Towards realizing nano-enabled precision delivery in plants.

Nanocarriers (NCs) that can precisely deliver active agents, nutrients and genetic materials into plants will make crop agriculture more resilient to climate change and sustainable. As a research field, nano-agriculture is still developing, with significant scientific and societal barriers to overcome. In this Review, we argue that lessons can be learned from mammalian nanomedicine. In particular, it may be possible to enhance efficiency and efficacy by improving our understanding of how NC properties affect their interactions with plant surfaces and biomolecules, and their ability to carry and deliver cargo to specific locations. New tools are required to rapidly assess NC-plant interactions and to explore and verify the range of viable targeting approaches in plants. Elucidating these interactions can lead to the creation of computer-generated in silico models (digital twins) to predict the impact of different NC and plant properties, biological responses, and environmental conditions on the efficiency and efficacy of nanotechnology approaches. Finally, we highlight the need for nano-agriculture researchers and social scientists to converge in order to develop sustainable, safe and socially acceptable NCs.

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.

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.

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.

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.

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.

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%.

Hydrophobic ion pairing: encapsulating small molecules, peptides, and proteins into nanocarriers.

Hydrophobic ion pairing has emerged as a method to modulate the solubility of charged hydrophilic molecules ranging in class from small molecules to large enzymes. Charged hydrophilic molecules are ionically paired with oppositely-charged molecules that include hydrophobic moieties; the resulting uncharged complex is water-insoluble and will precipitate in aqueous media. Here we review one of the most prominent applications of hydrophobic ion pairing: efficient encapsulation of charged hydrophilic molecules into nano-scale delivery vehicles - nanoparticles or nanocarriers. Hydrophobic complexes are formed and then encapsulated using techniques developed for poorly-water-soluble therapeutics. With this approach, researchers have reported encapsulation efficiencies up to 100% and drug loadings up to 30%. This review covers the fundamentals of hydrophobic ion pairing, including nomenclature, drug eligibility for the technique, commonly-used counterions, and drug release of encapsulated ion paired complexes. We then focus on nanoformulation techniques used in concert with hydrophobic ion pairing and note strengths and weaknesses specific to each. The penultimate section bridges hydrophobic ion pairing with the related fields of polyelectrolyte coacervation and polyelectrolyte-surfactant complexation. We then discuss the state of the art and anticipated future challenges. The review ends with comprehensive tables of reported hydrophobic ion pairing and encapsulation from the literature.

Rapid Recovery of Clofazimine-Loaded Nanoparticles with Long-Term Storage Stability as Anti-Cryptosporidium Therapy.

While the formulation of nanoparticle (NP) suspensions has been widely applied in materials and life science, the recovery of NPs from such a suspension into a solid state is practically important to confer long-term storage stability. However, solidification, while preserving the original nanoscale properties, remains a formidable challenge in the pharmaceutical and biomedical applications of NPs. Herein we combined flash nanoprecipitation (FNP) and spray-drying as a nanofabrication platform for NP formulation and recovery without compromising the dissolution kinetics of the active ingredient. Clofazimine was chosen to be the representative drug, which has been recently repurposed as a potential treatment for cryptosporidiosis. Clofazimine was encapsulated in NPs with low-cost surface coatings, hypromellose acetate succinate (HPMCAS) and lecithin, which were required by the ultimate application to global health. Spray-drying and lyophilization were utilized to produce dried powders with good long-term storage stability for application in hot and humid climatic zones. The particle morphology, yield efficiency, drug loading, and clofazimine crystallinity in the spray-dried powders were characterized. The in vitro release kinetics of spray-dried NP powders were compared to analogous dissolution profiles from standard lyophilized NP samples, crystalline clofazimine powder, and the commercially available formulation Lamprene. The spray-dried powders showed a supersaturation level of up to 60 times the equilibrium solubility and remarkably improved dissolution rates. In addition, the spray-dried powders with both surface coatings showed excellent stability during aging studies with elevated temperature and humidity, in view of the dissolution and release in vitro. Considering oral delivery for pediatric administration, the spray-dried powders show less staining effects with simulated skin than crystalline clofazimine and may be made into minitablets without additional excipients. These results highlight the potential of combining FNP and spray-drying as a feasible and versatile platform to design and rapidly recover amorphous NPs in a solid dosage form, with the advantages of satisfactory long-term storage stability, low cost, and easy scalability.

Pseudomonas aeruginosa pyocyanin production reduced by quorum-sensing inhibiting nanocarriers.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes a wide range of infections; it is becoming increasingly difficult to treat due to antibiotic resistance. Quorum-sensing (QS) based therapeutics, which function by disabling pathogen virulence without killing pathogens, are a promising class of drugs that may be used to treat bacterial infections without eliciting resistance development. The use of QS drugs to treat pulmonary P. aeruginosa infections, however, has been greatly limited due to the inability to deliver QS drugs at sufficiently high concentrations past physiological barriers such as pulmonary mucus. Here we apply a block copolymer-directed self-assembly process, Flash NanoPrecipitation, to develop a series of QS-active formulations that are fully water dispersible, stable, and mucus-penetrating. These formulations inhibit P. aeruginosa virulence without inhibiting cell growth. Particle size (70 nm-400 nm) and release rate (1 h-14 days) can be tuned by altering constructs' physical properties and formulation excipients. We also demonstrate, to the best of our knowledge, the first instance of a QS nanocarrier platform technology that can penetrate through human cystic fibrosis pulmonary mucus. This work highlights the need to incorporate nanoformulation strategies into the development of next-generation antimicrobial therapeutics.

Encapsulation of OZ439 into Nanoparticles for Supersaturated Drug Release in Oral Malaria Therapy.

Malaria poses a major burden on human health and is becoming increasingly difficult to treat due to the development of antimalarial drug resistance. The resistance issue is further exacerbated by a lack of patient adherence to multi-day dosing regimens. This situation motivates the development of new antimalarial treatments that are less susceptible to the development of resistance. We have applied Flash NanoPrecipitation (FNP), a polymer-directed self-assembly process, to form stable, water-dispersible nanoparticles (NPs) of 50-400 nm in size containing OZ439, a poorly orally bioavailable but promising candidate for single-dose malaria treatment developed by Medicines for Malaria Venture (MMV). During the FNP process, a hydrophobic OZ439 oleate ion paired complex was formed and was encapsulated into NPs. Lyophilization conditions for the NP suspension were optimized to produce a dry powder. The in vitro release rates of OZ439 encapsulated in this powder were determined in biorelevant media and compared with the release rates of the unencapsulated drug. The OZ439 NPs exhibit a sustained release profile and several-fold higher release concentrations compared to that of the unencapsulated drug. In addition, XRD suggests the drug was stabilized into an amorphous form within the NPs, which may explain the improvement in dissolution kinetics. Formulating OZ439 into NPs in this way may be an important step toward developing a single-dose oral malaria therapeutic, and offers the possibility of reducing the amount of drug required per patient, lowering delivery costs, and improving dosing compliance.

Hydrophobic Ion Pairing of Peptide Antibiotics for Processing into Controlled Release Nanocarrier Formulations.

Nanoprecipitation of active pharmaceutical ingredients (APIs) to form nanocarriers (NCs) is an attractive method of producing formulations with improved stability and biological efficacies. However, nanoprecipitation techniques have not been demonstrated for highly soluble peptide therapeutics. We here present a model and technique to encapsulate highly water-soluble biologic APIs by manipulating API salt forms. APIs are ion paired with hydrophobic counterions to produce new API salts that exhibit altered solubilities suitable for nanoprecipitation processing. The governing rules of ion pair identity and processing conditions required for successful encapsulation are experimentally determined and assessed with theoretical models. Successful NC formation for the antibiotic polymyxin B requires hydrophobicity of the ion pair acid to be greater than logP = 2 for strong acids and greater than logP = 8 for weak acids. Oleic acid with a logP = 8, and pKa = 5, appears to be a prime candidate as an ion pair agent since it is biocompatible and forms excellent ion pair complexes. NC formation from preformed, organic soluble ion pairs is compared to in situ ion pairs where NCs are made in a single precipitation step. NC properties, such as stability and release rates, can be tuned by varying ion pair molecular structure and ion pair-to-API molar ratios. For polymyxin B, NCs ≈ 100-200 nm in size, displaying API release rates over 3 days, were produced. This work demonstrates a new approach that enables the formation of nanoparticles from previously intractable compounds.

Zein Nanoparticles Uptake by Hydroponically Grown Soybean Plants.

In the interest of developing and characterizing a polymeric nanoparticle pesticide delivery vehicle to soybeans, zein nanoparticle (ZNP) uptake by the roots and biodistribution to the leaves of soybean plants was measured. Zein was tagged with fluorescein isothiocyanate (FITC) and made into nanoparticles (135 ± 3 nm diameter. 0.202 ± 0.034 PDI and 81 ± 4 mV zeta-potential at pH 6) using an emulsion-diffusion method. After 10 days of hydroponic exposure, association between particles and roots of plants was found to vary based on bulk nanoparticle concentration. While 0.37 mg NP/mg dry weight were detected in roots immersed in 0.88 mg NP/mL nanoparticle suspension, 0.58 mg NP/mg dry weight associated with roots immersed in a high dose nanoparticle suspension of 1.75 mg NP/mL at 10 days. Nanoparticle root uptake followed second order kinetics. A small amount of increased fluorescence was detected in the hydroponically exposed plant's leaves, suggesting that either small amounts of particles or other fluorescent contaminants of zein were up taken by the roots and biodistributed within the plant. To the authors' knowledge, this is the first study in which the uptake and time-dependent association between polymeric nanoparticles and soybeans are quantified.

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.