Non-Invasive Particle Size Monitoring of Polymer-Based Vectors for Cell Transfection in Process Vessels

Accurate monitoring of nanoparticle size and growth is essential to ensure scalability in the development of transfection vectors for gene delivery. This study highlights how Spatially Resolved-DLS (SR-DLS) measurements using a NanoFlowSizer (NFS) instrument allow tracking of the particle size in real-time during the complexation of nucleic acids and polyethyleneimine (PEI).

Monitoring particle size can be achieved through any transparent reactor vessel wall, regardless of size, design, or material. The utility of the NFS in monitoring such reactions is shown using a model system based on bovine serum albumin (BSA) and PEI under various experimental conditions.

By providing non-invasive, real-time measurements, the NFS facilitates the optimization of nanoparticle production parameters with full freedom in vessel geometry. These findings contribute valuable insights into the production of transfection complexes while safeguarding their sterility.

Introduction

Transfection is a critical mechanism in gene delivery, enabling the delivery of genetic material such as DNA or RNA into target cells.^1,2 Various transfection methods are available, including chemical, physical, and biological approaches, each with advantages and limitations.³ 

A key challenge in transfection is achieving precise control over the size and dispersity of transfection vectors, such as polymer or cation complexes with biomolecules, as well as liposomes, viruses, and other delivery systems. Smaller particles are more readily internalized by cells, while larger aggregates may hinder cellular uptake or trigger immune responses. Understanding these vectors' formation mechanisms, and complexation kinetics by monitoring their size is thus essential to ensure good transfection.⁴ Additionally, ensuring sterility during reagent preparation is crucial for an uncontaminated and reliable drug end product.

A well-established method for synthesizing transfection vectors is the complexation of a positively charged molecule—typically a polymer or lipid—with a negatively charged biomolecule, such as RNA or DNA.^5,6 

In this study, InProcess Instruments monitor the complexation of Bovine Serum Albumin (BSA), a protein with polyethyleneimine (PEI), a cationic polymer commonly used as a gene carrier.^7,8 The PEI/BSA complexes (Figure 1) serve as a representative model for transfection vectors.

Figure 1. Combination of BSA and PEI to generate nanoparticles via complexation. Image Credit: InProcess-LSP

To accurately characterize the size of PEI/BSA complexes in real time, we utilized the NanoFlowSizer (NFS), an innovative advanced DLS instrument developed by InProcess-LSP (Figure 2).

Unlike traditional DLS, which often requires sample dilution or extensive preparation, the NFS enables non-invasive, inline size measurement through Spatially Resolved Dynamic Light Scattering (SR-DLS).⁹ Furthermore, SR-DLS also enables measuring highly turbid samples and monitoring the dynamic size changes of nanoparticles during processes such as milling, emulsification, filtration, complexation, etc.

Figure 2. A) NanoFlowSizer system: probe unit, base unit, and dedicated PC. B) Integrated on a dedicated trolley. Image Credit: InProcess-LSP

Spatially Resolved Dynamic Light Scattering (SR-DLS): The Technique in Depth

The NanoFlowSizer (Figure 2) measures the size characteristics of particles from 5 nm to 5 µm via novel but now well-established SR-DLS.^9,10 The unique spatial resolution of the technique provides unprecedented capabilities in terms of measurement of extremely turbid suspensions and, particularly relevant for Process Analytical Technology (PAT) and in-line monitoring, the capability to measure in practically relevant flows.

Standard DLS is based on measuring the diffusion rate of suspended nanoparticles by analyzing the fluctuations of scattered (laser) light originating from many particles in a relatively large scattering volume. Due to the Brownian motion of the suspended particles, the scattered signal fluctuates at a rate proportional to the particle diffusion rate (Figure 3 A).

Using the correlation function of the scattered signal, the diffusion constant is measured and translated into the hydrodynamic size of the particles via the Stokes-Einstein relation.^11,12 Standard DLS measurement and analysis require a homogeneous suspension in which particle motion is limited to Brownian diffusion (static liquids without flow), and that has low levels of multiple scattering (low concentration samples).

Figure 3. A) Conventional dynamic light scattering (DLS) principle, B) Spatially resolved dynamic light scattering (SR-DLS) principle. Image Credit: InProcess-LSP

SR-DLS circumvents these limitations using Low Coherence Interferometry (LCI). In SR-DLS, near Infra-Red (~1300 nm) broadband light illuminates the samples and (180º) backscattered light is then mixed with the reference beam in an interferometer.

From the measured spectrum of this mixed signal, scattered signals from different depths in the sample can be resolved simultaneously. Typically, 1000 consecutive depths with a resolution of a few microns and a total depth in the sample of a few millimeters (Figure 3 B) are thus analyzed. This allows the NFS to measure the Particle Size Distribution (PSD) and the related Polydispersity Index (PDI) or the size covering 90 % of the particles (D90) in highly turbid suspensions and during flow.^9,13,14 

With a typical measurement time of 5 seconds, the NanoFlowSizer can be used as a real time PAT tool to monitor and control processes in which particle size is a Critical Quality Attribute (CQA).¹⁵

The NFS further enables measurements in any transparent geometry (Figure 4) through its adjustable focus and path length range and the collection of (180°) backscattered light. This renders the measurements geometry-independent, allowing accurate particle size characterization even through curved or irregular container boundaries. Examples are syringes, plastic IV bags, bottles, vials flow cells. The instrument thus also keeps the product in a sterile environment during analysis.^16,17

Figure 4. Various containers in which the NanoFlowSizer can perform non-invasive nanoparticle size measurement, whilst maintaining the sterility of the process. Image Credit: InProcess-LSP

Besides the spatially resolved analysis of the intensity of the scattered light, the NFS has recently been upgraded with two novel and powerful measurement modes.

One is phase-sensitive spatially resolved measurement of the full scattered electromagnetic field, called PhaSR-DLS.¹⁸ The PhaSR-DLS mode significantly enhances the sensitivity for small particles (e.g., proteins) and low concentration, reduces noise for enhanced detail of the particle size distribution (PSD), and extends measurements to larger particles at smaller concentrations.¹⁸ 

A second measurement mode recently introduced is Large Particle Detection (LPD) based on additional lateral scanning to yield images and statistics of single ‘large particles’ in the suspension that form the ‘minority’ tail of the PSD.¹⁹ The LPD mode in the NFS thus offers additional inline/PAT characterization of very rare aggregates or oversized particles in the suspension.

Investigating the Impact of Process Parameters on Transfection Complex Formation

Small particles are more likely to be taken up by cells via endocytosis, whereas larger particles or aggregates may be excluded from cellular entry.²⁰ Consequently, monitoring the transfection complexes' size during formation is crucial to guarantee their efficacy.

As will be shown, such monitoring can uniquely be performed using the NanoFlowSizer SR-DLS technology, as it avoids the need for sampling and dilution, and has the geometric measurement flexibility to non-invasively monitor complexation in any container, features that are lacking in standard DLS. In particular, the NFS is here employed to study the impact of several process parameters on the size and growth of BSA/PEI complexes.

The employed method for complex formation follows that by Hergli et al.²¹​

The BSA solution in a vial was positioned in front of the NFS and the PEI solution was then added dropwise. The complexation process was analyzed in real time for a range of different formulations and ‘process’ parameters that are expected to affect the complexation: (i) the molecular weight of the PEI polymer, (ii) the mass ratios of BSA:PEI,(iii) the addition of a NaCl salt and (iv) the total volume of the container.

Impact of Molecular Weight (Mw) of PEI, BSA:PEI Mass Ratio, Salt Addition and Volume of the Container

The molecular weight of PEI and, more generally, of any type of polymer can significantly influence the kinetics and final size of the complexation.^22,23 Using the NFS, various molecular weights were investigated for a BSA:PEI mass ratio of 20:1.

Figure 5 A shows that increasing the PEI molecular weight leads to the formation of larger particles for similar timeframes: complexing BSA with 2.5 kDa Mw PEI yields a particle size of around 200 nm (after 1 hour of monitoring) while utilizing PEI with Mw of 250 kDa triples the final particle size. This confirms that higher Mw polymers generate bigger complex particles and growth at a faster rate compared to lower Mw but otherwise identical polymers.

Besides the molecular weight of PEI, the mass ratio of polymer (in this case, PEI) to DNA is also known to change the size of the resulting particles.²⁴ The complexation kinetics were studied for four different BSA:PEI mass ratios in aqueous suspensions.

The results are shown in Figure 5 B and demonstrate that increasing the proportion of PEI in the suspension results in a larger final complex size after one hour and an accelerated growth rate of the BSA/PEI complexes. Doubling the amount of PEI can lead to a fourfold increase in the final size of the complex. This is likely due to the balance of charges between the positively charged PEI and the negatively charged BSA.^25,26

The growth curve for the 10:1 BSA:PEI mass ratio—and, to a lesser extent, other ratios—shows a swift growth rate in the initial minutes of the analysis.

Following this initial quick nucleation phase, there is an inflection point where the growth rate changes and becomes linear, likely due to the aggregation of already formed complexes or due to an Ostwald ripening phenomenon.²⁷ This results in clusters of varying sizes, and more heterogeneity in the suspensions, leading to fluctuations in the measured trends.

The measured polydispersity index (PDI) indeed progresses from 0.23±0.02 at the initial stages of the experiment to 0.55±0.04 after 30 minutes and progresses up to 0.65±0.05 after one hour. Similarly, the backscattered intensity detected increased from 4500±100 at the initial stages to 6000±300 after one hour, further confirming the presence of larger particles in the suspension. At these sizes (a couple of micrometers), the diffusion of the particles is also more difficult to measure as significant sedimentation is taking place. This leads to more fluctuations in the results.

Figure 5. Real-time complex hydrodynamic diameter (Z_av) of BSA/PEI complexes using the NFS . A) Different molecular mass of PEI and a fixed BSA:PEI mass ratio of 20:1. B) Different mass ratios of BSA:PEI with 250 kDa PEI. C) Different salt (NaCl) addition at 1000 seconds on PEI 2.5 k Da complexes and BSA: PEI mass ratio 50:1. D) Measurements were performed in Phosphate-buffered saline (PBS; pH 7.4, isotonic ionic strength, total 2 ml of sample. Image Credit: InProcess-LSP

In R&D and manufacturing of transfection complexes, growth of the complexes is typically done in the presence of salt that helps control their size by manipulating environmental conditions that affect the complexes’ stability and aggregation: Na⁺ and Cl^- ions compete with the cationic polymer (here PEI) and the negatively charged biomolecules (here BSA), which disrupts the electrostatic interactions that drive the complex growth.²⁸ Increasing the solution’s ionic strength by adding salt shields these charges and can prevent further aggregation or binding.^21,4

Figure 5 C shows in real time that after adding the salt solution without any stirring, the growth is immediately halted, and the complexes are disrupted, their size rapidly decreasing below 100 nm. For a higher salt content (40 mg), the complex size decreases even further than for an addition of 20 mg. It is likely that at these concentrations, there is too much NaCl and that it disrupts the PEI-BSA binding to the point that aggregation is minimal; some complexes may dissociate, causing the overall population’s particle size to decrease.²⁹

Lastly, studying reaction kinetics and nanoparticle size across different volumes is crucial when scaling up a process to ensure consistent product quality and efficiency. As volume increases, factors such as mixing efficiency and reactant diffusion can change, potentially altering the reaction rate, final size and overall uniformity of the suspension.

Understanding the influence of these factors at different scales allows for better optimization to maintain uniform particle size and reaction behavior, which is relevant for reproducibility and scalability in industrial production.³⁰ Therefore, different volumes have been investigated: 2 mL (vial) and 50 mL flask (Figure 5 D).

The results evidence that an increase in the volume in which the reaction takes place significantly increases the size of the particles after 1 hour as well as the reaction rate (from about 100 nm in the 2 mL container to about 2 µm in the 50 mL one). This suggests that scaling up the reaction results in the formation of larger particles.

These results also display a broader size distribution and more heterogeneous results when increasing the reaction volume and are confirmed by the measurement of the polydispersity index (PDI), which evolves from 0.25 at the beginning of the experiment to 1.1 after 30 minutes to 2.3 after one hour for the experiment performed in the 50 mL flask. The backscattered intensity also evolves from 4500 at the beginning to 5400 after one hour of measurement in the 50 mL flask, further confirming the presence of larger particles and a more heterogeneous suspension.

The NanoFlowSizer (NFS) then demonstrates its effectiveness as a tool for real-time monitoring of particle size during complexation processes, highlighting its potential for transfection vector development and real-time process monitoring. The NFS successfully tracked the size of BSA/PEI complexes in real time, measuring the impact of key parameters such as the mass ratio of BSA: PEI and the molecular weight of PEI on growth kinetics.

Additionally, it effectively monitored the influence of NaCl addition, showing that it can disrupt a particle’s stability, and displayed its ability to study the reaction for larger volumes, facilitating upscaling studies. This real-time capability is critical for monitoring upstream processes, controlling vector size and dispersity, improving transfection efficiency, and ensuring reliable gene delivery.

Monitoring of Nanoparticle Size Across Scalable Volumes and Challenging Geometries While Maintaining Sterility

Studying reaction kinetics and nanoparticle size across different volumes and containers is crucial when scaling up a process to ensure consistent product quality and efficiency. The NFS, thanks to the 180° backscattering detection and adaptable edge, allows the monitoring of the particle size inside a broad range of containers in a sterile manner. It can adapt to different geometries and materials such as glass vials and bottles (transparent and amber) ranging from a few mL to liters in volume, prefilled syringes, polycarbonate (PC) and polyethylene terephthalate (PET) vessels, IV bags, etc. as presented in Figure 6.

Figure 6. Various types of closed containers in which the NFS can perform measurements. A) Prefilled syringes, 10R vial, amber and glass bottles. B) IV bag C) Amber bottle, D) 10R vial on the sample holder. E) 250 mL IVLE bottle, taken from ^[17] F) PET container. Image Credit: InProcess-LSP

The NFS capabilities were demonstrated in a recently published white paper by measuring particle size inside two common but challenging geometries: large 20 liters of plastic containers of varying plastic composition (Figure 6 F) and closed, sterilizable IV bags (Figure 6 B).¹⁶ 

A silica suspension (1 % weight fraction in water) was measured in both types of containers. Two common materials for the 20-liter containers (Mixed4Sure) were chosen to highlight the material independence of the measurement: polycarbonate (PC) and polyethylene terephthalate (PET).

Independently of the plastic composition of the container, particles were precisely characterized at 120 ± 1 nm and 121 ± 1 nm, respectively, with PDI being 0.1 ± 0.1. The same suspension was tested in an IV bag (Flexboy), which is a very challenging geometry given its soft nature. Particle size was accurately measured at 122 ± 1 nm (PDI: 0.1 ± 0.1) inside the IV bag with a reliable reproducibility of the full PSD.¹⁶

The ability of the NFS to measure inside closed and sterile containers was also demonstrated in 2023 by Rooimans et al. (Figure 7 E).¹⁷ In this study, propofol emulsions (an anesthetic drug used to achieve sedation of patients, particularly critical during the COVID crisis) were analyzed non-destructively by diffuse reflectance near-infrared Spectroscopy (DR-NIRS) and simultaneously by SR-DLS. The mixing of propofol in a sealed IVLE bottle (250 mL) was studied, and the nanoparticle average size and PDI were obtained as a function of the propofol content.

These results further demonstrate the NFS versatility in measuring nanoparticle size and polydispersity across a variety of challenging geometries and container materials in a sterile manner. This ability makes it an invaluable tool for process monitoring and quality control.

Conclusion

The NanoFlowSizer directly addresses the challenges highlighted in transfection vector development by enabling real-time, non-invasive measurement of particle size and size distribution without the need for dilution or extensive sample preparation. During the synthesis of BSA/PEI transfection complexes, the NFS effectively monitored the impact of critical parameters, such as the molecular weight of PEI, the BSA-to-PEI mass ratio on the particle size and complexation kinetics. It was also demonstrated that the addition of NaCl disrupts the complexes and leads to a sudden decrease in particle size.

Additionally, the NFS demonstrated its reliability in upscaling studies by maintaining accurate measurements across a variety of container volumes and materials. Its ability to perform in challenging geometries, such as syringes, IV bags, and large-scale vessels, while preserving sterility ensures its versatility for both research and industrial applications.

By facilitating the optimization of process parameters and ensuring robust quality control, the NFS proves to be a valuable PAT tool. This technology advances the development of transfection vectors by supporting the production of highly uniform and scalable nanoparticle formulations, ultimately contributing to more efficient and reliable gene delivery processes.

Materials and Methods

The method for the complex formation was obtained from Hergli et al.²¹​ The BSA solution was prepared by dissolving 50 mg BSA in 10 mL PBS for a 5 mg/ml concentration. The PEI solution was prepared by dissolving 5 mg PEI in 10 mL demineralized H₂O for a 0.5 mg/ml concentration.

The analysis was started by placing the BSA solution in a 2R vial that was placed on the NFS (Figure 6). The PEI solution was then added dropwise to the BSA solution without stirring. The complexation was analyzed for 60 minutes, and several parameters that are assumed to influence the complex size and growth were investigated: (i) the molecular weight of the PEI polymer, (ii) the mass ratios of BSA:PEI,(iii) the addition of a NaCl salt and (iv) the total volume of the container.

1. PEI molecular weights of 2.5-25-100 and 250 kDa in a 2 mL 10R vial.
2. BSA:PEI mass ratio, 10:1-20:1-50:1 and 100:1 (PEI 250 kDa) in a 2 mL 10R vial.
3. Addition of 20 and 40 mg of NaCl (without stirring) to the suspension (PEI 2.5 kDa, BSA:PEI mass ratio 10:1) in a 2 mL 10R vial. 
4. Reaction volumes of 2 mL and 50 mL (PEI 2.5 k Da, BSA:PEI mass ratio of 50:1).

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This information has been sourced, reviewed and adapted from materials provided by InProcess-LSP.

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