Nanoparticle drug delivery formulations overcome many limitations associated with free therapeutics. However, determining the correct formulation can be difficult and often requires lengthy trial-and-error optimization. The NanoFabTx™ drug delivery formulation platform addresses these challenges by providing a highly flexible platform to synthesize, screen, and select the microparticle, nanoparticle, or liposome formulation that best fits your needs. The NanoFabTx™ drug delivery formulations kits and lipid mixes have been developed and tested by our R&D formulation scientists for both small molecule and nucleic acid delivery and include detailed protocols with step-by-step instructions to make synthesis simple.
The NanoFabTx™ platform has been designed to be synthesis-method agnostic. Nanoparticles can be generated via traditional nanoprecipitation using standard laboratory glassware, or via microfluidics using our NanoFabTx™ microfluidic device kits. Here, we demonstrate our NanoFabTx™ drug formulation screening kits and lipid mixes can also be used with Precision Nanosystems Inc.’s (PNI) NanoAssemblr® and NanoAssmeblr® Ignite microfluidic platform. Various PEG-PLGA nanoparticles were synthesized, optimized, screened, and selected using the NanoFabTX™ PEG-PLGA drug formulation screening kit (917796). Additionally, liposomes containing mRNA were prepared using our NanoFabTx™ DOTAP lipid mix (926027).
The NanoFabTx™ PEG-PLGA drug formulation screening kit (917796) is a ready-to-use nanoformulation kit for the synthesis of PEGylated PLGA nanoparticles. This kit provides four properly selected PEGylated PLGAs and stabilizer, allowing rapid screening of optimal formulation materials for enhanced drug delivery.
NanoFabTx™ PEG-PLGA FormulationPolymeric nanoparticles synthesized using the NanoFabTx PEG-PLGA drug formulation screening kit and NanoAssemblr®
Nanoparticles of uniform size (PDI ~0.06-0.09) and various formulations were generated using the NanoFabTx™ PEG-PLGA drug formulation screening kit (917796) and PNI’s NanoAssemblr®. The NanoFabTx™ PEG-PLGA drug formulation screening kit consists of four different PEG-PLGA formulations that can be screened and selected to best suit the researcher's needs. Here, nanoparticles of varying formulations were manufactured rapidly and with precise size control, eliminating lengthy trial-and-error optimization. The same flow rate ratio (1:1) and polymer concentration (2.5 wt%) were used to reach formulation.
Nanoparticle size was controlled by varying manufacturing parameters within the Precision Nanosystems software. A range of nanoparticle sizes (48 nm to 60 nm) were achieved with increased polymer concentration. While polymer concentration directly contributed to nanoparticle size, the PDI remained low, suggesting uniform size distribution.
Polymer Concentration (wt%)Controlled synthesis of PEG-PLGA nanoparticles using the NanoFabTx PEG-PLGA drug formulation screening kit and NanoAssemblr®
Flow rate ratio (FRR) is another manufacturing parameter that can be used to control nanoparticle size. Here, the flow rate ratio in the PNI software was changed from 1:1 to 1:3 and 1:5, respectively, while maintaining a total flow rate of 8 ml/min. PEG-PLGA nanoparticle size decreased with increased FRR. Nanoparticles generated at each FRR were uniform in size and resulted in a low PDI.
Flow Rate Ratio (Polymer:Stabilizer)NanoFabTx™ PEG-PLGA nanoparticle size can be controlled by flow rate ratio (FRR)
The NanoFabTx™ drug delivery formulation platform is designed to aid researchers in their formulation journey—from screening to preclinical development. Here, the NanoFabTx™ PEG-PLGA drug formulation screening kit was used with PNI’s screening and preclinical development NanoAssemblr® devices: the NanoAssemblr® and the NanoAssemblr® Ignite™.
PNI’s NanoAssemblr® is ideal for screening formulations because it requires minimal sample consumption, is easy to use, and rapidly produces nanoparticles of uniform size. The microfluidic technology can also be scaled up across the NanoAssemblr® Platform to accelerate future development. The NanoAssemblr® Ignite platform takes production from initial discovery stages to early preclinical formulation development. Here, NanoFabTx™ PEG-PLGA nanoparticle production by the NanoAssemblr® and NanoAssemblr® Ignite™ devices were compared.
Polymer ConcentrationNanoFabTx™ PEG-PLGA nanoparticles were synthesized using the NanoAssemblr® and NanoAssemblr® Ignite™
PEG-PLGA nanoparticles were generated at various polymer concentrations using the NanoAssemblr® and NanoAssemblr® Ignite™ instruments. Nanoparticle sizes ranged from 50nm to 175nm depending on polymer concentration. The flow rate parameters established using the NanoAssemblr® device were applied to the NanoAssemblr® Ignite™. The results suggest nanoparticles of similar size were generated between the two instruments and that the NanoFabTx™ formulation screening kits can easily be scaled up from early-stage screening to preclinical development.
The results above are indicative of the capabilities and flexibility of the NanoFabTX™ platform. Uniform nanoparticles were generated using the NanoAssemblr® instrument, and nanoparticle size could be adjusted in a controlled and precise manner. Additionally, NanoFabTx™ nanoparticle manufacturing could be scaled up across the NanoAssemblr® Platform.
The NanoFabTx™ DOTAP lipid mix (926027) is designed to prepare specifically sized cationic liposomes for gene delivery applications. The lipid mix contains rationally selected lipids to achieve a specific size range and positive charge for efficient complexation with negatively charged nucleic acids. Here, mRNA containing DOTAP liposomes were prepared using the NanoAssemblr® and NanoFabTx™ DOTAP lipid mix (926027).
Luciferase reporter mRNA was encapsulated in DOTAP liposomes using the NanoAssemblr® following the protocol provided below. Reproducible mRNA containing DOTAP liposomes were prepared of desired size (approximately 120nm) with an encapsulation efficiency of approximately 90%. Here, an N/P ratio of 5:1 and a flow rate ratio (FRR) of 3:1 was chosen and can be optimized depending on mRNA/nucleic acid and desired liposome size.
Luciferase Reporter mRNA-Containing Catatonic DOTAP Liposomes: mRNA containing NanoFabTx™ DOTAP liposomes prepared with the NanoAssemblr® Ignite
The in vitro transfection of mRNA-containing DOTAP liposomes was assessed. mRNA-containing DOTAP liposomes were delivered to HeLa cells. After 24 hours, the mRNA containing liposomes and empty liposomes were cytocompatible, resulting in 100% cell viability. Transfection of mRNA was evaluated via luciferase (luminescence) expression. mRNA delivered in the DOTAP liposomes expressed the targeted protein, whereas the untreated cells, and those treated with empty liposomes did not.
The protocol listed below provides step-by-step instructions on mRNA containing DOTAP liposomes using the NanoAssemblr® and NanoFabTX™ DOTAP lipid mix (926027). This protocol was optimized for a specific reporter mRNA strand as a model. It is suggested as a guide for your own optimization, and additional modifications may be necessary for other therapeutics/nucleic acids of interest.
We offer additional NanoFabTx™ formulation screening kits, lipid mixes, and microfluidic device kits. Our R&D scientists have carefully selected and curated each formulation kit and lipid mix to ensure reproducible and unform nanoparticle production to best suit researchers’ drug delivery applications. Each kit has been designed to be preparation method agnostic— nanoparticles can be generated with standard laboratory glassware or NanoAssemblr® instruments following the general protocols described here.
For material requirements, notes, reagent preparation, and drug quantification, please refer to the protocol included with the NanoFabTx™ PEG-PLGA drug formulation screening kit (917796).
For additional material requirements, notes, and reagent preparation, please refer to the protocol included with NanoFabTx™ DOTAP lipid mix (926027).
This is a generalized method (for guidance only) used to quantify the mRNA encapsulation efficiency of DOTAP lipid nanoparticles by Quant-it™ RiboGreen RNA Assay Kit. Note: the fluorescent sensitivity is instrument-dependent. If the fluorescence signal is too low, you can decrease the dilution ratio.
Encapsulation efficiency is calculated by:
EE (%) = encapsulated mRNA ÷ total mRNA added x 100
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