The Critical Transition from Soluble Complexes to Colloidal Aggregates of Polyelectrolyte Complexes at Non-Stoichiometric Charge Ratios.

The transition from soluble to colloidal polyelectrolyte complex normally occurs at a critical non-stoichiometric charge ratio. Here, it is demonstrated that the conventional batch mixing produces heterogeneous binding and complexation, which can easily mask this soluble-colloidal complex transition (sol-col transition) even for weakly binding polyelectrolytes like polyacrylic acid (PAA) and poly(diallyldimethylammonium chloride) (PDADMAC). When mixed efficiently using multi-inlet vortex mixer (MIVM), the sol-col transition occurs beyond a critical charge ratio (n-/n+) and the large colloidal complexes are formed through the aggregation of small primary complexes (as revealed by atomic force microscopy). Moreover, the sol-col transition occurs at a constant charge ratio below the overlapping concentration (c*) of the long host polyelectrolyte, but at lower charge ratios above c* due to chain entanglement. When adding NaCl to the solution, the sol-col transition charge ratio first decreases, then remained stable for a period, and finally increased and vanished at high ionic strength. When replacing NaCl with chaotropic salts, the sol-col transition occurs at lower charge ratios, while kosmotropes has little impact. The solvent quality and polymer hydrophobicity effects are also discussed. With the assistance of rapid mixing, this study provides a more reliable way of studying the sol-col transition of polyelectrolyte complexes.

Flash Technology-Based Self-Assembly in Nanoformulation: From Fabrication to Biomedical Applications.

Advances in nanoformulation have driven progress in biomedicine by producing nanoscale tools for biosensing, imaging, and drug delivery. Flash-based technology, the combination of rapid mixing technique with the self-assembly of macromolecules, is a new engine for the translational nanomedicine. Here, we review the state-of-the-art in flash-based self-assembly including theoretical and experimental principles, mixing device design, and applications. We highlight the fields of flash nanocomplexation (FNC) and flash nanoprecipitation (FNP), with an emphasis on biomedical applications of FNC, and discuss challenges and future directions for flash-based nanoformulation in biomedicine.

Continuous Production of Discrete Plasmid DNA-Polycation Nanoparticles Using Flash Nanocomplexation.

Despite successful demonstration of linear polyethyleneimine (lPEI) as an effective carrier for a wide range of gene medicine, including DNA plasmids, small interfering RNAs, mRNAs, etc., and continuous improvement of the physical properties and biological performance of the polyelectrolyte complex nanoparticles prepared from lPEI and nucleic acids, there still exist major challenges to produce these nanocomplexes in a scalable manner, particularly for lPEI/DNA nanoparticles. This has significantly hindered the progress toward clinical translation of these nanoparticle-based gene medicine. Here the authors report a flash nanocomplexation (FNC) method that achieves continuous production of lPEI/plasmid DNA nanoparticles with narrow size distribution using a confined impinging jet device. The method involves the complex coacervation of negatively charged DNA plasmid and positive charged lPEI under rapid, highly dynamic, and homogeneous mixing conditions, producing polyelectrolyte complex nanoparticles with narrow distribution of particle size and shape. The average number of plasmid DNA packaged per nanoparticles and its distribution are similar between the FNC method and the small-scale batch mixing method. In addition, the nanoparticles prepared by these two methods exhibit similar cell transfection efficiency. These results confirm that FNC is an effective and scalable method that can produce well-controlled lPEI/plasmid DNA nanoparticles.

Kinetically Controlled Polyelectrolyte Complex Assembly of microRNA-Peptide Nanoparticles toward Treating Mesothelioma.

Broad size distributions and poor long-term colloidal stability of microRNA-carrying nanoparticles, especially those formed by polyelectrolyte complexation, represent major hurdles in realizing their clinical translation. Herein, peptide design is used alongside optimized flash nanocomplexation (FNC) to produce uniform peptide-based miRNA particles of exceptional stability that display anticancer activity against mesothelioma in vitro and in vivo. Modulating the content and display of lysine-based charge from small intrinsically disordered peptides used to complex miRNA proves essential in achieving stable colloids. FNC facilitates kinetic isolation of the mechanistic steps involved in particle formation to allow the preparation of particles of discrete size in a highly reproducible, scalable, and continuous manner, facilitating pre-clinical studies. To the best of the authors knowledge, this work represents the first example of employing FNC to prepare polyelectrolyte complexes of miRNA and peptide. Encapsulation of these particles into an injectable hydrogel matrix allows for their localized in vivo delivery by syringe. A one-time injection of a gel containing particles composed of miRNA-215-5p and the peptide PKM1 limits tumor progression in a xenograft model of mesothelioma.

Osmundea sp. macroalgal polysaccharide-based nanoparticles produced by flash nanocomplexation technique.

The macroalgae-derived polysaccharides' biological potential has been explored due to their attractive intrinsic properties such as biocompatibility, biodegradability, and their ability to conjugate with other compounds. In particular, in the drug delivery systems field, the anionic macroalgae polysaccharides have been combined with cationic compounds through ionotropic gelation and/or bulk mixing. However, these techniques did not assure reproducibility, and the stability of nanoparticles is undesired. To overcome these limitations, herein, the polysaccharide extracted from Osmundea sp. was used to produce nanoparticles through the flash nanocomplexation technique. This approach rapidly mixed the negative charge of macroalgae polysaccharide with a positive chitosan charge on a millisecond timescale. Further, diclofenac (an anti-inflammatory drug) was also incorporated into complex nanoparticles. Overall, the gathered data showed that hydrodynamic diameter nanoparticles values lower than 100 nm, presenting a narrow size distribution and stability. Also, the diclofenac exhibited a targeted and sustained release profile in simulating inflammatory conditions. Likewise, the nanoparticles showed excellent biological properties, evidencing their suitability to be used to treat inflammatory skin diseases.

Mixing efficiency affects the morphology and compactness of chitosan/tripolyphosphate nanoparticles.

Due to the rapid reaction kinetics, the morphology of chitosan/tripolyphosphate nanoparticles was difficult to control. We mixed chitosan and tripolyphosphate through a multi-inlet vortex mixer at different mixing efficiency and characteristic mixing time (τmix). Below a critical τmix, the aggregation of primary chitosan/TPP particles can be ceased. The corresponding aggregation time of primary particles (τagg) was dependent on the ionic strength, the degree of deacetylation and molecular weight of chitosan. Moreover, the particle compactness was estimated from the hydrodynamic diameter and the turbidity using a model based on the Mie theory. Slow and nonhomogeneous mixing led to more compact nanoparticles, while rapid and homogeneous mixing produced nanoparticles with a higher swelling ratio. Besides, the ionic strength and polymer concentration could affect the internal structure of nanoparticles. This study revealed the significance of mixing on the internal structure of chitosan/TPP nanoparticles, which could guide the preparation of other ionically-crosslinked polysaccharide colloids.

Universal and Translational Nanoparticulate CpG Adjuvant.

CpG, an agonist of toll-like receptor 9 (TLR9), has become a novel adjuvant that substantially potentiates cellular immunity. However, this agonist may increase systemic toxicity by diffusing into blood after administration and is difficult to be internalized by immune cells to reach TLR9 located in endosomes as a result of the characteristics of negative charge of CpG. Here, we applied a scalable and controllable flash nanocomplexation technology to prepare nanoparticulate CpG adjuvant (npCpG), CpG encapsulated in a physical cross-linking network of protamine and TPP. The nanoadjuvant could redirect CpG into draining lymph nodes to reduce systemic diffusion to improve safety. Further, a combination of npCpG and influenza H1N1 hemagglutinin antigen showed excellent humoral and cellular immunity, evoking high levels of antibodies and cytokines and inducing a great expansion of splenocytes in immunized mice. Also, the nanoadjuvant combined with ovalbumin antigen led to a potent cytotoxic T-cell response, substantially inhibited tumor growth, and improved the survival rate of mice in a melanoma model. This study showed the universal performances of npCpG in infectious disease prevention and tumor immunotherapy to demonstrate the translational potential.

Scalable Manufacture of Curcumin-Loaded Chitosan Nanocomplex for pH-Responsive Delivery by Coordination-Driven Flash Nanocomplexation.

Metal coordination-driven nanocomplexes are known to be responsive to physiologically relevant stimuli such as pH, redox, temperature or light, making them well-suited for antitumor drug delivery. The ever-growing demand for such nanocomplexes necessitates the design of a scalable approach for their production. In this study, we demonstrate a novel coordination self-assembly strategy, termed flash nanocomplexation (FNC), which is rapid and efficient for the fabrication of drug-loaded nanoparticles (NPs) in a continuous manner. Based on this strategy, biocompatible chitosan (CS) and Cu2+ can be regarded anchors to moor the antitumor drug (curcumin, Cur) through coordination, resulting in curcumin-loaded chitosan nanocomplex (Cur-loaded CS nanocomplex) with a narrow size distribution (PDI < 0.124) and high drug loading (up to 41.75%). Owing to the excellent stability of Cur-loaded CS nanocomplex at neutral conditions (>50 days), premature Cur leakage was limited to lower than 1.5%, and pH-responsive drug release behavior was realized in acidic tumor microenvironments. An upscaled manufacture of Cur-loaded CS nanocomplex is demonstrated with continuous FNC, which shows an unprecedented method toward practical applications of nanomedicine for tumor therapy. Furthermore, intracellular uptake study and cytotoxicity experiments toward H1299 cells demonstrates the satisfied anticancer efficacy of the Cur-loaded CS nanocomplex. These results confirm that coordination-driven FNC is an effective method that enables the rapid and scalable fabrication of antitumor drugs.

Kinetic control of Phytic acid/Lixisenatide/Fe (III) ternary nanoparticles assembly process for sustained peptide release.

The preferable choice of sustained peptide delivery systems is generally polymer-based microspheres in which their large particle size, wide size distribution, low drug encapsulation efficacy, poor colloidal stability, and undesirable burst release eventually hinder their clinical translation. In this study, a nanoscale ternary Lixisenatide (Lix) sustained delivery system based on strong multivalent interactions (electrostatic and coordination complexation) among small molecular phytic acid (PA), Lix and Fe3+ was developed. Flash nanocomplexation (FNC) was utilized to facilitate the rapid and efficient mixing of the three components and kinetically control the assembly process that enabled dynamic balance of two competitive chemical reactions with different kinetic rates (slow chemical reaction of PA/Lix and fast chemical reaction of PA/Fe3+) to generate structural uniform ternary nanoparticles and avoid heterogeneous complexes. By tuning the mixing conditions (i.e., flow rate, mass ratio, concentration, pH value, etc.), the ternary PA/Lix/Fe3+ nanoparticles were assembled with reproducible production in a manner of high uniformity and scalability, achieving small size (∼50 nm), uniform composition (PDI: ∼0.12), favourable colloidal stability, high encapsulation efficiency (∼100%), and tunable drug release kinetics. The optimized formulation exhibited a minor Lix release (<20%) in the first day and extended peptide release period over 8 days. Unexpectedly, upon a single injection administration, the as-prepared formulation (600 µg/kg) rapidly brought the high BGL (∼30 mmol/L) back to normal range (<10 mmol/L) within the initial 6 h and achieved a 180 h glycemic control in T2D mouse model. Moreover, this sustained peptide delivery system demonstrated a repeatable hypoglycemic effects and significantly suppressed the pathological damage of major organs following multiple injection. This sustained peptide delivery system with aqueous, facile and reproducible preparation process possesses good biocompatibility, tunable release kinetics, and prolonged hypoglycemic effects, portending its great translational potential in the chronic disease treatment.

A Versatile and Robust Platform for the Scalable Manufacture of Biomimetic Nanovaccines.

Biomimetic strategies are useful for designing potent vaccines. Decorating a nanoparticulate adjuvant with cell membrane fragments as the antigen-presenting source exemplifies, such as a promising strategy. For translation, a standardizable, consistent, and scalable approach for coating nanoadjuvant with the cell membrane is important. Here a turbulent mixing and self-assembly method called flash nanocomplexation (FNC) for producing cell membrane-coated nanovaccines in a scalable manner is demonstrated. The broad applicability of this FNC technique compared with bulk-sonication by using ten different core materials and multiple cell membrane types is shown. FNC-produced biomimetic nanoparticles have promising colloidal stability and narrow particle polydispersity, indicating an equal or more homogeneous coating compared to the bulk-sonication method. The potency of a nanovaccine comprised of B16-F10 cancer cell membrane decorating mesoporous silica nanoparticles loaded with the adjuvant CpG is then demonstrated. The FNC-fabricated nanovaccines when combined with anti-CTLA-4 show potency in lymph node targeting, DC antigen presentation, and T cell immune activation, leading to prophylactic and therapeutic efficacy in a melanoma mouse model. This study advances the design of a biomimetic nanovaccine enabled by a robust and versatile nanomanufacturing technique.

Engineered therapeutic nanovaccine against chronic hepatitis B virus infection.

Chronic hepatitis B (CHB), caused by persistent hepatitis B virus (HBV) infection, significantly increases the risk of leading to liver diseases. Despite the successful development and implementation of HBV prophylactic vaccines for several decades, the development of therapeutic vaccine, a substantially potential strategy to eradicate HBV and achieve CHB cure, remains a great challenge. Herein, we applied flash nanocomplexation (FNC) technology to prepare nanovaccines with narrow size distribution and high encapsulation via the charge complexation between chitosan and heparin to encapsulate recombinant hepatitis B virus surface antigen (rHBsAg) or core antigen (rHBcAg), with CpG as adjuvant. The two nanovaccines enhanced the uptake of antigen and adjuvant into Raw264.7 cells and their co-administration further promoted maturation and activation of bone marrow-derived dendritic cells (BMDCs). Meanwhile, they exhibited excellent lymph nodes (LNs) targeting ability, draining to proximal and distal LNs with prolonged retention time, following subcutaneous injection. Co-administered nanovaccines could break immune tolerance and restore HBV-specific immune responses. In a mouse model of CHB, 90% and 80% of mice achieved hepatitis B virus surface antigen (HBsAg) seroclearance and hepatitis B virus surface antibody (HBsAb) seroconversion, respectively. Moreover, the vaccines induced long-term immune memory in HBV-cured mice to protect them from HBV reinfection. Thus, this work offers a promising and translational alternative for therapeutic CHB vaccine.

Augmenting Therapeutic Potential of Polyphenols by Hydrogen-Bonding Complexation for the Treatment of Acute Lung Inflammation.

Dysregulated inflammation is considered as an essential pathological process in inflammation-associated diseases, which would be aggravated by high levels of reactive oxygen species (ROS) generation inducing oxidative stress. Currently, extensive attention has been paid to polyphenolic compounds owing to their broad spectrum biological activities, such as antioxidant and anti-inflammatory effects, while their therapeutic potential has been compromised by the poor stability, short plasma half-life, and low bioavailability. Given that polyphenols have a wide range of structural characteristics and various physicochemical properties, there remains a real challenge toward green, mass production of universal nanocarriers for effective entrapment of these active pharmaceutical ingredients. In this study, we adopted a flash nanocomplexation (FNC) platform to prepare nanocomplexes comprising polyphenols and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) enabled by hydrogen bonding. We confirmed that the molecular structure of polyphenols has a great influence on their complexation with TPGS, and stable nanocomplexes were formed when the number of phenolic hydroxyl groups of polyphenols was above the value of 8. These hydrogen-bonded nanocomplexes produced by an FNC apparatus exhibited well-controlled quality with uniform size, good colloidal stability, and high batch-to-batch repeatability, thus improving the druggability as potent nanotherapeutics for antioxidant and anti-inflammatory applications. In vivo experiments indicated that the optimal nanocomplex (EGCG-NC) can be applied to ameliorate acute lung injury in a mice model after nasal administration. These results proved that polyphenols formulated with TPGS for nanocomplex formation through hydrogen-bonding complexation could augment their therapeutic potential for modulating hyperactive inflammation in the treatment of acute lung inflammation.

Effective encapsulation of curcumin in nanoparticles enabled by hydrogen bonding using flash nanocomplexation.

Nanoparticular system of a model small molecular drug curcumin (CUR) was prepared using a novel method, namely, flash nanocomplexation by hydrogen bonding interactions. The CUR-loaded nanoparticles (NPs) were fabricated by mixing CUR, tannic acid and polyvinyl alcohol (PVA) in aqueous solutions under turbulent condition through a three-inlet confined impinging jet (CIJ) device. Compared to bulk mixing, FNC has the advantage of scalability, reproducibility and without causing the variations by different mixing sequences. Three NPs with different drug loading levels were prepared by tuning the CUR feeding amount. In human prostate cancer PC3 cells, both cellular uptake and cytotoxicity of these NPs were negatively correlated with the drug loading level. These findings indicate that FNC is an easy and feasible method for small molecular drug delivery by hydrogen bonding interactions.

Sustained release of exendin-4 from tannic acid/Fe (III) nanoparticles prolongs blood glycemic control in a mouse model of type II diabetes.

Exendin-4 has been clinically adopted as an effective drug for treating type 2 diabetes (T2D), but its short circulation half-life in the blood requires two injections per day to maintain effective glycemic control. This significantly limits its clinical application. In this study, we developed a tannic acid/exendin-4/Fe3+ ternary nanoparticle system to provide sustained release of exendin-4 in vivo. The formation of these nanoparticles relies on TA/exendin-4 complexation and stabilization through TA-Fe3+ coordination, where the rapid reaction kinetics can benefit from efficient mixing of all three components. Adapting our recently developed flash nanocomplexation (FNC) method, we formulated nanoparticles with high encapsulation efficiency (~ 100%) of exendin-4, high payload capacity, and high degrees of uniformity and stability because the rapid turbulent mixing facilitated a homogeneous distribution of all three components in the complexation process. Intraperitoneal injection in mice showed that exendin-4 released from the nanoparticles had an AUC 7.2-fold higher than the free exendin-4 injection. Efficacy study in a T2D mouse model showed that the optimized formulation achieved a rapid reduction of the blood glucose level to the normal range within <12 h and maintained the same level for 72 h following a single intraperitoneal dose. The blood glucose level was maintained to below the therapeutic level (< 15 mmol/L) for 6 days, and the treatment led to reduced body weight with pathological and functional improvements in the kidney and liver. This tannic acid/exendin-4/Fe3+ ternary nanoparticle system holds translational potential in treating T2D, due to its improved treatment outcomes in terms of extended release of exendin-4, prolonged control of blood glucose level, reduced dosing frequency, and improved pathological indicators.

Uniform Core-Shell Nanoparticles with Thiolated Hyaluronic Acid Coating to Enhance Oral Delivery of Insulin.

Oral delivery of protein drugs is an attractive route of administration due to its convenience for repeated dosing and good patient compliance. However, currently oral protein therapeutics show very low bioavailability mainly due to the existence of hostile gastrointestinal (GI) environments, including mucus layers and intestinal epithelial barriers. Herein, using insulin as a model protein therapeutic, the core-shell nanoparticles with thiolated hyaluronic acid (HA-SH) coating (NPHA-SH ) are produced utilizing a two-step flash nanocomplexation process to enhance oral delivery efficiency of insulin. A positively charged nanoparticle core is first generated by electrostatic complexation between insulin and N-(2-hydroxypropyl)-3-trimethyl ammonium chloride modified chitosan (HTCC), followed by surface coating with HA-SH. The optimized NPHA-SH shows an average size of 100 nm with high encapsulation efficiency (91.1%) and loading capacity (38%). In vitro and ex vivo results confirm that NPHA-SH shows high mucus-penetration ability, improved intestinal retention and transepithelial transport property due to its thiolated surface and the ability of HA-SH coating to dissociate from the nanoparticle surface when across the mucosal layer. Oral administration of NPHA-SH to Type 1 diabetic rats yields high efficacy and an average relative bioavailability of 11.3%. These results demonstrate that the HA-SH coated core-shell nanoparticles are a promising oral delivery vehicle for protein therapeutics.

Size-controlled lipid nanoparticle production using turbulent mixing to enhance oral DNA delivery.

Lipid-based nanoparticles (LNPs) have been developed to address the transport and uptake barriers to enhance the delivery efficiency of plasmid DNA therapeutics. In these systems, plasmid DNA can be encapsulated through condensation by a cationic lipid to form lipo-complexes, or polycation following complexation into cationic liposomes to form lipo-polyplexes. Conventional methods for achieving these two DNA-delivering LNP vehicles suffer from significant batch-to-batch variation, poor scalability and complicated multi-step preparation procedures. Resultant nanoparticles often have uncontrollable size and surface charge with wide distribution, and poor stability when exposed to physiological media. Here we report a single-step flash nanocomplexation (FNC) process using turbulent mixing to prepare uniform lipo-complex or lipo-polyplex LNPs in a scalable manner, demonstrating excellent control over the nanoparticle size (from 40 to several hundred nm) and surface charge, with narrow size distribution. The FNC-produced LNPs could be purified and concentrated using a tangential flow filtration (TFF) process in a scalable manner. An optimized formulation of purified lipo-complex LNPs (DOTAP/Chol/DNA, 45 nm) showed significantly higher (5-fold in the lungs and 4-fold in the liver) transgene expression activity upon oral dosage than lipo-polyplex LNPs (DPPC/Chol/lPEI/DNA, 75 nm) or lPEI/DNA nanoparticles (43 nm). Repeated dosing (4 days, 150 µg/day) of the lipo-complex LNPs sustained the transgene activity over a period of one week without detectable toxicity in major organs, suggesting its potential for clinical translation. STATEMENT OF SIGNIFICANCE: We report a new method to prepare uniform size-controlled lipid-based DNA-loaded nanoparticles by turbulent mixing delivered by a multi-inlet vortex mixer. Two distinct compositions were successfully prepared: (1) lipo-complexes, through condensation of the plasmid DNA by cationic lipids; (2) lipo-polyplexes, by encapsulation of DNA/PEI together with neutral lipids. Comparing with conventional methods, which use multi-step processes with high batch-to-batch variations and poor control over nanoparticle characteristics, this method offers a single-step, continuous and reproducible assembly methodology that would promote the translation of such gene medicine products. Effective purification and concentration of nanoparticles were achieved by adopted tangential flow filtration method. Following oral gavage in mice, the lipo-complex nanoparticles showed the highest level of transgene expression in the lung and liver.

Scalable fabrication of size-controlled chitosan nanoparticles for oral delivery of insulin.

Controlled delivery of protein would find diverse therapeutic applications. Formulation of protein nanoparticles by polyelectrolyte complexation between the protein and a natural polymer such as chitosan (CS) is a popular approach. However, the current method of batch-mode mixing faces significant challenges in scaling up while maintaining size control, high uniformity, and high encapsulation efficiency. Here we report a new method, termed flash nanocomplexation (FNC), to fabricate insulin nanoparticles by infusing aqueous solutions of CS, tripolyphosphate (TPP), and insulin under rapid mixing condition (Re > 1600) in a multi-inlet vortex mixer. In comparison with the bulk-mixing method, the optimized FNC process produces CS/TPP/insulin nanoparticles with a smaller size (down to 45 nm) and narrower size distribution, higher encapsulation efficiency (up to 90%), and pH-dependent nanoparticle dissolution and insulin release. The CS/TPP/insulin nanoparticles can be lyophilized and reconstituted without loss of activity, and produced at a throughput of 5.1 g h-1 when a flow rate of 50 mL min-1 is used. Evaluated in a Type I diabetes rat model, the smaller nanoparticles (45 nm and 115 nm) control the blood glucose level through oral administration more effectively than the larger particles (240 nm). This efficient, reproducible and continuous FNC technique is amenable to scale-up in order to address the critical barrier of manufacturing for the translation of protein nanoparticles.