Hoang D. Lu

Modulating Vibrio cholerae Quorum-Sensing-Controlled Communication Using Autoinducer-Loaded Nanoparticles

The rise of bacterial antibiotic resistance has created a demand for alternatives to traditional antibiotics. Attractive possibilities include pro- and anti-quorum sensing therapies that function by modulating bacterial chemical communication circuits. We report the use of Flash NanoPrecipitation to deliver the Vibrio cholerae quorum-sensing signal CAI-1 ((S)-3-hydroxytridecan-4-one) in a water dispersible form as nanoparticles. The particles activate V. cholerae quorum-sensing responses 5 orders of magnitude higher than does the identically administered free CAI-1 and are diffusive across in vivo delivery barriers such as intestinal mucus. This work highlights the promise of combining quorum-sensing strategies with drug delivery approaches for the development of next-generation medicines.

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.

Narrow Absorption NIR Wavelength Organic Nanoparticles Enable Multiplexed Photoacoustic Imaging

Photoacoustic (PA) imaging is an emerging hybrid optical-ultrasound based imaging technique that can be used to visualize optical absorbers in deep tissue. Free organic dyes can be used as PA contrast agents to concurrently provide additional physiological and molecular information during imaging, but their use in vivo is generally limited by rapid renal clearance for soluble dyes and by the difficulty of delivery for hydrophobic dyes. We here report the use of the block copolymer directed self-assembly process, Flash NanoPrecipitation (FNP), to form series of highly hydrophobic optical dyes into stable, biocompatible, and water-dispersible nanoparticles (NPs) with sizes from 38 to 88 nm and with polyethylene glycol (PEG) surface coatings suitable for in vivo use. The incorporation of dyes with absorption profiles within the infrared range, that is optimal for PA imaging, produces the PA activity of the particles. The hydrophobicity of the dyes allows their sequestration in the NP cores, so that they do not interfere with targeting, and high loadings of >75 wt % dye are achieved. The optical extinction coefficients (ε (mL mg(-1) cm(-1))) were essentially invariant to the loading of the dye in NP core. Co-encapsulation of dye with vitamin E or polystyrene demonstrates the ability to simultaneously image and deliver a second agent. The PEG chains on the NP surface were functionalized with folate to demonstrate folate-dependent targeting. The spectral separation of different dyes among different sets of particles enables multiplexed imaging, such as the simultaneous imaging of two sets of particles within the same animal. We provide the first demonstration of this capability with PA imaging, by simultaneously imaging nontargeted and folate-targeted nanoparticles within the same animal. These results highlight Flash NanoPrecipitation as a platform to develop photoacoustic tools with new diagnostic capabilities.

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.

Copper Loading of Pre-Formed Nanoparticles for PET-Imaging Applications

Nanoparticles (NP) are promising contrast agents for positron emission tomography (PET) radionuclide imaging that can increase signal intensity by localizing clusters of PET radionuclides together. However, methods to load NPs with PET radionuclides suffer from harsh loading conditions or poor loading efficacies or result in NP surface modifications that alter targeting in vivo. We present the formation of water-dispersible, polyethylene glycol coated NPs that encapsulate phthalocyanines into NP cores at greater than 50 wt % loading, using the self-assembly technique Flash NanoPrecipitation. Particles from 70 to 160 nm are produced. Phthalocyanine NPs rapidly and spontaneously chelate metals under mild conditions and can act as sinks for PET radionuclides such as 64-Cu to produce PET-active NPs. NPs chelate copper(II) with characteristic rates of 1845 M-1 h-1 at pH 6 and 37 °C, which produced >90% radionuclide chelation within 1 h. NP physical properties, such as core composition, core fluidity, and size, can be tuned to modulate chelation kinetics. These NPs retain 64Cu even in the presence of the strong chelator ethylene diamine tetraacetic acid. The development of these constructs for rapid and facile radionuclide labeling expands the applications of NP-based PET imaging.

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.

Pseudomonas aeruginosa pyocyanin production reduced by quorum-sensing inhibiting nanocarriers

Graphical abstract Figure. No Caption available. Abstract 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.

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.

Photoacoustic assessment of nanoparticles distribution pattern in the mouse brain following blood-brain barrier (BBB) disruption

Focused ultrasound (FUS) with circulating microbubbles creates transient and reversible permeabilization of vasculatures which provides an essential means to deliver therapeutics and imaging agents, for instance across the BBB. We have previously shown that 3D color Doppler and photoacoustic imaging can monitor BBB disruption (BBBD) in mice. Here we study how changes in NPs size, concentration and dye properties play an important role in their spatial deposition.