hydrophobic counterions without alkyl tails did not form internal structures. It was also clear that the lipophilicity of the counterion does not dictate drug release. These findings confirm that the liquid crystalline phase behavior of the drug:counterion complex dictates drug release and significantly improves our understanding of the types of controlled release formulations that are possible using FNP with HIP.

Highly-loaded protein nanocarriers prepared by Flash NanoPrecipitation with hydrophobic ion pairing.

The efficient encapsulation of therapeutic proteins into delivery vehicles, particularly without loss of function, remains a significant research hurdle. Typical liposomal formulations achieve drug loadings on the order of 3-5% and encapsulation efficiencies around 50%. We demonstrate the encapsulation of model proteins with isoelectric points above and below pH 7 into nanocarriers (NCs) with protein loadings as high as 46% and encapsulation efficiencies above 95%. This is done by combining the continuous nanofabrication process Flash NanoPrecipitation (FNP) with the technique of hydrophobic ion pairing by forming and encapsulating an ionic complex within a nanocarrier stabilized by a block copolymer surface layer. We complex and encapsulate lysozyme with two anionic hydrophobic counterions, sodium oleate and sodium dodecyl sulfate, using either a pre-formed complex or in situ pairing. The strategy successfully forms NCs ~150 nm in diameter and achieves encapsulation efficiencies over 95%. Protein release rate from the NCs in physiological conditions and the bioactivity of released lysozyme are measured, and both are found to vary with the complexing counterion and the protein/counterion ratio used during formulation. Protein release on the time scale of weeks is observed, and up to 100% bioactivity is measured from released lysozyme. 16 quaternary ammonium cationic counterions are tested to encapsulate ovalbumin in 32 formulations. Of these, 19 successfully form ~150 nm NCs with loadings up to 29% and encapsulation efficiencies up to 88%. We divide the formulations into four regimes and identify chemical factors responsible for the success or failure of a given counterion to formulate NCs with the desirable size, loading, and encapsulation efficiency. A successful ovalbumin NC formulation was then tested in vivo in a mouse nasal vaccine model and found to induce a higher titer of OVA-specific IgG than unencapsulated ovalbumin. Taken together, these findings suggest that Flash NanoPrecipitation with hydrophobic ion pairing is an attractive platform for encapsulating high molecular weight proteins into NCs. In particular, the ability to tune protein release rate by varying the counterion or protein/counterion ratio used during formulation is a useful feature.

Internal liquid crystal structures in nanocarriers containing drug hydrophobic ion pairs dictate drug release.

HYPOTHESIS: Hydrophobic ion pairing (HIP), a solubility engineering technique in which ionic hydrophilic molecules are paired with a hydrophobic counterion, is an attractive strategy for encapsulating ionic water-soluble species into nanocarriers (NCs). Drug release from NCs containing HIP complexes is sensitive to ionic strength, pH, and drug:counterion charge ratio, but the exact mechanism for this was unknown, as was the underlying microstructure inside the NCs. We hypothesize that HIP complexes arrange into liquid crystalline structures in NC cores and that these structures are responsible for salt- and pH-dependent release. EXPERIMENT: A model hydrophobic ion pair from the cationic antimicrobial peptide polymyxin B sulfate and the anionic counterion sodium oleate is encapsulated into ~100 nm NCs formed using Flash NanoPrecipitation (FNP) and stabilized with an amphiphilic diblock copolymer, poly(caprolactone)-b-poly(ethylene glycol). Internal structures are observed using synchrotron small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) following NC formulation and are found to vary with polymyxin:oleate charge ratio. In vitro drug release is also measured with changes in pH and two charge ratio. FINDINGS: For a formulation containing a four-fold charge excess of oleate relative to polymyxin, internal structures rearranged from a lamellar phase into an inverse hexagonal phase. The hexagonal phase formation corresponds to a greatly reduced rate of polymyxin release, suggesting that the polymyxin was incorporated into the center of hexagonally-packed rods. When release tests were repeated using phosphate-buffered saline (PBS) at pH 2.0 to ensure protonation of the oleic acid, all internal structures were eliminated and release occurs much faster than at neutral pH, regardless of charge ratio. These findings shed light on the mechanism behind stimulus-responsive drug release from systems containing hydrophobic ion pairs and enable the rational design of controlled-release formulations by manipulating the formation and dynamics of liquid crystalline phases inside NCs.

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites.

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Insights into Hydrophobic Ion Pairing from Molecular Simulation and Experiment.

Hydrophobic ion pairing (HIP) is the process by which a charged hydrophilic molecule of interest is electrostatically coupled with an oppositely charged hydrophobic counterion to produce a complex with greater hydrophobicity than the original molecule. This process is of interest in drug delivery, but a molecular-based mechanistic understanding is still incomplete. In this work, we used molecular simulation and experiment to study a model system of Polymyxin B (drug) and oleic acid (hydrophobic counterion). Validation of the simulation system was performed by assessing HIP complex stability under various solvent conditions, and the results were found to be in good agreement with experiment. The effects of solvent composition, particle size, and charge ratio on the observed hydrophobicity, morphology, and stability were studied through the simulation of small HIP clusters. Microsecond simulation of a larger system was then used to characterize the kinetics of assembly. Particle formation over longer length (µm) and time scales (ms) was studied experimentally via flash nanoprecipitation, and the formation of electrostatically stabilized nanoparticles was observed. These results provide a mechanistic and morphological picture of the HIP event and will help inform the development of future formulations that utilize HIP.

Polymeric Nanocarriers With Mucus-Diffusive and Mucus-Adhesive Properties to Control Pharmacokinetic Behavior of Orally Dosed Cyclosporine A.

The present study develops cyclosporine A (CsA)-loaded polymeric nanocarriers with mucus-diffusive and mucus-adhesive potential to control pharmacokinetic behavior after oral administration for the treatment of inflammatory bowel diseases (IBD). CsA-loaded nanocarriers consisting of polystyrene-block-polyethylene glycol (PEG-CsA) and polystyrene-block-polyacrylic acid (PAA-CsA) were prepared by a flash nanoprecipitation. Both nanocarriers showed redispersibility from lyophilized powder back to uniform nanocarrier with a mean diameter of approximately 150 nm. The nanocarriers exhibited significantly improved release behavior of CsA under pH 6.8 condition compared. A test of mucodiffusion, using artificial mucus, demonstrated the mucus-diffusive and mucus-adhesive potential of PEG-CsA and PAA-CsA, respectively, dependent on the lack of electrostatic interactions between the surface-coated polymer and mucin. Oral administrations of PEG-CsA and PAA-CsA (10 mg-CsA/kg) in rats resulted in significant improvements of absorption, as evidenced by 50- and 25-fold higher bioavailability than crude CsA, respectively. PAA-CsA exhibited more sustained and slower absorption process of CsA than PEG-CsA because of the different diffusion behavior within the mucus layer. In the rat model of IBD, significant suppression of inflammatory symptoms could be achieved by oral treatment with both CsA nanoparticles. These polymeric nanocarriers are promising dosage options to control pharmacokinetic behavior of orally dosed CsA, contributing to the development of safe and effective treatment for IBD.

Solid-State Behavior and Solubilization of Flash Nanoprecipitated Clofazimine Particles during the Dispersion and Digestion of Milk-Based Formulations.

Clofazimine, a drug previously used to treat leprosy, has recently been identified as a potential new drug for the treatment for cryptosporidiosis: a diarrheal disease that contributes to 500 000 infant deaths a year in developing countries. Rapid dissolution and local availability of the drug in the small intestine is considered key to the treatment of the infection. However, the commercially available clofazimine formulation (Lamprene) is not well-suited to pediatric use, and therefore reformulation of clofazimine is desirable. Development of clofazimine nanoparticles through the process of flash nanoprecipitation (FNP) has been previously shown to provide fast and improved drug dissolution rates compared to clofazimine crystals and Lamprene. In this study, we investigate the effects of milk-based formulations (as possible pediatric-friendly vehicles) on the in vitro solubilization of clofazimine formulated as either lecithin- or zein/casein-stabilized nanoparticles. Milk and infant formula were used as the lipid vehicles, and time-resolved synchrotron X-ray scattering was used to monitor the presence of crystalline clofazimine in suspension during in vitro lipolysis under intestinal conditions. The study confirmed faster dissolution of clofazimine from all the FNP formulations after the digestion of infant formula was initiated, and a reduced quantity of fat was required to achieve similar levels of drug solubilization compared to the reference drug material and the commercial formulation. These attributes highlight not only the potential benefits of the FNP approach to prepare drug particles but also the fact that enhanced dissolution rates can be complemented by considering the amount of co-administered fat in lipid-based formulations to drive the solubilization of poorly soluble drugs.

Spray drying OZ439 nanoparticles to form stable, water-dispersible powders for oral malaria therapy.

BACKGROUND: OZ439 is a new chemical entity which is active against drug-resistant malaria and shows potential as a single-dose cure. However, development of an oral formulation with desired exposure has proved problematic, as OZ439 is poorly soluble (BCS Class II drug). In order to be feasible for low and middle income countries (LMICs), any process to create or formulate such a therapeutic must be inexpensive at scale, and the resulting formulation must survive without refrigeration even in hot, humid climates. We here demonstrate the scalability and stability of a nanoparticle (NP) formulation of OZ439. Previously, we applied a combination of hydrophobic ion pairing and Flash NanoPrecipitation (FNP) to formulate OZ439 NPs 150 nm in diameter using the inexpensive stabilizer hydroxypropyl methylcellulose acetate succinate (HPMCAS). Lyophilization was used to process the NPs into a dry form, and the powder's in vitro solubilization was over tenfold higher than unprocessed OZ439. METHODS: In this study, we optimize our previous formulation using a large-scale multi-inlet vortex mixer (MIVM). Spray drying is a more scalable and less expensive operation than lyophilization and is, therefore, optimized to produce dry powders. The spray dried powders are then subjected to a series of accelerated aging stability trials at high temperature and humidity conditions. RESULTS: The spray dried OZ439 powder's dissolution kinetics are superior to those of lyophilized NPs. The powder's OZ439 solubilization profile remains constant after 1 month in uncapped vials in an oven at 50 °C and 75% RH, and for 6 months in capped vials at 40 °C and 75% RH. In fasted-state intestinal fluid, spray dried NPs achieved 80-85% OZ439 dissolution, to a concentration of 430 µg/mL, within 3 h. In fed-state intestinal fluid, 95-100% OZ439 dissolution is achieved within 1 h, to a concentration of 535 µg/mL. X-ray powder diffraction and differential scanning calorimetry profiles similarly remain constant over these periods. CONCLUSIONS: The combined nanofabrication and drying process described herein, which utilizes two continuous unit operations that can be operated at scale, is an important step toward an industrially-relevant method of formulating the antimalarial OZ439 into a single-dose oral form with good stability against humidity and temperature.

Amorphous nanoparticles by self-assembly: processing for controlled release of hydrophobic molecules.

More than 40% of newly developed drug molecules are highly hydrophobic and, thus, suffer from low bioavailability. Kinetically trapping the drug as a nanoparticle in an amorphous state enhances solubility. However, enhanced solubility can be compromised by subsequent recrystallization from the amorphous state during drying processes. We combine Flash NanoPrecipitation (FNP) to generate nanoparticles with spray-drying to produce stable solid powders. We demonstrate that the continuous nanofabrication platform for nanoparticle synthesis and recovery does not compromise the dissolution kinetics of the drug. Lumefantrine, an anti-malaria drug, is highly hydrophobic with low bioavailability. Increasing the bioavailability of lumefantrine has the potential to reduce the dose and number of required administrations per treatment, thus reducing cost and increasing patient compliance. The low melting temperature of lumefantrine (Tm = 130 °C) makes the drying of amorphous nanoparticles at elevated temperatures potentially problematic. Via FNP, we produced 200-400 nm nanoparticles using hydroxypropyl methylcellulose acetate succinate (HPMCAS), lecithin phospholipid, and zein protein stabilizers. Zein nanoparticles were spray-dried at 100 °C and 120 °C to study the effect of the drying temperature. For zein powders, at two hours the dissolution kinetics under fasted conditions reached 85% release for the 100 °C sample, but only 60% release for the 120 °C sample. Powder X-ray diffraction, differential scanning calorimetry, and solid state nuclear magnetic resonance indicate that the lumefantrine in the nanoparticle core is amorphous for samples spray-dried at 100 °C. Dissolution under fed state conditions showed similar release kinetics for both temperatures, with 90-95% release at two hours. Zein and HPMCAS nanoparticles spray-dried at 100 °C showed release profiles in fasted and fed state media that are identical to those of lyophilized samples, i.e. those dried at cryogenic conditions where no transformation to the crystalline state can occur. Thus, spray drying 30 °C below the melting transition of lumefantrine is sufficient to maintain the amorphous state. These inexpensive formulations have potential to be developed into future therapies for malaria, and the results also highlight the potential of combining FNP and spray-drying as a versatile platform to assemble and rapidly recover amorphous nanoparticles in a solid dosage form.

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of ± 6%.