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\u27s 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\u27 knowledge, this is the first study in which the uptake and time-dependent association between polymeric nanoparticles and soybeans are quantified

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 p<i>K</i>_a = 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

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

Abstract 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

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

Rapid Recovery of Clofazimine-Loaded Nanoparticles with Long-Term Storage Stability as Anti-<i>Cryptosporidium</i> 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

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

Clofazimine, a lipophilic (log <i>P</i> = 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

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 <i>in vitro</i> 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

Targeted Antitubercular Peptide Nanocarriers Prepared by Flash NanoPrecipitation with Hydrophobic Ion Pairing

The encapsulation of therapeutics into nanocarriers with specialized surface chemistries for targeting applications in the body is a major goal in the field of drug delivery. Here the encapsulation of an antitubercular peptide, ecumicin, into monodisperse nanocarriers 60 nm in diameter using a combination of Flash NanoPrecipitation and hydrophobic ion pairing is demonstrated. The lead formulation achieves 70% ecumicin encapsulation efficiency and 24% loading by mass. In vivo single-dose oral (PO), subcutaneous (SC), and intraperitoneal (IP) pharmacokinetics (PK) are measured in mice, and the dose-normalized area under the curve (AUC) of ecumicin nanocarriers dosed IP exceeded the dose-normalized AUC of unencapsulated ecumicin dosed IP by a factor of 2.5. Next, variations of the lead formulation stabilized with a custom-synthesized poly(caprolactone)-block-poly(ethylene glycol)-hexamannose polymer at three levels of mannose surface coverage (0%, 4%, and 74% of polymer chains terminating in hexamannose) for targeting to macrophages are prepared. These formulations are evaluated against Mycobacterium tuberculosis in a macrophage culture at multiple concentrations and found to reduce colony-forming units (CFU) counts by up to 3.8-log10 units, with greater antitubercular ecumicin activity measured from formulations prepared with higher amounts of surface mannose coverage. Taken together, these results suggest that Flash NanoPrecipitation with hydrophobic ion pairing is an effective method for encapsulating ionizable peptide therapeutics into macrophage-targeted formulations for improved PK and targeted macrophage uptake in the body